InteractiveFly: GeneBrief

Ribosomal protein S6 kinase : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The phosphorylation of ribosomal protein S6 (RPS6 or simply S6) is a rapid and highly conserved cellular growth response that is observed during development and/or in response to a variety of extracellular stimuli. This phosphorylation is correlated with regulation of mRNA translation which, in turn, may influence cell size, cell proliferation or differentiation. The kinase responsible for the phosphorylation of RPS6 in mammalian cells is the serine/threonine kinase p70^S6k (S6k).

Increased RPS6 phosphorylation has been found to be paralleled by the selective translational up-regulation of a class of mRNAs containing an oligopyrimidine tract at their transcriptional start site, termed the 5'TOP. The genes representing this family of mRNAs are small in number, containing no more than 200 members, but can account for up to 20% of total cellular mRNA. 5'TOP mRNAs encode for a number of components of the translational apparatus, including ribosomal proteins and translation elongation factors. Recent studies have shown that the up-regulation of these mRNAs is largely suppressed by the antibiotic rapamycin, a bacterial macrolide. In parallel, rapamycin also abolishes S6K activation, and subsequent mitogen-induced S6 phosphorylation. More importantly, the suppressive effects of rapamycin on 5'TOP translation can be rescued by coexpression of recombinant S6K1 rapamycin-resistant mutants, providing a causal link between S6K1 activation and translational up-regulation of 5'TOP mRNAs. The effects of S6K activation on the translation of 5'TOP mRNAs are thought to be mediated through the increased phosphorylation of S6. S6 itself has been mapped by protein-protein and RNA-protein cross-linking studies to the mRNA-tRNA binding site of the 40S ribosome, suggesting that it might have a role in the regulation of mRNA translation. Following mitogenic stimulation, up to 5 mol of phosphate is incorporated in an ordered fashion into mammalian S6. In mammals, the phosphorylation sites have been mapped to a short sequence at the carboxy terminus of the molecule, with phosphorylation progressing in an ordered fashion from Ser236, Ser235, Ser240, Ser244 and Ser247 (Radimerski, 2000 and references therein).

Like RPS6 phosphorylation, activation of S6k, the protein that targets RPS6, is a highly conserved mitogenic response. Activation of S6k is regulated by phosphatidylinositol 3-kinase (PI3K: see Drosophila Phosphotidylinositol 3 kinase 92E). The PI3K inhibitor wortmannin abrogates the mitogen-stimulated activation of S6k. A separate pathway contributing to S6k activation involves the FKBP12-rapamycin (RAP)-associated protein (FRAP, also known as mTOR, RAFT, and RAPT) and has been demonstrated using the immunosuppressant RAP. RAP, through complex formation with FKBP12 and FRAP/mTor causes G₁-phase arrest in T lymphocytes and other hematopoietic tissues. Many of the effects of RAP correlate with the inactivation of S6k and inhibition of protein synthetic activities. FRAP/mTor activity is regulated directly by Protein phosphatase 2A (Drosophila homolog: Twins) which also interacts directly with the S6k. S6k is activated by PP2A inhibition of FRAP/mTor (Peterson, 1999).

Inhibition of p70^S6k activation results in the reduction of RPS6 phosphorylation that apparently leads to the selective inhibition of protein synthesis and a delay or arrest at the G₁/S phase of the cell cycle in certain cell types. The requirement for p70^S6k activity during G₁ phase was shown in experiments using neutralizing p70^S6k antibodies that prevented both the activation of protein synthesis and the serum-induced entry of cells into S phase. S6k (RPS6-p70-protein kinase) has been identified in Drosophila (Stewart, 1996 and Watson, 1996), and has been found to regulate cell size in a cell-autonomous manner without impinging on cell number (Montagne, 1999). One of the activating kinases of mammalian p70^S6k has been identified: mammalian target of rapamycin (mTOR). The Drosophila mTor homolog, dTor, similarly acts to activate Drosophila S6k (Zhang, 2000).

Drosophila deficient in the S6k gene exhibit an extreme delay in development and a severe reduction in body size. These flies have smaller cells rather than fewer cells. The effect is cell-autonomous, displayed throughout larval development, and distinct from that of ribosomal protein mutants (Minute mutants). A female sterile mutant, fs(3)07084, was found to contain a P-element insertion in the 5' noncoding region of the Drosophila S6k gene. Only 25% of the expected number of homozygous flies emerge as adults, with a 3-day delay and reduced body size. This phenotype is rescued either by excision of the P element or by a Drosophila S6K or mammalian S6K transgene. Northern (RNA) blot analysis and sequencing of a reverse transcriptase polymerase chain reaction product (RT-PCR) from homozygous mutant flies reveals the presence of anomalous transcripts, suggesting that S6k expression may persist in homozygous mutant flies. More severe alleles were generated by imprecise P-element excisions, removing part of the S6k gene. Most of these flies die as larvae, with the lethality rescued by expression of Drosophila S6k or mammalian S6K transgenes. One of the excisions, dS6K^l-1, removes part of the first exon, including a portion of the catalytic domain. The few surviving dS6K^l-1 homozygous flies emerge after a 5-day delay, live no longer than 2 weeks, and display a severe reduction in body size, with all body parts apparently affected to the same extent. Thus, loss of S6k function induces female sterility, a strong developmental delay, a severe reduction in growth, and often death (Montagne, 1999).

Since mammalian S6Ks control the synthesis of ribosomal proteins, it was hypothesized that the dS6K^l-1 phenotype might be equivalent to that of Minutes. The Minute M(3)95A, harbors a P-element insertion that severely reduces the expression of ribosomal protein S3. However, analysis of M(3)95A and two other Minutes shows no effect on size, although all display a developmental delay and slender bristles. In contrast, the bristles of homozygous dS6K^l-1 flies are proportional to body size. To determine whether the reduction in body size of homozygous dS6K^l-1 flies is due to a decrease in cell number, cells in wings and ommatidia in eyes of wild-type and dS6K mutant flies were compared. The cell density is greater in wings of homozygous dS6K^l-1 flies (as represented by each hair) than in wild-type flies. The difference in cell size is almost 30%, and flies homozygous for partial loss of function dS6K⁰⁷⁰⁸⁴ display an intermediate cell size. However, the total number of cells in wings remain constant. Analysis of eyes reveals a similar phenotype with reduced size but no effect on the number of ommatidia. Thus, in S6k mutants the decrease in the rate of proliferation is probably attributable to a reduction in ribosomal protein synthesis, whereas the effect on cell size may be due to the absence of S6 phosphorylation and an altered pattern of translation (Montagne, 1999).

The reduction in cell size of dS6K^l-1 flies indicates either that cells are proliferating at a smaller size or that flies emerge from the extensive developmental delay before completion of the last round of cell growth. To examine these possibilities, proliferating epithelial cells from the imaginal wing disc of larvae were analyzed at the end of the third instar. Imaginal discs give rise to the adult structures. At the end of the third instar, wing disc cells still require two mitotic cell cycles before they differentiate. Comparison of wing discs from homozygous dS6K^l-1 and wild-type larvae reveals that mutant discs are substantially smaller in size. Analysis of single cells from discs with a fluorescence-activated cell sorter (FACS) confirms that, on average, cells derived from S6k mutants are smaller than wild-type cells. There was no apparent difference between the distributions of S6k mutant and wild-type cells within each phase of the cell cycle, implying that the dS6K^l-1 loss-of-function mutation affects all stages of the cell cycle. Analysis of disc cells during puparium formation, when proportionally more cells are present in G₂ phase, also shows no detectable difference in the cell cycle distribution of mutant and wild-type cells. In addition, the number of wing disc cells present in somatically induced clones, marked by ectopic expression of beta-galactosidase, is reduced in mutant versus wild-type larvae. Consistent with this, cell cycle times are 12.5 ±1 hours and 24 ± 4 hours for wild-type and mutant wing disc cells, respectively. Thus, loss of Drosophila S6k function leads to cell proliferation at a smaller size and at a reduced rate, without affecting any specific stage of the cell cycle (Montagne, 1999).

S6k mutants may affect cell size through the loss of a humoral factor that regulates cell growth. To examine this possibility, genetically marked homozygous mutant cells were generated in a heterozygous mutant background by somatic recombination. At the wing margin, homozygous S6k mutant sensory bristles, identified by a yellow (y minus) marker, are reduced in size compared with their neighbors. In eyes, homozygous S6k mutant photoreceptor and pigment cells are marked by a white (w minus) mutation and recognized by the absence of red pigment, appearing as dark spots in photoreceptor cells. Again, only mutant cells are reduced in size, indicating that S6k acts in a cell-autonomous manner. Because dS6K mutations affect size in a cell-autonomous manner, expression of an extra copy of the wild-type gene in a specific compartment might positively affect growth. A compartment represents an independent unit of growth and size control, thought to be analogous to a mammalian organ. The wing disc is composed of a dorsal compartment and a ventral compartment that fold in an apposed manner at the wing margin to generate the flattened wing blade. Because the apterous promoter is only functional in the dorsal compartment of the wing disc, it was coupled to the GAL4 transcription factor to induce an extra copy of the S6k gene linked to a UAS responsive element. An increase in cell size of less than 1% should alter the morphology of the adult wing blade. In all UAS S6k lines examined, S6k protein expression was increased and the wing was convex and bent downward. The phenotype can be explained by an increase in the size of the dorsal versus the ventral wing surface, forcing the wing blade to curve down to accommodate the greater surface. Therefore, increased expression of S6k positively affects growth in a cell-autonomous and compartment-dependent manner (Montagne, 1999).

S6Ks appear to be downstream effectors of the phosphatidylinositide-3OH kinase (PI3K) signaling pathway. However, activated or dominant interfering alleles of PI3K affect cell number and cell size. This would imply that S6Ks reside on a branch of the PI3K signaling pathway that controls cell growth and size but not cell number. Overexpression of the cell cycle regulator E2F in the posterior compartment of the wing disc increases cell number without affecting final compartment size. These findings are consistent with the hypothesis that compartments, like organs, adjust their final mass independent of cell number. However, the Drosophila S6k phenotypes suggest that cell size participates in the control of compartment size. Indeed, S6Ks are thought to play a critical role in organ hypertrophy, where the organ increases in size as a function of demand (Montagne, 1999).

It is concluded that flies deficient in S6k exhibit an extreme delay in development and a severe reduction in body size. These flies have smaller cells rather than fewer cells. The effect is cell-autonomous, displayed throughout larval development, and distinct from that of ribosomal protein mutants. Thus, the S6k gene product regulates cell size in a cell-autonomous manner without impinging on cell number (Montagne, 1999).

S6 kinase localizes to the presynaptic active zone and functions with PDK1 to control synapse development

The dimensions of neuronal dendrites, axons, and synaptic terminals are reproducibly specified for each neuron type, yet it remains unknown how these structures acquire their precise dimensions of length and diameter. Similarly, it remains unknown how active zone number and synaptic strength are specified relative the precise dimensions of presynaptic boutons. This paper demonstrates that S6 kinase (S6K) localizes to the presynaptic active zone. Specifically, S6K colocalizes with the presynaptic protein Bruchpilot (Brp) and requires Brp for active zone localization. Evidence is provided that S6K functions downstream of presynaptic PDK1 to control synaptic bouton size, active zone number, and synaptic function without influencing presynaptic bouton number. It was further demonstrated that PDK1 is also a presynaptic protein, though it is distributed more broadly. A model is presented in which synaptic S6K responds to local extracellular nutrient and growth factor signaling at the synapse to modulate developmental size specification, including cell size, bouton size, active zone number, and neurotransmitter release (Cheng, 2011).

Dendrite diameter, the size of a dendritic field, and the area of the presynaptic nerve terminal are all reproducibly specified for a given cell type. The size and complexity of a neuron far exceeds that of any other cell type, suggesting that there may be unique solutions to the challenge of controlling and coordinating the growth of the many different and distinct features of neuronal architecture. Specification of neuronal dimension is further complicated because it is intimately associated with the electrochemical function of the neuron. Indeed, a specific form of neuronal growth, isoelectronic growth, has been observed in both invertebrates and vertebrates in which dendrite diameter grows precisely as the square of dendrite length, thereby maintaining the cable properties of individual dendrites. This type of growth is fundamentally different from most types of neuronal growth that are characterized experimentally, including axon extension, dendrite branching, spine formation, and synapse expansion. It remains unknown how the dimensions of individual neuronal compartments can be precisely specified and how these growth-related parameters are coordinated with neuronal function. Mechanistically, very few studies report the identification of genes that specifically control neuronal dimensions without otherwise perturbing the ability of these structures to form properly (Cheng, 2011).

Candidates for the control of neuronal dimension are genes associated with the regulation of cell size, including mammalian target of rapamycin, PDK1, and S6 kinase (S6K). S6K is necessary for long-term facilitation, the early phase of long-term potentiation, learning, and activity-dependent neuronal sprouting during epilepsy. S6K also influences the growth of dendritic arbors in cultured hippocampal neurons. However, there are conflicting studies regarding whether S6K influences bouton number at the Drosophila melanogaster neuromuscular junction (Cheng, 2011).

The formation of complex neural circuitry is largely determined by the sequential processes of axon guidance, target recognition, and the activity-dependent refinement of synaptic connectivity. However, the dimensions of individual neuronal compartments, including the length and diameter of dendrites, axons, and presynaptic boutons are also reproducibly specified for each cell type, and these parameters strongly influence neuronal function. It was previously established that PDK1 and S6K control cell size, but it has remained unclear how these potent signaling molecules influence synaptic growth. This study provides evidence that signaling through PDK1 and S6K is specifically required to control synaptic bouton size without strongly influencing the proliferation of bouton number or the length of the nerve terminal. Many genes have been identified that perturb synapse morphology at the Drosophila NMJ when mutated. In most cases, however, the gross morphology of the synapse is perturbed, indicating that the cellular mechanics of nerve terminal extension are altered. In contrast, signaling via PDK1 and S6K primarily control the dimensions of individual synaptic boutons without otherwise altering the appearance of the presynaptic terminal (Cheng, 2011).

How are changes in compartment dimensions (cell diameter, axon diameter, and bouton diameter) mechanistically executed downstream of PDK1 and S6K? It is particularly interesting that both PDK1 and S6K are distributed throughout the cell, being present in axons, presynaptic terminals, and in the case of S6K, at the active zone. This distribution suggests that PDK1 and S6K could exert local effects that contribute to the specification of cell shape. Indeed, there is a correlation between impaired S6K localization at the active zone in the brp mutant and decreased bouton size as well as decreased axon size. Future experiments will be necessary to determine whether the changes in compartment size are a direct consequence of S6K mislocalization or whether size is influenced by altered synaptic transmission in the brp mutant. It is interesting to speculate about the cell biological processes that might function downstream of S6K to influence cellular dimensions. A previous study has provided evidence that the submembranous skeleton composed of spectrin and ankyrin provides structural integrity and modulates the shape of axons and presynaptic terminals, in part through organization of the underlying microtubule cytoskeleton (Pielage, 2008). However, a connection between PDK1, S6K, and the submembranous spectrin/ankyrin skeleton remains to be identified. In other systems, S6K has been shown to interact with spinophillin/neurabin, which is an F-actin- and protein phosphatase 1-binding protein linked to the control of dendritic spine size in vertebrates and active zone integrity in Caenorhabditis elegans. A link between S6K and neurabin/spinophillin could provide a mechanism to link local nutrient detection to the modulation of the neuronal cytoskeleton and cell shape (Cheng, 2011).

It remains unknown how active zone number is specified for a given cell type. The neuromuscular synapse, the Calyx of Held, and other large, powerful synapses harbor a robust and reproducibly large number of active zones. In contrast, other neuronal cell types make synaptic connections composed of fewer active zones, many forming a single release site within a synaptic bouton. Genes have been identified that negatively regulate active zone assembly. Other genes have been identified that are necessary for correct placement of active zones, active zone assembly, and active zone dimension. In contrast, the specification of total active zone number has remained less well defined. Target-derived growth factors clearly influence the growth and development of the presynaptic nerve terminal and have been shown to influence total active zone number. These signaling systems will activate downstream intracellular signaling cascades, such as MAPK signaling, recently implicated in the specification of active zone number at the Drosophila NMJ (Wairkar, 2009). However, it also seems likely that the control of active zone number during development will be more complex than simple specification based upon the quantity of a target-derived trophic signal. It is speculated that there will exist cell type-specific programs that interface with growth factor signaling to determine characteristic active zone densities. Cellular metabolic signaling might be one such cell type-specific parameter, including the actions of S6K and PDK1 (Cheng, 2011).

It is particularly remarkable that presynaptic overexpression of S6K and PDK1 are sufficient to increase active zone number at the NMJ. These data indicate that synapse assembly during neuromuscular development is a process that can be driven by signaling that originates within the motoneuron. It is speculated that the localization of S6K at the active zone may be critical in this regard. In some respects, it seems counterintuitive that a signaling system coupled to the metabolism of the motoneuron (PDK1-S6K) would be able to determine active zone number and, therefore, the level of postsynaptic excitation. Therefore, in keeping with well-established trophic mechanisms, it is predicted that S6K and PDK1 normally function downstream of muscle-derived factors, including nutrients and growth factors, that couple the needs of the muscle to the insertion of active zones by the motoneuron (Cheng, 2011).

There is increasing evidence that local protein synthesis plays a prominent role within postsynaptic dendrites. In contrast, the evidence for presynaptic protein translation is less abundant, including an apparent absence of polyribosomes within axons and nerve terminals. The most convincing evidence for local presynaptic protein translation is observed in Aplysia, in which serotonin-dependent long-term facilitation can be induced in synaptic compartments separated from the soma. Interestingly, two recent studies implicate S6K in presynaptic, translation-dependent long-term facilitation in Aplysia. The localization of S6K to the presynaptic active zone, where additional RNA-interacting signaling molecules have recently been identified, is intriguing. In Drosophila, the translational repressors nanos and pumillio have been implicated in the regulation of neuromuscular growth, membrane excitability, and postsynaptic glutamate receptor abundance. Nanos is present presynaptically, and recent data demonstrate that loss of nanos leads to an increase in total bouton number and an increase in the total active zone number. In a separate study, the IGF-II RNA-binding protein (Imp-GFP) was observed to traffic to the presynaptic terminal, and loss of Imp caused a decrease in bouton number. Although none of these data provide direct evidence for local presynaptic protein translation being important for presynaptic development or function, there is an accumulation of data suggesting that this may be a realistic possibility. Perhaps local protein translation, downstream of nutrient and growth factor signaling, could help independently shape the dimensions of each neuronal compartment (soma, axon, dendrite, and nerve terminal) and, thereby, fine tune the input-output properties of neurons during development. In this regard, it is particularly interesting that the synaptic localization of S6K by Brp seems to be important for size regulation at the synapse and perhaps throughout the cell (Cheng, 2011).

CycD/Cdk4 and discontinuities in Dpp signaling activate TORC1 in the Drosophila wing disc

The molecular mechanisms regulating animal tissue size during development are unclear. This question has been extensively studied in the Drosophila wing disc. Although cell growth is regulated by the kinase TORC1, no readout exists to visualize TORC1 activity in situ in Drosophila. Both the cell cycle and the morphogen Dpp are linked to tissue growth, but whether they regulate TORC1 activity is not known. This study developed an anti-phospho-dRpS6 antibody that detects TORC1 activity in situ. Unexpectedly, it was found that TORC1 activity in the wing disc is patchy. This is caused by elevated TORC1 activity at the cell cycle G1/S transition due to CycD/Cdk4 phosphorylating TSC1/2.TORC1 is also activated independently of CycD/Cdk4 when cells with different levels of Dpp signaling or Brinker protein are juxtaposed. This study has thereby characterize the spatial distribution of TORC1 activity in a developing organ (Romero-Pozuelo, 2017).

During animal development, tissues increase tremendously in mass, yet stop growing at very stereotyped sizes in a robust manner. For instance, the Drosophila wing is specified as a cluster of circa 50 cells, which increases in mass ~500-fold before terminating growth. Once growth has ceased, the left and right wings of an individual fly are virtually identical in size, to within 1%, illustrating the robustness of this process. How animal tissue size is regulated is a fundamental open question in developmental biology (Romero-Pozuelo, 2017).

As mitotically growing tissues develop, two independent cellular processes occur in a coordinated manner: proliferation and cell growth. By itself, proliferation -- the division of cells -- does not lead to mass accumulation. This was nicely shown in the Drosophila wing where overexpression of E2F speeds up the cell cycle, but leads to a normally sized tissue containing more, smaller cells. For a tissue to grow, cells need to accumulate biomass. The mechanisms interconnecting cell proliferation and cell growth are not completely understood. In organisms from yeast to humans, growth is in large part regulated by the target of rapamycin complex 1 (TORC1) kinase. TORC1 promotes biomass accumulation by promoting anabolic metabolic pathways such as protein, lipid, and nucleotide biosynthesis, while repressing catabolic processes such as autophagy. Hence, to understand tissue growth it would be of interest to study the spatial distribution of TORC1 activity in a developing tissue. This line of investigation has been hampered, however, by the lack of readouts for TORC1 activity that can be used in situ (Romero-Pozuelo, 2017).

One signaling pathway that strongly affects tissue size is the Dpp pathway. Dpp is expressed and secreted by a stripe of cells in the medial region of the wing imaginal disc, and forms an extracellular morphogen gradient that both helps to pattern the wing and affects its size. In the absence of Dpp signaling during development, only small rudimentary wings are formed. In contrast, overexpression of Dpp leads to strong tissue overgrowth, in particular along the axis of the morphogen gradient. Several models have been proposed for how Dpp signaling regulates wing size. The exact mechanism by which Dpp regulates tissue size, however, is an unresolved issue. Dpp signaling acts to repress expression of a transcription factor called Brinker. Brinker appears to mediate most of the size effects of Dpp signaling. When Brinker is genetically removed, Dpp signaling becomes dispensable for wing growth. Given that Dpp signaling promotes tissue growth, an open question is whether Dpp signaling promotes TORC1 activity (Romero-Pozuelo, 2017).

Thia study examined whether Dpp signaling promotes TORC1 activity in the Drosophila wing disc. To this end, a phospho-RpS6 (pS6) antibody was developed that allows TORC1 activity to be assayed in situ in tissue. This reagent reveals unexpectedly that TORC1 activity in the growing wing disc is neither uniform nor graded, but is instead patchy. This patchiness is mediated via CycD/Cdk4 and the tuberous sclerosis 1 (TSC1)-TSC2 complex in response to cell cycle stage. Using this pS6 antibody, this study found that TORC1 activity is also induced by discontinuities in Dpp signaling or discontinuities in Brinker levels. It is proposed that these discontinuous conditions may be analogous to regenerative conditions that happen in the wing disc in response to tissue damage. In sum, this work reveals the pattern of TORC1 activity in the context of a developing organ (Romero-Pozuelo, 2017).

TORC1 activity in the wing disc is modulated by the cell cycle, with cells in early S phase showing the highest TORC1 activity. Interestingly, an accompanying paper finds similar results in the Drosophila eye disc (Kim, 2017). This might reflect a metabolic requirement by early S-phase cells for large amounts of nucleotide biosynthesis, an anabolic process promoted by TORC1. Indeed, in various contexts S6K and TORC1 activity were found to be required for the transition from G1 to S. Connections between mechanistic TOR (mTOR) and the cell cycle have previously been found in cultured cells. In human fibroblasts, mTOR shuttles in and out of the nucleus in a cell cycle-dependent manner, peaking in the nucleus shortly before S phase. The relevance of this subcellular relocalization to what is observe in this study, however, is unclear. In fibroblasts, S6K1 activity was found to be highest during early G1, whereas in HeLa cells it was found to be highest during M phase. In sum, it is unclear to what extent cells in culture recapitulate endogenous development, or whether the influence of the cell cycle on TORC1 activity is very context dependent. The TSC1/2 complex has been reported to be phosphorylated by cell cycle-dependent kinases.TSC1 is phosphorylated on Thr417 by Cdk1 during the G2/M transition. This inhibitory phosphorylation would lead to elevated TORC1 activity during G2/M, which does not fit with what was observe here, and thus might be relevant in a different developmental context. Instead, this study found that TSC2 can be phosphorylated by the CycD/Cdk4 complex on Ser1046, and possibly other sites as well, and that this leads to activation of TORC1. This fits with several observations in the literature. Firstly, in U2OS cells the TSC complex was also found to bind cyclin D, leading to its phosphorylation at unknown sites. In U2OS cells, this causes destabilization of the Tsc1 and Tsc2 proteins, which was not observed in this study. Secondly, Tsc1/2 and CycD/Cdk4 were previously found to interact genetically in Drosophila: The reduced tissue growth caused by Tsc1 + Tsc2 overexpression was found to be fully suppressed by expression of CycD + Cdk4. This fits well with the current data suggesting that CycD/Cdk4 directly inhibits the TSC complex via phosphorylation. Thirdly, Cyclin D and Cdk4 were previously reported in Drosophila to promote cell and tissue growth, fitting with activation of the TORC1 complex by CycD/Cdk4. It is worth noting that some patchy TORC1 activity is still seen in CycD- or Cdk4-null discs and in discs with the single phospho-site mutations in TSC2. Hence it is possible that Cdk4 may not be the only factor regulating TORC1 activity in response to the cell cycle, and that Cdk4 might phosphorylate TSC2 on additional sites (Romero-Pozuelo, 2017).

What are the roles of CycD/Cdk4 in cell cycle progression and cell growth? Whereas mammals have three cyclin D genes, CycD1-3, and two CycD binding kinases, Cdk4 and Cdk6, Drosophila has a single CycD, a single Cdk4, and no Cdk6. Hence Drosophila provides an opportunity to elucidate the function of the CycD/Cdk4 complex without difficulties arising from redundancy. Indeed, results in Drosophila clearly show that CycD/Cdk4 promotes cell growth and not cell cycle progression. Both CycD- and Cdk4-null animals are viable, and fluorescence-activated cell sorting (FACS) analysis of null cells revealed that they have a normal cell cycle profile, indicating that they are dispensable for normal cell cycle progression. Instead, Cdk4- and CycD-null animals are 10%-20% smaller than controls, indicating that they promote cell growth. The finding that CycD/Cdk4 activates TORC1 during the G1/S transition can provide one mechanism by which the CycD/Cdk4 complex promotes growth. Hence, from these data it is proposed that in Drosophila the CycD/Cdk4 complex is not part of the core machinery required for cell cycling, but is rather an effector 'side branch' activated at G1/S to promote cell growth. Data from the mouse suggest something similar. CycD1, CycD2, and CycD3 knockout mice are all viable. One could imagine this to be due to redundancy between these three genes, but actually CycD1, CycD2, CycD3 triple-knockout mice survive to mid-gestation, and the triple-knockout mouse embryonic fibroblasts proliferate relatively normally. The mid-gestation lethality of the triple knockouts appears to be due to specific effects in hematopoietic and myocardial cells. Hence, cyclins D1-D3 are also dispensable for cell cycle progression in mice. Interestingly, CycD1 knockout mice and CycD1, CycD2 double-knockout mice are viable but have reduced body size, reminiscent of the size phenotype observed in CycD knockout flies. In sum, despite CycD/Cdk4 being claimed in most reviews on the cell cycle as playing an important role in G1/S progression, it appears that this complex may function rather to promote cell growth in a cell cycle-dependent manner (Romero-Pozuelo, 2017).

Does Dpp control growth in the wing? When discontinuities in Dpp activity or in Brinker levels were genetically induce, activation was observed of TORC1 at the site of discontinuity. Hence, Dpp signaling per se does not appear to activate TORC1; rather, the comparison between high Dpp signaling and low Dpp signaling cells does. In an unperturbed disc, no pattern of pS6 staining was observed that correlates with the Dpp activity gradient, which is highest medially and drops toward the anterior and posterior extremities. This might be due to the fact that in an unperturbed disc the Dpp and Brinker gradients are smooth and do not have such discontinuities. A similar effect of Dpp was previously observed on cell prolife ration, except that in this case the effect of the Dpp discontinuity was very transient, lasting only a few hours after clone induction, whereas the effect seen on growth is sustained. Dpp signaling is, nonetheless, required for growth, because in the absence of Dpp, small vestigial wings are formed. Hence one interpretation might be that low levels of Dpp signaling are continuously required for growth, but that Dpp signaling becomes instructive for tissue growth only when discontinuities in the gradient arise, perhaps as a result of tissue damage or cell delamination, to initiate a regenerative response (Romero-Pozuelo, 2017).

One additional interesting non-autonomous phenomenon observed is that sometimes when a region of the wing disc has high pS6 levels, the rest of the disc loses its typically patchy pS6 pattern and becomes pS6 negative. This phenomenon is not understood, and future work will be necessary to understand it molecularly (Romero-Pozuelo, 2017).

REGULATION

Functional characterization of Drosophila Sk6

To determine whether the Drosophila S6k cDNA encodes a protein product equivalent to the endogenous protein, Drosophila Schneider line 2 (S2) cells were transiently transfected with a Drosophila S6k construct containing an myc epitope-tag at its N terminus. Following transfection, protein products were visualized by Western blot analysis, employing the monoclonal antibody 9E10. A protein band with Mr 70 kDa was specifically detected in extracts from transiently transfected cells, but was absent in extracts from control cells. To determine whether an equivalent protein could be detected in extracts of S2 cells, a rabbit polyclonal antibody (D20) was generated against a peptide representing amino acids 1-19 of the Drosophila S6k N terminus. This antibody specifically recognizes an endogenous protein of Mr 70 kDa in S2 cells, which comigrates with the kinase produced ectopically from transient transfection of the cDNA in S2 cells that is detected with either the D20 antiserum or the 9E10 antibody. Longer exposures of the film do not reveal additional bands that might be suggestive of an isoform equivalent to the mammalian p85 s6k. Preincubation of the D20 antiserum with the antigenic peptide prevents binding to Drosophila S6k. Thus, the Drosophila S6k cDNA encodes a protein that is antigenically equivalent to a Drosophila protein that migrates at a similar Mr (Stewart, 1996).

Activation of the rat p70 s6k is associated with phosphorylation of 10 residues, 5 of which are conserved in Drosophila S6k. By mutational analysis, 3 of the 10 sites, T229,S371, and T389, have been argued to be critical for p70 s6k activation. These sites are conserved in Drosophila S6k as T238, S380, and T398. In addition, the principal target of rapamycin-induced p70 s6k dephosphorylation and inactivation is T389, with T229 acting as a secondary target. However, for rapamycin to exert this inhibitory response on mammalian p70 s6k, the macrolide requires the acidic residues at the N terminus. This may be a feature also conserved in Drosophila S6k. To examine whether S2 cells contain S6 kinase activity and, if so, whether this activity is sensitive to rapamycin, mammalian 40S ribosomes were incubated with extracts from S2 cells treated with cycloheximide, an agent known to activate mammalian p70 s6k, in the absence or presence of the macrolide. Mammalian 40S ribosomes were employed in these assays because they were found to be as good a substrate as their Drosophila counterparts. Cycloheximide treatment of S2 cells increases S6 kinase activity 2.2-fold over basal levels and rapamycin pre-treatment prevents this increase, reducing activity below basal levels. However, a significant amount of rapamycin insensitive kinase activity toward S6 is still apparent (Stewart, 1996).

To distinguish between these rapamycin sensitive and insensitive S6 kinase activities and to determine whether either represents Drosophila S6k, extracts from cycloheximide-stimulated S2 cells, pretreated with or without rapamycin, were fractionated by Mono Q chromatography. The fractions eluted from the Mono Q column were monitored by A280 and subjected to an in vitro S6 kinase assay. Two peaks of S6 kinase activity emerged from the column. The first peak containing less S6 kinase activity eluted at 0.13 M NaCl, and was rapamycin insensitive. The second peak of activity eluted at 0.29 M NaCl, contained approximately 15-fold more S6 kinase activity, and was completely abolished by rapamycin pretreatment. To assess whether the S6 kinase activity in this peak is attributable to Drosophila S6k, Fast Flow Q Sepharose was used to concentrate the fractions 16-18 containing the rapamycin sensitive S6 kinase activity. The concentrated samples from cells treated with cycloheximide either in the absence or presence of rapamycin pretreatment, were either immunoprecipitated and assayed for S6 kinase activity or analyzed on Western blots employing the D20 antibody. The D20 antibody specifically immunoprecipitates native Drosophila S6k kinase activity from the sample stimulated with cycloheximide, but no activity could be detected from the sample pretreated with rapamycin prior to the addition of cycloheximide. Western blot analysis of these fractions reveals a slower electrophoretic mobility on SDSy PAGE for the active Drosophila S6k as compared with the inactive kinase. This altered electrophoretic mobility is similar to the mobility shift induced by phosphorylation of the mammalian p70 s6k, an effect that is ablated by rapamycin treatment. Thus, cycloheximide treatment induces the activation of Drosophila S6k, which is apparently regulated by phosphorylation and is rapamycin sensitive (Stewart, 1996).

To determine whether Drosophila S6k is regulated in a manner similar to the mammalian enzyme, HA-epitope-tagged Drosophila S6k was transiently expressed in NIH 3T3 cells. The addition of PDGF led to the decreased electrophoretic mobility of HA-S6k as determined by immunoblot analysis. The kinetics of the mobility shift are similar to that of mammalian p70^S6k, which exhibits multiple size forms in response to PDGF that are attributable to phosphorylation. This mobility shift coincides with the activation of HA-S6k, as measured by S6k's ability to phosphorylate RPS6. Futhermore, pretreatment with wortmannin or RAP potently inhibits PDGF-dependent phosphorylation and activation of HA-S6K. Thus these results indicate that the upstream regulatory elements present in mammalian cells are capable of activating Drosophila S6K and that activated S6K can use mammalian RPS6 as a substrate. Expression of another epitope-tagged S6k (myc-S6k) in COS cells confirms that the components required for the regulation of Drosophila S6K are present in other mammalian cell types (Watson, 1996).

Scylla decreases S6K but not PKB activity

Diverse extrinsic and intrinsic cues must be integrated within a developing organism to ensure appropriate growth at the cellular and organismal level. In Drosopohila, the insulin receptor/TOR/S6K signaling network plays a fundamental role in the control of metabolism and cell growth. scylla and charybdis (a. k. a. charybde), two homologous genes identified as growth suppressors in an EP (enhancer/promoter) overexpression screen, act as negative regulators of growth. The genes are named after mythological monsters that lived in the Strait of Messina between Sicily and Italy, posing a threat to the passage of ships. The simultaneous loss of both genes generates flies that are more susceptible to reduced oxygen concentrations (hypoxia) and that show mild overgrowth phenotypes. Conversely, either scylla or charybdis overactivation reduces growth. Growth inhibition is associated with a reduction in S6K but not PKB/Akt activity. Together, genetic and biochemical analysis places Scylla/Charybdis downstream of PKB and upstream of TSC1. Furthermore, scylla and charybdis are induced under hypoxic conditions and scylla is a target of Drosopohila HIF-1 (hypoxia-inducible factor-1: Similar) like its mammalian counterpart RTP801/REDD1, thus establishing a potential cross-talk between growth and oxygen sensing (Reiling, 2004).

To test whether the placement of Scylla between PKB and TSC can be corroborated biochemically, the effect of scylla overexpression on PKB and S6K activity was examined. PKB activity of adult female heads overexpressing scylla or charybdis was tested in conjunction with PKB and PDK1 under control of the GMR-Gal4 enhancer. The same experimental setup has previously been used to demonstrate that PDK1 increases PKB activity. Total fly head protein was extracted and PKB activity was assayed by incorporation of ³²P-labeled phosphate into a synthetic PKB substrate (Crosstide, CT). Although scylla/charybdis overexpression substantially suppresses the PKB/PDK1-induced bulging eye phenotype, PKB activity is not reduced in these eyes. Moreover, PKB activity is also unaffected in a scylla^-/- background (Reiling, 2004).

These results are consistent with the placement of Scylla downstream of PKB. To test the effect of Scylla on S6K activity, second instar larvae expressing scylla under the control of Act5C-Gal4 were collected, and larval extracts were assayed for S6K activity. On average, S6K activity was down-regulated by >50%. Although there may also be a slight reduction in total S6K protein levels, this effect cannot account for the much stronger reduction in S6K activity. Taken together with the genetic evidence, these results strongly support the argument that Scylla acts between PKB and TSC to regulate S6K activity. Furthermore, Brugarolas (2004) provide direct biochemical evidence that a functional TSC complex is required for RTP801/REDD1 to affect S6 phosphorylation. Altogether, these data indicate that Scylla functions upstream of TSC (Reiling, 2004).

dTOR and PDK1 and their interaction with Sk6

Genetic studies in Drosophila underscore the importance of the insulin-signaling pathway in controlling cell, organ and animal size. Effectors of this pathway include Chico (the insulin receptor substrate homolog), PI(3)K, PKB, PTEN, and S6k. Mutations in any of these components have a striking effect on cell size and number, with the exception of S6k. Mutants in S6k affect cell size but not cell number, seemingly consistent with arguments that S6k is a distal effector in the signaling pathway, directly controlled by Target of rapamycin (Tor), a downstream effector of PI(3)K and PKB. Unexpectedly, recent studies showed that S6k activity is unimpaired in chico-deficient larvae, suggesting that S6k activation may be mediated through the PI(3)K docking sites of the Drosophila insulin receptor. It has been shown genetically, pharmacologically and biochemically that S6k resides on an insulin signaling pathway distinct from that of PKB, and surprisingly also from that of PI(3)K. More striking, despite PKB-PI(3)K-independence, S6k activity is dependent on the Drosophila homolog of the phosphoinositide-dependent protein kinase 1, PDK1, demonstrating that both PDK1 (as well as Tor) mediated S6K activation is phosphatidylinositide-3,4,5-trisphosphate (PIP3)-independent (Radimerski, 2002a).

Regulation of cellular growth by the drosophila target of rapamycin dTOR

The TOR protein kinases (TOR1 and TOR2 in yeast; mTOR/FRAP/RAFT1 in mammals) promote cellular proliferation in response to nutrients and growth factors, but their role in development is poorly understood. The Drosophila TOR homolog dTOR is required cell autonomously for normal growth and proliferation during larval development, and for increases in cellular growth caused by activation of the phosphoinositide 3-kinase (PI3K) signaling pathway. As in mammalian cells, the kinase activity of dTOR is required for growth factor-dependent phosphorylation of S6 kinase in vitro, and overexpression of S6k in vivo can rescue dTOR mutant animals to viability. Loss of dTOR also results in cellular phenotypes characteristic of amino acid deprivation, including reduced nucleolar size, lipid vesicle aggregation in the larval fat body, and a cell type-specific pattern of cell cycle arrest that can be bypassed by overexpression of the S-phase regulator cyclin E. These results suggest that dTOR regulates growth during animal development by coupling growth factor signaling to nutrient availability (Zhang, 2000).

The Drosophila genome encodes four members of the PIK-related family: mei41, which encodes an ATR/ATM homolog required for cell cycle arrest in response to DNA damage (Hari, 1995); CG6535, a second ATM-related gene of unknown function; CG4549, whose closest relative encodes the nonsense-mediated mRNA decay protein SMG-1 in C. elegans, and the dTOR. A fifth member of this family, the DNA-dependent protein kinase, is not found in Drosophila or C. elegans (Zhang, 2000).

Using a combination of cDNA library screening and RACE (5' rapid amplification of cDNA ends), overlapping cDNAs have been isolated that together contain a large ORF of 2471 amino acids with strong similarity to mammalian mTOR and to TOR1 and TOR2 from budding yeast. Subsequently, the identical ORF was identified by computational analysis of the annotated Drosophila genome (CG5092). Sequence comparisons reveal that dTOR is 56% and 38% identical to human mTOR and yeast TOR2, respectively, with the highest levels of identity in the carboxy-terminal region containing the putative kinase and rapamycin/FKBP12-binding domains (73% identity with mTOR over the carboxy-terminal 675 amino acids). Additional structural motifs were also found to be well-conserved, including a series of HEAT repeats in the amino-terminal half of the protein, a domain shown to bind the peripheral membrane protein gephyrin, and a short sequence at the extreme carboxyl terminus of essential but unknown function that is highly conserved amongst PIK-related family members. Interestingly, sites of autophosphorylation and phosphorylation by Akt/PKB are not conserved in dTOR (Zhang, 2000).

To begin a mutational analysis of dTOR, the Berkeley Drosophila Genome Project P-element database was searched and two independent homozygous lethal lines bearing P insertions in the dTOR gene were identified (designated here as dTOR^P1 and dTOR^P2). Mobilization of these elements restores viability to each chromosome, indicating that the insertions are responsible for the associated lethality. Comparison of the insertion sites of dTOR^P1 and dTOR^P2 with the dTOR transcription unit reveal that the P-elements are inserted at 24 and 74 bases downstream of the dTOR transcription start site, respectively, and would likely interfere with normal dTOR expression (Zhang, 2000).

To generate additional dTOR alleles, a series of deletions spanning the dTOR gene was generated by imprecise mobilization of the P-elements. One such mutant, designated dTOR^DeltaP, was selected for further analysis. Sequence analysis reveals that the dTOR^DeltaP deletion originates at the dTOR^P2 insertion site and extends 3514 bp downstream, removing the dTOR translation start site and amino-terminal 902 codons, and thus likely represents a null allele of dTOR. This was confirmed by the absence of detectable dTOR protein in immunoblots of dTOR^DeltaP larval extracts. A 9.4-kb genomic rescue construct encompassing the dTOR gene and no other predicted transcription units restores full viability and fertility to dTOR^DeltaP homozygotes (Zhang, 2000).

dTOR^DeltaP homozygotes hatch at normal rates, but grow more slowly than normal and eventually arrest during larval development, reaching only 24% the mass of wild-type controls. Larvae homozygous for dTOR^P1 or dTOR^P2 alleles display a less severe phenotype, eventually growing to approximately 40% and 79% the mass of wild type, respectively, indicating that these alleles likely retain partial dTOR function. In each case, the mutants remain viable and active during an extended larval period of ~30 d, and eventually die without pupating. Larvae heterozygous for dTOR grow at a rate indistinguishable from wild-type controls under normal culture conditions, but are hypersensitive to low concentrations of rapamycin. Thus, dTOR encodes a rapamycin-sensitive protein required for normal growth during larval development (Zhang, 2000).

Overall growth of an organism is generally accompanied by increases in cell number (proliferation), cell size (hypertrophy), or both. To determine how mutations in dTOR inhibit growth, these parameters were examined in a number of tissues. The effect of dTOR on cell size was analyzed in marked clones of dTOR^DeltaP homozygous cells, which were generated by FLP/FRT-mediated mitotic recombination in dTOR^DeltaP heterozygous animals. Examination of adult cuticular structures reveal that dTOR homozygous mutant cells are markedly reduced in size. For example, bristles of the wing margin that lack dTOR were both thinner and shorter than adjacent wild-type cell. Area measurements of mutant clones in the wing epithelium show that dTOR^DeltaP mutant cells are approximately half (56%) the size of controls. Similar effects have been observed in the eye, abdomen, and notum (Zhang, 2000).

To determine whether loss of dTOR affects the size of actively proliferating cells, dTOR mutant clones were examined in the developing imaginal discs, epithelial primordia that proliferate mitotically to give rise to adult structures. Imaginal wing discs containing GFP-marked clones of dTOR^DeltaP homozygous cells were dissociated into single cells, which were then analyzed by flow cytometry. The mean forward light scatter value (a measure of cell size) of dTOR mutant cells is decreased by 30% compared to wild-type control cells from the same discs. This decrease in cell size is observed in all phases of the cell cycle. Thus, loss of dTOR causes a cell autonomous reduction in the size of both proliferating and postmitotic cells (Zhang, 2000).

Fluorescence-activated cell sorter (FACS) analysis has also revealed that the cell cycle phasing of dTOR cells differs significantly from that of controls, with relatively more cells in G₁, and fewer in S and G₂ phases. This is consistent with the ability of rapamycin to induce G₁ arrest in yeast and in mammalian cell culture. To measure proliferation rates of dTOR mutant cells, the number of cells in dTOR^DeltaP clones were compared with that of their wild-type sister clones (twin spots). Clones of dTOR^DeltaP mutant cells are similar in size to their twin spots at 48 h after induction, but by 72-96 h they contain significantly fewer cells, indicating that loss of dTOR leads to a reduced rate of cell proliferation. In addition, lone twin spots lacking a corresponding mutant sister clone have occasionally been observed at 96 h after induction, indicating that at some frequency dTOR^DeltaP homozygous cells are eliminated from the disc epithelium. Because dTOR^DeltaP cells remain viable for weeks in the context of a homozygous mutant animal, the loss of dTOR^DeltaP mutant clones is likely the result of cell competition with adjacent wild-type cells, which is known to occur for cells with a growth disadvantage (Zhang, 2000).

Growth properties of cells in the salivary glands of homozygous dTOR^DeltaP larvae were also examined. The salivary gland is comprised of two cell types: polytene gland cells that undergo multiple rounds of endoreduplication to generate giant nuclei with a ploidy of up to 2048 C, and imaginal ring cells that remain diploid and cycle mitotically. Loss of dTOR affects both cell types. The endoreplicative cells in dTOR^DeltaP salivary glands undergo only four to five rounds of replication before entering quiescence, reaching a ploidy of 16-32C and a size ~10% that of wild type. The imaginal rings in dTOR^DeltaP larvae contain approximately fivefold fewer cells than wild type. Together, these results indicate that dTOR is required to promote cell cycle progression in both mitotic and endoreplicative cells, and acts primarily at the G₁/S transition (Zhang, 2000).

The cell autonomous reduction in the size of dTOR mutant cells is reminiscent of mutations in components of the PI3K/S6K signaling pathway. Mutations in Pten, the fly homolog of the PTEN tumor suppressor, cause activation of this pathway, leading to increased cell growth. To determine whether dTOR is required for PI3K-dependent signaling, the growth properties of cells lacking both Pten and dTOR were examined. Clonal loss of Pten causes enlargement of imaginal and adult cells, and increases the percentage of cells in the S and G₂ phases of the cell cycle. In contrast, cells carrying null alleles of both Pten and dTOR are indistinguishable from cells lacking dTOR alone, with a similar reduction in cell size and accumulation in G₁. Loss of dTOR also prevents the increased proliferation caused by mutations in Pten. Cells mutant for weaker alleles of Pten and dTOR (MGH1 and P2, respectively) are intermediate in size, indicating that dTOR^P2 cells retain partial signaling function. It is concluded that dTOR is epistatic to Pten, and therefore, that dTOR functions at a step downstream of or in parallel to PI3K signaling (Zhang, 2000).

In further tests of dTOR's role in PI3K/S6k signaling, it was found that rapamycin inhibits the serum-dependent phosphorylation of Drosophila p70^S6k (S6k) expressed in S₂ cells. Dephosphorylation of S6k by rapamycin is prevented by cotransfection of a dTOR point mutant containing a Ser¹⁹⁵⁶ to Thr substitution (dTOR^RR, which confers rapamycin resistance to mammalian and yeast TOR proteins. Expression of dTOR^RR carrying an additional point mutation in a residue crucial for kinase activity (dTOR^RRKD fails to protect S6k from rapamycin-induced dephosphorylation, indicating that the kinase function of dTOR is required to maintain S6k phosphorylation (Zhang, 2000).

To determine whether these biochemical interactions between dTOR and S6k are relevant to their functions in vivo, tests were performed for genetic interactions between them. Remarkably, constitutive overexpression of Drosophila S6k or human p70^S6K1 is able to rescue dTOR^P2/P2 and dTOR^P1/P2 flies to viability. The greatest degree of rescue is provided by a mutant version of p70^S6K1, in which four mitogen-induced phosphorylation sites are mutated to aspartate and glutamate residues (mutant D4). Expression of this construct allowed 74% of expected dTOR^P1/P2 progeny to survive to adulthood, whereas no dTOR^P1/P2 animals survived in the absence of S6k overexpression. dTOR flies rescued by S6k overexpression are slightly smaller than wild-type controls, but are fertile and develop at a similar rate as wild type. Although overexpression of S6k does not rescue dTOR^DeltaP null mutants to adulthood, it does enable them to progress to the pupal stage. Overexpression of S6k in wild-type larvae also confers significant resistance to rapamycin. Again, constitutively active p70^S6K1 provides the greatest degree of rapamycin resistance. Together, these results indicate that a major function of dTOR is to maintain levels of active S6k sufficient for normal growth (Zhang, 2000).

Like dTOR mutants, wild-type larvae deprived of amino acids enter an extended larval period with little or no growth. Amino acid deprivation also causes a series of distinctive cellular phenotypes including a reduction in nucleolar area, changes in morphology of the larval fat body, and a cell type-specific cell cycle arrest. Since TOR proteins have been proposed to be regulated in response to amino acid levels, it was of interest to examine whether loss of dTOR mimics these cellular effects (Zhang, 2000).

The nucleolus is the major cellular site of ribosomal assembly, and its size has been shown to correlate with protein synthetic capacity and proliferation rate. To measure nucleolar size in wild-type and dTOR mutant cells, wing imaginal discs containing clones of dTOR^DeltaP homozygous cells were labeled with an antibody against the nucleolar protein fibrillarin, and examined by confocal sectioning. The nucleolar area in clones of dTOR mutant cells in the wing imaginal disc is approximately half that of surrounding wild-type cells, consistent with a role for dTOR in ribosome biogenesis (Zhang, 2000).

During metamorphosis or starvation, stores of protein, lipid, and glycogen are mobilized from adipose cells of the larval fat body and are used by other tissues as an energy source in place of dietary nutrients. These metabolic effects are visible as changes in appearance of fat body cells. The major visible change in fat body cells in larvae deprived of amino acids is an aggregation of lipid vesicles, and this effect is indistinguishable from that caused by loss of dTOR (Zhang, 2000).

Within 48-72 h of amino acid withdrawal, endoreplicative cells become quiescent, whereas mitotic neuroblasts of the central nervous system continue to cycle for at least 8 d in the absence of amino acids. This pattern of cell cycle responses is distinct from that caused by complete inhibition of protein synthesis, which causes all larval cells to arrest DNA synthesis. To determine whether loss of dTOR causes a cell cycle response similar to that elicited by starvation, cell cycle behavior of these cell types was examined in dTOR^DeltaP larvae at multiple stages of development. At 3-4 d after egg deposition (AED), both endoreplicative and mitotic tissues were found to cycle normally in dTOR^DeltaP homozygotes, as measured by incorporation of the nucleotide analog BrdU. In contrast, by 5-6 d AED all endoreplicative tissues including the gut, fat body, and salivary glands fail to incorporate BrdU, whereas neuroblasts continued to cycle. A similar pattern is observed at 10 d AED. Presumably the appearance of this cell cycle arrest at 4-5 d AED results from the perdurance of maternal stores of dTOR mRNA or protein until this time. The cell cycle arrest of dTOR endoreplicative cells can be bypassed by overexpression of the G₁/S regulators cyclin E or dE2F/dDP, as has been demonstrated previously for endoreplicative cells arrested by amino acid withdrawal. Thus, amino acid insufficency and loss of dTOR each cause similar growth arrests, changes in cell morphology, and cell type-specific patterns of G₁ arrest (Zhang, 2000).

In budding yeast, TOR proteins govern S-phase entry in response to nutrient levels by regulating translation of the G₁/S regulator Cln3 (Barbet, 1996 and Polymenis, 1997). Drosophila cyclin E has been proposed to play a role analogous to Cln3, and its abundance increases in response to growth stimuli such as overexpression of activated Ras. Because cyclin E overexpression is able to bypass the cell cycle arrest in dTOR mutants, whether loss of dTOR affects cyclin E expression was examined. Immunoblot analysis of whole larval extracts has revealed that the level of cyclin E protein is reduced ~30-fold in dTOR^DeltaP mutants, as compared to wild-type larvae of similar stage (Zhang, 2000).

Clonal induction of cells lacking cyclin E in the wing imaginal disc results in a G₁ arrest within one to two cell divisions. In contrast, cells mutant for dTOR continue to cycle slowly for several days, and give rise to clones containing multiple cells, indicating that dTOR mutant cells retain at least partial cyclin E activity. Accordingly, cyclin E protein is reduced but not eliminated in dTOR mutant clones in the wing disc. Although 72-h dTOR^DeltaP clones containing little or no detectable cyclin E immunoreactivity are often observed, many dTOR clones were found containing cells with apparently normal cyclin E levels. It is concluded that the observed reduction in cyclin E protein levels in dTOR^DeltaP larval extracts is due largely to reduced cyclin E levels in endoreplicating cells, which comprise the majority of larval mass, resulting in a G₁ arrest in this cell type. In contrast, dTOR may be required in imaginal cells to maintain normal rates of cyclin E accumulation, rather than absolute levels, consistent with the reduced rate of cell division and extended G₁ phase observed in dTOR mutant clones (Zhang, 2000).

dTOR mutants differ from the known PI3K pathway mutants in several important respects. (1) The growth defects caused by loss of dTOR are more severe than those arising from mutations in components of the PI3K signaling pathway. (2) Null mutations in the PI3K subunits Dp110 or p60 allow growth to the third instar larval stage, and chico (IRS-1) and S6k mutants survive to adulthood. At least in the case of chico and S6k, many cells are able to cycle normally throughout development. In contrast, animals lacking dTOR reach only the size of second instar larvae, at which point they undergo cell cycle arrest. (3) Whereas overexpression of Dp110, Akt, or S6k leads to increased growth rate and cell size, dTOR overexpression inhibits growth and reduces cell size. Although variations in genetic background may partially account for some of these phenotypic differences, these results are inconsistent with dTOR acting as an integral component of a linear PI3K/Akt/S6k pathway, and instead argue that dTOR may converge upon this pathway in response to a distinct set of cues (Zhang, 2000 and references therein).

In yeast, TOR1 and TOR2 regulate cell growth directly in response to levels of nutrients such as amino acids, rather than in response to intercellular signals. Similarly, human mTOR may also be regulated by nutrient levels. These considerations prompted a comparison of the phenotypes of dTOR mutant animals with the physiological changes caused by nutrient deprivation. By the three criteria examined -- nucleolar size, fat body vesicle formation, and endoreplicative cell cycle arrest -- loss of dTOR precisely mirrors the effects of starvation. An efficient explanation of these results is that dTOR is required for normal responses to changes in nutrient levels. This would be consistent with a model in which full activation of growth targets such as S6k requires two distinct inputs: growth factor-mediated intercellular signals through PI3K, and nutrient-sensing signals through TOR. In this view, TOR proteins may act as part of a checkpoint to attenuate growth factor signaling when local conditions are unfavorable for cell growth (Zhang, 2000).

How TOR proteins might be regulated by nutrient levels is unclear. In the case of amino acids, recent evidence suggests that the primary signal may be uncharged tRNA, which increases in abundance when amino acid levels are low. Interestingly, other members of the PIK-related kinase family such as ATM and DNA-PK also act as checkpoint proteins that are regulated by specific nucleic acid structures. In addition, a recently described member of this family, SMG-1, is involved in the degradation of aberrant mRNAs containing inappropriate nonsense codons. Thus, regulation by nucleic acid may be a common feature of this kinase family (Zhang, 2000 and references therein).

Activation of p70^S6K is a common response to virtually all mitogenic stimuli. Phosphorylation of the target of p70^S6K, ribosomal protein S6, leads to the selective increase in translation of a subset of transcripts that contain an oligopyrimidine tract at their 5' termini (5' TOP). This class of messages encodes ribosomal proteins and translation elongation factors, and thus p70^S6K activation leads to increased ribosomal biogenesis. The demonstration in this study that overexpression of S6k can rescue dTOR mutants to viability indicates that S6k is a critical effector of dTOR, and that one essential function of dTOR is to regulate the activity of S6k (Zhang, 2000).

Despite the central role of S6k in mediating the functions of dTOR, several lines of evidence indicate that dTOR has additional S6k-independent roles. (1) dTOR mutant phenotypes are more severe than those of S6k; lack of dTOR results in growth arrest at the second instar larval stage, whereas S6k mutant animals survive to adulthood, albeit with a delayed development and decreased body size. (2) Although S6k overexpression suppresses the lethality of dTOR mutants, this is only the case for hypomorphic dTOR allelic combinations; dTOR null animals expressing S6k advance only to the pupal stage. (3) dTOR flies rescued by S6k do not grow to the full size of wild-type controls. Thus, some dTOR functions are not fully rescued by S6k. As noted above, in mammalian cells mTOR also stimulates translation through phosphorylation and inactivation of 4E-BP1, an inhibitory binding factor of the translation initiation factor eIF4E. Drosophila eIF4E mutants display a severe growth arrest phenotype, and thus aspects of dTOR function that are not rescued by S6k may reflect diminished eIF4E activity. However, neither overexpression of eIF4E nor mutations in 4E-BP1 detectably alleviate dTOR mutant phenotypes (Zhang, 2000).

In addition to effects on translation, studies in budding yeast have found that inactivation of TOR modulates the level of a number of growth-related or nutrient-regulated transcripts, including those encoding ribosomal proteins (Barbet, 1996; Zaragoza, 1998; Beck, 1999; Cardenas, 1999; Hardwick, 1999; Powers, 1999). A transcriptional role for TOR in higher eukaryotes has been reported as well. Moreover, recent studies have found that mTOR can interact with a number of additional signaling factors including STAT3, protein kinase C, c-Abl, and 14-3-3 (Parekh, 1999; Kumar, 2000a and b; Mori, 2000; Yokogami, 2000). Rapamycin has also recently been shown to disrupt microtubule assembly and function in yeast, independent of its effects on translation (Choi, 2000). Thus, the inability of S6k overexpression to fully supplant dTOR may be due to a requirement for dTOR in multiple cellular functions (Zhang, 2000).

The reduced growth of dTOR mutants reflects reductions in both cell size and cell number. Because direct inhibition of proliferation increases rather than decreases cell size, it is proposed that the primary function of dTOR is to promote cell growth, and that the decreased proliferation of dTOR mutant cells is a secondary effect in response to their reduced rate of growth. The accumulation of dTOR mutant cells in the G₁ phase of the cell cycle suggests that the G₁/S transition is particularly sensitive to growth rate. This is consistent with previous observations that stimulation of cell growth by overexpression of dMyc, PI3K, or activated Ras promotes progression through the G₁/S but not G₂/M transition (Zhang, 2000 and references therein).

A factor likely to be involved in coupling growth and division rates is the G₁/S regulator cyclin E. Cyclin E is rate limiting for G₁/S progression in imaginal discs, and its levels increase by a post-transcriptional mechanism in response to stimulation of cell growth. The demonstration that dTOR is required for normal accumulation of cyclin E suggests that translational control is likely to play an important role in cyclin E regulation. In budding yeast, translation of the G₁ cyclin Cln3 is regulated by a leaky scanning mechanism involving a small upstream ORF (Polymenis, 1997). In this regard, it is interesting to note that the 5'-untranslated region of the Drosophila cyclin E message contains multiple upstream ORFs. Thus, the mechanisms connecting G₁/S progression to cell growth may be conserved between yeast and multicellular organisms (Zhang, 2000).

Gigas negatively regulates S6k

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in one of two tumor suppressor genes, TSC1 and TSC2. Absence of Drosophila Tsc1 and/or Tsc2 (Gigas) leads to constitutive S6k activation and inhibition of PKB, the latter effect being relieved by loss of S6K. In contrast, the Pten tumor suppressor, a negative effector of PI3K, has little effect on S6k, but negatively regulates PKB (Akt1). More importantly, reducing S6k signaling rescues early larval lethality associated with loss of Tsc1/2 function, arguing that the S6k pathway is a promising target for the treatment of TSC (Radimerski, 2002b).

To determine whether loss of Tsc1/2 or Pten directly affected S6k activity, each was depleted in Drosophila Kc167 cells by dsRNAi. Quantitative Real Time PCR showed that such treatment strongly reduced levels of both transcripts. Compared with control cells, depletion of Tsc1 increases S6k activity and T398 phosphorylation, consistent with the reduced electrophoretic mobility of S6k. These results are in agreement with recent findings in TSC1 null mammalian cells (Kwiatkowski, 2002). Insulin treatment of either control cells or Tsc1-depleted cells did not significantly increase these responses beyond that of Tsc1 depletion alone, indicating that loss of Tsc function leads to full S6k activation. RAD001, a rapamycin derivative, blocks S6k activity in both control and Tsc1-depleted cells treated with insulin. However, it was consistently noted that the RAD001 block of insulin-induced S6k activation is not as strong in Tsc1-depleted cells, suggesting that not all the effects of Tsc on S6k are dependent on Tor, the Drosophila target of rapamycin. Similar results to those described here were obtained by Tsc2 depletion. In addition, the effects appear specific, since Tsc1 depletion has no effect on the basal activity of other AGC-kinase family members, such as PKB or Drosophila atypical PKC. However, insulin-induced PKB activation and S505 phosphorylation are repressed in Tsc1-depleted cells as compared with control cells, consistent with S6k acting in a negative feedback loop to dampen PKB signaling. In contrast to loss of Tsc1, depletion of Pten has little effect on S6k activity and T398 phosphorylation, whereas it leads to elevated levels of both basal and insulin-stimulated PKB activity and S505 phosphorylation. Thus, loss of Tsc1/2, but not Pten, leads to constitutive S6k activation (Radimerski, 2002b).

To determine whether the findings above could be corroborated in the animal, S6k activity was measured in extracts of Tsc1, Pten, and S6k null larvae. The results show that S6k activity in extracts derived from Tsc1 null larvae is strongly increased over that of wild-type larvae, whereas it is slightly increased in larvae lacking Pten. The opposite was found for PKB activity, which is strongly repressed in Tsc1 null larvae, and up-regulated in Pten-deficient larvae. Hence, it cannot be excluded that reduced PKB activity contributes to larval lethality of Tsc mutants. Given that loss of Tsc function leads to increased S6k activity, it was reasoned that ectopic expression of Tsc1/2, but not Pten, would inhibit S6k activity. To test this hypothesis, both tumor suppressors were expressed ubiquitously in larvae using the GAL4/UAS system, such that the GAL4 promoter chosen in each case led to developmental arrest at late larval second instar. Extracts from larvae overexpressing Tsc1/2 display strongly reduce S6k activity, whereas those from Pten overexpressing larvae have normal levels of S6k activity. In contrast, PKB activity is strongly suppressed in Pten overexpressing larvae and little affected in extracts from larvae overexpressing Tsc1/2. These data corroborate previous findings that S6k and PKB act in parallel signal transduction pathways (Radimerski, 2002a), and provide compelling evidence that they are negatively controlled by distinct tumor suppressor genes (Radimerski, 2002b).

Despite the fact that S6k and PKB act in parallel signaling pathways, loss of Tsc1/2 function leads to inhibition of PKB activity, suggesting cross-talk between the two pathways. Compatible with such a model, recent studies have shown that rapamycin treatment of adipocytes inhibits a negative feedback loop, which normally functions to dampen insulin-induced PKB activation. Since RAD001 inhibits S6k activity (Radimerski, 2002a) and increases PKB activity (Radimerski, 2002a), it raised the possibility that the effects of Tsc mutants on PKB are mediated through S6k. Consistent with this hypothesis, inhibition of PKB activity due to loss of Tsc function was relieved in the absence of S6k. Similar results were obtained by using dsRNAi in cell culture. Thus, the suppression of PKB by loss of Tsc function requires S6k (Radimerski, 2002b).

To genetically test the specificity of Tsc1/2 and Pten tumor suppressor function, either Tsc1 or Pten were removed in cells giving rise to the adult eye structure, by inducing mitotic recombination with the FLP/FRT system under the control of the eyeless promoter. In a wild-type genetic background, loss of either Tsc1 or Pten within the developing eye causes strong overgrowth of the head. Eye overgrowth by removal of Tsc1 is strongly suppressed in a genetic background null for S6k, as is ommatidia size, in agreement with a previous report analyzing double mutant clones of Tsc2 and S6k in the eye (Potter, 2001). In contrast, removal of Pten in the eyes of S6k null flies still induces overgrowth of clones with enlarged ommatidia. These findings are supported by results showing that eye overgrowth by removal of Tsc1 is still observed in clones devoid of PKB function (Potter, 2001) and overgrowth by removal of Pten is suppressed in a viable PKB mutant genetic background (Stocker, 2002). Thus, Tsc1/2 appears to be specific for the S6k-signaling pathway, whereas Pten antagonizes PI3K signaling to counteract PKB activation by decreasing PIP3 levels (Radimerski, 2002b).

Since Tsc1/2 loss-of-function overgrowth in clones is suppressed by removing S6k, it was reasoned that reducing increased S6k activity in Tsc1 loss-of-function larvae might rescue second larval instar lethality. Consistent with this, feeding Tsc1 null larvae low doses of RAD001, which induces a developmental delay of 3 d in wild-type larvae, allowed them to reach late wandering third larval instar. The Tsc1 null larvae died shortly after pupation, presumably because wandering third instar larvae stop feeding and thus failed to receive the drug during pupal stages. To circumvent the problem of feeding, attempts were made to reduce S6k signaling by reducing the dosage of the gene. Compared with wild-type pupae, S6k null larvae are significantly reduced in size, and lack of one allele of Tsc1 in this background has no significant effect on the S6k null phenotype. Strikingly, the second instar lethality caused by lack of both Tsc1 alleles is rescued to early pupal stages in the S6k null background; however, these larvae are still small and severely delayed in development. In contrast, larvae in which one allele of S6k has been removed in a Tsc1 null background develop to early pupal stages with little developmental delay, although they are now significantly larger than wild type. On the basis of these latter findings, it was reasoned that further reduction of S6k signaling, but not its abolishment, may allow Tsc1 null animals to develop beyond early pupal stages. To test this in the Tsc1 null background, either one allele of Tor bearing a mutation in the kinase domain was used alone or in combination with one null allele of S6k. Tsc1 null larvae bearing one kinase mutant Tor allele survived with higher frequency to pupae than animals with one null allele of S6k, with a few emerging as adults. However, genetically lowering S6k signaling further by combining the Tor and S6k loss-of function alleles, results in more than 60% of animals surviving to the adult stage. The rescued females and males were slightly larger than wild-type flies, with overall patterning appearing normal. Furthermore, the rescued females were semifertile when crossed to wild-type males, whereas the rescued males were fully fertile when crossed to wild-type females. Similarly, animals lacking Tsc2 function were rescued to viability by the same genetic approach applied above. Importantly, flies lacking one S6k allele and bearing one kinase mutant Tor allele display no obvious mutant phenotype. Therefore, lowering but not abolishing S6k signaling is sufficient to allow development of Drosophila lacking Tsc function (Radimerski, 2002b).

Taken together, these results demonstrate that the tumor suppressor Tsc1/2 is a critical component in controlling S6k activation. Interestingly, this effect may be Tor independent, as insulin-induced S6k activation is more elevated in Tsc1/2-depleted cells pretreated with RAD001 than in control cells, and in preliminary studies, clonal overgrowth in the eye induced by loss of Tsc1 is not suppressed in a semiviable, heterorallelic Tor mutant background. Overexpression of Tsc1/2 selectively suppresses the S6k-signaling pathway, whereas Pten operates on the dPI3K-signaling pathway. Double mutations for Pten and Tsc1 are additive for clonal overgrowth, compatible with S6k and PKB independently mediating growth. Nevertheless, inhibition of PKB by loss of Tsc function shows that there is negative cross-talk between the two signaling pathways. Given this negative cross-talk, the observation that in double mutant clones growth is additive, suggests that in the absence of Pten, inhibition of PKB by loss of Tsc is circumvented. However, despite the observation that double mutations for Pten and Tsc1 are additive for clonal overgrowth, overgrowth induced by absence of Pten is suppressed in clones mutant for Tor. Since S6k does not prevent such overgrowth, it is possible that this suppression actually represents an intermediate phenotype, or that Pten negatively acts on a Tor target distinct from S6k. At this point, it is important to gain a deeper knowledge of the molecular mechanisms by which Tsc1/2 acts to suppress S6k function and how the signaling components of these two pathways cross-talk with one another (Radimerski, 2002b).

Recently, a successful Phase I clinical trial was completed for a rapamycin analog in the treatment of solid tumors. The results of the trial demonstrated that the drug was efficacious at subtoxic doses, and suggested that specific tumor types may be more sensitive to inhibition by rapamycin than others. The question that arose from the trial is, which tumors would be susceptible to rapamycin treatment? Here, it has been demonstrated for the first time in vivo that a mild reduction in S6k signaling, which alone has no blatant phenotype, is sufficient to restore viability of flies devoid of Tsc function. Thus, these findings imply that rapamycin or its derivatives might be very promising pharmaceutical agents in the treatment of tumors arising from TSC (Radimerski, 2002b).

Rheb, a target of Gigas, functions upstream of S6K

Insulin signalling is a potent inhibitor of cell growth and has been proposed to function, at least in part, through the conserved protein kinase TOR (target of rapamycin). Recent studies suggest that the tuberous sclerosis complex Tsc1-Tsc2 may couple insulin signalling to Tor activity. However, the regulatory mechanism involved remains unclear, and additional components are most probably involved. In a screen for novel regulators of growth, Rheb (Ras homolog enriched in brain), a member of the Ras superfamily of GTP-binding proteins, was identified. Increased levels of Rheb in Drosophila promote cell growth and alter cell cycle kinetics in multiple tissues. In mitotic tissues, overexpression of Rheb accelerates passage through G1-S phase without affecting rates of cell division, whereas in endoreplicating tissues, Rheb increases DNA ploidy. Mutation of Rheb suspends larval growth and prevents progression from first to second instar. Genetic and biochemical tests indicate that Rheb functions in the insulin signalling pathway downstream of Tsc1-Tsc2 and upstream of TOR. Levels of rheb mRNA are rapidly induced in response to protein starvation, and overexpressed Rheb can drive cell growth in starved animals, suggesting a role for Rheb in the nutritional control of cell growth (Saucedo, 2003).

To examine how Rheb interfaces with TOR, advantage was taken of a biochemical readout of TOR function: S6K phosphorylation. Tagged S6K and Rheb were transfected into Drosophila S2 cells and activation of S6K was measured using a phospho-specific antibody (Thr 398) that correlates with kinase activity. Overexpression of Rheb increases S6K phosphorylation. Although S6K is normally inactivated in response to amino-acid starvation, Rheb-mediated phosphorylation of S6K persists in the absence of amino acids. Loss of Tsc1 or Tsc2 has been shown to cause a similar, nutrient-insensitive increase in S6K activity. RNA interference (RNAi) was used to examine the relationship between Tsc2 and Rheb in modulating S6K function. Whereas loss of Tsc2 results in persistent phosphorylation of S6K in media free of amino acids, loss of Rheb abolishes S6K phosphorylation regardless of the presence of amino acids. In the absence of both Tsc2 and Rheb, S6K remains dephosphorylated. These experiments indicate that Rheb is epistatic to Tsc2, required for S6K phosphorylation, and, presumably, S6K activity (Saucedo, 2003).

Signaling from Akt to FRAP/TOR targets both 4E-BP and S6K in Drosophila melanogaster

The eIF4E-binding proteins (4E-BPs) interact with translation initiation factor 4E to inhibit translation. Their binding to eIF4E is reversed by phosphorylation of several key Ser/Thr residues. In Drosophila, S6 kinase (dS6K) and a single 4E-BP (d4E-BP) are phosphorylated via the insulin and target of rapamycin (TOR) signaling pathways. Although S6K phosphorylation is independent of phosphoinositide 3-OH kinase (PI3K) and serine/threonine protein kinase Akt, that of 4E-BP is dependent on PI3K and Akt. This difference prompted an examination of the regulation of d4E-BP/Thor in greater detail. Analysis of d4E-BP phosphorylation using site-directed mutagenesis and isoelectric focusing-sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the regulatory interplay between Thr37 and Thr46 of d4E-BP is conserved in flies and that phosphorylation of Thr46 is the major phosphorylation event that regulates d4E-BP activity. RNA interference (RNAi) was used to target components of the PI3K, Akt, and TOR pathways. RNAi experiments directed at components of the insulin and TOR signaling cascades show that d4E-BP is phosphorylated in a PI3K- and Akt-dependent manner. Surprisingly, RNAi of dAkt also affects insulin-stimulated phosphorylation of dS6K, indicating that dAkt may also play a role in dS6K phosphorylation (Miron, 2003).

Is d4E-BP regulated by a PI3K/Akt-independent pathway similar to that described for dS6K? Analysis of signaling to d4E-BP using RNAi indicates that it is not. It is more likely that d4E-BP is a direct downstream target of the dInR-dPI3K-dPTEN-dAkt-dTSC-dTOR signaling cascade. Thus, a linear pathway from InR to Akt that is important for 4E-BP regulation is conserved between Drosophila and mammals (Miron, 2003)

dPDK1 is critical for regulating growth by phosphorylating dAkt and dS6K. RNAi of dPDK1 does not significantly affect insulin-induced phosphorylation of d4E-BP. However, consistent with the direct phosphorylation of dS6K by dPDK1, the phosphorylation of dS6K at Thr398 is completely blocked by RNAi of PDK1. Thus, the results favor a model in which d4E-BP regulation is effected through dAkt, even when dPDK1 levels are dramatically reduced, whereas dS6K requires both dAkt and dPDK1. The differential effects of dPDK1 RNAi on d4E-BP and dS6K phosphorylation can be explained as follows: dPDK1 levels may be reduced below a threshold that is required to phosphorylate dS6K but is still adequate to activate dAkt, allowing d4E-BP phosphorylation. Since dS6K requires direct phosphorylation by dPDK1, it may be more susceptible to variations in its levels. In contrast, d4E-BP, which relies on a signal relayed by dAkt, may be less affected by variations in dPDK1. In mammalian PDK1-hypomorphic mutants, a kinase activity that is 10-fold lower than normal still results in normal Akt and S6K1 activation, yet these animals are greatly reduced in size. This observation supports the notion that reduced PDK1 activity may differentially activate downstream targets (Miron, 2003).

In Drosophila, coexpression of dS6K with dPI3K does not cause additive cellular overgrowth, unlike coexpression of dAkt and dPI3K. RNAi of dPTEN in Kc 167 cells and overexpression of dPTEN in Drosophila larvae had little effect on dS6K activity. Moreover, removal of both dS6K and dPTEN in cell clones does not prevent the dPTEN-dependent overgrowth phenotype. Together, these results and the results of dPI3K and dPTEN RNAi experiments would seemingly support the notion that dS6K-dependent cell growth is not influenced by dPI3K and dPTEN. However, a different effect of dPTEN RNAi on dS6K has been reported in another study: increase in dS6K phosphorylation following RNAi of dPTEN. Consistent with this observation RNAi directed against dPI3K and dPTEN has been shown to modulate dS6K phosphorylation. A reasonable explanation for these discrepancies is that the knockdown of dPI3K and dPTEN achieved in the current experiments was not sufficient to completely deplete these proteins and affect dS6K phosphorylation (Miron, 2003 and references therein).

The role of dAkt in regulating dS6K is subject to debate. In Drosophila, Akt plays a predominant role in mediating the effects of increased PIP₃ levels, and all Akt-mediated growth signals are thought to be transduced via Tsc1/2. Tsc2 is directly phosphorylated by Akt, implying that S6K is downstream of Akt in the PI3K signaling pathway. The observation that RNAi of dAkt reduces dS6K phosphorylation at Thr398 supports a direct link among dAkt, dTSC, and dS6K but contradicts the finding that TSC modulates dS6K activity in a dAkt-independent manner. Recent data also support the conclusion of a link between dAkt and dS6K. Clones of cells doubly mutant for dPTEN and dTsc1 display an additive overgrowth phenotype, suggesting that the tumor suppressors act on two independent pathways, from dPTEN to dAkt and from dTSC to dS6K. The findings demonstrate clear effects of dPTEN, dAkt, and dTSC on d4E-BP, which does not preclude the possibility that two pathways regulate d4E-BP; however, a simpler interpretation is that a single pathway is important for its regulation. A possibility is that d4E-BP requires higher dAkt activity than dS6K in order to be phosphorylated. In circumstances of low PI3K activation, low levels of PIP₃ are produced, resulting in weaker dAkt activity that is sufficient for dS6K activation but not for d4E-BP phosphorylation. A differential threshold of activation could be the source of the discrepancies between the current results and those of others. This model is strongly supported by recent data showing that in cells lacking both Akt1 and Akt2 isoforms, the low level of Akt activity remaining is sufficient for robust S6K1 phosphorylation, but phosphorylation of 4E-BP1 is dramatically reduced (Miron, 2003 and references therein).

Alternatively, the results could also be explained by the existence of a negative feedback loop between dPI3K and dS6K that dampens insulin signaling by suppressing dAkt activity. This negative feedback loop has been described. Similar observations were made in mammals; insulin-induced activation of Akt is inhibited in Tsc2-deficient mouse embryonic fibroblasts. Thus, depletion of dAkt may trigger this negative feedback loop, which diminishes dS6K phosphorylation and activation. Interestingly, engagement of this feedback mechanism can also provide an explanation for the reduction in total d4E-BP levels observed in dPDK1 RNAi-treated cells. Under these conditions, the reduction of dS6K signaling is accompanied by a concomitant reduction in growth signaling on the dPI3K-dAkt branch of the pathway. Thus, a reduced level of d4E-BP is required to accommodate the reduced need for deIF4E inhibition (Miron, 2003).

MAP4K3 regulates body size and metabolism in Drosophila

The TOR pathway mediates nutrient-responsive regulation of cell growth and metabolism in animals. TOR Complex 1 activity depends, among other things, on amino acid availability. MAP4K3 was recently implicated in amino-acid signaling in cell culture. This study reports the physiological characterization of MAP4K3 mutant flies. Flies lacking MAP4K3 have reduced TORC1 activity detected by phosphorylation of S6K and 4EBP. Furthermore MAP4K3 mutants display phenotypes characteristic of low TORC1 activity and low nutrient availability, such as reduced growth rate, small body size, and low lipid reserves. The differences between control and MAP4K3 mutant animals diminish when animals are reared in low-nutrient conditions, suggesting that the ability of TOR to sense amino acids is most important when nutrients are abundant. Lastly, physical interaction is shown between MAP4K3 and the Rag GTPases raising the possibility they might be acting in one signaling pathway (Bryk, 2010).

The multiprotein complex TORC1, containing TOR kinase, is a central regulator of cellular growth and metabolism in animals. It is activated by a number of inputs relating to cellular energy and nutrient status. These include insulin, glucose, cellular energy levels and amino acid availability. In response, TORC1 activates protein synthesis via a number of mechanisms including activation of the ribosomal S6 kinase (S6K), repression of the translational inhibitor 4E-BP, and promotion of ribosome biogenesis via myc. In particular since TORC1 is a master regulator of protein biosynthesis, its regulation by amino acids, the building blocks of proteins, likely constitutes an important regulatory feedback mechanism. Furthermore, the importance of amino acid signaling to TOR is highlighted by the observation that circulating amino acids are elevated in humans with obesity, where they have been shown to activate TORC1 activity and modulate glucose metabolism. Despite this, understanding of the molecular mechanism by which amino acids regulate TOR remains fragmentary (Bryk, 2010).

Three protein complexes have recently been implicated in the activation of TORC1 in response to amino acids. The human class III PI3K (phosphoinositide 3-kinase) hVps34 is activated by amino acids via a calcium dependent mechanism (Gulati, 2008). This leads to accumulation of phosphatidylinositol 3-phosphate (PI(3)P) in cells, which is thought to cause the recruitment of proteins recognizing PI(3)P to early endosomes, forming an intracellular signaling platform that leads to TORC1 activation. This feature of the pathway may be specific for vertebrates, as flies mutant for Vps34 have been reported to not have TORC1 signaling defects. Recently, two groups discovered that Rag GTPases mediate amino acid signaling to TORC1. The emerging picture is that amino acids change the GDP/GTP loading of the Rag GTPases, thereby stimulating the binding of Rag heterodimeric complexes to TORC1. This in turn causes TORC1 to change its intracellular localization, perhaps relocalizing it to vesicles containing the activator Rheb. This mechanism appears to be evolutionarily conserved from flies to humans (Bryk, 2010).

The third protein recently identified as a mediator of amino acid signaling to TOR is MAP4K3 (Findlay, 2007); in HeLa cells the kinase activity of MAP4K3 is activated in the presence of amino acids. In turn, MAP4K3 is required for TOR to phosphorylate its targets S6K and 4E-BP1 in response to amino acid sufficiency. The cell-culture data also suggest this mechanism is conserved from flies to humans as knockdown of Drosophila MAP4K3 causes a reduction in TOR activity (Bryk, 2010).

Although considerable progress has been made, important questions remain unanswered. The role of TORC1 activity in vivo has been well studied in flies and mice, but fundamental issues regarding the regulation of TOR by amino acids have thus far only been explored in vitro in cell culture. To assess the functional significance of the ability of TORC1 to sense amino acids in the organismal context, use has been made of a Drosophila mutant for MAP4K3 (CG7097). dMAP4K3 mutant flies have reduced TOR activity, detected by phosphorylation of TOR targets. dMAP4K3 mutants are viable but display physiological aberrations emblematic of animals starved of nutrients: MAP4K3 mutant flies have retarded growth, reduced size, and low lipid reserves. Both the biochemical results and the phenotypes indicate that MAP4K3 modulates, but is not absolutely required, for TOR activity in vivo. This is similar to what is observed with other modulators of the pathway, such as Melted. Unexpectedly, the function of MAP4K3 is most required when nutrient conditions are rich (Bryk, 2010).

Recent reports have shown that not all components identified in cell culture as regulators of TORC1 activity also affect TORC1 in vivo in an animal model. The purpose of this study was two-fold: (1) to analyze whether MAP4K3 regulates TORC1 activity in vivo in the fly, and (2) to study the physiological consequences for the organism when the ability of TORC1 to sense amino acids is impaired (Bryk, 2010).

Biochemical evidence is presented that TORC1 activity is reduced in MAP4K3 mutant animals, consistent with published cell-culture data showing that MAP4K3 is required for full TORC1 activation (Findlay, 2007). Furthermore, MAP4K3 mutants have defects typical of reduced TORC1 activity. They are delayed in their development due to a reduced rate of growth. They eventually pupate leading to adults of reduced size and their tissues are comprised of cells that are smaller than normal. Furthermore, MAP4K3 mutants have significantly reduced triglyceride stores compared to controls. These physiological effects are similar to the phenotypes observed with mutants for other regulators of TOR, such as Melted. Melted mutant flies are also 10% smaller than controls and are significantly leaner (Bryk, 2010).

As a whole, the MAP4K3- mutant phenotypes emulate the physiological effects observed when flies are grown on conditions of limiting food. When wildtype larvae are put on a low-nutrient diet, they are delayed in pupation and yield animals of small size that are lean. Thus loss of MAP4K3 activity phenocopies a reduced nutrient environment, consistent with MAP4K3 playing a role in the ability of animals to sense their nutrient conditions. This suggests the ability of TORC1 to sense amino acids is most important when nutrient conditions are rich, allowing animals to accelerate their growth accordingly. In contrast, on a low-nutrient diet, control and MAP4K3 mutant flies grow equally slowly consistent with TOR activity being low in both groups. This parallels nicely the results reported in cell culture by (Findlay, 2007): In the absence of amino acids, both control and MAP4K3 knockdown cells have low TOR activity whereas in the presence of amino acids, TOR is activated strongly in control cells but only weakly in MAP4K3 knockdown cells (Bryk, 2010).

Unexpectedly, it was found that MAP4K3 mutant animals are viable, although they have an elevated mortality rate compared to controls. This suggests that the amino acid sensing pathway might only modulate TORC1 activity. If TORC1 activity were completely blunted in MAP4K3 mutants, the animals would be dead, as is the case for TOR or Rheb mutants. Consistent with this, residual TORC1 activity was observed in MAP4K3 mutants, as detected by phosphorylation of the TORC1 targets S6K and 4EBP. This also parallels results from cell culture. The results presented in Findlay (2007) are obtained with cells starved of serum and consequently of insulin signaling. In the presence of insulin signaling, which resembles the physiological situation more closely, MAP4K3 mutant cells still retain residual TOR activity, similar to what was observed in vivo in this study. Consistent with these findings, it was observed that the Rheb expression is able to drive tissue growth also in the absence of MAP4K3 (Bryk, 2010).

Both MAP4K3 and the Rag GTPases have recently been shown to be required for amino acids to stimulate TORC1 activity. While studying dMAP4K3, it was noticed that dMAP4K3 binds physically to the Rag GTPases, suggesting they might act together as components of a single signaling pathway. This interaction is likely specific for several reasons: (1) no binding of MAP4K3 to another GTPase, Rheb, could be detected, (2) binding of MAP4K3 to the RagA/C complex was significantly stronger than binding of an unrelated HA-tagged protein, HA-medea (3) MAP4K3 bound FLAG-RagC significantly stronger than FLAG-RagA showing that MAP4K3 distinguishes between two Rag proteins and (4) binding of MAP4K3 to RagC depended on its GDP/GTP state (Bryk, 2010).

Further studies will be required to test whether this interaction is important for TORC1 to sense amino acids. The data suggest that MAP4K3 might be functioning upstream of the Rag GTPases, and not downstream since activated RagA does not require MAP4K3 to promote tissue growth in vivo. This raises the possibility that the Rag GTPases may be substrates for MAP4K3 phosphorylation. Indeed, RagC is phosphorylated in vivo in Kc167 cells on Ser388. If MAP4K3 were to phosphorylate RagC, this would provide a mechanism for regulation of the Rag GTPases, which to date is mysterious. Work in the near future should shed further light on this issue (Bryk, 2010).

In summary, this study has characterized the physiological function of MAP4K3 in Drosophila, and shown that it modulates TORC1 activity, tissue growth and lipid metabolism in the animal. Physical interaction data hints at a possible link between MAP4K3 and the Rag GTPases. The organismal function of amino acid sensing by TORC1 is mainly required to spur growth when nutrient conditions are rich (Bryk, 2010).

ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase

It has been proposed that cell growth and autophagy are coordinated in response to cellular nutrient status, but the relationship between them is not fully understood. This study characterized the fly mutants of Autophagy-specific gene 1 (ATG1), an autophagy-regulating kinase, and it was found that ATG1 is a negative regulator of the target of rapamycin (TOR)/S6 kinase (S6K) pathway. The Drosophila studies have shown that ATG1 inhibits TOR/S6K-dependent cell growth and development by interfering with S6K activation. Consistently, overexpression of ATG1 in mammalian cells also markedly inhibits S6K in a kinase activity-dependent manner, and short interfering RNA-mediated knockdown of ATG1 induces ectopic activation of S6K and S6 phosphorylation. Moreover, ATG1 specifically inhibits S6K activity by blocking phosphorylation of S6K at Thr 389. Taken together, these genetic and biochemical results strongly indicate crosstalk between autophagy and cell growth regulation (Lee, 2007).

Previous studies have demonstrated that homozygous EP3348 flies, in which a single P element is inserted into the 5' untranslated region of DmATG1, show larval/pupal lethality. However, after the genomic background of the EP3348 line was cleared by four backcrosses with w1118 flies, about 30% of homozygous mutants were found to develop to adults. To confirm that the P-element insertion in EP3348 hampers transcription of DmATG1 (Scott, 2004), quantitative real-time reverse transcriptase-PCR (qRT-PCR), was performed. This showed highly reduced DmATG1 expression in the mutant. Therefore, this cleared EP3348 allele was named DmATG1¹.

The mitotic phenotypes in the larval brain and imaginal discs of DmATG1 mutants, DmATG1¹ and DmATG1^Δ3d (a null allele of DmATG1 were examined. However, no notable mitotic defects could be found in the mutants compared with the wild-type control (w1118. Consistently, DmATG1 mutants had no gross chromosomal abnormalities in the neuroblast cells of third instar larval brains (Lee, 2007).

Next, defects in autophagy in DmATG1¹ flies were examined using toluidine blue-azure II staining and transmission electron microscopy (TEM) analyses. As expected, DmATG1¹ flies showed marked defects in the induction of autophagy under conditions of starvation. This is highly consistent with the previous study using DmATG1^Δ3d (Scott, 2004). These results strongly supported that DmATG1¹ is a new hypomorphic mutant allele of DmATG1 (Lee, 2007).

To understand further the in vivo roles of ATG1 in Drosophila, double-mutant lines were generated for dTOR^P1 (a loss-of-function mutant allele of dTOR and DmATG1 mutants (DmATG1¹, DmATG1^Δ3d and EP3348). Homozygous dTOR^P1 mutants showed growth arrest in the second/early third instar larval stage and markedly delayed growth. Surprisingly, homozygous dTOR^P1 larvae with a heterozygous genetic background of DmATG1¹ or DmATG1^Δ3d not only grew faster than homozygous dTOR^P1, but also extended their developmental stage to the mid-late third instar larval stage. Furthermore, the double-homozygous mutants between dTOR^P1 and various DmATG1 mutants survived up to the mid-late third instar larval stage, which is inconsistent with the previous results (Scott, 2004; Lee, 2007).

In addition, it was found that another phenotype of dTOR^P1 mutants, lipid vesicle aggregation in the fat body, was also suppressed by a reduction of the gene dosage of DmATG1. These results implicated that ATG1 negatively mediates the developmental and physiological roles of TOR in Drosophila (Lee, 2007).

Since dTOR regulates cell growth in a cell-autonomous manner, whether DmATG1 is also involved in this role of dTOR was examined. The cell and nuclear sizes of the salivary gland cells were markedly reduced in dTOR^P1 larvae. Intriguingly, the heterozygous genetic background of DmATG1¹ or DmATG1^Δ3d partly rescued the reduced cell and nuclear size phenotype of dTOR^P1, strongly implicating that DmATG1 mediates the crucial function of dTOR in cell growth regulation. This is further supported by recent results, that DmATG1-null cells have a relative growth advantage over wild-type cells when treated with rapamycin, a specific inhibitor of dTOR (Lee, 2007).

To examine the possibility that the suppression of dTOR^P1 phenotypes by DmATG1 mutation resulted from altered auto-phagic activities, the genetic interactions were investigated between dTOR and other autophagy-related genes, such as ATG6 (Beclin1) and UVRAG (UV radiation resistance associated gene), which are known to form a complex regulating autophagosome formation (Liang, 2006). As a result, ATG6 and UVRAG mutations did not suppress the developmental delay and cell growth defects of dTOR mutants, showing that the interaction between dTOR and DmATG1 is not caused indirectly by uncontrolled regulation of autophagy (Lee, 2007).

To determine the functional interaction between ATG1 and S6K, a downstream effector of TOR, double-mutant lines were generated between DmATG1¹ and dS6K mutants -- a hypomorphic allele, dS6K⁰⁷⁰⁸⁴, and a null allele, dS6K^l−1. Using TEM analyses, it was observed that a reduction of dS6K gene dosage did not rescue the defects in autophagosome formation in starved DmATG1¹ homozygous larvae. However, surprisingly, the reduced gene dosage of dS6K increased the eclosion rate of homozygous DmATG1¹ in a dS6K gene dosage-dependent manner. Although the possibility that S6K promotes autophagy as reported previously (Scott, 2004) cannot be excluded, these data indicate that dS6K has an important role in DmATG1-dependent developmental processes (Lee, 2007).

Next, a biochemical analysis was conducted to examine the effect of loss of DmATG1 on dS6K activation. As a result, it was found that dS6K was markedly activated (~threefold increase) in homozygous DmATG1¹ larvae and pupae, measured by the phosphorylation of dS6K at the Thr 398 site, compared with the wild-type controls. Notably, the increased level of dS6K phosphorylation in DmATG1¹ mutants was about one-third of that in flies overexpressing Rheb. Consistent with this, DmATG1 overexpression almost completely inhibited dS6K Thr 398 phosphorylation in Drosophila (Scott, 2007). These results strongly suggest an important role of ATG1 in the regulation of S6K (Lee, 2007).

To extend these findings to the mammalian system and also to investigate further the molecular mechanism of the interaction between ATG1 and TOR/S6K, the effect of ATG1 on the activity of S6K was examined in mammalian cells. There are two isoforms of ATG1 in mammals, which are named UNC-51-like kinase (ULK) 1 and 2. However, according to the agreement on gene nomenclature made by researchers in the field of autophagy, they were renamed ATG1α and ATG1β, respectively. Nutrient deprivation of HEK293T cells abolished the phosphorylation of S6K at both Thr 229 and Thr 389 sites, which represents the activation status of S6K . When nutrients including amino acids and glucose (DMEM) were added back to the cells, the phosphorylation of both sites in S6K was strongly induced. However, co-expression of wild-type mouse ATG1α (ATG1α WT) strongly inhibited S6K activity induced by DMEM. On the contrary, a kinase-dead form of ATG1α (ATG1α KI) was not able to block the nutrient-induced activation of S6K, showing that ATG1α inhibits S6K in a kinase activity-dependent manner. Consistently, epidermal growth factor (EGF)-stimulated S6K activation was also inhibited by ATG1α. Furthermore, ATG1β, another isoform of ATG1, has the same inhibitory effect on S6K phosphorylation as ATG1α. These data strongly suggest that ATG1 regulates the activities of upstream kinases or phosphatases of S6K, which affect both Thr 229 and Thr 389 phosphorylation (Lee, 2007).

As ATG1 is a crucial regulator of autophagy in yeast and Drosophila, whether overexpression of ATG1 can induce autophagy in mammalian cells was tested. Nutrient deprivation was able to induce autophagy in MCF-7 cells, whereas overexpression of ATG1 did not induce autophagy in MCF-7 and HEK 293T cells, indicating that the inhibition of TOR/S6K by ATG1 is not an indirect consequence of an ectopic induction of autophagy. This was further supported by the observation that overexpression of ATG6 and UVRAG did not inhibit the phosphorylation of S6K , which is also highly consistent with the above Drosophila data (Lee, 2007).

Then, short interfering RNA (siRNA) targeting was used for ATG1α and ATG1β messenger RNA to confirm that ATG1 inhibits S6K activity. The efficacy of siRNA was verified by qRT–PCR using ATG1-specific primers. Transfection of ATG1α and ATG1β siRNA to HEK 293T cells led to increased phosphorylation of S6K Thr 389 and S6 (the only proven in vivo substrate of S6K) Thr 235/236. This result was further supported by immunocytochemistry by using phosphospecific S6 antibody; ATG1 siRNA transfection alone induced phosphospecific immunostaining of S6 in starved cells. Taken together, these results clearly demonstrate that ATG1 inhibits S6K and S6 in vivo (Lee, 2007).

Notably, the level of S6K activation by ATG1 siRNA was about 5% of that by nutritional stimulation. This weak activation of S6K resulted from partial gene knockdown by RNAi. Consistent with this conclusion, more pronounced activation of dS6K was observed in DmATG1 mutants in Drosophila, which contains only a single orthologue of ATG1 (Lee, 2007).

S6K is in the AGC kinase family, which also includes Akt and p90 ribosomal S6 kinase (RSK). These kinases are regulated by a similar mechanism in which both phosphorylation at their activation loop and a hydrophobic motif next to the kinase domain are required for full activation. 3-Phosphoinositide-dependent kinase 1 (PDK1) is a kinase responsible for phosphorylation at the activation loop of AGC kinases. In the case of S6K, PDK1 directly phosphorylates the Thr 229 residue at the activation loop of S6K, which is strictly dependent on the previous phosphorylation of Thr 389 at the hydrophobic motif. These motifs are well conserved among the family members in different species. Therefore, whether ATG1α also affects the phosphorylation of Akt and RSK was examined. Interestingly, the phosphorylation of Akt and RSK was not affected by ATG1α, with or without stimulation by insulin and EGF. These data indicate that ATG1α specifically modulates S6K activity (Lee, 2007).

Next, to understand the molecular mechanism of the specific regulation of S6K by ATG1, whether ATG1 affects the phosphorylation of Thr 229 in S6K was investigated by using an S6K mutant that specifically mimics the phosphorylated form of S6K, Thr 389 Glu. As a result, Thr 229 phosphorylation of the S6K Thr 389 Glu mutant was not affected by wild-type ATG1. Because Akt was not inhibited by ATG1, it is unlikely that ATG1 regulates Thr 389 phosphorylation of S6K by inhibiting the PDK1/Akt signalling module. Therefore, it is believed that ATG1 modulates S6K activity by affecting S6K Thr 389-specific kinases or phosphatases (Lee, 2007).

In summary, under nutrient-rich conditions, activation of TOR leads to inhibition of ATG1, which facilitates S6K Thr 389 phosphorylation and the subsequent phosphorylation of Thr 229 by PDK1 to activate S6K fully. Consequently, activated S6K promotes cell growth. However, under conditions of starvation, TOR becomes quiescent and ATG1 can inhibit S6K by blocking Thr 389 phosphorylation. This nutrient-dependent signalling switch operated by TOR and ATG1 is highly consistent with that in yeast. The observations described in this study clearly show the presence of crosstalk between ATG1 and S6K signalling, in which ATG1 specifically inhibits S6K. This study also showed that this is evolutionarily conserved between Drosophila and mammals. It is believed that these biochemical data and the fly system will be useful in future studies that address the detailed molecular mechanism of crosstalk between the two nutrition-dependent physiological processes -- autophagy and cell growth (Lee, 2007).

AKT and TOR signaling set the pace of the circadian pacemaker

The circadian clock coordinates cellular and organismal energy metabolism. The importance of this circadian timing system is underscored by findings that defects in the clock cause deregulation of metabolic physiology and result in metabolic disorders. On the other hand, metabolism also influences the circadian clock, such that circadian gene expression in peripheral tissues is affected in mammalian models of obesity and diabetes. However, to date there is little to no information on the effect of metabolic genes on the central brain pacemaker which drives behavioral rhythms. This study found that the AKT and TOR-S6K pathways, which are major regulators of nutrient metabolism, cell growth, and senescence, impact the brain circadian clock that drives behavioral rhythms in Drosophila. Elevated AKT or TOR activity lengthens circadian period, whereas reduced AKT signaling shortens it. Effects of TOR-S6K appear to be mediated by SGG/GSK3beta, a known kinase involved in clock regulation. Like SGG, TOR signaling affects the timing of nuclear accumulation of the circadian clock protein Timeless. Given that activities of AKT and TOR pathways are affected by nutrient/energy levels and endocrine signaling, these data suggest that metabolic disorders caused by nutrient and energy imbalance are associated with altered rest:activity behavior (Zheng, 2010).

There are several possible mechanisms by which nutrient and energy metabolism could affect peripheral clocks. Local physiological factors dependent on metabolic activity could influence the expression of core clock components and of nuclear receptors that regulate clock gene expression. Indeed, cellular redox state, AMPK activity, NAD+ levels, and SIRT1 activities appear to feed into the circadian clock in peripheral tissues such as the liver. AMPK, which acts upstream of TSC in mammals, directly phosphorylates Cryptochrome in peripheral tissues. However, prior to this work, there was no known mechanism for the modulation of the central pacemaker by nutrient-sensing pathways. This study identifies such a mechanism by demonstrating that metabolic genes such as AKT and TOR-S6K act in the central pacemaker cells in the brain. The lengthened circadian period caused by high-fat diet in mammals is likely mediated by these molecules. This conclusion is further supported by a recent cell-culture-based genome-wide RNAi study that implicated the PI3K-TOR pathway in the regulation of circadian period. In addition, another ribosomal S6 kinase (S6KII) was found to influence the circadian clock through its interaction with casein kinase 2β. Importantly, daily fasting:feeding cycles driven by the central clock regulate circadian gene transcription in the liver, whereas clock function in the liver contributes to energy homeostasis. It is speculated that metabolic stress or energy imbalance affects AKT and TOR-S6K signaling, resulting in general circadian disruption, which in turn exacerbates metabolic deregulation and, consequently, facilitates the development of metabolic syndromes prevalent in modern society (Zheng, 2010).

A mitochondrial ATP synthase subunit interacts with TOR signaling to modulate protein homeostasis and lifespan in Drosophila

Diet composition is a critical determinant of lifespan, and nutrient imbalance is detrimental to health. However, how nutrients interact with genetic factors to modulate lifespan remains elusive. This study investigated how diet composition influences mitochondrial ATP synthase subunit d (ATPsyn-d) in modulating lifespan in Drosophila. ATPsyn-d knockdown extends lifespan in females fed low carbohydrate-to-protein (C:P) diets but not the high C:P ratio diet. This extension is associated with increased resistance to oxidative stress; transcriptional changes in metabolism, proteostasis, and immune genes; reduced protein damage and aggregation, and reduced phosphorylation of S6K and ERK in TOR and mitogen-activated protein kinase (MAPK) signaling, respectively. ATPsyn-d knockdown did not extend lifespan in females with reduced TOR signaling induced genetically by Tsc2 overexpression or pharmacologically by rapamycin. These data reveal a link among diet, mitochondria, and MAPK and TOR signaling in aging and stresses the importance of considering genetic background and diet composition in implementing interventions for promoting healthy aging (Sun, 2014).

Dietary nutrients are among the most critical environmental factors that modulate healthspan and lifespan. Nutrient imbalance is a major risk factor to human health and common among old people. Dietary restriction (DR), by reducing the amount of all or specific nutrients, is a potent nongenetic intervention that promotes longevity in many species. In general, protein restriction is more effective in influencing lifespan than sugar or calorie restriction in Drosophila. However, increasing evidence indicates that the composition of dietary nutrients, such as carbohydrate-to-protein (C:P) ratio, is more critical than individual nutrients in affecting health and lifespan. Optimal lifespan peaks at the C:P ratio 16:1 in Drosophila and 9:1 in Mexican fruit fly. A recent study in mice shows that lifespan is primarily regulated by the C:P ratio in the diet and tends to be longer with higher C:P ratios. Diet composition is also critical for DR to promote longevity in nonhuman primate rhesus monkeys. Two major nutrient-sensing pathways are known to modulate lifespan. One is target-of-rapamycin (TOR) signaling that mostly senses cellular amino acid content and the other is insulin/insulin-like signaling that primarily responds to circulating glucose and energy levels. Excessive carbohydrate and protein intake both contribute to development of insulin resistance and diabetes in animal models and humans. Dietary macronutrients, such as sugar, protein, and fat, may interact with each other to influence nutrient-sensing pathways and consequently health outcome. It is thus critical to take into account diet composition in elucidating molecular mechanisms of aging and in developing effective interventions for promoting healthy aging (Sun, 2014).

Aging is associated with transcriptional and translational changes in many genes and proteins. Some age-related changes are evolutionarily conserved, and many function in nutrient metabolism, such as mitochondrial electron transfer chain (ETC) genes, many of which are downregulated with age in worms, flies, rodents, and humans. Knocking down ETC genes affects lifespan in yeast, worms, and flies. Mitochondrial genes also play a key role in numerous age-related diseases, such as Parkinson's and Alzheimer's disease. However, how mitochondrial genes interact with nutrients to modulate lifespan and health-span remains incompletely elucidated. Understanding gene-environment interactions will be a key to tackle aging and age-related diseases (Sun, 2014).

ATP synthase subunit d (ATPsyn-d) is a component of ATP synthase, ETC complex V, and is known to modulate lifespan in C. elegans. How ATPsyn-d modulates lifespan and whether it functions in modulating lifespan in other species remain to be determined (Sun, 2014).

Given the importance of nutrients as environmental factors in modulating lifespan, this study has investigated whether and how ATPsyn-d interacts with dietary macronutrients to modulate lifespan in Drosophila. ATPsyn-d was found to interact with dietary macronutrients to influence accumulation of oxidative damage and protein aggregates; resistance to oxidative stress; and expression of numerous genes involved in metabolism, proteolysis, and innate immune response and more importantly to modulate lifespan. Moreover, ATPsyn-d affects mitogen-activated protein (MAP) kinase (MAPK) signaling and genetically interacts with TOR signaling to influence lifespan of flies in a diet-composition-dependent manner. This study reveals the critical interaction between mitochondrial genes and nutritional factors and the underlying mechanisms involving TOR signaling in modulating lifespan (Sun, 2014).

Considering the essential role of mitochondrial function in metabolism and aging, this study investigated how diet composition influences the function of ATPsynd, a component of mitochondrial ATP synthase, in aging and the underlying mechanisms. ATPsyn-d knockdown extends lifespan in Drosophila under low sugar-high protein diets, but not under a high sugar-low protein diet. Lifespan extension induced by ATPsyn-d knockdown is associated with increased resistance to oxidative stress and improved protein homeostasis. Furthermore, evidence is provided suggesting ATPsyn-d modulates lifespan through genetically interacting with TOR signaling. Knocking down of atp-5, the worm ATPsynd, extends lifespan in C. elegans, along with the current data suggesting a conserved role of ATPsyn-d in modulating lifespan. Altogether, these findings reveal a connection among diet, mitochondrial ATP synthase, and MAPK and TOR signaling in modulating lifespan and shed light on the molecular mechanisms underlying the impact of diet composition on lifespan (Sun, 2014).

The following model is proposed to explain how ATPsyn-d interacts with dietary macronutrients to modulate lifespan, considering the genetic interaction between ATPsyn-d and TOR signaling and the fact that suppression of TOR signaling by altering expression of its components, such as Tsc1/Tsc2, S6K, and 4E-BP, activates autophagy, improves proteostasis, and promotes longevity in high-protein diets, but not necessarily low-protein diets. It is postulated that TOR signaling is regulated by ATPsyn-d and perhaps other mitochondrial proteins. ATPsyn-d knockdown reduces MAPK signaling and probably affects other signaling pathways, which may consequently decrease TOR signaling to extend lifespan in Drosophila fed high-protein diets, such as SY1:9 and SY1:1, but not low protein diets. It is possible that diet-dependent response is due to knockdown of ATPsyn-d protein to different extent by RNAi under different dietary conditions. This is not likely the case. The amount of ATPsyn-d knockdown is not much different between flies on sugar (S) and yeast (Y) SY1:9 and SY9:1, although lifespan is not increased by ATPsyn-d knockdown for flies under SY9:1. Therefore, variations in ATPsyn-d knockdown under current experimental conditions unlikely contribute significantly to diet-dependent lifespan extension. Consistent with this model, ATPsyn-d knockdown increases resistance to acute oxidative stress, reduces cellular oxidative damage, and improves proteostasis in Drosophila. Reduced oxidative damage by ATPsynd knockdown may lead to decreased MAPK signaling, which in turn modulates TOR signaling and proteostasis (Sun, 2014).

Another likely scenario would be that ATPsyn-d and TOR signaling form a positive but vicious feedback loop through MAPK signaling to induce molecular, metabolic, and physiological changes detrimental to lifespan. This vicious cycle can be disrupted by high-C:P diet, knockdown of mitochondrial genes, or suppression of TOR signaling pharmacologically by rapamycin or genetically by Tsc2 overexpression. Consistent with this possibility is that ATPsyn-d knockdown reduces phosphorylation of S6K, a component of TOR signaling, and increases expression of genes involved in maintaining proteostasis and possibly autophagy, which are regulated by TOR signaling. The level of pS6K reflects the strength of TOR signaling, and reduction- of-function mutants of S6K are known to extend lifespan in several species. ATPsyn-d may genetically interact with TOR signaling to modulate lifespan by influencing protein levels of both S6K and pS6K, although it does not necessarily affect the pS6K/S6K ratio, which may not be a reliable indicator for the strength of TOR signaling under the three SY diets due to the change of S6K level. Furthermore, ATPsyn-d knockdown reduces oxidative damage and polyubiquitinated protein aggregates, which are biomarkers of aging. Rapamycin reduces lifespan extension induced by ATPsyn-d knockdown, which may be due to exacerbation of some detrimental effects of reduced TOR signaling. However, this observation further supports the connection between ATPsyn-d and TOR signaling. Although both rapamycin and ATPsyn-d knockdown reduce pS6K level, ATPsyn-d knockdown, but not rapamycin, decreases S6K level, suggesting ATPsyn-d knockdown and rapamycin affect TOR signaling in different manners. Further studies are warranted to clarify the epistatic relationship between ATPsyn-d and TOR signaling (Sun, 2014).

Increasing evidence has demonstrated the importance of diet composition or carbohydrate to protein ratio in modulating lifespan and health. Nutrient geometry studies conducted in Drosophila have shown that C:P ratio in the diet is far more important in determining lifespan than calorie content or single macronutrient. A recent tour de force nutrient geometry study in mice has confirmed and expanded the view on the critical role of C:P ratio in regulating lifespan and cardiometabolic health to mammals. An important implication from nutrient geometric studies is that diet composition would have a significant impact on the effectiveness of inventions for promoting healthy aging by genetic, pharmaceutical, or nutraceutical approaches (Sun, 2014).

This indeed is the case, although evidence comes from only a handful of studies. Rapamycin feeding extends lifespan in yeast, worms, flies, and mice. Although rapamycin feeding has been shown to extend lifespan of flies under a broad range of diets, some studies have shown that rapamycin feeding does not extend lifespan in flies under high carbohydrate-low protein diets. Supplementation of a nutraceutical derived from cranberry extends lifespan in female flies under a high-C:P-ratio diet, but not a low-C:P-ratio diet. Suppression of TOR signaling by overexpression of Tsc1/Tsc2 extends lifespan in flies under relatively higher-protein diets, but not under low-protein diets, although those studies focused on the variation of protein concentration instead of C:P ratio. Consistent with the link between ATPsyn-d and TOR signaling, ATPsyn-d knockdown extends lifespan in female flies under low sugar-high protein diets, but not high sugar-low protein diet, likely due to the fact that TOR signaling is already low under the high sugar-low protein diet. It was further shown that rapamycin feeding extends lifespan in wild-type female flies, but not in ATPsyn-d knockdown flies (Sun, 2014).

Aging is associated with profound decline in protein homeostasis, and many longevity-related pathways, such as TOR and insulin-like signaling, modulate lifespan through improving proteostasis. Suppression of TOR signaling extends lifespan through decreasing protein translation and increasing autophagy, key processes for maintaining proteostasis. This study found that ATPsyn-d knockdown reduces the level of 4-HNE (an α, β-unsaturated hydroxyalkenal that is produced by lipid peroxidation) protein adducts; a biomarker for lipid protein oxidation; and the level and aggregation of polyubiquitinated protein, a biomarker for proteostasis and aging. ATPsyn-d is a key component of mitochondrial ATP synthase complex. Along with the link between ATPsyn-d and TOR signaling, these data suggest that mitochondrial ATP synthase is critical for maintaining proteostasis and modulating lifespan. This notion is further supported by a recent study showing that α-ketoglutarate, an intermediate in the TCA cycle, suppresses mitochondrial ATP synthase probably by binding to ATP synthase subunit b (ATPsyn-b) and also inhibits TOR signaling to extend lifespan in C. elegans. However, it remains to be determined whether suppression of ATP synthase by α-ketoglutarate results in inhibition of TOR signaling in C. elegans or any other species. It is also likely that ATPsyn-d and ATPsyn-b influences ATP synthase and TOR signaling through different mechanisms, because α-ketoglutarate reduces cellular ATP level in C. elegans, whereas ATPsyn-d knockdown does not significantly change or even increase ATP level in Drosophila. This also suggests that lifespan extension is not necessarily associated with decreased ATP level, which is supported by a study in Drosophila showing that any change of ATP level is not correlated with any change of lifespan induced by knockdown of a number of mitochondrial genes. Nevertheless, these studies suggest that ATP synthase is a key and conserved player linking dietary nutrients from TOR signaling to proteostasis and lifespan (Sun, 2014).

Similar to many longevity-related mutants, lifespan extension induced by ATPsyn-d knockdown is associated with reduced oxidative damage and increased resistance to oxidative stress. ATPsyn-d knockdown increases lifespan and resistance to paraquat, an acute oxidative stress response, under SY1:9 or SY1:1. However, ATPsyn-d knockdown increases resistance to paraquat but does not extend lifespan in female flies under SY9:1. In addition, ATPsyn-d knockdown decreases 4-HNE level, an indicator of accumulated oxidative damage, under SY1:9, but not SY1:1. These indicate that the effect of ATPsyn-d knockdown on oxidative damage and lifespan depends on diet composition, suggesting that oxidative stress resistance is at most partially responsible for lifespan extension. This should not be surprising because it is consistent with numerous studies in the literature showing that stress resistance does not always result in lifespan extension despite the strong link between oxidative stress and aging (Sun, 2014).

The role of mitochondrial genes in modulating lifespan is complex. Knockdown of some electron transfer chain (ETC) genes increases lifespan whereas knockdown of others decreases or does not alter lifespan in C. elegans and Drosophila. This study reveals another layer of complexity regarding the role of ETC genes in lifespan modulation, namely the impact of diet composition. These findings indicate that ATPsyn-d knockdown promotes longevity at least partially through TOR signaling. TOR signaling senses cellular amino acid content and regulates numerous biological processes, including translation, autophagy, and lifespan. 4E-BP, a translational repressor in TOR signaling, mediates lifespan extension induced by dietary restriction (Sun, 2014).

Activated 4E-BP suppresses general translation but selectively increases translation of some mitochondrial ETC genes, the latter of which results in increased mitochondrial biogenesis and potentially lifespan. Lifespan extension induced by dietary restriction is suppressed by knocking down ETC genes regulated by 4E-BP. The findings by Zid suggest that increased protein expression of some ETC genes is associated with lifespan extension induced by dietary restriction. However, unlike those ETC genes, ATPsyn-d knockdown extends instead of decreases lifespan under high-protein diets. Therefore, it is likely that ETC genes can be categorized into two groups: one selectively upregulated by activated 4E-BP and the other insensitive to activated 4E-BP, the latter of which may include ATPsyn-d. The two groups of ETC genes may interact with dietary macronutrients to modulate lifespan perhaps through different modes of action. Future studies are warranted to investigate the dichotomous role of translation of ETC genes in modulating lifespan (Sun, 2014).

DEVELOPMENTAL BIOLOGY

The S6k gene gives rise to two major transcripts that are expressed throughout development, suggesting a continuous requirement for this enzyme. Two major transcripts of 3.0 and 5.0 kb are found at abundant steady-state levels in whole animal poly(A)⁺ or total RNA. Two smaller transcripts (<3.0 kb) are expressed at lower levels at all tested developmental stages. All of these transcripts hybridize with probes that include the catalytic domain, C-terminal domain and the 3' UTR. Only the two major transcripts are detected with 5'-UTR probes common to class 1 and class 2 S6k cDNAs that also share a complete ORF. These 5'-UTR probes did not detect the smaller transcripts, suggesting that they have different N-terminal sequences. The smaller S6k transcripts are the predominant forms found in cultured air8 blood cells, which are derived from RpS6^air8 mutant larvae suggesting that the S6k gene gives rise to tissue-specific expression patterns. An alternative explanation is that the S6k transcripts expressed in air8 cultured cells are influenced by the RpS6^air8 mutation (Watson, 1996).

TSC1/2 tumour suppressor complex maintains Drosophila germline stem cells by preventing differentiation

Tuberous sclerosis complex human disease gene products TSC1 and TSC2 form a functional complex that negatively regulates target of rapamycin (TOR), an evolutionarily conserved kinase that plays a central role in cell growth and metabolism. This study describes a novel role of TSC1/2 in controlling stem cell maintenance. In the Drosophila ovary, disruption of either the Tsc1 or Tsc2 gene in germline stem cells (GSCs) leads to precocious GSC differentiation and loss. The GSC loss can be rescued by treatment with TORC1 inhibitor rapamycin, or by eliminating S6K, a TORC1 downstream effecter, suggesting that precocious differentiation of Tsc1/2 mutant GSC is due to hyperactivation of TORC1. One well-studied mechanism for GSC maintenance is that BMP signals from the niche directly repress the expression of a differentiation-promoting gene bag of marbles (bam) in GSCs. In Tsc1/2 mutant GSCs, BMP signalling activity is downregulated, but bam expression is still repressed. Moreover, Tsc1 bam double mutant GSCs could differentiate into early cystocytes, suggesting that TSC1/2 controls GSC differentiation via both BMP-Bam-dependent and -independent pathways. Taken together, these results suggest that TSC prevents precocious GSC differentiation by inhibiting TORC1 activity and subsequently differentiation-promoting programs. As TSC1/2-TORC1 signalling is highly conserved from Drosophila to mammals, it could have a similar role in controlling stem cell behaviour in mammals, including humans (Sun 2010).

TSC1/2 is known to regulate cell growth via inhibition on TORC1. This study demonstrates that it also functions by inhibiting the activity of TORC1 to maintain GSCs. Treatment with rapamycin, a TORC1-specific inhibitor, can completely rescue GSC loss in Tsc1 mutants. In addition, eliminating S6K, which functions downstream of TORC1 in regulating protein translation, could also completely rescue GSC loss in Tsc2 mutants. Interestingly, the daughters of Tor mutant GSCs can differentiate into germline cyst properly, indicating that TOR is normally not required for differentiation, but its hyperactivation in Tsc1/2 mutants drives precocious GSC differentiation. The simplest explanation of the delayed cystoblast differentiation in rapamycin-treated females might be a non-specific effect of drug treatment. However, it is also possible that TORC1 inhibition by rapamycin might cause repression of some, but not all, aspects of TOR function, which leads to uncoordinated development and/or differentiation of cystoblasts in response to GSC division. Consistently, accumulated cystoblasts where also observed when overexpressing both Tsc1 and Tsc2 in the germline. Together with the observation that TSC1/2-TORC1 signaling controls cell growth of germline cysts, this study suggests that TSC1/2-TORC1 may serve as a signaling integration point that orchestrates germline division, differentiation and development in order to control egg production in response to the local micro-environment and the system environment of the animals (Sun 2010).

In the Drosophila ovary, BMP signaling from the niche directly suppresses bam expression in GSCs to prevent differentiation. This signaling is crucial for GSC maintenance. As revealed by pMad expression, BMP signaling activity is significantly downregulated in Tsc1 mutant GSCs. This study also demonstrated that downregulation of pMad in Tsc1 mutant GSCs is mediated by TORC1 hyperactivation, as rapamycin treatment is able to restore the downregulated pMad level. However, TOR is not required for proper BMP signaling activity because pMad expression is not altered in rapamycin-treated germaria. Therefore, only TORC1 hyperactivation could inhibit BMP signaling in GSCs through unknown mechanisms, and this inhibitory effect occurs specifically in GSCs, as BMP signaling activity is not altered in Tsc1 mutant imaginal disc cells (Sun 2010).

Logically, bam expression could be derepressed in Tsc1 mutant GSCs as a consequence of BMP pathway downregulation. Surprisingly, no significant upregulation of bam-GFP expression could be detected in mutant GSCs, although in other GSCs that were compromised by BMP signaling, such as tkv mutant and mad mutant GSCs, bam transcription is significantly upregulated. Nevertheless, there might still be residual BMP signaling activities in Tsc1/2 mutant GSCs that are sufficient to suppress bam expression. Consistent with this notion is the observation that bam-GFP is not obviously upregulated in aged GSCs, even if BMP signaling activity has been significantly reduced. Together with the observation that bam mutation could not rescue the differentiation of Tsc1 mutant germ cells, it is suggested that the compromised BMP signaling activity may not be primarily responsible for Tsc1/2 mutant GSC loss. It is not clear why the effect of TSC1/2 on BMP signaling occurs specifically in GSCs. Possibly, Tsc1/2 mutant GSCs, once induced, have already primed for differentiation through a Bam-independent mechanism, which may trigger a positive feedback signal to inhibit BMP signaling activity, in order to facilitate differentiation (Sun 2010).

This study also reveals a BMP-Bam-independent mechanism that probably underlies the major role of TSC1/2-TORC1 signaling in GSC maintenance. The phenotype of Tsc1 bam double mutant germ cells differs from the bam alone mutant germ cells, as the double mutant GSCs can still become lost from the niche over time and undergo further differentiation into early cystocytes. Interestingly, the phenotype of Tsc1/2 mutant GSCs is similar to that of pelota (pelo) mutants. Pelo encodes a translational release factor-like protein and may regulate GSC maintenance at the translational level. In pelo mutant GSCs, there is also a downregulation of BMP signaling but no obvious upregulation of bam expression, and bam pelo double mutant germ cells are able to undergo similar limited differentiation into cystocytes, suggesting that TSC1/2 and Pelo might function in the same or parallel pathway to control GSC differentiation. It is proposed that similar to Pelo, TSC1/2 might function in a parallel pathway with the BMP-Bam pathway to control GSC differentiation, possibly by regulating the translation of differentiation-related mRNAs (Sun 2010).

Pum and Nos, which are known to function together to repress translation of the target mRNAs in embryos, are also essential for GSC maintenance. Recent genetic and biochemical studies suggest that Bam/Bgcn may directly inhibit the function of Pum/Nos to allow cystoblast differentiation. However, BMP signaling activation is able to prevent differentiation of nos mutant primordial germ cells, indicating that Pum/Nos could also function in parallel with the BMP-Bam pathway to control germ cell differentiation. In the future, it would be important to determine the functional relationships between the TSC1/2-TORC1 pathway, Pelo and Pum/Nos in regulating GSCs, and whether these factors, together with the microRNA pathway, target similar mRNAs to control GSC differentiation (Sun 2010).

This study has identified a novel role of TSC1/2 in controlling GSC maintenance and differentiation in the Drosophila ovary. Increasing evidence also suggests similar roles for TSC1/2-TOR signaling in regulating adult stem cell differentiation in mammals. For example, TSC1/2-mTOR signaling is also required for maintaining the quiescence of haematopoietic stem cells (HSCs), as Tsc1 deletion drives HSCs from quiescence to rapid cycling, which compromises HSC self-renewal. Thus, TSC1/2-TOR signaling could have an evolutionarily conserved role in regulating stem cell maintenance and differentiation from Drosophila to mammals (Sun 2010).

Anaplastic lymphoma kinase spares organ growth during nutrient restriction in Drosophila

Developing animals survive periods of starvation by protecting the growth of critical organs at the expense of other tissues. This study used Drosophila to explore the as yet unknown mechanisms regulating this privileged tissue growth. As in mammals, it was observed in Drosophila that the CNS is more highly spared than other tissues during nutrient restriction (NR). Anaplastic lymphoma kinase (Alk) efficiently protects neural progenitor (neuroblast) growth against reductions in amino acids and insulin-like peptides during NR via two mechanisms. First, Alk suppresses the growth requirement for amino acid sensing via Slimfast/Rheb/TOR complex 1. And second, Alk, rather than insulin-like receptor, primarily activates PI3-kinase. Alk maintains PI3-kinase signaling during NR as its ligand, Jelly belly (Jeb), is constitutively expressed from a glial cell niche surrounding neuroblasts. Together, these findings identify a brain-sparing mechanism that shares some regulatory features with the starvation-resistant growth programs of mammalian tumors (Cheng, L. Y. 2011).

This study found that CNS progenitors are able to continue growing at their normal rate under fasting conditions severe enough to shut down all net body growth. Jeb/Alk signaling was identified as a central regulator of this brain sparing, promoting tissue-specific modifications in TOR/PI3K signaling that protect growth against reduced amino acid and Ilp concentrations. These findings highlight that a 'one size fits all' wiring diagram of the TOR/PI3K network should not be extrapolated between different cell types without experimental evidence. The two molecular mechanisms by which Jeb/Alk signaling confers brain sparing is discussed, and how they may be integrated into an overall model for starvation-resistant CNS growth (Cheng, L. Y. 2011).

One mechanism by which Alk spares the CNS is by suppressing the growth requirement for amino acid sensing via Slif, Rheb, and TORC1 components in neuroblast lineages. An important finding of this study is that in the presence of Alk signaling Tor has no detectable growth input (evidence from Tor clones), but in its absence (evidence from UAS-AlkDN; Tor clones) Tor reverts to its typical role as a positive regulator of both growth and proliferation. The growth requirement for Slif/TORC1 is clearly much less in the CNS than in other tissues such as the wing disc but a low-level input cannot be ruled out due to possible perdurance inherent in any clonal analysis. Although Slif, Rheb, Tor, and Raptor mutant neuroblast clones attain normal volume, this reflects increased cell numbers offset by reduced average cell size. Atypical suppression of proliferation by TORC1 has also been observed in wing discs, where partial inhibition with rapamycin advances G2/M progression (Cheng, L. Y. 2011).

Alk signaling in neuroblast lineages does not override the growth requirements for all TOR pathway components. The downstream effectors S6k and 4E-BP retain functions as positive and negative growth regulators, respectively. 4E-BP appears to be particularly critical in the CNS as mutant animals have normal mass, but mutant neuroblast clones are twice their normal volume. In many tissues, 4E-BP is phosphorylated by nutrient-dependent TORC1 activity. In CNS progenitors, however, 4E-BP phosphorylation is regulated in an NR-resistant manner by Alk, not by TORC1. Hence, although the pathway linking Alk to 4E-BP is not yet clear, it makes an important contribution toward protecting CNS growth during fasting (Cheng, L. Y. 2011).

A second mechanism by which Alk spares CNS growth is by maintaining PI3K signaling during NR. Alk suppresses or overrides the genetic requirement for InR in PI3K signaling, which may or may not involve the direct binding of intracellular domains as reported for human ALK and IGF-IR (Shi, 2009). Either way, in the CNS, glial Jeb expression stimulates Alk-dependent PI3K signaling and thus neural growth at similar levels during feeding and NR. In contrast, in tissues such as the salivary gland, where PI3K signaling is primarily dependent upon InR, falling insulin-like peptides concentrations during NR strongly reduce growth (Cheng, L. Y. 2011).

The finding that Alk signals via PI3K during CNS growth differs from the Ras/MAPK transduction pathway described in Drosophila visceral muscle. However, a link between ALK and PI3K/Akt/Foxo signaling during growth is well documented in humans, both in glioblastomas and in non-Hodgkin lymphoma. Similarities with mammals are less obvious with regard to Alk ligands, as there is no clear Jeb ortholog and human ALK can be activated, directly or indirectly, by the secreted factors Pleiotrophin and Midkine (Cheng, L. Y. 2011).

A comparison of these results with those of previous studies indicates that CNS super sparing only becomes fully active at late larval stages. Earlier in larval life, dietary amino acids are essential for neuroblasts to re-enter the cell cycle after a period of quiescence. This nutrient-dependent reactivation involves a relay whereby Slif-dependent amino acid sensing in the fat body stimulates Ilp production from a glial cell niche (Sousa-Nunes, 2011). In turn, glial-derived Ilps activate InR and PI3K/TOR signaling in neuroblasts thus stimulating cell cycle re-entry. Hence, the relative importance of Ilps versus Jeb from the glial cell niche may change in line with the developmental transition of neuroblast growth from high to low nutrient sensitivity (Cheng, L. Y. 2011).

The results of this study suggest a working model for super sparing in the late-larval CNS. Central to the model is that Jeb/Alk signaling suppresses Slif/ RagA/Rheb/TORC1, InR, and 4E-BP functions and maintains S6k and PI3K activation, thus freeing CNS growth from the high dependence upon amino acid sensing and Ilps that exists in other organs. The CNS also contrasts with other spared diploid tissues such as the wing disc, in which PI3K-dependent growth requires a positive Tor input but is kept in check by negative feedback from TORC1 and S6K. Alk is both necessary (in the CNS) and sufficient (in the salivary gland) to promote organ growth during fasting. However, both Alk manipulations produce organ-sparing percentages intermediate between the 2% salivary gland and the 96% neuroblast values, arguing that other processes may also contribute. For example, some Drosophila tissues synthesize local sources of Ilps that could be more NR resistant than the systemic supply from the IPCs. In mammals, this type of mechanism may contribute to brain sparing as it has been observed that IGF-I messenger RNA (mRNA) levels in the postnatal CNS are highly buffered against NR. It will also be worthwhile exploring whether mammalian neural growth and brain sparing involve Alk and/or atypical TOR signaling. In this regard, it is intriguing that several studies show that activating mutations within the kinase domain of human ALK are associated with childhood neuroblastomas. In addition, fetal growth of the mouse brain was recently reported to be resistant to loss of function of TORC1. Finally, a comparison between the current findings and those of a cancer study, highlights that insulin/IGF independence and constitutive PI3K activity are features of NR-resistant growth in contexts as diverse as insect CNS development and human tumorigenesis (Cheng, L. Y. 2011).

Regulation of cuticle pigmentation in Drosophila by the nutrient sensing insulin and TOR signaling pathways

Insect pigmentation is a phenotypically plastic trait that plays a role in thermoregulation, desiccation tolerance, mimicry, and sexual selection. The extent and pattern of pigmentation of the abdomen and thorax in Drosophila melanogaster is affected by environmental factors such a growth temperature and access to the substrates necessary for melanin biosynthesis. This study aimed to determine the effect of nutritional status during development on adult pigmentation and test whether nutrient sensing through the Insulin/IGF and target of rapamycin (TOR) pathways regulates the melanization of adult cuticle in Drosophila. Flies reared on low quality food were shown to exhibit decreased pigmentation, which can be phenocopied by inhibiting expression of the Insulin receptor (InR) throughout the entire fly during mid to late pupation. The loss of Insulin signaling through PI3K/Akt and FOXO in the epidermis underlying the developing adult cuticle causes a similar decrease in adult pigmentation, suggesting that Insulin signaling acts in a cell autonomous manner to regulate cuticle melanization. In addition, TOR signaling increases pigmentation in a cell autonomous manner, most likely through increased S6K activity. These results suggest that nutrient sensing through the Insulin/IGF and TOR pathways couples cuticle pigmentation of both male and female Drosophila with their nutritional status during metamorphosis (Shakhamtsir, 2013).

An investigation of nutrient-dependent mRNA translation in Drosophila larvae

The larval period of the Drosophila life cycle is characterized by immense growth. In nutrient rich conditions, larvae increase in mass approximately two hundred-fold in five days. However, upon nutrient deprivation, growth is arrested. The prevailing view is that dietary amino acids drive this larval growth by activating the conserved insulin/PI3 kinase and Target of rapamycin (TOR) pathways and promoting anabolic metabolism. One key anabolic process is protein synthesis. However, few studies have attempted to measure mRNA translation during larval development or examine the signaling requirements for nutrient-dependent regulation. This work addresses this issue. Using polysome analyses, it was observed that starvation rapidly (within thirty minutes) decreased larval mRNA translation, with a maximal decrease at 6-18 hours. By analyzing individual genes, it was observed that nutrient-deprivation led to a general reduction in mRNA translation, regardless of any starvation-mediated changes (increase or decrease) in total transcript levels. Although sugars and amino acids are key regulators of translation in animal cells and are the major macronutrients in the larval diet, this study found that they alone were not sufficient to maintain mRNA translation in larvae. The insulin/PI3 kinase and TOR pathways are widely proposed as the main link between nutrients and mRNA translation in animal cells. However, this study found that genetic activation of PI3K and TOR signaling, or regulation of two effectors - 4EBP and S6K - could not prevent the starvation-mediated translation inhibition. Similarly, it was shown that the nutrient stress-activated eIF2α kinases, GCN2 and PERK, were not required for starvation-induced inhibition of translation in larvae. These findings indicate that nutrient control of mRNA translation in larvae is more complex than simply amino acid activation of insulin and TOR signaling (Nagarajan, 2014: PubMed).

Diacylglycerol lipase regulates lifespan and oxidative stress response by inversely modulating TOR signaling in Drosophila and C. elegans

Target of rapamycin (TOR) signaling is a nutrient-sensing pathway controlling metabolism and lifespan. Although TOR signaling can be activated by a metabolite of diacylglycerol (DAG), phosphatidic acid (PA), the precise genetic mechanism through which DAG metabolism influences lifespan remains unknown. DAG is metabolized to either PA via the action of DAG kinase or 2-arachidonoyl-sn-glycerol by diacylglycerol lipase (DAGL). This study reports that in Drosophila and Caenorhabditis elegans, overexpression of diacylglycerol lipase (DAGL/inaE/dagl-1) or knockdown of diacylglycerol kinase (DGK/rdgA/dgk-5) extends lifespan and enhances response to oxidative stress. Phosphorylated S6 kinase (p-S6K) levels are reduced following these manipulations, implying the involvement of TOR signaling. Conversely, DAGL/inaE/dagl-1 mutants exhibit shortened lifespan, reduced tolerance to oxidative stress, and elevated levels of p-S6K. Additional results from genetic interaction studies are consistent with the hypothesis that DAG metabolism interacts with TOR and S6K signaling to affect longevity and oxidative stress resistance. These findings highlight conserved metabolic and genetic pathways that regulate aging (Lin, 2014).

EFFECTS OF MUTATION

The adaptation of growth in response to nutritional changes is essential for the proper development of all organisms. The identification of the Drosophila homolog of the target of rapamycin (TOR), a candidate effector for nutritional sensing, is described. Genetic and biochemical analyses indicate that dTOR impinges on the insulin signaling pathway by autonomously affecting growth through modulating the activity of Drosophila S6k. However, in contrast to other components in the insulin signaling pathway, partial loss of dTOR function preferentially reduces growth of the endoreplicating tissues. These results are consistent with dTOR residing on a parallel amino acid sensing pathway (Oldham, 2000).

A tissue-specific genetic screen has been carried out for recessive mutations affecting cell growth and proliferation in the Drosophila compound eye. In this screen, genetically mosaic flies are generated in which the eye and head capsule are homozygous for a randomly induced mutation, while the rest of the body and the germ line are heterozygous and, thus, phenotypically wild type. Remarkably, mosaic flies containing a mutation in a growth-promoting gene have eye and head structures that are strongly reduced in size relative to their wild-type sized heterozygous bodies and are termed pinhead flies. Two EMS-induced pinhead mutations (2L1 and 2L19) map to chromosomal position 34A, where the Drosophila homolog of TOR (dTOR) is located. These mutations fail to complement two lethal P-element insertions, EP(2)2353 and l(2)k17004, located 262 and 211 bp, respectively, upstream of the putative translation start site of dTOR. The dTOR genomic region spans ~10 kb and is composed of seven exons (7.4 kb cDNA) encoding a 2480-amino acid protein with a predicted M_w of 282 kD. The dTOR protein exhibits 35%, 38%, 35%, and 41% overall amino acid identity to yeast TOR1, TOR2, C. elegans TOR, and Arabidopsis thaliana TOR, respectively. The overall identity with mTOR is significantly higher (56%) and is especially conserved in the kinase and FRB domains (74% and 77%). Sequence analysis of DNA from flies heterozygous for the EMS-induced dTOR mutations reveal two nucleotide substitutions. The lesion of dTOR^2L1 results in a change of a proline to a leucine at amino acid position 2303 (P2303L). The location of this mutation within a highly conserved region of the kinase domain implies that the kinase activity of dTOR is critical for its function, as has been shown for the yeast TORs. In contrast, the lesion in dTOR^2L19 is an arginine changed to a nonsense mutation at amino acid residue 248 (R248Stop), giving rise to a stop codon. This mutation would be predicted to result in a short, truncated protein and should thus be a complete loss-of-function mutation (Oldham, 2000).

The phenotypes associated with the complete loss of dTOR function are remarkably similar to phenotypes associated with mutations in the Insulin-like receptor (Inr) pathway. (1) Strong dTOR mutants arrest development at a similar stage as do strong mutants in the Inr pathway or amino acid-starved larvae with little detectable imaginal tissue. (2) dTOR mutant clones have a significant proliferative disadvantage similar to Inr pathway mutant clones. Clones of dTOR null mutant cells, although severely affected, are not cell lethal. Similarly, in most mammalian cell types, rapamycin decreases but does not abolish cell growth, except for IL2-mediated T-cell proliferation. (3) The strict autonomous control of cell growth without disturbing the specification and differentiation is also seen with the dTOR mutants. Indeed, loss of dTOR function in clones of homozygous mutant cells in the adult eye show that only the mutant cells, as exemplified by the dark, circular rhabdomeres, are severely reduced in size. Analysis of imaginal wing disc cells at the end of the third larval instar by fluorescence-activated cell sorting (FACS), confirms that cells from the weak heteroallelic combination, dTOR^2L1/dTOR^l(2)k17004, are smaller than those of wild type. The effect on cell size is more pronounced in G₁ than G₂, consistent with mTOR function having a predominante role on cell growth during G₁ (Oldham, 2000).

Despite this observation, there is no apparent difference between the distribution of dTOR mutant and wild-type cells within each phase of the cell cycle. Although the similarities of the loss-of-function mutant phenotypes of dTOR and other components of the Inr pathway are consistent with a model in which dTOR acts downstream of dPI3K, the analysis of partial loss-of-function dTOR mutants and the biochemical analysis of Drosophila S6K activity indicates a more complex relationship between dTOR and the Inr pathway (Oldham, 2000).

Genetic interactions between dTOR mutants and other mutations in the insulin pathway were examined. Drosophila Pten encodes a negative effector of insulin signaling, and eyes and heads lacking Pten function are significantly larger than wild-type heads. Removal of dTOR function strongly reduces the size of Pten mutant heads, suggesting that dTOR is required for the increased growth generated by the loss of Pten function (Oldham, 2000).

In vertebrates, S6K activity is blocked by rapamycin, an inhibitor of TOR. Therefore, Drosophila S6K activity was examined in immunoprecipitates of extracts from larvae mutant for dTOR, S6K, chico, and larvae treated with rapamycin or deprived of amino acids. A severe reduction in the phosphorylation of ribosomal protein S6 was observed in extracts from strong dTOR^2L1/dTOR^2L19 mutant larvae. This was not caused by a reduction in Drosophila S6k protein as shown by Western blotting of these extracts. In addition, the S6k protein is up-regulated in the dTOR mutant larvae and amino acid-starved larvae. In all cases, Western blot analysis has shown equivalent amounts of initiation factor 4E (eIF-4E). S6k activity is not detected in S6k^l-1 null mutants and is severely reduced when wild-type larvae are starved for amino acids or treated with rapamycin. Higher doses of rapamycin blocks development during early larval stages, leading to lethality. Analysis of the weak dTOR^2L1/dTOR^l(2)k17004 or dTOR^2L1/dTOR^EP(2)2353 heteroallelic combinations also reveal a reduction in S6k activity in the third larval instar and an up-regulation of the protein as compared with wild-type flies. The surprising fact that dTOR mutants and amino acid starvation result in an up-regulation of S6k levels suggests that dTOR and amino acids may negatively control the protein levels of S6k. Unexpectedly, S6k activity as well as protein levels are unaffected in chico mutants. It may be that Inr does not signal to S6k or that S6k resides on a parallel pathway that bifurcates upstream of Chico. In support of the latter possibility, Inr has been shown to genetically interact with PI3K independently of Chico, presumably through docking sites for the p60 adaptor of PI3K in the Inr C-terminal tail. This result suggests that there is a S6k independent pathway for growth control and that the reduced Inr-mediated PI3K signaling in a chico mutant is sufficient for S6k activation (Oldham, 2000).

The biochemical differences between the ability of Chico and dTOR to activate S6k argue for a more complex relationship between the Inr pathway and dTOR. Given the low number of pharate adults, the weights of dTOR, S6k^l-1, and chico mutants were compared at an early pupal stage. The weight of the dTOR mutant pupae is more similar to S6k than to chico mutant pupae. Thus, in the absence of S6k function or the presence of reduced dTOR levels, cellular growth rates are diminished but larvae pupariate at a larger size as a result of a longer developmental delay. Importantly, S6k mutant flies have cells that are smaller but of the normal number. However, in chico mutants, pupariation is initiated at a much smaller size. The result is that chico mutants emerge after only a 2-d delay and are smaller than dTOR and S6k mutants because of fewer and smaller cells. Therefore, while insulin signaling controls cell size and cell number, S6k primarily controls cell size. It will be of interest to know whether dTOR is also limited to controlling only cell size (Oldham, 2000).

Larvae are composed of mitotic cells, largely represented by the imaginal discs, and of endoreplicating tissues, which form larval structures like the gut, fat body, and salivary glands. An increase in DNA ploidy of larval cells is required for the ~200-fold increase in mass obtained by the larvae during the 5-d period between the completion of embryogenesis and the beginning of pupation. During starvation, larvae sacrifice their endoreplicating tissue to maintain the growth and proliferation of the mitotic cells that are required to form the reproductive adult. Furthermore, S6k activity is reduced in starved larvae and dTOR mutants. These observations prompted an analysis of the mitotic and endoreplicating tissues of dTOR, S6k, and chico mutant larvae just before pupariation. Strong dTOR and PI3K mutants, as well as amino acid-starved larvae, are incapable of growth and have barely detectable imaginal and endoreplicative tissues. Surprisingly, the wing discs of the weak dTOR heteroallelic combination are of approximately equivalent size to that of wild-type larvae, whereas those of S6k^l-1 mutants are reduced. However, the amount of endoreplicating tissue in the dTOR mutant as compared to wild-type larvae is severely decreased. This is clearly demonstrated by comparing the salivary glands of dTOR mutant and wild-type larvae. In contrast, the size of endoreplicating tissue and imaginal discs in S6k null mutants as well as chico null mutants is reduced to approximately the same extent. Staining of the salivary glands with DAPI and phalloidin reveals that the size of the nuclei and, thus, the degree of endoreplication is severely reduced in S6k, chico, and dTOR mutants. The difference in size between dTOR and S6k mutant salivary glands is largely caused by a very pronounced reduction in cytoplasmic volume in dTOR mutants. The nuclear to cytoplasmic ratio is higher in dTOR salivary glands than in y w, S6k, or chico mutant salivary glands. Thus, it appears that partial loss of dTOR function permits the growth of imaginal tissue to wild-type size, while endoreplicating tissue is disproportionally reduced, a phenotype distinct from S6k mutants. Consistent with this finding, the lethality of the different dTOR mutants could not be rescued by constitutive expression of a S6K1 variant, D₃E-E₃₈₉, which exhibits high basal activity in the absence of mitogens under the control of the alpha-tubulin promoter, which rescues all aspects of the S6k^l-1 null phenotype. Therefore, S6k-independent processes must contribute to the weak dTOR phenotype (Oldham, 2000).

The effect of rapamycin and amino acids on translation in mammals is mediated through the S6Ks and the 4E-BPs. Unlike the other elements in the PI3K signaling pathway, absence of amino acids blocks both S6K activation and 4E-BP phosphorylation. Indeed, a mutant of S6K1, lacking a portion of both its amino and carboxyl termini, is resistant to rapamycin but still sensitive to the fungal metabolite wortmannin, an inhibitor of PI3K. This suggests that the PI3K-dependent signal to S6K activation does not involve TOR. This same mutant is also unaffected by amino acid withdrawal, consistent with the role of mTOR as an amino acid checkpoint in S6K activation. Although there is some controversy concerning the ability of mitogens to activate mTOR, the in vitro activity of mTOR from cultured cells toward either itself, S6K1, or 4E-BP1 is unaffected by mitogens. Thus, mTOR may act as a permissive signal that primes 4E-BP phosphorylation and S6K activation by the PI3K signaling pathway if amino acids, and possibly other nutrients, are at sufficient levels. Likewise, in Drosophila larvae, amino acids are necessary, but not sufficient, for imaginal disc and endoreplicating tissue proliferation, compatible with dTOR acting in a parallel pathway involved in amino acid sensing. The fact that chico mutant larvae have normal levels of S6k activity and that the dTOR larval phenotypes with respect to the imaginal discs and endoreplicating tissues are so distinct compared with other mutants in the Inr pathway, supports the possibility that dTOR is not responsive to insulin signaling (Oldham, 2000 and references therein).

It is well established in yeast that TOR is an important mediator of nutrient limitation, and it has been proposed that TOR acts as an amino acid effector to coordinate the response of yeast to different nutritional conditions (Barbet, 1996). Indeed, the similarities between dTOR mutant larvae and larvae deprived of amino acids are striking. Therefore, it is likely that dTOR also functions as an amino acid sensor in multicellular organisms. The fact that yeast and Arabidopsis do not have an insulin system suggests that TOR may be an ancestral and widespread nutritional sensor. To provide additional levels of control, it may have been integrated into the insulin system later to respond to different modes of nutrient deprivation with different developmental responses (Oldham, 2000 and references therein).

Insulin treatment of Drosophila Kc 167 cells induces the multiple phosphorylation of a Drosophila ribosomal protein, as judged by its decreased electrophoretic mobility on two-dimensional polyacrylamide gels. The extent to which insulin induces this response is potentiated by cycloheximide and blocked by pretreatment with rapamycin. Isolation and mass spectrometric analysis have revealed that the multiply phosphorylated protein is the larger of two Drosophila melanogaster orthologs of mammalian 40S ribosomal protein S6, termed here DS6A. Proteolytic cleavage of DS6A (derived from stimulated Kc 167 cells), with the endoproteinase Lys-C releases a number of peptides, one of which contains all the putative phosphorylation sites. Conversion of phosphoserines to dehydroalanines with Ba(OH)(2) shows that the sites of phosphorylation reside at the carboxy terminus of DS6A. The sites of phosphorylation have been identified by Edman degradation after conversion of the phosphoserine residues to S-ethylcysteine as Ser(233), Ser(235), Ser(239), Ser(242), and Ser(245). Phosphopeptide mapping of individual phosphoderivatives, isolated from two-dimensional polyacrylamide gels, indicate that DS6A phosphorylation, in analogy to mammalian S6 phosphorylation, appears to proceed in an ordered fashion (Oldham, 2000 and references therein).

Precise body and organ sizes in the adult animal are ensured by a range of signaling pathways. Rheb (Ras homolog enriched in brain), a novel, highly conserved member of the Ras superfamily of G-proteins, promotes cell growth. Overexpression of Rheb in the developing fly causes dramatic overgrowth of multiple tissues: in the wing, this is due to an increase in cell size; in cultured cells, Rheb overexpression results in accumulation of cells in S phase and an increase in cell size. Rheb is required in the whole organism for viability (growth) and for the growth of individual cells. Inhibition of Rheb activity in cultured cells results in their arrest in G1 and a reduction in size. These data demonstrate that Rheb is required for both cell growth (increase in mass) and cell cycle progression; one explanation for this dual role would be that Rheb promotes cell cycle progression by affecting cell growth. Consistent with this interpretation, flies with reduced Rheb activity are hypersensitive to rapamycin, an inhibitor of the growth regulator target of rapamycin (TOR), a kinase required for growth factor-dependent phosphorylation of ribosomal S6 kinase (S6K). In cultured cells, the effect of overexpressing Rheb was blocked by the addition of rapamycin. These results imply that Rheb is involved in TOR signaling (Patel, 2003). Additional studies show that Rheb functions downstream of the tumor suppressors Tsc1 (tuberous sclerosis 1)-Tsc2, with Tsc2 functioning as a GAP for Rheb (Saucedo, 2003; Zhang, 2003), and that a major effector of Rheb function in controlling growth is, in fact, ribosomal S6 kinase (Stocker, 2003). It is still not clear, however, how Rheb signals to TOR (Zhang, 2003).

Given the similarities between Rheb and mutants in the InR and TOR signalling pathways, it is conceivable that Rheb represents a novel component of one of these growth control pathways. To test this possibility, a detailed epistasis analysis was performed. Examined first was whether the negative regulators of InR and TOR signalling (PTEN and Tsc1-Tsc2, respectively) could counteract the effects of Rheb overexpression. All overexpression experiments were performed in the eye using the GMR-Gal4 driver line. Expression of either PTEN or Tsc1-Tsc2 alone results in a very similar size reduction of the ommatidia when compared with control ommatidia. However, whereas expression of PTEN has no influence on the increase in ommatidial size caused by Rheb overexpression, co-expression of Tsc1-Tsc2 results in ommatidia of approximately wild-type size, indicating that the activities of Rheb and Tsc1-Tsc2 can counteract each other. Next, the enlarged ommatidia phenotype of GMR-Rheb was assayed in a number of mutant backgrounds. Reducing the activity of Drosophila protein kinase B (PKB) has no effect on ommatidial size. Similar results were obtained with hypomorphic mutations in InR and Dp110, respectively. In contrast, ommatidial size is dominantly reduced by a mutation in TOR (TOR^2L1), and a suppression to wild-type size is observed in a S6K mutant background. Thus, the Rheb overexpression phenotype is dependent on TOR and S6K function, but is independent of InR signal strength. Finally, the behaviors of Rheb PTEN and Rheb Tsc1 double mutants were examined. The phenotypic consequences were assayed in mosaic animals using the ey-Flp method. As expected, the Rheb PTEN double-mutant tissue clearly displays a Rheb phenotype. The Rheb Tsc1 mutant tissue also resembles Rheb single mutants, indicating that Rheb is epistatic over (functions downstream of) Tsc1 (Stocker, 2003).

Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway

In many species, reducing nutrient intake without causing malnutrition extends lifespan. Like DR (dietary restriction), modulation of genes in the insulin-signaling pathway, known to alter nutrient sensing, has been shown to extend lifespan in various species. In Drosophila, the target of rapamycin (TOR) and the insulin pathways have emerged as major regulators of growth and size. Hence, the role of TOR pathway genes in regulating lifespan has been examined by using Drosophila. Inhibition of TOR signaling pathway by alteration of the expression of genes in this nutrient-sensing pathway, which is conserved from yeast to human, extends lifespan in a manner that may overlap with known effects of dietary restriction on longevity. In Drosophila, TSC1 and TSC2/Gigas (tuberous sclerosis complex genes 1 and 2) act together to inhibit TOR (target of rapamycin), which mediates a signaling pathway that couples amino acid availability to S6 kinase, translation initiation, and growth. Overexpression of dTsc1, dTsc2, or dominant-negative forms of dTOR or dS6K all cause lifespan extension. Modulation of expression in the fat is sufficient for the lifespan-extension effects. The lifespan extensions are dependent on nutritional condition, suggesting a possible link between the TOR pathway and dietary restriction (Kapahi, 2004).

The Drosophila homologs of human Tsc1 (Hamartin) and Tsc2 (tuberin) function in vivo as a complex that controls growth and size in a cell-autonomous manner. To examine their role in regulating lifespan, dTsc1 and dTsc2 were overexpressed through the ubiquitously expressed driver, daughterless (da-GAL4). Overexpression in transgenic flies carrying UAS constructs containing dTsc1 or dTsc2 extends mean lifespan at 29°C by 14% and 12%, respectively. Since GAL4 enhancer traps generally yield stronger effects at 29°C, most of the experiments were performed at that temperature (Kapahi, 2004).

dTsc1 and dTsc2 physically interact with dTOR, which is conserved from yeast to human as a nutrient sensor. Loss of dTsc1 in Drosophila eye leads to an increase in cell size, provided that dTOR is present. Surprisingly, however, dTOR overexpression causes a reduction in cell size, a phenotype similar to dTOR loss-of-function mutations, perhaps due to titration of cofactors required for TOR signaling. The effect of dTOR on lifespan was examined by using three UAS. One carries the full-length wild-type TOR gene. The second carries FRB, the 11 kDa FKBP12-rapamycin binding domain, which has been shown to prevent S phase entry when injected into human osteosarcoma cells. The third carries TED (toxic effector domain), containing the 754 amino acid central region, which inhibits cell growth and arrests cells in G1 when overexpressed in yeast (Hennig, 2002). Ubiquitous overexpression with the da-GAL4 driver of UAS-dTOR^FRB led to a mean lifespan increase at 29°C of 24%. However, overexpression of UAS -dTOR^WT or UAS-dTOR^TED prevented eclosion to adulthood (Kapahi, 2004).

S6 kinase activation upon phosphorylation has been implicated in mediating the downstream effects of TOR on translation initiation in flies and mammals. S6 kinase phosphorylation of ribosomal protein S6 is accompanied by upregulation of a class of mRNAs containing an oligopyrimidine tract at their transcriptional start site termed 5'TOP (Thomas, 2002). Some 200 genes, most of which encode components of the translational apparatus including ribosomal proteins and elongation factors, have this sequence and can account for about 20% of total cellular mRNA. Flies carrying homozygous mutations in dS6K show a developmental delay and a reduction in body size. The stimulation of dS6K phosphorylation by dTOR is abrogated when dTsc1 and dTsc2 are overexpressed. Furthermore, flies with reduced dTSC1 show increased dS6 kinase activation, and genetic reduction of S6 kinase level can rescue the lethality caused by loss of function of dTsc1 (Kapahi, 2004).

The role of S6 kinase in regulating lifespan was examined by using dominant-negative and constitutively active constructs. The dominant-negative effect was achieved by replacing the conserved lysine in the ATP binding site by glutamine (UAS-dS6K^KQ), which causes cell-size reduction. The constitutively active form was generated by replacing the phosphorylation sites of S6 kinase by acidic amino acids (UAS-dS6K^STDETE), causing an autonomous cell size increase. By using da-GAL4 to drive ubiquitous overexpression of the dominant-negative form, a mean lifespan increase of 22% at 29°C was observed. Conversely, overexpression of the constutively active form of S6 kinase caused a mean lifespan decrease of 34% at 29°C. Overexpression of dTsc2 and dTOR^FRB was also tested at 25°C and led to a 20% and 26% increase in mean lifespan increase, respectively (Kapahi, 2004).

To determine which tissues are responsible for the lifespan extension, various GAL4 drivers with specific GAL4 expression pattern were employed to overexpress dTsc2 via a UAS promoter. Overexpression in the eye by using the driver gmr-GAL4 or in the nervous system by using appl-GAL4 did not extend lifespan. In contrast, by using the drivers 24B-GAL4 and PO188-GAL4, enhancer traps that are predominantly expressed in the muscle and fat, results in mean lifespan extensions of 27% and 37%, respectively, at 29°C. The fat-specific drivers DJ634-GAL4 and PO163-GAL4, when used to overexpress dTsc2, also led to a mean lifespan extension of 22% and 31%, respectively, at 29°C. Using DJ634-GAL4 to overexpress the dominant-negative form of TOR (UAS-dTOR^FRB) or of S6 kinase (UAS-UAS-dS6K^KQ) also led to mean lifespan increases of 30% and 29%, respectively, at 29°C. These results indicate that manipulation of the TSC, TOR, and S6 kinase genes in the fat tissue is sufficient for their lifespan extension effects in Drosophila (Kapahi, 2004).

Amino acids have been shown to activate dS6k via TOR, an effect that can be abrogated in the presence of increased levels of dTsc1 and dTsc2. Since nutrients in the diet can modulate lifespan and because the TOR pathway is a critical mediator of nutrient signaling, it was asked whether the observed lifespan-extension effects are dependent on nutrient conditions. This was tested with overexpression of dTsc2 by using the ubiquitously expressing da-GAL4 driver. Flies were allowed to develop to adulthood under standard laboratory food and then maintained on specially prepared food containing various concentrations of yeast extract. At high concentrations of yeast extract, which may be regarded as the opposite of dietary restriction, the lifespan of control flies (da-GAL4/+) is severely reduced. However, overexpression of dTsc2 protects the fly from the deleterious effects of rich food, as if mimicking the effect of dietary restriction. Similar results were observed by overexpression of the dominant-negative form of S6 kinase (Kapahi, 2004).

Recent evidence from Drosophila suggests that signaling through TSC is both parallel to and interacting with the insulin pathway. This is supported by the finding that heterozygosity of dTsc1 or dTsc2 is sufficient to rescue the lethality of loss-of-function dInR mutants. However, the finding that loss-of-function mutations of dTsc1 and dPTEN, a phosphatase that negatively regulates the insulin-signaling pathway, cause cell autonomous and additive increases in cell size suggests that they may be in parallel pathways. Furthermore, in Drosophila, dPTEN loss of function, which leads to an increase in cell size, is only slightly suppressible by loss of function of dFOXO, a fly homolog of C. elegans daf-16. However, the increase in cell size resulting from dTsc1 is enhanced by dFOXO loss of function. Interestingly, unlike long-lived daf-2 mutants, the lifespan extension due to TOR deficiency in C. elegans is not suppressible by a daf-16 mutation. However, the TOR mutant animals do not further extend lifespan in a daf-2 background, leading to the possibility that TOR may be acting downstream or separately from daf-16 to exert its lifespan effects (Kapahi, 2004).

Lifespan extension has been linked with other phenotypes, including stress resistance, metabolic rate, lipid level, reproductive capacity, and body size. The long-lived strains described above with their respective controls for resistance to starvation were compared but no significant differences were found. Similarly, no significant differences were observed for weight and lipid content among these strains. It may be that lifespan extension can be produced by mild modulation of these genes, whereas effects on other phenotypes require severe perturbations. While lifespan extension is observed by using the da-GAL4 driver to overexpress dTsc1 or dTsc2 alone, simultaneous overexpression of dTsc1 and dTsc2 prevented eclosion to adulthood. Similarly, no change in size is observed if dTsc1 or dTsc2 alone are overexpressed in the eye, but a cell-autonomous decrease in size is seen when both are overexpressed simultaneously. Lifespan extension by chico is semidominant, but its effect on body size is recessive. Dominant effects on lifespan are observed with the genes Inr, EcR, Indy, and Rpd3, but their effects on lifespan can be uncoupled from other phenotypes such as fecundity, stress resistance, or lipid accumulation (Kapahi, 2004).

In humans, mutations in TSC1 and TSC2 lead to tuberous sclerosis, a common disorder characterized by the presence of benign tumors in various tissues, with some having large cells. DR in mice has been shown to protect against age-related tumorigenesis. These results suggest a link between lifespan extension by DR and the activities of genes in the TOR pathway. Hence, it is conceivable that the protective effects of DR on tumorigenesis and age-related decline might come from inhibition of such nutrient-responsive pathways (Kapahi, 2004).

These results show that upregulation of dTsc2 in the fat is sufficient for lifespan extension effects in Drosophila. Reduction of daf-2 levels in the C. elegans nervous system has been shown to be sufficient for lifespan extension. However, the lifespan extensions due to mutations in the insulin pathway or germline ablation in C. elegans are dependent on daf-16 activity in the intestine, the fat storage tissue in C. elegans. In Drosophila, the fat body has been proposed to modulate insulin signaling in peripheral tissues by secretion of dALS (acid-labile subunit), which, in mammals, forms a ternary complex with insulin-like growth factor, leading to an extension of the half-life of its ligand. Recently, mice with FIRKO (fat-specific insulin receptor knockout) have been shown to live 18% longer than controls . Hence, it is possible that secondary endocrine signals downstream of the insulin and TOR signaling pathways are released from the fat, and these affect the rate of aging in other tissues. Juvenile hormone and ecdysone are two such endocrine signals that have been implicated in regulating lifespan in conjunction with the insulin pathway in Drosophila (Kapahi, 2004).

Role and regulation of starvation-induced autophagy in the Drosophila fat body

In response to starvation, eukaryotic cells recover nutrients through autophagy, a lysosomal-mediated process of cytoplasmic degradation. Autophagy is known to be inhibited by TOR signaling, but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear. Signaling through TOR and its upstream regulators PI3K and Rheb is necessary and sufficient to suppress starvation-induced autophagy in the Drosophila fat body. In contrast, TOR's downstream effector S6K promotes rather than suppresses autophagy, suggesting S6K downregulation may limit autophagy during extended starvation. Despite the catabolic potential of autophagy, disruption of conserved components of the autophagic machinery, including ATG1 and ATG5, does not restore growth to TOR mutant cells. Instead, inhibition of autophagy enhances TOR mutant phenotypes, including reduced cell size, growth rate, and survival. Thus, in cells lacking TOR, autophagy plays a protective role that is dominant over its potential role as a growth suppressor (Scott, 2004).

Autophagy likely evolved in single-cell eukaryotes to provide an energy and nutrient source allowing temporary survival of starvation. In yeast, Tor1 and Tor2 act as direct links between nutrient conditions and cell metabolism. These proteins sense nutritional status by an unknown mechanism, and effect a variety of starvation responses including changes in transcriptional and translational programs, nutrient import, protein and mRNA stability, cell cycle arrest, and induction of autophagy. Autophagy thus occurs in the context of a comprehensive reorganization of cellular activities aimed at surviving low nutrient levels (Scott, 2004).

In multicellular organisms, TOR is thought to have retained its role as a nutrient sensor but has also adopted new functions in regulating and responding to growth factor signaling pathways and developmental programs. Thus in a variety of signaling, developmental, and disease contexts, TOR activity can be regulated independently of nutritional conditions. In these cases, autophagy may be induced in response to downregulation of TOR despite the presence of abundant nutrients and may potentially play an important role in suppressing cell growth rather than promoting survival. Identification of the tumor suppressors PTEN, and TSC1 and TSC2 as positive regulators of autophagy provides correlative evidence supporting such a role for autophagy in growth control. Alternatively, since TOR activity is required for proper expression and localization of a number of nutrient transporters, inactivation of TOR may lead to reduced intracellular nutrient levels, and autophagy may therefore be required under these conditions to provide the nutrients and energy necessary for normal cell metabolism and survival (Scott, 2004).

The results presented here provide genetic evidence that under conditions of low TOR signaling, autophagy functions primarily to promote normal cell function and survival, rather than to suppress cell growth. This conclusion is based on the finding that genetic disruption of autophagy does not restore growth to cells lacking TOR, but instead exacerbates multiple TOR mutant phenotypes. It is important to note that mutations in TOR do not disrupt larval feeding, and thus disruption of autophagy is detrimental in TOR mutants despite the presence of ample extracellular nutrients. The finding that autophagy is critical in cells lacking TOR further supports earlier studies suggesting that inactivation of TOR causes defects in nutrient import, resulting in an intracellular state of pseudo-starvation (Scott, 2004).

Can the further reduction in growth of TOR mutant cells upon disruption of autophagy be reconciled with the potential catabolic effects of autophagy? TOR regulates the bidirectional flow of nutrients between protein synthesis and degradation through effects on nutrient import, autophagy, and ribosome biogenesis. When TOR is inactivated, rates of nutrient import and protein synthesis decrease, resulting in a commensurate reduction in mass accumulation and cell growth. In addition, autophagy is induced to maintain intracellular nutrient and energy levels sufficient for normal cell metabolism. When autophagy is experimentally inhibited in cells lacking TOR, this reserve source of nutrients is blocked, leading to a further decrease in energy levels, protein synthesis, and growth. It is noted that autophagy may have additional functions in cells with depressed TOR signaling, including recycling of organelles damaged by the absence of TOR activity, or selective degradation of cell growth regulators, analogous to the regulatory roles of ubiquitin-mediated degradation (Scott, 2004).

Autophagy is required for normal developmental responses to inactivation of insulin/PI3K signaling in the nematode C. elegans. In response to starvation or disruption of insulin/PI3K signaling, C. elegans larvae enter a dormant state called the dauer. Autophagy has been observed in C. elegans larvae undergoing dauer formation: disruption of a number of ATG homologs interfers with normal dauer morphogenesis. Importantly, simultaneous disruption of insulin/PI3K signaling and autophagy genes results in lethality, similar to the results presented in this study. Thus despite significant differences in developmental strategies for surviving nutrient deprivation, autophagy plays an essential role in the starvation responses of yeast, flies, and worms (Scott, 2004).

The prevailing view that S6K acts to suppress autophagy was founded on correlations between induction of autophagy and dephosphorylation of rpS6 in response to amino acid deprivation or rapamycin treatment. However, the genetic data presented in this study argue strongly against a role for S6K in suppressing autophagy: unlike other positive components of the TOR pathway, null mutations in S6K do not induce autophagy in fed animals. It is suggested that the observed correlation between S6K activity and suppression of autophagy is due to common but independent regulation of S6K and autophagy by TOR. Thus, autophagy suppression and S6K-dependent functions such as ribosome biogenesis represent distinct outputs of TOR signaling (Scott, 2004).

How might TOR signal to the autophagic machinery, if not through S6K? In yeast, this is accomplished in part through regulation of Atg1 kinase activity and ATG8 gene expression (Kamada, 2000 and Kirisako, 1999). The demonstration of a role for Drosophila ATG1 and ATG8 homologs [see TG8a (CG32672) and ATG8b (CG12334)] in starvation-induced autophagy, and the genetic interaction observed between ATG1 and TOR, are consistent with a related mode of regulation in higher eukaryotes. However, it is noted that other components of the yeast Atg1 complex such as Atg17 and Atg13, whose phosphorylation state is rapamycin sensitive, do not have clear homologs in metazoans, indicating that differences in regulation of autophagy by TOR are likely (Scott, 2004).

In addition to excluding a role for S6K in suppression of autophagy, these results reveal a positive role for S6K in induction of autophagy. S6K may promote autophagy directly, through activation of the autophagy machinery, or indirectly through its effects on protein synthesis. The latter possibility is consistent with previous reports that protein synthesis is required for expansion and maturation of autophagosomes. Interestingly, despite being required for autophagy, S6K is downregulated under conditions that induce it, including chronic starvation and TOR inactivation. Consistent with this, it was found that lysotracker staining is significantly weaker in chronically starved animals or in TOR mutants than in wild-type animals starved 3-4 hr. Furthermore, expression of constitutively activated S6K has no effect in wild-type, but restores lysotracker staining in TOR mutants to levels similar to those of acutely starved wild-type animals. It is suggested that downregulation of S6K may limit rates of autophagy under conditions of extended starvation or TOR inactivation and that this may protect cells from the potentially damaging effects of unrestrained autophagy (Scott, 2004).

Co-culture and conditioned media experiments have shown that the Drosophila fat body is a source of diffusible mitogens. The fat body has also been shown to act as a nutrient sensor through a TOR-dependent mechanism and to regulate organismal growth through effects on insulin/PI3K signaling. The results in this study extend these findings by showing that this endocrine response is accompanied by the regulated release of nutrients through autophagic degradation of fat body cytoplasm. Preventing this reallocation of resources, either through constitutive activation of PI3K or through inactivation of ATG genes, results in profound nutrient sensitivity. Thus, in response to nutrient limitation, the fat body is capable of simultaneously restricting growth of peripheral tissues through downregulation of insulin/PI3K signaling and providing these tissues with a buffering source of nutrients necessary for survival through autophagy (Scott, 2004).

Regulation of hunger-driven behaviors by neural ribosomal S6 kinase in Drosophila

Hunger elicits diverse, yet coordinated, adaptive responses across species, but the underlying signaling mechanism remains poorly understood. This study reports on the function and mechanism of the Drosophila insulin-like system in the central regulation of different hunger-driven behaviors. Overexpression of Drosophila insulin-like peptides (DILPs) in the nervous system of fasted larvae suppresses the hunger-driven increase of ingestion rate and intake of nonpreferred foods (e.g., a less accessible solid food). Moreover, up-regulation of Drosophila p70/S6 kinase activity in DILP neurons leads to attenuated hunger response by fasted larvae, whereas its down-regulation triggered fed larvae to display motivated foraging and feeding. Finally, evidence is provided that neural regulation of food preference but not ingestion rate may involve direct signaling by DILPs to neurons expressing neuropeptide F receptor 1, a receptor for neuropeptide Y-like neuropeptide F. This study reveals a prominent role of neural Drosophila p70/S6 kinase in the modulation of hunger response by insulin-like and neuropeptide Y-like signaling pathways (Wu, 2006).

The relatively simple Drosophila larva offers a genetically tractable model to define and characterize different neuronal signaling pathways that constitute a complete central feeding apparatus. Younger third-instar larvae forage actively and use their mouth hooks for food intake. Larvae normally feed on liquid food, and their food ingestion can be quantified by measuring the contraction rate of the mouth hooks. This study examined how food deprivation affects larval feeding response to a liquid (e.g., 10% glucose-agar paste) and less accessible solid food (e.g., 10% glucose agar blocks). To extract embedded glucose from the solid food, larvae have to pulverize the food by scraping agar surface with mouth hooks. Unless stated otherwise, synchronized third-instar larvae (74 h after egg laying) were used for the assays (Wu, 2006).

When fed ad libitum, normal larvae (w¹¹¹⁸) display significant feeding activity in the liquid food with an average mouth-hook contraction frequency of ~30 times in a 30-s test period; in contrast, these larvae declined the solid food. However, larvae withheld from food (on a wet tissue) for 40 or 120 min display increased intake of both liquid and solid foods. For example, larvae fasted for 120 min show a 100% and >500% increase in mouth-hook contraction rate in liquid and solid food, respectively. Thus, deprivation not only enhances feeding rate in a graded fashion, but also triggers motivated foraging on the less accessible food normally rejected by fed larvae. In addition, larvae display virtually identical feeding responses to liquid and solid foods containing 10% glucose, apple juice, or 10% glucose/yeast under deprived and nondeprived conditions. Therefore, these paradigms appear to provide a general assessment of larval feeding response (Wu, 2006).

dS6K is a cell-autonomous effector of nutrient-sensing pathways. This study investigated a possible role of neural dS6K in coupling peripheral physiological hunger signals and neuronal activities critical for hunger-driven behaviors. The transcripts of dilp1, dilp2, dilp3, and dilp5 are predominantly expressed in two small clusters of medial neurosecretory cells that project to the ring gland, the fly heart, and the brain lobes. A gal4 driver containing a 2-kb fragment from the dilp2 promoter (dilp2-gal4) was generated that directs the specific expression of a GFP reporter in those cells. Using dilp2-gal4, two transgenes, UAS-dS6K^DN, encoding a dominant negative, and UAS-dS6K^ACT, a constitutively active form of dS6K, were expressed. When fed ad libitum, control larvae (w x UAS-dS6K^DN or UAS-dS6K^ACT) behave like w larvae. However, dilp2-gal4 x UAS-dS6K^DN larvae displayed a 50% increase in the rate of liquid-food intake and significant feeding of the solid food. Conversely, fasted larvae overexpressing dS6K activity (dilp2-gal4 x UAS-dS6K^ACT) showed attenuated feeding response to both liquid and solid foods. These findings reveal that dS6K in DILP neurons mediates hunger regulation of approaching/consumptive behaviors, controlling both quality and quantity of food for ingestion. The body size and the developmental rate of all four groups of larvae were measured, and no significant differences were detected (Wu, 2006).

DILPs act as neurohormones in Drosophila larvae. Down-regulation of dS6K activity in DILP neurons may reduce DILP release, thereby promoting increased food intake that is normally triggered only by hunger. A corollary of this interpretation is that overproduction of DILPs in the nervous system should interfere with hunger response by deprived animals. To test this idea, a neural-specific elav-gal4 driver was used to direct dilp expression in the larval nervous system. Three UAS-dilp lines (UAS-dilp2, UAS-dilp3, and UAS-dilp4) were chosen for the analysis. The elav-gal4 x UAS-dilp2 and UAS-dilp4 larvae displayed normal feeding response when fed ad libitum. However, the same larvae fasted for 120 min displayed significantly attenuated feeding rates, similar to those of dilp2-gal4 x UAS-dS6K^ACT larvae. For example, the comparative analysis of the elav-gal4 x UAS-GFP control and elav-gal4 x UAS-dilp2 and UAS-dilp4 experimental larvae showed that the latter were ~30% and 33–45% lower in the ingestion rate of the liquid and solid food, respectively; surprisingly, elav-gal4 x UAS-dilp3 and UAS-GFP larvae showed virtually identical feeding responses. Therefore, DILP2 and DILP4 negatively regulate hunger-driven feeding activities. Taken together, these results suggest that a high level of dS6K activity in DILP neurons may suppress hunger response by reducing DILP release (Wu, 2006).

Attempts were made to delineate the signaling mechanism that couples the dS6K activity in DILP neurons with its broad impact on hunger-driven feeding activities. A previous study showed that fasted larvae ablated of NPF or its receptor (NPFR1) neurons are deficient in motivated feeding of the less-preferred solid food but normal in feeding of richer liquid food. It was of interest to enquire whether the NPF/NPFR1 neuronal pathway might be one of the downstream effectors of the DILP pathway. To test this hypothesis, the function of three components of the dInR signaling pathway were analyzed in NPFR1 neurons: dInR, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase (dPTEN), and phosphatidylinositol 3-kinase (dPI3K). Five different transgenes were used: UAS-dInR^ACT and UAS-dInR^DN encode a constitutively active and a dominant-negative form of dInR, respectively; UAS-Dp110 and UAS-dPI3K^DN encode a catalytic subunit and a dominant-negative form of dPI3K, respectively; and UAS-dPTEN encodes a functional enzyme. When fed ad libitum, npfr1-gal4 x UAS-dInR^DN, UAS-dPTEN, or UAS-dPI3K^DN larvae display hyperactive feeding of the solid food, similar to w larvae deprived for 40 min. In contrast, fasted larvae overexpressing dInR or dPI3K (npfr1-gal4 x UAS-dInR^ACT or UAS-Dp110) display attenuated feeding response to the solid food. Importantly, larvae with up- or down-regulated dInR signaling in NPFR1 neurons do not exhibit significant changes in the intake rate of the richer liquid food relative to the paired controls. Taken together, these findings suggest that the dInR pathway negatively regulates the activity of NPFR1 neuron and mediates the DILP-regulated change in food preference but not ingestion rate. Furthermore, the results suggest that NPFR1 neurons are the direct targets of DILPs (Wu, 2006).

A possible role of dS6K in hunger regulation of the functioning of NPFR1 neurons was evaluated, by expressing UAS-dS6K^DN and UAS-dS6K^ACT using npfr1-gal4. When fed ad libitum, npfr1-gal4 x UAS-dS6K^DN larvae display hyperactive feeding of the solid food, similar to npfr1-gal4 x UAS-dInR^DN larvae. However, these larvae, unlike dilp2-gal4 x UAS-dS6K^DN animals, display no increases in the ingestion rate of the richer liquid food. Conversely, fasted larvae overexpressing dS6K (npfr1-gal4 x UAS-dS6K^ACT) display attenuated feeding response to the solid food. These findings suggest that dS6K also negatively regulates the activity of NPFR1 neurons in food preference, but does not mediate the regulation of feeding rate by DILP signaling (Wu, 2006).

The food response was evaluated of the solid and liquid food by larvae overexpressing an npfr1 cDNA under the control of an npfr1-gal4 driver. In the presence of the liquid food, both experimental (npfr1-gal4 x UAS-npfr1) and control larvae (e.g., npfr1-gal4 x UAS-ANF-GFP), fed or fasted, show similar intake rates and comparable increases in feeding response to hunger. However, when forced to feed on the solid food, fed experimental larvae exhibit significant intake of the solid food (30 times per 30 s), whereas fed controls rejected the same food. Thus, NPFR1 overexpression selectively promotes change in food preference without increasing ingestion rate. It was also observed that the feeding responses of NPFR1-overexpressing larvae and controls fasted for 120 min were indistinguishable. Thus, the effect of NPFR1 overexpression on food preference is detectable only in fed or mildly fasted larvae, suggesting hunger-activated NPFR1 signaling approaches a plateau in severely fasted animals (Wu, 2006).

npfr1 activity was selectively knocked down by expressing npfr1 dsRNA in the nervous system. The UAS-npfr1^dsRNA lines were previously used to functionally disrupt npfr1 activity. It was found that 120-min fasted larvae expressing npfr1 dsRNA in NPFR1 or the nervous system (npfr1-gal4, elav-gal4, or appl-gal4 x UAS-npfr1^dsRNA) were deficient in motivated feeding of the solid but not liquid food. In contrast, all control larvae, including those expressing npfr1^dsRNA in muscle cells (MHC82-gal4 x UAS-npfr1^dsRNA), showed normal feeding responses. These results indicate that neural NPFR1 mediates hunger regulation of food selection (Wu, 2006).

A potential problem of the previous transgenic studies is that NPF/NPFR1 signaling is likely to be disrupted in a relatively early stage of larval development. Conceivably, the NPF/NPFR1 neuronal pathway could be essential for ad libitum or hunger-driven feeding of richer liquid foods, but such an activity might be masked by some yet-unidentified compensatory mechanism triggered by its early loss. To test this idea, attempts were made to disrupt NPF/NPFR1 neuronal signaling in a temporally controlled manner by expressing a temperature-sensitive allele of shibire (shi^ts1) driven by npf-gal4 or npfr1-gal4. The shi^ts1 allele encodes a semidominant-negative form of dynamin that blocks neurotransmitter release at a restrictive temperature (>29°C). At the permissive temperature of 23°C, 120-min-fasted experimental larvae (npf-gal4 and npfr1-gal4 x UAS-shi^ts1) and paired controls (y w x UAS-shi^ts1 and npf-gal4 and npfr1-gal4 x w¹¹¹⁸) displayed normal feeding responses to both liquid and solid foods. However, if larvae were incubated at 30°C for 15 min, controls still displayed normal feeding activities, whereas the experimental larvae showed attenuated feeding response to the solid but not liquid food. Therefore, there was no detectable developmental or physiological compensation for the loss of NPF signaling in Drosophila larvae. These results also suggest that the NPF/NPFR1 neuronal pathway is acutely required to initiate and maintain larval hunger response. The foraging activity of the experimental larvae was completely restored when the assay temperature was reduced to 23°C, suggesting that the NPF system can modulate the intensity and duration of feeding response (Wu, 2006).

This study has shown that dS6K regulates different, yet coordinated, behaviors controlling quantitative and qualitative aspects of hunger-adaptive food response. Evidence is provided that dS6K mediates hunger regulation of two opposing insulin- and NPY-like signaling activities, dynamically modifying larval food preference and feeding rate based on the nutritional state. For example, hunger stimuli may cause a reduction of dS6K activity in DILP neurons, resulting in the suppression of DILP signaling that negatively regulates a downstream NPF/NPFR1-dependent and another NPF-independent neuronal pathway. The DILP/NPFR1 neuronal pathway selectively mediates hunger-adaptive change in food preference, possibly by overriding the high threshold of food acceptance set by a separate default pathway, enabling hungry animals to be receptive to less preferred foods. The NPF/NPFR1-independent pathway promotes a general increase in the ingestion rate of preferred/less preferred foods, enabling animals to compete effectively for limited food sources. This study also implicates the presence of a separate default pathway for mediating the selective intake of preferred foods (baseline feeding) in larvae fed ad libitum. This default pathway may be largely insensitive to DILP or NPF signaling, because overexpression of dS6K, DILPs, or NPFR1 in nondeprived larvae does not affect ad libitum feeding in the liquid food. It is suggested that the conserved S6K pathway may be critical for regulating behavioral adaptation to hunger in diverse organisms, including humans, and its components are potential drug targets for appetite control (Wu, 2006).

The functional differences of DILP1–7 have not been reported previously. In this study, dilp2, dilp3, and dilp4 were shown to be functionally distinct. DILP2 and DILP3 both are produced in the same medial neurosecretory cells. However, unlike DILP2, DILP3 is apparently not involved in suppressing deprivation-motivated feeding. It is still unclear whether the differential activities of DILP2 and DILP3 reflect their structural divergence or are caused by the presence of yet-unidentified dInR isoforms. DILP4 is not expressed within the two medial clusters of DILP neurons. Under acute deprivation, the level of dilp4 transcripts showed a 5-fold reduction in the larval CNS. Thus, it is possible that DILP4 may play a localized role in promoting feeding response inside the CNS (Wu, 2006).

Feeding is a reward-seeking behavior, and deprivation strengthens the reinforcing effect (reward value) of food. These studies suggest a previously uncharacterized role of the DILP/dInR signaling pathway in regulating an animal's perception of food quality. The DILP/NPF neural network may regulate an animal's incentive to acquire lower-quality foods by modifying the reward circuit. This hypothesis is interesting in light of the findings that foods and abused substances may act on the same reward circuit, and highly palatable foods can reduce drug-seeking behaviors. It is also possible that the DILP/NPF system might represent a specialized neural circuit that positively alters the reward value of lesser-quality foods. Conceivably, a better understanding of the action of this signaling system may provide fresh insights into neural mechanisms for controlling eating and drug-seeking behaviors (Wu, 2006).

Given its prominent role in behavioral adaptation to hunger, the insulin/NPY-like neural network is likely of primary importance to animal evolution. In addition, insulin and NPY family molecules have been found in a wide range of animals from humans to worms. Therefore, the insulin/NPY-like network may be a useful model for studying comparatively how diverse animals have evolved distinct ways of adapting an ancestral neural system to suit their respective lifestyles (Wu, 2006).

Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase

Cellular growth and proliferation are coordinated during organogenesis. Misregulation of these processes leads to pathological conditions such as cancer. Tuberous sclerosis (TSC) is a benign tumour syndrome caused by mutations in either TSC1 or TSC2 tumour suppressor genes. Studies in Drosophila and other organisms have identified TSC signalling as a conserved pathway for growth control. Activation of the TSC pathway is mediated by Rheb (Ras homologue enriched in brain), a Ras superfamily GTPase. Rheb is a direct target of TSC2 Gigas in Drosophila) and is negatively regulated by its GTPase-activating protein activity. However, molecules required for positive regulation of Rheb have not been identified. This study shows that a conserved protein, translationally controlled tumour protein (TCTP), is an essential new component of the TSC-Rheb pathway. Reducing Drosophila TCTP (dTCTP) levels reduces cell size, cell number and organ size, which mimics Drosophila Rheb (dRheb) mutant phenotypes. dTCTP is genetically epistatic to Tsc1 and dRheb, but acts upstream of dS6k, a downstream target of dRheb. dTCTP directly associates with dRheb and displays guanine nucleotide exchange activity with it in vivo and in vitro. Human TCTP (hTCTP) shows similar biochemical properties compared to dTCTP and can rescue dTCTP mutant phenotypes, suggesting that the function of TCTP in the TSC pathway is evolutionarily conserved. These studies identify TCTP as a direct regulator of Rheb and a potential therapeutic target for TSC disease (Hsu, 2007).

S6k is identified in a screen for genes that function in leg disc regeneration in Drosophila

Many diverse animal species regenerate parts of an organ or tissue after injury. However, the molecules responsible for the regenerative growth remain largely unknown. The screen reported in this study aimed to identify genes that function in regeneration and the transdetermination events closely associated with imaginal disc regeneration using Drosophila melanogaster. A collection of 97 recessive lethal P-lacZ enhancer trap lines were screened for two primary criteria: first, the ability to dominantly modify wg-induced leg-to-wing transdetermination and second, for the activation or repression of the lacZ reporter gene in the blastema during disc regeneration. Of the 97 P-lacZ lines, six genes (Krüppelhomolog- 1, rpd3, jing, combgap, Aly and S6 kinase) were identified that met both criteria. Five of these genes suppress, while one enhances, leg-to-wing transdetermination and therefore affects disc regeneration. Two of the genes, jing and rpd3, function in concert with chromatin remodeling proteins of the Polycomb Group (PcG) and trithorax Group (trxG) genes during Drosophila development, thus linking chromatin remodeling with the process of regeneration (McClure, 2008).

There are three different mechanisms that organisms use to re-grow and replace lost or damaged body parts, and often, more than one mechanism can function within different tissues of the same organism. Muscle and bone, for example, repair themselves by activating a resident stem cell population, while the liver regenerates by compensatory proliferation of normally quiescent differentiated cells. Appendage/fin regeneration in lower vertebrates occurs by a process termed epimorphic regeneration, which proceeds in three distinct stages: (1) wound healing and migration of the surrounding epithelial cells to form the wound epidermis, (2) formation of the regeneration blastema -- a mass of undifferentiated and proliferating cells of mesenchymal origin and (3) regenerative outgrowth and pattern re-formation. Whether these diverse modes of regeneration share a common molecular and genetic basis is not known (McClure, 2008).

Regeneration in the Drosophila imaginal discs, the primordia of the adult fly appendages, closely parallels epimorphic limb/fin regeneration in lower vertebrates. Cells in the imaginal discs are rigidly determined to form specific adult structures (e.g., legs and wings) by the third larval instar. If the discs are fragmented at this time and cultured in vivo, they will regenerate. Disc regeneration begins 12 h after wounding, when transient heterotypic contacts are made between peripodial (squamous epithelium) and columnar cells (disc proper) near the cut edges of the wound. These initial contacts involve microvilli-like extensions and provide temporary wound closure. Then, approximately 24 h after wounding, homotypic cell contacts (between columnar or between squamous cells) are made involving the close apposition of cell membranes and cellular bridges, which eventually (48 h after wounding) restore the physical continuity of the disc. Before and during wound healing, cell division is randomly distributed throughout the disc. However, once completed (36-48 h after wounding), division is observed only in cells near the wound site. These cells are known as the regeneration blastema. Thus, like appendage regeneration in lower vertebrates, disc regeneration involves wound healing followed by blastema formation (McClure, 2008).

Blastema cells are responsible for the regeneration and repatterning of the entire missing disc fragment. Thus, these cells exhibit remarkable developmental plasticity. For example, in anterior- only leg disc fragments, some blastema cells will switch to posterior identity and establish a novel posterior compartment in the regenerate. This anterior/posterior conversion occurs during heterotypic wound healing, when hedgehog (hh)- expressing peripodial cells induce ectopic engrailed (en) expression in the apposing anterior columnar cells. In addition, the disc blastema, like its vertebrate counterpart, is able to form a normal regenerate (complete leg disc and adult leg) when isolated from the remaining disc fragment. Regenerative plasticity is also observed when a few blastema cells switch fate to that of another disc type (e.g., leg-to-wing), in a phenomenon known as transdetermination. Transdetermination events are closely associated with regenerative disc growth. Clonal analysis, for example, has shown that blastema cells first regenerate the missing disc structures, and only then, are they competent to transdetermine (McClure, 2008).

Little is known about how the regeneration blastema forms in the fragmented leg disc, although ectopic Wingless (Wg/Wnt1) expression is detected along the cut site, both prior to and during blastema formation. Wg is a developmental signal in many different tissues and animals; in flies Wg patterns all of the imaginal discs, functioning as both a morphogen and mitogen to regulate disc cell fate and growth. In lower vertebrates, Wnt ligands are key regulators of blastema formation during epimorphic regeneration. Thus, activation of Wg within the disc blastema is potentially important for regeneration. This idea is consistent with the observation that ubiquitous expression of wg during the second or third larval instars, in unfragmented leg discs, is sufficient to induce a regeneration blastema in the proximodorsal region of the disc, known as the weak point. Moreover, ubiquitous expression of wg mimics the pattern deviations associated with leg disc fragmentation and subsequent regeneration, including the duplication of ventral with concomitant loss of dorsal pattern elements and leg-to-wing transdetermination events. Thus, leg disc regeneration can be examined using two experimental protocols: fragmentation or ubiquitous wg expression. However, it is important to point out that only fragmentation-induced regeneration involves wound healing (McClure, 2008).

Precisely which molecules and signaling pathways are required for the process of regeneration remain poorly understood, partly because the organisms historically used to study regeneration (e.g., newts and salamanders) have been refractory to genetics and molecular manipulations. Recently, however, the use of new genetic techniques together with 'regeneration' model systems -- such as planarians, hydra and zebrafish have given researchers the opportunity to examine the mechanisms of regeneration and to identify the genes, proteins and signaling pathways that regulate different regenerative processes. For example, a large scale RNAi-based screen was performed to survey gene function in planarian tissue homeostasis and regeneration. Out of ~1000 genes examined, RNAi knock-down of 240 displayed regeneration-related phenotypes, including defects in wound healing, blastema formation and blastema cell differentiation. Despite these studies, however, it remains unclear whether regeneration requires only the modulation of genes expressed at the time of injury, the reactivation of earlier developmental genes and/or signaling pathways, or the activation of novel genes specific to the process of regeneration. Thus, a major interest in the field of regenerative biology is the identification of gene products that regulate blastema formation, blastema growth and regenerative cellular plasticity. A genetic screen, using wg-induced leg disc regeneration, aimed at identifying genes that regulate cellular plasticity and regeneration using Drosophila was carried out prothoracic leg discs. A collection of 97 recessive lethal P-element lacZ (PZ) insertion lines were screened for ectopic lacZ expression during wg-induced leg disc regeneration, and six genes were identified that function in wg-induced leg disc regeneration, including genes with functional ties to Wg signaling as well as chromatin remodeling proteins (McClure, 2008).

This study consisted of an enhancer trap screen designed to identify genes with changed gene expression during leg disc regeneration as well as required for regenerative proliferation and growth. The screen identified 19 genes that when heterozygous mutant (PZ/+), dominantly modify wg-induced leg-to-wing transdetermination, which serves as a functional assay for disc regeneration. Of the 19 genes, 37% are transcription factors or involved in transcriptional regulation (tai, Krh1, ken, jing, combgap (cg), rpd3 and Aly), 21% function in cell cycle regulation and growth (oho23B, S6k, polo and cycA), 10.5% play a role in protein secretion (Secβ61 and Syx13), and 31% are of other or unknown function [l(3)01629, CG30947, l(2)00248, l(3)05203, l(3)01344, Nup154]. The identification of transcription factors as the most frequent class of genes that modify wg-induced leg disc regeneration was similarly observed in a DNA microarray screen designed to identify genes enriched in leg disc cells that transdetermine to wing (Klebes, 2005). Together, these findings strongly suggest that transcription factors and their downstream targets play a prominent role in disc cell plasticity (McClure, 2008).

Using lacZ expression analyses, together with whole mount in situ hybridization experiments, the expression patterns of the 19 genes that modified wg-induced leg-to-wing transdetermination were verified. This analysis identified several different expression patterns upon wg-induced regeneration, including a loss of gene expression, ubiquitous expression and genes with expression limited to the regeneration blastema. Such observations indicate that a complex change of gene expression, both negative and positive, mediates the process of epimorphic regeneration. Six (jing, Alyi cg, rpd3, Kr-h1 and S6k) of the 19 modifiers displayed expression limited to the regeneration blastema, indicating that novel markers of regeneration and transdetermination have been identified. The blastema-specific expression patterns of jing, Aly, cg, Kr-h1, rpd3 and S6k raised the intriguing possibility that these genes may be functionally involved in the formation, cell proliferation or maintenance of the blastema during disc regeneration. Indeed, upon ubiquitous wg expression jing/+ animals rarely formed a regeneration blastema, indicating that two wild-type copies of jing are required for the initiation of the regenerative process. In contrast, Aly/+ and cg/+ animals formed a normal blastema, but only after a one-day delay. Therefore, two wild-type copies of the Aly and cg genes are required for the proper timing of regeneration. In addition, it was found that the frequency of blastema formation was reduced in rpd3/+ animals, implicating this gene in the process of regeneration. Interestingly, heterozygous mutations in all four of these genes (jing, Aly, cg and rpd3) strongly suppress wg-induced leg-to-wing transdetermination. It is speculated that the transdetermination frequency declines in these mutant animals because the initiation and/or timing of blastema formation is delayed. This idea is consistent with all previous work which has shown that blastema cells are only competent to transdetermine after they have regenerated the missing disc structures. Heterozygous mutations in Kr-h1 and S6k did not significantly alter the formation of the wg-induced regeneration blastema, however, these genes did affect regeneration-induced transdetermination. Such results suggest that Kr-h1 and S6k specifically function to modulate the cell fate changes that occur as a consequence of regeneration (McClure, 2008).

Investigations into the molecular basis of transdetermination have shown that inputs from the Wg, Decapentapelagic (Dpp) and Hedgehog (Hh) signaling pathways activate key selector genes out of their normal developmental context, such as ectopic Vg activation in the leg disc, which then drives cell-fate switches. Several of the genes identified in this screen have functional ties to Wg, Dpp and Hh signaling pathways. For example, Cg is a zinc-finger transcription factor that is required for proper transcriptional regulation of the Hh signaling effector gene Cubitus interruptus (Ci). In cg mutant wing and leg discs, Ci expression is lowered in the anterior compartment, resulting in the ectopic activation of wg and dpp and significant disc overgrowth. Another gene identified in this screen -- ken, functions in concert with Dpp to direct the development of the Drosophila terminalia. Further characterizations of whether these genes and other modifiers of transdetermination and regeneration affect Wg, Dpp and Hh expression and/or signaling may shed light on the regulation of regeneration and regeneration-induced proliferation and cell fate plasticity (McClure, 2008).

Regulation of neurogenesis and epidermal growth factor receptor signaling by the Insulin receptor/Target of rapamycin pathway in Drosophila

Determining how growth and differentiation are coordinated is key to understanding normal development, as well as disease states such as cancer, where that control is lost. Growth and neuronal differentiation are coordinated by the insulin receptor/target of rapamycin (TOR) kinase (InR/TOR) pathway. The control of growth and differentiation diverge downstream of TOR. TOR regulates growth by controlling the activity of S6 kinase (S6K) and eIF4E. Loss of s6k delays differentiation, and is epistatic to the loss of tsc2, indicating that S6K acts downstream or in parallel to TOR in differentiation as in growth. However, loss of eIF4E inhibits growth but does not affect the timing of differentiation. This study shows that there is crosstalk between the InR/TOR pathway and epidermal growth factor receptor (EGFR) signaling. InR/TOR signaling regulates the expression of several EGFR pathway components including pointedP2 (pntP2). In addition, reduction of EGFR signaling levels phenocopies inhibition of the InR/TOR pathway in the regulation of differentiation. Together these data suggest that InR/TOR signaling regulates the timing of differentiation through modulation of EGFR target genes in developing photoreceptors (McNeill, 2008).

Tight coordination of growth and differentiation is essential for normal development. InR/TOR signaling controls the timing of neuronal differentiation in the eye and leg in Drosophila. This study demonstrates that the InR/TOR pathway regulates neuronal differentiation in an S6K-dependent, but 4EBP/eIF4E-independent manner. It has previously been impossible to determine whether InR/TOR signaling was acting downstream or in parallel to the EGFR/MAPK pathway. Using argos and rho as reporters this study shows that the InR/TOR pathway is able to regulate EGFR/MAPK signaling downstream of MAPK. Moreover, pntP2 expression is up- and downregulated by activation or inhibition of InR/TOR signaling, respectively, and InR/TOR and EGFR pathways interact through pntP2. Taken together these data suggest that temporal control of differentiation by the InR/TOR pathway is achieved by modulation of EGFR pathway transcriptional targets in differentiating PRs (McNeill, 2008).

TOR is part of two multimeric complexes (TORC1 and TORC2) and is a core component of the InR pathway. TORC1 activity is regulated by nutrient and energy levels providing a conduit for hormonal and catabolic cellular inputs. Growth is regulated by two downstream targets of TORC1: S6K and 4EBP. The current data demonstrate that upstream of TORC1, differentiation and growth are regulated by the same factors. Downstream of TORC1, differentiation and growth differ significantly in that loss of s6k, but not eIF4E (or overexpression of 4EBP) affects differentiation. eIF4E regulates 7-methyl-guanosine cap-dependent translation and is the rate-limiting factor in translation initiation. The finding that eIF4E does not affect differentiation suggests that the temporal control of differentiation is not based on a translation initiation-dependent mechanism. Strikingly, loss of s6k blocks the precocious differentiation induced by loss of tsc2. Given the relatively weak effects of loss of s6k this may seem surprising. However, the degree of suppression is similar to the effect of loss of s6k on the overgrowth phenotype caused by loss of tsc2, namely, tsc2, s6k double-mutant cells are the same size as wild-type cells. Although loss of eIF4E has no affect on differentiation it may act redundantly with another factor, such as s6k. Testing this hypothesis though is technically challenging since the Drosophila genome contains eight different eIF4E isoforms. It will be interesting in future to test whether any of these isoforms regulate differentiation or alternatively whether eIF4E and s6k act redundantly. Although further work is required to determine the precise relationship between S6K and the InR/TOR pathway, the data point to a critical role of S6K in coordinating neuronal differentiation and growth (McNeill, 2008).

As in other neuronal systems, differentiation of PRs in the Drosophila eye occurs in a stereotyped manner. The advantage of the Drosophila retina as an experimental system is that the PRs differentiate spatiotemporally. Using this feature, as well as a series of cell-type-specific antibodies, this study has demonstrated that InR/TOR signaling is selective in the cell-types that it affects. The differentiation of PRs 2/5, 3/4, and 8 are unaffected by perturbations in InR/TOR signaling, whereas PRs 1, 6, and 7 and cone cells are dependent on this pathway for temporal control of differentiation. Interestingly the affected cells all differentiate after the second mitotic wave. However, regulators of the cell cycle do not affect the temporal control of differentiation. Why then are PRs 1, 6, and 7 and cone cells specifically affected? In cells with increased InR/TOR signaling, the expression of argos, rho, and pntP2 is precocious and increased throughout the clone, suggesting that the upregulation of EGFR signaling occurs in all cells. However, decreasing EGFR activity using a hypomorphic pntP2 allele specifically affects the differentiation of PRs 1, 6, and 7 and cone cells. Interestingly, pntP2 expression in differentiated cells is also restricted to PRs 1, 6, and 7 and cone cells. These observations suggest that differentiation of PRs 1, 6, and 7 and cone cells is critically dependent on EGFR levels signaling through pntP2. Therefore, although activation of InR/TOR signaling causes upregulation of EGFR transcriptional targets in all cells as they differentiate, the phenotypic effect is seen only in PRs 1, 6, and 7 and cone cells since these cells are highly sensitive to EGFR activity signaling through pntP2. This possibility is supported by the fact that precocious differentiation caused by overexpression of Dp110 can be suppressed by the simultaneous reduction of pntP2 levels. The complete suppression of the Dp110 differentiation phenotype by simultaneous reduction of pntP2 strongly suggests that pntP2 acts downstream of Dp110 and InR/TOR signaling in a pathway that regulates the temporal control of differentiation. It has been suggested that later differentiating PRs require higher levels of EGFR activity than their earlier differentiating neighbors. In particular, the activation of PR 7 requires both EGFR and Sevenless RTKs. In the case of InR/TOR pathway activation it may be that, through its regulation of EGFR downstream targets, the 'second burst' of RTK activity is enhanced causing PRs 1, 6, and 7 and cone cells to differentiate precociously. There may also be other as yet unidentified factors through which the InR/TOR pathway controls the expression of Aos and rho in PRs 2-5 and 8 (McNeill, 2008).

Activation of insulin and insulin-like growth factor receptors in mammalian systems is well known to elicit a response via the Ras/MAPK pathway. However, loss of the InR in the Drosophila eye does not result in a loss of PRs, a hallmark of the Ras pathway, nor does mutation of the putative Drk binding site in chico affect the function of the Drosophila IRS. In accordance with these data no change is seen in dpERK staining when the InR/TOR pathway is activated in the eye disc. Rather than a direct activation of Ras signaling by the InR, the data suggest that in the developing eye crosstalk between these pathways occurs at the level of regulation of the expression of EGFR transcriptional outputs. The most proximal component of the EGFR pathway that is regulated by InR/TOR signaling is pntP2. However, the data suggest that temporal control of PR differentiation requires concerted regulation of EGFR transcriptional outputs, since overexpression of pntP2 alone is not sufficient to cause precocious differentiation, whereas overexpression of activated EGFR is sufficient. Interestingly, microarray analyses of Drosophila and human cells have shown that the InR/TOR pathway regulates the expression of hundreds of genes. The mechanism by which this transcriptional control is exerted has yet to be elucidated. It will be interesting in future to determine the extent of transcriptional crosstalk between InR/TOR and EGFR pathways in developing neurons (McNeill, 2008).

Drosophila RSK negatively regulates bouton number at the neuromuscular junction

Ribosomal S6 kinases (RSKs) are growth factor-regulated serine-threonine kinases participating in the RAS-ERK signaling pathway. RSKs have been implicated in memory formation in mammals and flies. To characterize the function of RSK at the synapse level, the effect was investigated of mutations in the rsk gene on the neuromuscular junction (NMJ) in Drosophila larvae. Immunostaining revealed transgenic expressed RSK in presynaptic regions. In mutants with a full deletion or an N-terminal partial deletion of rsk, an increased bouton number was found. Restoring the wild-type rsk function in the null mutant with a genomic rescue construct reverted the synaptic phenotype, and overexpression of the rsk-cDNA in motoneurons reduced bouton numbers. Based on previous observations that RSK interacts with the Drosophila ERK homologue Rolled, genetic epistasis experiments were performed with loss- and gain-of-function mutations in Rolled. These experiments provided evidence that RSK mediates its negative effect on bouton formation at the Drosophila NMJ by inhibition of ERK signaling (Fischer, 2009).

This study investigated the effect of rsk loss of function mutations in Drosophila, and found higher numbers of synaptic boutons in these mutants. The effect could be rescued by transgenic rsk expression. Vice versa overexpression of RSK reduced bouton numbers. Furthermore, removal of one allele of the Drosophila erk/rl gene normalized the effect of rsk loss of function on bouton formation, indicating that RSK mediates its effect through ERK/RL. Indeed, RSK and ERK/RL proteins interact directly with each other, and this interaction is abolished in the rl^Sem mutant. Furthermore, rl^Sem mutants show enhanced bouton numbers, similarly as rsk mutants, indicating that RSK negatively regulates ERK/RL activity at the NMJ and thus modulates bouton formation (Fischer, 2009).

A role of vertebrate RSK2 in inhibition of the RAS/ERK pathway has been proposed in several studies, but different underlying mechanisms have been suggested. In isolated mouse motoneurons, RSK2 is a negative regulator of axon growth by inhibiting ERK phosphorylation. In skeletal muscles of RSK2 knock-out mice, increased ERK activation has been observed. This could be explained by lack of inhibition of the ERK pathway via RAS guanine exchange factor SOS. In Drosophila, this inhibition seems not to occur through SOS. Knockdown of RSK2 leads to increased ERK phosphorylation in PC12 cells and cortical neurons. Moreover basal and 5HT2A receptor-mediated ERK 1/2 phosphorylation is increased in RSK2 knock-out fibroblasts These data are consistent with the current results showing that RSK interacts with ERK/RL and that this interaction leads to inhibition of ERK/RL activity in bouton formation at the NMJ (Fischer, 2009).

Previous studies on RSK and RL in the developing eye and wing imaginal disc provided evidence that RSK inhibits translocation of ERK/RL from the cytoplasm to the nucleus and thereby controls RL dependent gene transcription. However, the NMJ constitutes a separate part of the cell, and it is also conceivable that the effects of RSK and RL are mediated locally and do not involve nuclear translocation of these proteins. RSK seems to be present in the presynapse, but its distribution is diffuse and not restricted to active zones. This corresponds to the known distribution of RL in axon terminals. Thus, it is possible that RSK determines the localization of RL within synaptic boutons. Interestingly, an antibody that only recognizes active, phosphorylated RL showed a restricted localization to spots most likely corresponding to active zones. Thus one could speculate that RSK binds ERK/RL in axon terminals, thus inhibiting its activation, and only ERK/RL that is unbound can be activated by phosphorylation and move to active zones (Fischer, 2009).

In conclusion, these data indicate that RSK negatively regulates bouton formation at the NMJ, and that negative regulation of RL signaling is involved in this effect. Thus, Drosophila RSK seems to have a similar function as the RSK2 isoform in vertebrates. Therefore, the memory defects observed in flies, mice, and human CLS patients with mutations in rsk could be caused by dysregulated synapse architecture, as observed in the Drosophila model (Fischer, 2009).

The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila

S6 kinases (S6Ks) act to integrate nutrient and insulin signaling pathways and, as such, function as positive effectors in cell growth and organismal development. However, they also have been shown to play a key role in limiting insulin signaling and in mediating the autophagic response. To identify novel regulators of S6K signaling, a Drosophila-based, sensitized, gain-of-function genetic screen was used. Unexpectedly, one of the strongest enhancers to emerge from this screen was the nuclear receptor (NR), Drosophila hormone receptor 3 (DHR3), a critical constituent in the coordination of Drosophila metamorphosis. This study demonstrates that DHR3, through dS6K, also acts to regulate cell-autonomous growth. Moreover, the ligand-binding domain (LBD) of DHR3 was shown to be essential for mediating this response. Consistent with these findings, an endogenous DHR3 isoform was identified that lacks the DBD. These results provide the first molecular link between the dS6K pathway, critical in controlling nutrient-dependent growth, and that of DHR3, a major mediator of ecdysone signaling, which, acting together, coordinate metamorphosis (Montagne, 2009).

By using Drosophila genetics and a gain-of-function strategy, the NR, DHR3, was identified as an enhancer of a dS6K-regulated growth phenotype. This effect can be mediated by an isoform of DHR3 lacking the DBD. Moreover, using a revertant screening strategy, LBD-specific DHR3 mutants were generated, and it was demonstrated that the LBD of DHR3 is necessary to maintain normal growth and dS6K activity. In contrast to the role DHR3 plays in transcriptional regulation affecting the onset of metamorphosis, these studies indicate that it also plays a role in regulating cell-autonomous growth. These effects are most likely mediated through dS6K, as the ability of ectopically expressed DHR3-RS to drive growth in the dorsal wing blade is blunted in Drosophila deficient for dS6K. Consistent with these findings, it has been demonstrated that dS6K also controls cell growth in a cell-autonomous manner. However, the effect on cell size is more pronounced in dS6K mutants than in the DHR3-mutant clones described in this study. This may reflect the fact that dS6K activity is blunted, but not abolished, in DHR3 LBD-mutant larvae. Compatible with this hypothesis, it was found that in a dS6K P-element-induced mutant (P{PZ}S6K[07084]) no dS6K protein could be detected; however, this mutation induced a much less severe phenotype as compared with the dS6K^l-1 null mutation. In homozygous DHR3 mutant eyes both the size and the number of ommatidia were decreased, whereas in dS6K mutant flies the size reduction of the eye was only due to a decrease in ommatidia size but not number. This difference might be attributed to the experimental settings. In the current study, DHR3 mutant eyes were generated by mitotic recombination in a heterozygous Minute background, whose developmental delay is less than two days. In contrast, the size and number of ommatidia in dS6K mutant eyes were measured in homozygous mutant flies that exhibit a five-day delay at eclosion. The longer time for the latter to emerge as adults allows additional cell divisions to proceed, leading to a higher number of ommatidia (Montagne, 2009).

Previous studies demonstrated that DHR3 participates in a hierarchal regulatory circuit in response to ecdysone signaling, but also acts in a negative feedback loop to repress ecdysone receptor-mediated signaling. Prothoracic gland production of ecdysone is mediated by the brain neuropeptide prothoracicotropic hormone (PTTH). Recent studies in Drosophila have shown that genetic ablation of PTTH-producing neurons induces a delay in larval development and results in larger adult flies as a direct consequence of reduced levels of ecdysone. Interestingly, in the tobacco hornworm, Manduca sexta, PTTH-induced ecdysone production is paralleled by the phosphorylation of the Manduca orthologue of Drosophila ribosomal protein S6. Moreover, this process is sensitive to rapamycin, and a burst of dS6K activity is observed at early pupation. As the body size of the adult fly appears to be determined by growth regulators, including dS6K, as well as by hormones that control the timing of developmental windows, such as PTTH, the results suggest that the DHR3/dS6K regulatory module acts to integrate these two processes (Montagne, 2009).

These studies supports the existence of a novel DHR3 polypeptide devoid of a DBD, DHR3-PS. Nonetheless, although DHR3-PS is sufficient to potentiate a dS6K-dependent growth phenotype, it is not possible to exclude that the other DBD-containing DHR3 isoforms also contribute to dS6K activation. In general, DHR3, like other NRs, is a transcription factor composed of four elements: a modulator domain, the DBD, the hinge region, and the LBD. The DBD of NRs typically consists of two zinc fingers, with the first being critical for conferring DNA-binding specificity. Like DHR3-PS, NRs lacking a DBD have been previously reported. Notably, in Drosophila, the NR E75B, a DHR3 partner, lacks one of the 2 zinc fingers that is required to form a functional DBD. However, E75B, through its ability to interact with DHR3, modulates DHR3 transcriptional activity in a gas-responsive manner (Reinking, 2005). Like the putative DHR3-PS, the NR short heterodimer partner (SHP) in mammals is also devoid of DBD, but, as with E75B, it interacts with other NRs to modulate their transcriptional activity. It is unlikely that DHR3-PS behaves as a dominant-interfering effector of full-length DHR3 as ectopic DHR3-PS expression induces growth, whereas DHR3-RNAi inhibits growth. However, DHR3 also heterodimerizes with two NRs: E75 and the ecdysone receptor. Thus, in the case of E75, ectopically expressed DHR3-PS may act to decrease the levels of free E75, leaving full-length DHR3 free to increase the transcription of target genes. In contrast, DHR3-PS binding to the ecdysone receptor could counteract the negative growth regulation mediated by ecdysone signaling. However, it should be noted that the negative effects of ecdysone are humoral and mediated by dFOXO-inactivation within the fat body, whereas, as this study has shown, DHR3 regulates growth in a cell-autonomous manner. Moreover, dFOXO subcellular distribution was not altered in DHR3 mutant clones in third instar wing imaginal discs, indicating that the DHR3 cell-autonomous effect on cell growth is not mediated by the PKB/dFOXO signaling (Montagne, 2009).

In contrast to acting as a dominant-interfering isoform, the results presented in this study also suggest that DHR3 activates dS6K through a non-genomic mechanism, an effect of NRs that does not require the DBD function. Such a model is supported by NR responses whose kinetics are too rapid to be explained by de novo transcription and translation of a gene product. Indeed, nongenomic effects typically occur within minutes following addition of the cognate ligand and are resistant to transcriptional inhibitors. In the case of DHR3, it is experimentally difficult to address this question as the ligand for DHR3 is unknown, and a genetic endpoint is being scored for, resulting from events induced much earlier in larval development. It has been demonstrated that vitamin D3 and all-trans-retinoic acid both induce activation of S6K1 within minutes of administration to cells. Moreover, in the case of vitamin D3, it was shown that these effects were mediated through protein phosphatases PP1 and PP2A in a vitamin D3 receptor (VDR)-dependent manner. VDR appears to directly interact with the catalytic subunits of PPI and PP2A, and vitamin D3 acts to disrupt this interaction and enhance an interaction between VDR and S6K1, stabilizing S6K1 in its phosphorylated active state. However, depleting DHR3 levels by RNA interference blunts both dS6K T398 and d4E-BP T37/T46 phosphorylation, suggesting that DHR3 acts upstream or at the level of dTORC1. Identification of potential partners for DHR3-PS may be useful in determining, at the molecular level, the mechanism by which DHR3 controls cell growth and dS6K activity (Montagne, 2009).

The data further support the notion that a ligand exists for DHR3, and that the ligand is required for many of the pleiotropic activities of DHR3. Those NRs that bind steroid hormones are, in general, high-affinity receptors, whereas the low-affinity NRs bind ligands that are present in high concentration, such as dietary nutrients. The observation that an NR, generated by fusing the DHR3 LBD with the DBD of Gal4, is transcriptionally active in a number of specific embryonic and larval tissues suggests that such a ligand is widely present. Given the role of dTOR/dS6K as a nutritional effector, it is interesting to note that the chimeric DHR3/Gal4 NR is active in organs that provide basal nutrients, in particular, in a group of cells of the larval midgut, which are essential for the transfer of nutrients to the hemolymph. Importantly, the mammalian orthologues to DHR3 and its partner E75 are retinoid-related orphan receptor (ROR)α and Rev-erb (NR1D)α, respectively. As in Drosophila, the NR1D subgroup functions as dominant transcriptional silencers by inhibiting transactivation mediated by RORα. Interestingly, it was recently reported that RORα-deficient mice, like S6K1-deficient mice, exhibit reduced fat-pad mass, smaller adipocytes, and resistance to diet-induced obesity. Moreover, in solving the X-ray structure of the RORα LBD, it was revealed that cholesterol was bound in the ligand-binding pocket. While the Drosophila NR, DHR96, has recently been shown to bind cholesterol thereby modulating cholesterol homeostasis, this does not exclude the possibility that DHR3 could also bind cholesterol. However, the predicted models of the structure of DHR3 indicate that the size of the ligand-binding pocket is smaller than those of either RORα or RORβ. Given the role of the mTOR/S6K1 nutrient-responsive pathway in mammals, it raises the possibility that DHR3 is a low-affinity receptor for an abundant nutrient ligand. Identification of this specific ligand constitutes the next issue to investigate (Montagne, 2009).

Rapamycin activation of 4E-BP prevents parkinsonian dopaminergic neuron loss

Mutations in PINK1 and parkin cause autosomal recessive parkinsonism, a neurodegenerative disorder characterized by the loss of dopaminergic neurons. To highlight potential therapeutic pathways, this study identified factors that genetically interact with parkin/PINK1. Overexpression of the translation inhibitor 4E-BP can suppress all pathologic phenotypes including degeneration of dopaminergic neurons in Drosophila. 4E-BP is activated in vivo by the TOR inhibitor rapamycin, which can potently suppress pathology in PINK1/parkin mutants. Rapamycin also ameliorates mitochondrial defects in cells from parkin-mutant patients. Recently, 4E-BP was shown to be inhibited by the most common cause of parkinsonism, dominant mutations in LRRK2. This study further shows that loss of the Drosophila LRRK2 homolog activates 4E-BP and is also able to suppress PINK1/parkin pathology. Thus, in conjunction with recent findings these results suggest that pharmacologic stimulation of 4E-BP activity may represent a viable therapeutic approach for multiple forms of parkinsonism (Tain, 2009).

This study used Drosophila as a model system to uncover genetic suppressors in order to understand the pathogenic mechanisms and to highlight putative therapeutic pathways for PD. Thor, the sole Drosophila homolog of mammalian 4E-BP1, has been identified as a genetic modifier of parkin. In the present study, the genetic interaction of Thor with parkin and PINK1 was investigated. While loss-of-function mutations in Thor dramatically decrease parkin and PINK1 mutant viability, overexpression of 4E-BP is able to suppress PINK1 and parkin mutant phenotypes, including degeneration of dopaminergic neurons. These results suggest that 4E-BP acts to mediate or promote a survival response implemented upon loss of parkin or PINK1 (Tain, 2009).

4E-BP1 is an inhibitor of 5' cap-dependent protein translation, which is known to play an important role in cellular response to changes in environmental conditions such as altered nutrient levels and various physiological stresses. It has been demonstrated that Drosophila 4E-BP is important for survival under a wide variety of stresses including starvation, oxidative stress, unfolded protein stress and immune challenge. Such a response pathway represents a likely target for possible manipulation by therapeutics. Fenetic evidence supports this idea, hence, this study attempted to validate whether this represented a viable therapeutic target (Tain, 2009).

4E-BP activity is regulated post-translationally by the TOR signaling pathway. Activated TOR hyper-phosphorylates 4E-BP inhibiting it leading to promotion of 5' cap-dependent translation. Rapamycin is a small molecule inhibitor of TOR signaling and has been shown to lead to 4E-BP hypo-phosphorylation. Genetic evidence suggested that administration of rapamycin to parkin/PINK1 mutants should relieve 4E-BP inhibition and confer a protective effect. Exposing mutant animals to rapamycin during development caused an increase in hypo-phosphorylated 4E-BP and, remarkably, was sufficient to suppress all pathologic phenotypes, including muscle degeneration, mitochondrial defects and locomotor ability. Continued administration of rapamycin during aging also completely suppressed progressive degeneration of dopaminergic neurons (Tain, 2009).

To validate this pathway as a viable target for therapy, the studies were extended to human tissue. There is growing evidence that mitochondrial dysfunction is a key pathologic event across the spectrum of parkinsonism. Mitochondrial defects have been demonstrated in a number of cell lines derived from patients with parkin mutations. This study shows that rapamycin is also capable of ameliorating mitochondrial bioenergetic and morphological defects in parkin-deficient PD patient cell lines. Thus, the results provide strong support for the proposition that modulating 4E-BP mediated translation by pharmaceuticals such as rapamycin can be efficacious in vivo and is relevant to human pathophysiology (Tain, 2009).

TOR signaling regulates a number of downstream effectors other than 4E-BP, for example, up-regulation of S6 kinase promoting protein synthesis and cell proliferation, and down-regulation of autophagy likely through inhibition of ATG1. The coordinated regulation of these pathways serves to optimize cellular activity in response to vital changes such as nutrient availability and environmental stresses. Stimulation of autophagy under nutrient-deprived conditions is a survival mechanism that recycles essential metabolic components, but this mechanism also promotes the degradation of aggregated or misfolded proteins. Thus, the potential therapeutic effects of rapamycin have been widely promoted as a strategy to combat a number of neurodegenerative diseases including PD primarily for its perceived role in promoting autophagic clearance of aggregated proteins. However, recent studies have provided compelling evidence that the pro-survival effects of rapamycin can be mediated in the absence of autophagy by reducing protein translation. This study has demonstrated that genetic ablation of 4E-BP is sufficient to completely abrogate any beneficial effects of rapamycin in vivo while inhibiting Atg5, a key mediator of autophagy, does not diminish the efficacy of rapamycin-mediated protection. Together, these results indicate that in this instance the major protective effects of rapamycin treatment are mediated through regulated protein translation, with little or no contribution from autophagy (Tain, 2009).

A switch from cap-dependent to cap-independent translation is likely to effect widespread changes in the proteome, particularly the induction of pro-survival factors including chaperones, anti-oxidants and detoxifying enzymes. In support of this, it was shown that transgenic or rapamycin-induced 4E-BP activation leads to increased protein levels of GstS1, a major detoxification enzyme in Drosophila. Interestingly, it was previously shown that transgenic overexpression of Drosophila GstS1 is able to suppress dopaminergic neuron loss in parkin mutants. Elucidating the global changes in response to 4E-BP activation will be crucial to understanding the exact molecular mechanisms of neuro-protection but currently remains unresolved (Tain, 2009).

The potential importance of 4E-BP modulation as a therapeutic target is underscored by recent findings that report the most common genetic cause of PD, dominant mutations in LRRK2, inhibit 4E-BP function through direct phosphorylation. Expression of these mutations causes disruption of dopaminergic neurons in Drosophila and mouse, however, in striking similarity to the current results, overexpression of 4E-BP can circumvent the pathogenic effects of mutant LRRK2 and prevent neurodegeneration (Imai, 2008) in Drosophila. This study shows that loss of Drosophila LRRK leads to activation of 4E-BP and can suppress pathology in PINK1 and parkin mutants. These data further support a link between LRRK2 and 4E-BP activity and a common cause of PD. Thus, the results indicate that promoting 4E-BP activity may be beneficial in preventing neurodegeneration in multiple forms of parkinsonism. Since 4E-BP activity can be manipulated by small molecule inhibitors such as rapamycin, this pathway represents a viable therapeutic target. It will be particularly interesting to determine whether rapamycin is efficacious in ameliorating pathologic phenotypes in the recently reported LRRK2 transgenic mouse model, but further studies will be necessary to determine whether pharmacologic modulation of 4E-BP function is therapeutically relevant in all forms of parkinsonism including sporadic PD (Tain, 2009).

Reduction of protein translation and activation of autophagy protect against PINK1 pathogenesis in Drosophila melanogaster

Mutations in PINK1 and Parkin cause familial, early onset Parkinson's disease. In Drosophila, PINK1 and Parkin mutants show similar phenotypes, such as swollen and dysfunctional mitochondria, muscle degeneration, energy depletion, and dopaminergic (DA) neuron loss. PINK1 and Parkin have been shown to genetically interact with the mitochondrial fusion/fission pathway, and PINK1 and Parkin have been proposed to form a mitochondrial quality control system that involves mitophagy. However, the in vivo relationships among PINK1/Parkin function, mitochondrial fission/fusion, and autophagy remain unclear; and other cellular events critical for PINK1 pathogenesis remain to be identified. This study shows that PINK1 genetically interacte with the protein translation pathway. Enhanced translation through S6K activation significantly exacerbates PINK1 mutant phenotypes, whereas reduction of translation shows suppression. Induction of autophagy by Atg1 overexpression also rescues PINK1 mutant phenotypes, even in the presence of activated S6K. Downregulation of translation and activation of autophagy are already manifested in PINK1 mutant, suggesting that they represent compensatory cellular responses to mitochondrial dysfunction caused by PINK1 inactivation, presumably serving to conserve energy. Interestingly, the enhanced PINK1 mutant phenotype in the presence of activated S6K can be fully rescued by Parkin, apparently in an autophagy-independent manner. These results reveal complex cellular responses to PINK1 inactivation and suggest novel therapeutic strategies through manipulation of the compensatory responses (Liu, 2010; full text of article)

Previously, PINK1 and Parkin have been suggested to interact with mitochondrial fusion/fission machinery and the autophagy pathway. This study found that PINK1 also genetically interacts with the protein translation pathway. Increased global protein translation with S6K or eIF4E over expression (OE) exacerbates PINK1 mutant phenotypes, while decreased translation has the opposite effects. Overexpression of constitutively active S6Ks dramatically enhances muscle and DA neuron degeneration in PINK1 mutant flies, which can be mitigated by the co-expression of RpS6 RNAi or RpS9 RNAi, supporting that the TOR/S6K pathway modifies PINK1 mutant phenotypes through regulating global translation. Recently, it has been reported that pathogenic leucine-rich repeat kinase 2 (LRRK2), which represents the most frequent molecular lesions found in Parkinson's disease, promotes 4E-BP phosphorylation, resulting in increased eIF4E-mediated translation, enhanced sensitivity to oxidative stress, and DA neuron loss. Taken together, these results support the idea that deregulated protein translation is generally involved in the pathogenesis of Parkinson's disease (Liu, 2010).

Deregulated translation affects Parkinson's disease pathogenesis most likely at the level of energy metabolism, since protein translation is a very energy-consuming process, of which ribosomal biogenesis is the most costly, consuming approximately 80% of the energy in proliferating cells. This study shows that forced upregulation of ribosomal biogenesis in the fly muscle by the overexpression of constitutively active S6K is well tolerated in WT flies; however, such manipulation in PINK1 RNAi flies completely abolishes their flight ability, depletes ATP in the muscle and enhances muscle and DA neuron degeneration. The tolerance of increased protein translation by wild type flies is probably due to the existence of an intact mitochondrial quality control system containing PINK1 and Parkin, which can either eliminate damaged mitochondria generated during elevated energy production or minimize damages caused by increased ROS generated during energy production. However, in PINK1 or Parkin mutants that lack a functional mitochondrial quality control system, increased protein translation and the corresponding energy demand will translate into increased ROS generation, accumulation of dysfunctional mitochondria, and eventual energy depletion and tissue degeneration. Since downregulation of translation through knockdown of S6K, RpS6, or RpS9 is beneficial to PINK1 mutant flies, and S6K activity is already tuned down in PINK1 mutant flies, reduction of translation likely represents one of the cellular compensatory responses to the energy deficit caused by mitochondrial dysfunction in PINK1 mutants. Interestingly, partial reduction of S6K activity prolonges fly lifespan, whereas increased S6K activity has the opposite effects on longevity. The effects of S6K on animal lifespan and PINK1 mutant phenotypes can both be explained by the energy metabolism hypothesis and they offer a tantalizing link between aging and the pathogenesis of Parkinson's disease (Liu, 2010).

Supporting the energy metabolism model, it was shown that downregulation of protein translation by knocking down positive regulators of translation (S6K, RpS6, RpS9) or overexpressing a negative regulator (4E-BP) could rescue PINK1 mutant phenotypes. These manipulations presumably act by preserving cellular energy and reducing the workload and ROS production of mitochondria. Previously, 4E-BP OE was suggested to rescue PINK1 mutant phenotype by upregulating Cap-independent translation of stress related genes, including antioxidant genes, and boosting antioxidant gene activity has been suggested as a therapeutic strategy in the PINK1 and Parkin models of Parkinson's disease. This study found that although overexpression of antioxidant genes, such as Catalase, GTPx-1, SOD and GstS1, all showed some degree of rescue of PINK1 mutant phenotypes, their effects were in general weaker than that of Atg1 OE, Parkin OE, or Marf RNAi, particularly in the PINK1 RNAi/S6K-TE OE background. These data suggest that increasing autophagy and mitochondrial fission might be better choices to combat PINK1-related Parkinson's disease (Liu, 2010).

Autophagy is a conserved cellular process through which cytoplasmic content or defective intracellular organelles can be eliminated or recycled. Although autophagy is usually induced under adverse conditions to provide means for survival, basal level of autophagy in the cell is just as critical to the physiological health of the organism, since defects in autophagy are frequently associated with cancer, neurodegeneration, and aging. The induction of autophagy leads to the de novo formation of double membrane structure called isolation membrane, which expands to form a sealed compartment named autophagosome that will engulf materials destined for degradation. The large size of mitochondria likely poses a challenge for the autophagy machinery, as engulfment of an entire mitochondrion requires a significant amount of building materials for autophagosome formation. This is especially the case in PINK1 mutant where dysfunctional mitochondria becomes grossly swollen or aggregated. Previously, it has been shown that increased mitochondrial fission or Parkin OE could efficiently rescue the enlarged mitochondria phenotype in PINK1 mutants. The rescuing effect by increased mitochondrial fission could be due to the fact that it decreases mitochondrial size and makes it easier for the autophagosome to engulf the entire mitochondrion during mitophagy. In addition, increased mitochondrial fission could facilitate the segregation of the healthy part of a mitochondrion from the unhealthy part, thus enhancing the selective elimination of dysfunctional mitochondria through mitophagy. Supporting the mitophagy model, Parkin has been proposed to promote the efficient removal of damaged mitochondria by selectively ubiquitinating proteins on damaged mitochondria. A key prediction of the mitophagy model is that the protective effects of Parkin OE and increased mitochondrial fission as in the case of Marf RNAi will depend on the autophagy pathway. Surprisingly, this study found that blocking autophagy through Atg1 RNAi or Atg18 RNAi failed to block Parkin OE or Marf RNAi's rescuing abilities in PINK1 mutant, although Atg18 RNAi was effective in blocking the rescuing ability of Atg1 OE. This result suggests that the rescuing effect of Parkin OE or Marf RNAi is not entirely dependent on autophagy, and that other processes are likely involved. For example, Parkin has been suggested to promote mitochondrial biogenesis and regulate protein translation. Further studies are needed to elucidate the exact molecular functions of Parkin that are critically involved in mitochondrial function and tissue maintenance in vivo (Liu, 2010).

Given the well-established catabolic role of autophagy in degrading cytoplasmic contents, it helps recycle nutrients and provide energy source needed for survival under harsh conditions. In PINK1 mutants that suffer energy deficit due to mitochondrial dysfunction, induction of autophagy would present as a compensatory response to cope with the limited energy supply. Indeed, this study found that basal autophagy is induced in PINK1 mutant, and further increase of autophagy through Atg1 OE protects against PINK1 pathogenesis. Thus, decreased translation and increased autophagy both represent compensatory responses in PINK1 mutant flies, and further augmentation of these responses can effectively protect against the toxic effects of PINK1 inactivation. A previous study in cultured mammalian cells also indicated that autophagy is induced in response to PINK1 inactivation. Thus, the in vivo compensatory responses revealed in this study are likely relevant to PINK1 pathogenesis in mammals. Pharmacological interventions that promote these responses offer potential new treatment strategies for Parkinson's disease (Liu, 2010).

Insulin/IGF-regulated size scaling of neuroendocrine cells expressing the bHLH transcription factor dimmed in Drosophila

Neurons and other cells display a large variation in size in an organism. Thus, a fundamental question is how growth of individual cells and their organelles is regulated. Is size scaling of individual neurons regulated post-mitotically, independent of growth of the entire CNS? Although the role of insulin/IGF-signaling (IIS) in growth of tissues and whole organisms is well established, it is not known whether it regulates the size of individual neurons. The role of IIS in the size scaling of neurons in the Drosophila CNS was studied. By targeted genetic manipulations of insulin receptor (dInR) expression in a variety of neuron types it was demonstrated that the cell size is affected only in neuroendocrine cells specified by the bHLH transcription factor DimmedD (Dimm). Several populations of Dimm-positive neurons tested displayed enlarged cell bodies after overexpression of the dInR, as well as PI3 kinase and Akt1 (protein kinase B), whereas Dimm-negative neurons did not respond to dInR manipulations. Knockdown of these components produce the opposite phenotype. Increased growth can also be induced by targeted overexpression of nutrient-dependent TOR (target of rapamycin) signaling components, such as Rheb (small GTPase), Tor and S6K (S6 kinase). After Dimm-knockdown in neuroendocrine cells manipulations of dInR expression have significantly less effects on cell size. It was also shown that dInR expression in neuroendocrine cells can be altered by up or down-regulation of Dimm. This novel dInR-regulated size scaling is seen during postembryonic development, continues in the aging adult and is diet dependent. The increase in cell size includes cell body, axon terminations, nucleus and Golgi apparatus. It is suggested that the dInR-mediated scaling of neuroendocrine cells is part of a plasticity that adapts the secretory capacity to changing physiological conditions and nutrient-dependent organismal growth (Luo, 2013).

EVOLUTIONARY HOMOLOGS

Insect S6 kinases

The insect prothoracic glands are the source of steroidal molting hormone precursors and the glands are stimulated by a brain neuropeptide, prothoracicotropic hormone (PTTH). PTTH acts via a cascade including Ca2+/calmodulin activation of adenylate cyclase, protein kinase A, and the subsequent phosphorylation of a 34 kDa protein (p34) hypothesized, but not proven, to be the S6 protein of the 40S ribosomal subunit. The immunosuppressive macrolide, rapamycin, is a potent inhibitor of cell proliferation, a signal transduction blocker, and also prevents ribosomal S6 phosphorylation in mammalian systems. Rapamycin inhibits PTTH-stimulated ecdysteroidogenesis in vitro by the prothoracic glands of the tobacco hornworm, Manduca sexta, with half-maximal inhibition at a concentration of about 5 nM. At concentrations above 5 nM, there is a 75% inhibition of ecdysteroid biosynthesis. Similar results are observed with the calcium ionophore (A23187), a known stimulator of ecdysteroidogenesis. Most importantly, the inhibition of ecdysteroid biosynthesis is accompanied by the specific inhibition of the phosphorylation of p34, indicating that p34 indeed is ribosomal protein S6. In vivo assays reveal that injection of rapamycin into day 6 fifth instar larvae results in a decreased hemolymph ecdysteroid titer and a dose-dependent delay in molting and metamorphosis. When S6 kinase (S6K) activity is examined using rapamycin-treated prothoracic glands as the enzyme source and a synthetic peptide (S6-21) or a 40S ribosomal subunit fraction from Manduca tissues as substrate, the data reveal that rapamycin inhibits S6K activity. It is concluded that S6 kinase plays a role in prothoracicotropic hormone stimulation of insect prothoracic glands by targeting ribosomal protein S6 (Song, 1994).

Phosphorylation of ribosomal protein S6 is requisite for prothoracicotropic hormone (PTTH)-stimulated specific protein synthesis and subsequent ecdysteroidogenesis in the prothoracic glands of the tobacco hornworm, Manduca sexta. To better understand the role of S6 in regulating ecdysteroidogenesis, S6 cDNA was isolated from a Manduca prothoracic gland cDNA library and sequenced. The deduced protein is comprised of 253 amino acids, has a molecular weight of 29,038, and contains four copies of a 10-amino acid motif defining potential DNA-binding sites. This Manduca S6 possesses a consensus recognition sequence for the p70(s6k) binding domain as well as six seryl residues at the carboxyl-terminal sequence of 17 amino acids. Phosphoamino acid analysis reveals that the phosphorylation of Manduca prothoracic gland S6 is limited exclusively to serine residues. Although alterations in the quantity of S6 mRNA throughout the last larval instar and early pupal-adult development are not well correlated with the hemolymph ecdysteroid titer, developmental expression and phosphorylation of S6 are temporally correlated with PTTH release and the hemolymph ecdysteroid titer. These data provide additional evidence that S6 phosphorylation is a critical element in the transduction pathway leading to PTTH-stimulated ecdysteroidogenesis (Song, 1997).

A cDNA encoding the Drosophila melanogaster p90 ribosomal S6 kinase II (RSK) has been isolated from an eye-antennal imaginal disc library and sequenced. The conceptually translated protein is 60%-63% identical to vertebrate RSK homologs and contains a perfectly conserved mitogen-activated protein kinase phosphorylation site. The gene was mapped to the base of the X chromosome in division 20 by in situ hybridization to polytene chromosomes (Wassarman, 1994).

Frizzled 2 is a key component in the regulation of TOR signaling-mediated egg production in the mosquito Aedes aegypti

The Wnt signaling pathway was first discovered as a key event in embryonic development and cell polarity in Drosophila. Recently, several reports have shown that Wnt stimulates translation and cell growth by activating the mTOR pathway in mammals. Previous studies have demonstrated that the Target of Rapamycin (TOR) pathway plays an important role in mosquito vitellogenesis. However, the interactions between these two pathways are poorly understood in the mosquito. In this study, it was hypothesized that factors from the TOR and Wnt signaling pathways interacted synergistically in mosquito vitellogenesis. The results showed that silencing Aedes aegypti Frizzled 2 (AaFz2), a transmembrane receptor of the Wnt signaling pathway, decreased the fecundity of mosquitoes. AaFz2 was highly expressed at the transcriptional and translational levels in the female mosquito 6 h after a blood meal, indicating amino acid-stimulated expression of AaFz2. Notably, the phosphorylation of S6K, a downstream target of the TOR pathway, and the expression of vitellogenin were inhibited in the absence of AaFz2. A direct link was found in this study between Wnt and TOR signaling in the regulation of mosquito reproduction (Weng, 2015).

The Target of Rapamycin pathway antagonizes pha-4/FoxA to control development and aging in C. elegans

FoxA factors are critical regulators of embryonic development and postembryonic life, but little is know about the upstream pathways that modulate their activity. C. elegans pha-4 encodes a FoxA transcription factor that is required to establish the foregut in embryos and to control growth and longevity after birth. The AAA+ ATPase homolog ruvb-1 has been identified as a potent suppressor of pha-4 mutations. This study shows that ruvb-1 is a component of the Target of Rapamycin (TOR) pathway in C. elegans (CeTOR). Both ruvb-1 and let-363/TOR control nucleolar size and promote localization of box C/D snoRNPs to nucleoli, suggesting a role in rRNA maturation. Inactivation of let-363/TOR or ruvb-1 suppresses the lethality associated with reduced pha-4 activity. The CeTOR pathway controls protein homeostasis and also contributes to adult longevity. This study found that pha-4 is required to extend adult lifespan in response to reduced CeTOR signaling. Mutations in the predicted CeTOR target rsks-1/S6 kinase or in ife-2/eIF4E also reduce protein biosynthesis and extend lifespan, but only rsks-1 mutations require pha-4 for adult longevity. In addition, rsks-1, but not ife-2, can suppress the larval lethality associated with pha-4 loss-of-function mutations. In conclusion this data suggests that pha-4 and the CeTOR pathway antagonize one another to regulate postembryonic development and adult longevity. A model is suggested in which nutrients promote TOR and S6 kinase signaling, which represses pha-4/FoxA, leading to a shorter lifespan. A similar regulatory hierarchy may function in other animals to modulate metabolism, longevity, or disease (Sheaffer, 2008).

Structure of p70S6K

The mammalian target of rapamycin (mTOR) controls the translation machinery via activation of S6 kinases 1 and 2 (S6K1/2) and inhibition of the eukaryotic initiation factor 4E (eIF4E) binding proteins 1, 2, and 3 (4E-BP1/2/3). S6K1 and 4E-BP1 are regulated by nutrient-sensing and mitogen-activated pathways. The molecular basis of mTOR regulation of S6K1 and 4E-BP1 remains controversial. A conserved TOR signaling (TOS) motif has been identified in the N terminus of all known S6 kinases and in the C terminus of the 4E-BPs; the TOS motif is crucial for phosphorylation and regulation S6K1 and 4E-BP1 activities. Deletion or mutations within the TOS motif significantly inhibit S6K1 activation and the phosphorylation of its hydrophobic motif, Thr389. In addition, this sequence is required to suppress an inhibitory activity mediated by the S6K1 C terminus. The TOS motif is essential for S6K1 activation by mTOR, since mutations in this motif mimic the effect of rapamycin on S6K1 phosphorylation, and render S6K1 insensitive to changes in amino acids. Furthermore, overexpression of S6K1 with an intact TOS motif prevents 4E-BP1 phosphorylation by a common mTOR-regulated modulator of S6K1 and 4E-BP1. It is concluded that S6K1 and 4E-BP1 contain a conserved five amino acid sequence (TOS motif) that is crucial for their regulation by the mTOR pathway. mTOR seems to regulate S6K1 by two distinct mechanisms. The TOS motif appears to function as a docking site for either mTOR itself or a common upstream activator of S6K1 and 4E-BP1 (Schalm, 2002).

Germline signaling mediates the synergistically prolonged longevity produced by double mutations in daf-2 and rsks-1 in C. elegans

Inhibition of DAF-2 (insulin-like growth factor 1 [IGF-1] receptor) or RSKS-1 (S6K), key molecules in the insulin/IGF-1 signaling (IIS) and target of rapamycin (TOR) pathways, respectively, extend lifespan in Caenorhabditis elegans. However, it has not been clear how and in which tissues they interact with each other to modulate longevity. This study demonstrates that a combination of mutations in daf-2 and rsks-1 produces a nearly 5-fold increase in longevity that is much greater than the sum of single mutations. This synergistic lifespan extension requires positive feedback regulation of DAF-16 (FOXO) via the AMP-activated protein kinase (AMPK) complex. Furthermore, germline was identified as the key tissue for this synergistic longevity. Moreover, germline-specific inhibition of rsks-1 activates DAF-16 in the intestine. Together, these findings highlight the importance of the germline in the significantly increased longevity produced by daf-2 rsks-1, which has important implications for interactions between the two major conserved longevity pathways in more complex organisms (Chen, 2013).

IIS and TOR pathways play conserved roles in modulating lifespan in multiple species. However, it is unclear how they might interactively modulate aging. This study set out to address this question by constructing a daf-2 rsks-1 double mutant, which has reduced function of IIS and an important branch of the TOR pathway. Surprisingly, the daf-2 rsks-1 double mutant showed a nearly 5-fold lifespan extension. This phenotype is defined as a synergistic lifespan extension, based on the observation that the longevity of the daf-2 rsks-1 double mutant is beyond the combined effects of rsks-1 and daf-2 single mutants. This synergistic longevity phenotype cannot be explained by the hypothesis that daf-2 and rsks-1 function in parallel to modulate lifespan independently, since an additive effect would be expected under such an assumption (Chen, 2013).

The synergistic longevity phenotype is different from what we previously reported; i.e., that rsks-1 RNAi further extended the daf-2 lifespan by 24%. One major difference in the experimental procedures used is that in the previous study, daf-2 animals were treated with rsks-1 RNAi only during adulthood, whereas in the current work, a double mutant was made that carries the putative null allele of rsks-1 throughout life (Chen, 2013).

When daf-2 animals were treated with rsks-1 RNAi for two generations, resulting in a more complete reduction in rsks-1 mRNA levels, a 54% further lifespan extension was observed. These results suggest that inhibition of rsks-1 during development is critical for the synergistic longevity phenotype. Consistently, inhibition of the RSKS-1 upstream activator LET-363/CeTOR in daf-2 during adulthood led to a 17% additive lifespan extension. Since let-363 is an essential gene, inhibition of which during development leads to larval arrest, a pharmaceutical approach was used to inhibit let-363 by treating animals with rapamycin. Rapamycin treatment throughout life extended the lifespan of N2 and daf-2 animals by 26% and 45%, respectively. There are multiple possible reasons why rapamycin treatment could not extend the lifespan of daf-2 animals as much as the rsks-1 deletion mutant did. One possibility is that rapamycin treatment did not fully block RSKS-1, which is required for the synergistic longevity. Another possibility is that since rapamycin treatment at this dosage has been shown to inhibit both TOR complex 1 and complex 2 activities (Robida-Stubbs et al., 2012), the drug might also affect other lifespan-determinant genes. Nevertheless, these results are consistent with the idea that inhibiting rsks-1 in daf-2 during development leads to a synergistic lifespan extension (Chen, 2013).

Previous studies showed that null mutants of age-1, which encodes a catalytic subunit of the phosphatidylinositol-3-kinase (PI3K) in the IIS pathway, exhibit an exceptional lifespan extension in a DAF-16-dependent manner (Chen, 2013).

Since the daf-2 mutations that were used in this study are not null alleles, one possible explanation for the synergistic longevity produced by daf-2 rsks-1 is that the rsks-1 deletion makes daf-2 mutant phenotypes more severe. This is unlikely to be true, because many aging-related phenotypes of daf-2 are not enhanced by the rsks-1 deletion.rsks-1 does not affect daf-2-mediated dauer arrest, and rsks-1 has a minor or even opposite effect on most stress resistance. Understanding why these phenotypes are uncoupled from the synergistically prolonged longevity produced by daf-2 rsks-1 will help to elucidate the basic mechanisms of aging (Chen, 2013).

TOR plays a conserved role in dietary restriction (DR)-mediated lifespan extension. The effect of nutrients on the synergistic longevity was tested using the DR-FD regimen (FD stands for food deprivation). The rsks-1 single mutant did not show a lifespan extension under DR, which is consistent with the idea that DR and reduced TOR signaling function through overlapping mechanisms to extend lifespan. Interestingly, the synergistic longevity produced by daf-2 rsks-1 is nutrient independent, suggesting that rsks-1 functions through unidentified mechanisms to further extend the lifespan of daf-2 animals (Chen, 2013).

To better understand the molecular mechanisms of the synergistic longevity produced by daf-2 rsks-1, this study set out to identify critical mediators by testing known regulators of IIS or rsks-1. Heat-shock factor 1 (HSF-1) is critical for daf-2-mediated lifespan extension. Inhibition of hsf-1 almost completely abolished the lifespan extension produced by daf-2 rsks-1. Lifespan extension via genetic or pharmaceutical inhibition of TOR requires the IIS downstream transcription factor SKN-1. Surprisingly, inhibition of skn-1 by RNAi had little effect on the synergistic longevity produced by daf-2 rsks-1. Similarly, inhibition of PHA-4, a FOXA transcription factor that is required for the rsks-1 single mutant-mediated lifespan extension, did not affect the lifespan of daf-2 rsks-1. This is further evidence that the mechanism of the synergistic longevity in the daf-2 rsks-1 double mutant is distinct from the lifespan extension caused by the single mutants (Chen, 2013).

Microarray studies were performed and genes were identified that are differentially expressed in daf-2 rsks-1. A genetic screen using RNAi helped identify the AMPK complex (see Drosophila AMP-activated protein kinase alpha subunit) as the key mediator of the synergistic longevity produced by daf-2 rsks-1. Quantitative analysis of the lifespan data indicated that suppression of the daf-2 rsks-1 lifespan by inhibition of AMPK was not due to general sickness. Instead, inhibition of AMPK suppressed the synergy part of the lifespan extension. Further analysis identified a positive feedback regulation of DAF-16 via AMPK in the daf-2 rsks-1 mutant. AMPK plays important roles in various cellular functions. Under energy-starved conditions, AMPK is activated to promote catabolism and thus ATP production. Further characterization of the role of AMPK in metabolism will enhance understanding of the synergistic longevity produced by daf-2 rsks-1 (Chen, 2013).

Both IIS and signals from the reproductive system have endocrine functions. Modulation of these pathways in one tissue leads to nonautonomous activation of DAF-16 in the intestine. To better understand how aging is coordinately modulated across multiple tissues, the involvement of key regulators of the daf-2 rsks-1-mediated synergistic longevity were tested by tissue-specific RNAi. It was found that rsks-1, daf-16, and aak-2 function in the germline to regulate the synergistic lifespan extension, which can also be suppressed by a genetic mutation that causes germline overproliferation or by inhibition of key mediators of germline signaling. In addition, inhibiting rsks-1 in the germline leads to nonautonomous activation of DAF-16 in the intestine. Previous studies on the tissue-specific requirements of key longevity determinants, including DAF-16, mainly employed transgenic rescue approaches. However, the traditional microinjection method creates transgenic lines with a high copy number of transgenes, which will be silenced in the germline. The results indicate that the germline is an important tissue for integrating signals from the IIS pathway and S6K for lifespan determination (Chen, 2013).

Similar to the rsks-1 single mutant, daf-2 rsks-1 animals showed significantly delayed, prolonged, and overall reduced reproduction. This is consistent with a recent study showing that RSKS-1 acts in parallel with the IIS pathway to play an essential role in establishing the germline stem cell/progenitor pool. Interestingly, RSKS-1 functions cell autonomously to regulate establishment of the germline progenitor. This effect is independent of its known suppressors in the regulation of lifespan. These findings suggest that the synergistic longevity of daf-2 rsks-1 cannot simply be linked with its functions in germline development and reproduction (Chen, 2013).

In C. elegans, the intestine carries out multiple nutrient-related functions and is the site for food digestion and absorption, fat storage, and immune response. DAF-16 is one of the essential transcription factors that function in the intestine to modulate lifespan. It was found that intestinal-specific inhibition of daf-16, aak-2, or hsf-1 largely abolishes the synergistic lifespan extension of daf-2 rsks-1. However, knockdown of rsks-1 in the intestine only has an additive effect on daf-2 lifespan, suggesting that rsks-1 may function through nonautonomous mechanisms to activate DAF-16 (Chen, 2013).

The hypodermis is considered as part of the epithelial system in C. elegans. It is involved in basic body plan establishment, cell fate specification, axon migration, apoptotic cells removal, and fat storage. Hypodermis-specific knockdown of rsks-1 in daf-2 also leads to synergistic lifespan extension, and that hypodermis-specific knockdown of daf-16 significantly reduces the synergistic lifespan extension. These results provide evidence for the important role of the hypodermis in lifespan determination. In future studies, it will be interesting to examine which biological functions of the hypodermis are involved in regulating the synergistic longevity by daf-2 rsks-1 (Chen, 2013).

Previous studies showed that muscle decline is one of the major physiological causes of aging in C. elegans. Neither rsks-1 nor the downstream regulators daf-16, hsf-1, and aak-2 seem to function in the muscle to modulate the synergistic lifespan extension. However, the possibility that these regulators may function in other tissues to nonautonomously regulate muscle functions in daf-2 rsks-1 cannot be ruled out. Characterization of age-dependent muscle decline in daf-2 rsks-1 will help to elucidate whether muscle functions are important for the synergistic lifespan extension (Chen, 2013).

There are limitations to assessing tissue-specific involvement of key regulators in lifespan determination by RNAi, such as uncertainty of knockdown efficiency and potential leakiness. It has been reported that in rrf-1 mutants, RNAi can be processed in certain somatic tissues, including the intestine, at least for the genes tested. However, the critical function of rsks-1 in the germline is unlikely to be an artifact, as rsks-1 knockdown in the intestine of daf-2 animals did not lead to synergistic lifespan extension. Moreover, inhibition of certain strong suppressors of daf-2 rsks-1 (e.g., hsf-1) in the intestine, but not in the germline, significantly decreased the synergistic lifespan extension produced by daf-2 rsks-1. Further analyses with single-copied, isoform-specific transgenic rescue will help to quantitatively determine the tissue-specific involvement of key regulators in the synergistic lifespan extension produced by daf-2 rsks-1 (Chen, 2013).

It has not been clear whether DAF-16 is quantitatively more active or is uniquely activated in certain tissues, such as the germline of daf-2 rsks-1. Although the AMPK-mediated positive feedback regulation of DAF-16 was identified based on genes that are expressed to a greater extent in daf-2 rsks-1 animals, it is speculated that the double mutant has some unique properties, as shown in dauer formation and various stress-tolerance assays. The data from the phenotypic analysis of the double mutant and epistasis analysis of tissue requirement of DAF-16 suggest that with the rsks-1 deletion, DAF-16 plays a more important role in certain tissues, such as the germline, to further extend the lifespan of daf-2. Characterization of the genes that are uniquely upregulated in daf-2 rsks-1 or those that are regulated independently of DAF-16 will help distinguish these models (Chen, 2013).

In conclusion, this study found that the daf-2 rsks-1 double mutant shows a synergistic lifespan extension, which is achieved through positive feedback regulation of DAF-16 by AMPK. Tissue- specific epistasis analysis suggests that this enhanced activation of DAF-16 is initiated by signals from the germline, and that the germline tissue may play a key role in integrating the interactions between daf-2 and rsks-1 to cause a synergistic lifespan extension. Since DAF-2, RSKS-1, AMPK, and DAF-16 are highly conserved molecules, similar regulation may also exist in mammals. Further characterization of the daf-2 rsks-1-mediated synergistic longevity will contribute to a better understanding of the molecular mechanisms of aging and age-related diseases (Chen, 2013).

Mutation of p70S6K

The p70 S6 kinase (p70s6k) gene was disrupted in murine embryonic stem cells to determine the role of this kinase in cell growth, protein synthesis, and rapamycin sensitivity. p70s6k-/- cells proliferate at a slower rate than parental cells, suggesting that p70s6k has a positive influence on cell proliferation but is not essential. In addition, rapamycin inhibits proliferation of p70s6k-/- cells, indicating that other events inhibited by the drug, independent of p70s6k, also are important for both cell proliferation and the action of rapamycin. In p70s6k-/- cells, which exhibit no ribosomal S6 phosphorylation, translation of mRNA encoding ribosomal proteins is not increased by serum nor specifically inhibited by rapamycin. In contrast, rapamycin inhibits (1) phosphorylation of initiation factor 4E-binding protein 1 (4E-BP1); (2) general mRNA translation, and (3) overall protein synthesis in p70s6k-/- cells, indicating that these events proceed independently of p70s6k activity. This study localizes the function of p70s6k to ribosomal biogenesis by regulating ribosomal protein synthesis at the level of mRNA translation (Kawasome, 1998).

The p70s6k/p85s6k signaling pathway plays a critical role in cell growth by modulating the translation of a family of mRNAs termed 5'TOPs, which encode components of the protein synthetic apparatus. Homozygous disruption of the p70s6k/p85s6k gene does not affect viability or fertility of mice, but it has a significant effect on animal growth, especially during embryogenesis. Surprisingly, S6 phosphorylation in liver or in fibroblasts from p70s6k/p85s6k-deficient mice proceeds normally in response to mitogen stimulation. Furthermore, serum-induced S6 phosphorylation and translational up-regulation of 5'TOP mRNAs are equally sensitive to the inhibitory effects of rapamycin in mouse embryo fibroblasts derived from p70s6k/p85s6k-deficient and wild-type mice. A search of public databases has identified a novel p70s6k/p85s6k homolog that contains the same regulatory motifs and phosphorylation sites known to control kinase activity. This newly identified gene product, termed S6K2, is ubiquitously expressed and displays both mitogen-dependent and rapamycin-sensitive S6 kinase activity. More striking, in p70s6k/p85s6k-deficient mice, the S6K2 gene is up-regulated in all tissues examined, especially in thymus, a main target of rapamycin action. The finding of a new S6 kinase gene, which can partly compensate for p70s6k/p85s6k function, underscores the importance of S6K function in cell growth (Shima, 1998).

Activation of 40S ribosomal protein S6 kinases (S6Ks) is mediated by anabolic signals triggered by hormones, growth factors, and nutrients. Stimulation by any of these agents is inhibited by the bacterial macrolide rapamycin, which binds to and inactivates the mammalian target of rapamycin, an S6K kinase. In mammals, two genes encoding homologous S6Ks, S6K1 and S6K2, have been identified. Mice deficient for S6K1 or S6K2 are born at the expected Mendelian ratio. Compared to wild-type mice, S6K1(-/-) mice are significantly smaller, whereas S6K2(-/-) mice tend to be slightly larger. However, mice lacking both genes showed a sharp reduction in viability due to perinatal lethality. Analysis of S6 phosphorylation in the cytoplasm and nucleoli of cells derived from the distinct S6K genotypes suggests that both kinases are required for full S6 phosphorylation but that S6K2 may be more prevalent in contributing to this response. Despite the impairment of S6 phosphorylation in cells from S6K1(-/-)/S6K2(-/-) mice, cell cycle progression and the translation of 5'-terminal oligopyrimidine mRNAs were still modulated by mitogens in a rapamycin-dependent manner. Thus, the absence of S6K1 and S6K2 profoundly impairs animal viability but does not seem to affect the proliferative responses of these cell types. Unexpectedly, in S6K1(-/-)/S6K2(-/-) cells, S6 phosphorylation persists at serines 235 and 236, the first two sites phosphorylated in response to mitogens. In these cells, as well as in rapamycin-treated wild-type, S6K1(-/-), and S6K2(-/-) cells, this step is catalyzed by a mitogen-activated protein kinase (MAPK)-dependent kinase, most likely p90rsk. These data reveal a redundancy between the S6K and the MAPK pathways in mediating early S6 phosphorylation in response to mitogens (Pende, 2004).

Kinases directly targeting p70S6K

Protein kinase B and p70 S6 kinase are members of the cyclic AMP-dependent/cyclic GMP-dependent/protein kinase C subfamily of protein kinases and are activated by a phosphatidylinositol 3-kinase-dependent pathway when cells are stimulated with insulin or growth factors. Both of these kinases are activated in cells by phosphorylation of a conserved residue in the kinase domain [Thr-308 of protein kinase B (PKB) and Thr-252 of p70 S6 kinase] and another conserved residue located C-terminal to the kinase domain (Ser-473 of PKB and Thr-412 of p70 S6 kinase). Thr-308 of PKBalpha and Thr-252 of p70 S6 kinase are phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) in vitro. Recent work has shown that PDK1 interacts with a region of protein kinase C-related kinase-2, termed the PDK1 interacting fragment (PIF). Interaction with PIF converts PDK1 from a form that phosphorylates PKB at Thr-308 alone to a species capable of phosphorylating Ser-473 as well as Thr-308. This suggests that PDK1 may be the enzyme that phosphorylates both residues in vivo. PDK1 is capable of phosphorylating p70 S6 kinase at Thr-412 in vitro. The effect of PIF on the ability of PDK1 to phosphorylate p70 S6 kinase has been examined. Surprisingly, PDK1 bound to PIF is no longer able to interact with or phosphorylate p70 S6 kinase in vitro at either Thr-252 or Thr-412. The expression of PIF in cells prevents insulin-like growth factor 1 from inducing the activation of the p70 S6 kinase and its phosphorylation at Thr-412. Overexpression of PDK1 in cells induces the phosphorylation of p70 S6 kinase at Thr-412 in unstimulated cells, and a catalytically inactive mutant of PDK1 prevents the phosphorylation of p70 S6K at Thr-412 in insulin-like growth factor 1-stimulated cells. These observations indicate that PDK1 regulates the activation of p70 S6 kinase and provides evidence that PDK1 mediates the phosphorylation of p70 S6 kinase at Thr-412 (Balendran, 1999).

Activation of the protein p70s6k by mitogens leads to increased translation of a family of messenger RNAs that encode essential components of the protein synthetic apparatus. Activation of the kinase requires hierarchical phosphorylation at multiple sites, culminating in the phosphorylation of the threonine in position 229 (Thr229), in the catalytic domain. The homologous site in protein kinase B (PKB), Thr308, has been shown to be phosphorylated by the phosphoinositide-dependent protein kinase PDK1. A regulatory link between p70s6k and PKB has been demonstrated, since PDK1 was found to selectively phosphorylate p70s6k at Thr229. More importantly, PDK1 activates p70s6k in vitro and in vivo, whereas the catalytically inactive PDK1 blocks insulin-induced activation of p70s6k (Pullen, 1998).

The complex of rapamycin with its intracellular receptor, FKBP12, interacts with RAFT1/FRAP/mTOR (the in vivo rapamycin-sensitive target) and a member of the ataxia telangiectasia mutated (ATM)-related family of kinases that share homology with the catalytic domain of phosphatidylinositol 3-kinase. The function of RAFT1 in the rapamycin-sensitive pathway and its connection to downstream components of the pathway, such as p70 S6 kinase and 4E-BP1, are poorly understood. RAFT1 directly phosphorylates p70S6k, 4E-BP1, and 4E-BP2. Serum stimulates RAFT1 kinase activity with kinetics similar to those of p70S6k and 4E-BP1 phosphorylation. RAFT1 phosphorylates p70S6k on Thr-389, a residue whose phosphorylation is rapamycin-sensitive in vivo and necessary for S6 kinase activity. RAFT1 phosphorylation of 4E-BP1 on Thr-36 and Thr-45 blocks its association with the cap-binding protein, eIF-4E, in vitro, and phosphorylation of Thr-45 seems to be the major regulator of the 4E-BP1-eIF-4E interaction in vivo. RAFT1 phosphorylates p70(S6k) much more effectively than 4E-BP1, and the phosphorylation sites on the two proteins show little homology. This raises the possibility that, in vivo, an unidentified kinase analogous to p70S6k is activated by RAFT1 phosphorylation and acts at the rapamycin-sensitive phosphorylation sites of 4E-BP1 (Burnett, 1998).

p70 S6 kinase alpha (p70alpha) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or in response to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70alpha activation in a manner that can be overcome by coexpression of p70alpha with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. A mammalian recombinant p70alpha polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70alpha phosphorylation in vitro is accompanied by a substantial restoration in p70alpha kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70alpha by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro results in a synergistic stimulation of p70alpha activity to levels similar to those attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined (Isotani, 2000).

PDK1-catalyzed phosphorylation of Thr-252 on the p70alpha activation loop is a critical and probably final step in the physiologic activation of p70alpha in vivo. The ability of PDK1 to phosphorylate Thr-252 is regulated primarily by the accessibility of the p70alpha activation loop to PDK1, which in turn is controlled by a series of prior p70alpha phosphorylations. Phosphorylation of the multiple Ser/Thr-Pro motifs clustered in the pseudosubstrate autoinhibitory segment of the p70alpha carboxyl-terminal tail serves to disocclude the catalytic domain, greatly enhancing access to PDK1. A similar effect can be achieved by deletion of the p70alpha carboxyl-terminal tail (to give p70alpha-DeltaCT104). At any level of PDK1 activity, the extent of Thr-252 phosphorylation of p70alpha-DeltaCT104 is substantially greater than with a similar amount of full-length p70alpha polypeptide. In addition, the S6 kinase activity generated by any extent of PDK1-catalyzed Thr-252 phosphorylation is significantly higher for p70alpha-DeltaCT104 as compared with full-length p70alpha. Displacement of the p70alpha carboxyl-terminal tail is also necessary for the phosphorylation of Thr-412 in vivo, and modification of Thr-412 itself significantly enhances the ability of PDK1 to phosphorylate Thr-252. In addition, the simultaneous phosphorylation of Thr-412 and Thr-252 appears to generate a synergistic activation of p70alpha. Thus, the substitution of Thr-412 by Glu in p70alpha-DeltaCT104 alone gives a 6-fold increase in S6 kinase activity, and the PDK1 catalyzed phosphorylation of p70alpha-DeltaCT104 Thr-252 alone gives a 15-fold increase, but the two modifications together give at least a 240-fold increase in S6 kinase activity over the unmodified p70alpha-DeltaCT104 polypeptide. The importance of the strong positively cooperative effect of Thr-252 and Thr-412 phosphorylation for the physiologic activation of p70alpha is illustrated by the response of p70alpha to the inhibitors rapamycin and wortmannin; these agents each cause a rapid dephosphorylation of Thr-412 but a slower and lesser dephosphorylation of Thr-252. Despite the preservation of Thr-252 phosphorylation, S6 kinase activity in the presence of rapamycin or wortmannin declines in parallel to Thr-412 dephosphorylation. In view of the ability of mTOR to catalyze the in vitro phosphorylation of p70alpha Thr-412 as well as sites within the autoinhibitory segment in the p70alpha carboxyl-terminal tail and the potential effects of such phosphorylations on the response of p70alpha to PDK1, the p70alpha activation achieved in vitro by mTOR or PDK1 alone have been compared to that achieved by sequential phosphorylation by mTOR and PDK1 and to that achieved in vivo by stimulation of the cells with 10% serum. mTOR alone increases the S6 kinase activity of p70alpha in vitro by more than 10-fold, whereas PDK1 alone hardly activates p70alpha, presumably reflecting the relatively poor access of Thr-252 to PDK1 in full-length, inactive p70alpha. In contrast, phosphorylation of p70alpha by PDK1 after a prior phosphorylation by mTOR increases the p70alpha activity by 10-fold over that engendered by mTOR alone, to a level roughly 70-fold greater than that generated by PDK1 acting alone. Moreover, the S6 kinase activity generated in vitro by the sequential action of mTOR and PDK1 is indistinguishable from that achieved in vivo by stimulation of cells with 10% serum (Isotani, 2000).

mTOR and raptor are components of a signaling pathway that regulates mammalian cell growth in response to nutrients and growth factors. A member of this pathway, a protein named GbetaL, has been identified that binds to the kinase domain of mTOR and stabilizes the interaction of raptor with mTOR. Like mTOR and raptor, GbetaL participates in the control of cell size and in nutrient- and growth factor-mediated signaling to S6K1, a downstream effector of mTOR. The binding of GbetaL to mTOR strongly stimulates the kinase activity of mTOR toward S6K1 and 4E-BP1, an effect reversed by the stable interaction of raptor with mTOR. Interestingly, nutrients and rapamycin regulate the association between mTOR and raptor only in complexes that also contain GbetaL. Thus, it is proposed that the opposing effects on mTOR activity of the GbetaL- and raptor-mediated interactions regulate the mTOR pathway (Kim, 2003).

PP2A restrains the function of p70s6k

The FKBP12-rapamycin-associated protein (FRAP; also called RAFT1/mTOR) regulates translation initiation and entry into the cell cycle. Depriving cells of amino acids or treating them with the small molecule rapamycin inhibits FRAP and results in rapid dephosphorylation and inactivation of the translational regulators 4E-BP1(eukaryotic initiation factor 4E-binding protein 1) and p70s6k. Data published recently have led to the view that FRAP acts as a traditional mitogen-activated kinase, directly phosphorylating 4E-BP1 and p70s6k in response to mitogenic stimuli. Evidence indicates that FRAP controls 4E-BP1 and p70s6k phosphorylation indirectly by restraining a phosphatase. A calyculin A-sensitive phosphatase is required for the rapamycin- or amino acid deprivation-induced dephosphorylation of p70s6k, and treatment of Jurkat I cells with rapamycin increases the activity of the protein phosphatase 2A (PP2A) toward 4E-BP1. PP2A associates with p70s6k but not with a mutated p70s6k that is resistant to rapamycin- and amino acid deprivation-mediated dephosphorylation. FRAP also is shown to phosphorylate PP2A in vitro, consistent with a model in which phosphorylation of PP2A by FRAP prevents the dephosphorylation of 4E-BP1 and p70s6k, whereas amino acid deprivation or rapamycin treatment inhibits FRAP's ability to restrain the phosphatase (Peterson, 1999).

On TGF-beta binding, the TGF-beta receptor directly phosphorylates and activates the transcription factors Smad2/3, leading to G₁ arrest. Evidence is presented for a second, parallel, TGF-beta-dependent pathway for cell cycle arrest, achieved via inhibition of p70^s6k. TGF-beta induces association of its receptor with protein phosphatase-2A (PP2A)-Balpha. Concomitantly, three PP2A-subunits, Balpha, Abeta, and Calpha, associate with p70^s6k, leading to its dephosphorylation and inactivation. Although either pathway is sufficient to induce G₁ arrest, abrogation of both, the inhibition of p70^s6k, and transcription through Smad proteins is required for release of epithelial cells from TGF-beta-induced G₁ arrest. TGF-beta thereby modulates the translational and posttranscriptional control of cell cycle progression (Petritsch, 2000).

On receptor activation, PP2A-Balpha specifically binds the activated TbetaRI and is catalytically activated by TGF-beta. PP2A-Balpha then recruits PP2A-Abeta and PP2A-C to bind and dephosphorylate p70^s6k. Complexes containing at the same time PP2A, TbetaRI and p70^s6k, could not be detected, indicating that on activation the phosphatase is released from the receptor to bind to the target molecule. Immunolocalization of the endogenous proteins supports this model. p70^s6k activity controls the translational upregulation of proteins important for G₁/S progression and is itself essential for cell cycle progression. Most of the transcripts isolated to date represent ribosomal proteins and elongation factors of protein synthesis. TGF-beta-induced inactivation of p70^s6k leads to the translational regulation of a group of cell cycle regulators for G₁ progression. It remains unclear, however, if the repression of those cell cycle regulators result from global repression of protein translation or represent a class of specifically translationally repressed mRNAs. It is conceivable that the regulation of crucial components of the cell cycle machinery is mediated at the transcriptional, translational, and posttranslational levels (Petritsch, 2000).

Expression of the regulatory subunit PP2A-Balpha itself appears to be a prerequisite for the PP2A-mediated inhibition of p70^s6k by TGF-beta. Cells with nondetectable PP2A-Balpha expression remain solely responsive to TGF-beta-mediated transcriptional responses. p70^s6k is not inhibited by TGF-beta in these cells; this reflects the differential sensitivity of epithelial cells and mesenchymal cells to growth inhibitory effects of TGF-beta. The chromosomal localization of PP2A-Balpha has not been investigated; PP2A-Abeta, however, has been mapped to a human tumor suppressor locus on 11q22-24 and appears to be mutated in a subset of human lung tumors. It is tempting to speculate that mutations of the regulatory subunits of PP2A in human tumors abolish the regulation of p70^s6k to TGF-beta and confer a selective advantage to growing tumors (Petritsch, 2000).

The insulin pathway and other signaling upstream of p70sk6

The 70 kDa ribosomol S6 kinase (pp70S6k) plays an important role in the progression of cells through G1 phase of the cell cycle. However, little is known of the signaling molecules that mediate its activation. Rho family G proteins regulate pp70S6k activity in vivo. Activated alleles of Cdc42 and Rac1, but not RhoA, stimulate pp70S6k activity in multiple cell types. Activation requires an intact effector domain and isoprenylation of Cdc42 and Rac1. Coexpression of Dbl, an exchange factor for Cdc42, also activates pp70S6k. Growth factor-induced activation of pp70S6k is abrogated by dominant negative alleles of Cdc42 and Rac1. In addition, Cdc42 and Rac1 form GTP-dependent complex with the catalytically inactive form of pp70S6k in vitro and in vivo, suggesting a mechanism by which these G proteins activate pp70S6k (Chou, 1996).

This paper reviews signaling upstream of p70S6K and the role of p70S6K in controlling translation and cell growth. The highly homologous 40S ribosomal protein S6 kinases (S6K1 and S6K2) play a key role in the regulation of cell growth by controlling the biosynthesis of translational components which make up the protein synthetic apparatus, most notably ribosomal proteins. In the case of S6K1, at least eight phosphorylation sites are believed to mediate kinase activation in a hierarchical fashion. Activation is initiated by phosphatidylinositide-3OH kinase (PI3K)-mediated phosphorylation of key residues in the carboxy-terminus of the kinase, allowing phosphorylation of a critical residue residing in the activation loop of the catalytic domain by phosphoinositide-dependent kinase 1 (PDK1). The kinases responsible for phosphorylating the carboxy-terminal sites have yet to be identified. Additionally, S6 kinases are under the control of the PI3K relative, mammalian Target Of Rapamycin (mTOR), which may serve an additional function as a checkpoint for amino acid availability (Dufner, 2000).

After an initial burst of cell proliferation, the type 1 insulin-like growth factor receptor (IGF-IR) induces granulocytic differentiation of 32D IGF-IR cells, an interleukin-3-dependent murine hemopoietic cell line devoid of insulin receptor substrate-1 (IRS-1). The combined expression of the IGF-IR and IRS-1 (32D IGF-IR/IRS-1 cells) inhibits IGF-I-mediated differentiation, and causes malignant transformation of 32D cells. Because of the role of IRS-1 in changing the fate of 32D IGF-IR cells from differentiation (and subsequent cell death) to malignant transformation, differences in IGF-IR signaling between 32D IGF-IR and 32D IGF-IR/IRS-1 cells were examined. A focus was placed on p70S6K, which is activated by the IRS-1 pathway. The ectopic expression of IRS-1 and the inhibition of differentiation correlates with a sustained activation of p70S6K and an increase in cell size. Phosphorylation in vivo of threonine 389 and, to a lesser extent, of threonine 421/serine 424 of p70S6K seems to be a requirement for inhibition of differentiation. A role for IRS-1 and p70S6K in the alternative between transformation or differentiation of 32D IGF-IR cells was confirmed by evidence that inhibition of p70S6K activation or IRS-1 signaling, by rapamycin or okadaic acid, induces differentiation of 32D IGF-IR/IRS-1 cells. Expression of myeloperoxidase mRNA (a marker of differentiation, which sharply increases in 32D IGF-IR cells), does not increase in 32D IGF-IR/IRS-1 cells, suggesting that the expression of IRS-1 in 32D IGF-IR cells causes the extinction of the differentiation program initiated by the IGF-IR, while leaving intact its proliferation program (Valentinis, 2000).

The c-Abl protein-tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. The rapamycin and FKBP-target 1 (RAFT1), also known as FKBP12-rapamycin-associated protein (FRAP, mTOR), regulates the p70S6 kinase (p70S6k) and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). The present results demonstrate that c-Abl binds directly to RAFT1 and phosphorylates RAFT1 in vitro and in vivo. c-Abl inhibits autophosphorylation of RAFT1 and RAFT1-mediated phosphorylation p70S6k. The functional significance of the c-Abl-RAFT1 interaction is further supported by the finding that eIF4E-dependent translation in mouse embryo fibroblasts from Abl(-/-) mice is significantly higher than that compared in wild-type cells. The results also demonstrate that exposure of cells to ionizing radiation is associated with c-Abl-mediated binding of 4E-BP1 to eIF4E and inhibition of translation. These findings with the c-Abl tyrosine kinase represent the first demonstration of a negative physiologic regulator of RAFT1-mediated 5' cap-dependent translation (Kumar, 2000b).

Extracellular zinc ion may play a role in the activation of p70S6k, which in turn functions in regulating the progression of cells from the G(1) to S phase of the cell cycle. Treatment of Swiss 3T3 cells with zinc sulfate leads to the activation and phosphorylation of p70S6k in a dose-dependent manner. The activation of p70S6k by zinc treatment is biphasic, the early phase being at 30 min followed by the late phase at 120 min. The zinc-induced activation of p70S6k is partially inhibited by down-regulation of phorbol 12-myristate 13-acetate-responsive protein kinase C (PKC) by chronic treatment with phorbol 12-myristate 13-acetate, but this is not significant. Moreover, Go6976, a specific calcium-dependent PKC inhibitor, does not significantly inhibit the activation of p70S6k by zinc. These results demonstrate that the zinc-induced activation of p70S6k is not related to PKC. Also, extracellular calcium is not involved in the activation of p70S6k by zinc. Further characterization of the zinc-induced activation of p70S6k using specific inhibitors of the p70S6k signaling pathway, namely rapamycin, wortmannin, and LY294002, has shown that zinc acts upstream of mTOR/FRAP/RAFT and phosphatidylinositol 3-kinase (PI3K), because these inhibitors cause the inhibition of zinc-induced p70S6k activity. In addition, Akt, the upstream component of p70S6k, is activated by zinc in a biphasic manner, as is p70S6k. Moreover, dominant interfering alleles of Akt and PDK1 block the zinc-induced activation of p70S6k, whereas the lipid kinase activity of PI3K is potently activated by zinc. Taken together, these data suggest that zinc activates p70S6k through the PI3K signaling pathway (Kim, 2000).

Expression of the insulin-like growth factor-binding protein 5 (IGFBP-5) gene in vascular smooth muscle cells is up-regulated by IGF-I through an IGF-I receptor-mediated mechanism. The possible involvement of the mitogen-activated protein kinase (MAPK) and PI 3-kinase signaling pathways in mediating IGF-I-regulated IGFBP-5 gene expression has been examined. The addition of Des(1-3)IGF-I, an IGF analog with reduced affinity to IGFBPs, results in a transient activation of p44 and p42 MAPK. Inhibition of the MAPK activation by PD98059, however, does not affect IGF-I-stimulated IGFBP-5 expression. Des(1-3)IGF-I treatment also strongly activates PI 3-kinase. This activation is probably mediated through IRS-1, because IGF-I stimulation results in a significant increase in IRS-1- but not IRS-2-associated PI 3-kinase activity. This activation occurs within 5 min and is sustained at high levels for over 6 h. Likewise, Des(1-3)IGF-I causes a long lasting activation of PKB/Akt and p70s6k. When LY294002 and wortmannin, two specific inhibitors of PI 3-kinase, are added with Des(1-3)IGF-I, the IGF-I-regulated IGFBP-5 expression is negated. The addition of rapamycin, which inhibits IGF-I-induces p70s6k activation, significantly inhibits IGF-I-regulated IGFBP-5 gene expression. These results suggest that the action of IGF-I on IGFBP-5 gene expression requires the activation of the PI 3-kinase-PKB/Akt-p70s6k pathway but not the MAPK pathway in vascular smooth muscle cells (Duan, 1999).

Ligation of the B cell antigen receptor (BCR) induces a cascade of signaling pathways that lead to clonal expansion, differentiation, or abortive activation-induced apoptosis of B lymphocytes. BCR-mediated cross-linking induces the rapid phosphorylation of protein tyrosine kinases. However, the pathways leading to the activation of downstream serine/threonine kinases such as mitogen-activated protein kinase, p90Rsk, and p70S6 kinase (p70S6k) that mediate reorganization of the actin cytoskeleton, cell cycle progression, gene transcription, and protein synthesis have not been delineated. Cross-linking of BCR leads to activation of p70S6k in B lymphocytes. Multiple protein tyrosine kinase-dependent signal transduction pathways induced by BCR lead to the activation of p70S6k. These distinct pathways exhibit different thresholds with respect to the extent of receptor cross-linking required for their activation. Activation of p70S6k by suboptimal doses of anti-Ig is Syk-dependent and is mediated by protein kinase C and phosphoinositol 3-kinase. Moreover, the activation of p70S6k results in phosphorylation of S6 protein which is important for ribosomal protein synthesis and may be coupled to BCR-induced protein and DNA synthesis in primary murine B cells (Hsiu-Ling, 1999).

The present study identifies the operation of a signal tranduction pathway in mammalian cells that provides a checkpoint control, linking amino acid sufficiency to the control of peptide chain initiation. Withdrawal of amino acids from the nutrient medium of CHO-IR cells results in a rapid deactivation of p70 S6 kinase and dephosphorylation of eIF-4E BP1, which becomes unresponsive to all agonists. Readdition of the amino acid mixture quickly restores the phosphorylation and responsiveness of p70 and eIF-4E BP1 to insulin. Increasing the ambient amino acids to twice that usually employed increases basal p70 activity to the maximal level otherwise attained in the presence of insulin and abrogates further stimulation by insulin. Withdrawal of most individual amino acids also inhibits p70, although with differing potency. Amino acid withdrawal from CHO-IR cells does not significantly alter insulin stimulation of tyrosine phosphorylation, phosphotyrosine-associated phosphatidylinositol 3-kinase activity, c-Akt/protein kinase B activity, or mitogen-activated protein kinase activity. The selective inhibition of p70 and eIF-4E BP1 phosphorylation by amino acid withdrawal resembles the response to rapamycin, which prevents p70 reactivation by amino acids, indicating that mTOR is required for the response to amino acids. A p70 deletion mutant, p702-46/CT104, that is resistant to inhibition by rapamycin (but sensitive to wortmannin) is also resistant to inhibition by amino acid withdrawal, indicating that amino acid sufficiency and mTOR signal to p70 through a common effector, which could be mTOR itself, or an mTOR-controlled downstream element, such as a protein phosphatase (Hara, 1998).

Tuberous sclerosis (TSC) is a autosomal dominant genetic disorder caused by mutations in either TSC1 or TSC2, and characterized by benign hamartoma growth. A murine model of Tsc1 disease was developed by gene targeting. Tsc1 null embryos die at mid-gestation from a failure of liver development. Tsc1 heterozygotes develop kidney cystadenomas and liver hemangiomas at high frequency, but the incidence of kidney tumors is somewhat lower than in Tsc2 heterozygote mice. Liver hemangiomas are more common, more severe and cause higher mortality in female than in male Tsc1 heterozygotes. Tsc1 null embryo fibroblast lines have persistent phosphorylation of the p70S6K (S6K) and its substrate S6; this phosphorylation is sensitive to treatment with rapamycin, indicating constitutive activation of the mTOR-S6K pathway due to loss of the Tsc1 protein, hamartin. Hyperphosphorylation of S6 is also seen in kidney tumors in the heterozygote mice, suggesting that inhibition of this pathway may have benefit in the control of TSC hamartomas (Kwaitkowski, 2002).

The S/T-protein kinases activated by phosphoinositide 3-kinase (PI3K) regulate a myriad of cellular processes. An approach using a combination of biochemistry and bioinformatics can identify substrates of these kinases. This approach identifies the tuberous sclerosis complex-2 gene product, tuberin, as a potential target of Akt/PKB (see Drosophila Akt1). Upon activation of PI3K, tuberin is phosphorylated on consensus recognition sites for PI3K-dependent S/T kinases. Moreover, Akt/PKB can phosphorylate tuberin in vitro and in vivo. S939 and T1462 of tuberin are PI3K-regulated phosphorylation sites and T1462 is constitutively phosphorylated in PTEN-/- tumor-derived cell lines. Finally, a tuberin mutant lacking the major PI3K-dependent phosphorylation sites can block the activation of S6K1, suggesting a means by which the PI3K-Akt pathway regulates S6K1 activity (Manning, 2002).

Serine/threonine (S/T) protein kinases can account for much of the functional diversity of PI3K signaling. Akt/protein kinase B and the 70 kDa-S6 kinase 1 (S6K1) are the best characterized of the PI3K-regulated S/T kinases. The mitogen-stimulated activation of both of these kinases is blocked by PI3K-specific inhibitors. Akt contains a PH domain that is specific to PtdIns-3,4P2 and PtdIns-3,4,5P3. Akt is thereby recruited to these PI3K-generated second messengers and to the PDK1 protein kinase, which also specifically binds to these lipids. PDK1 then phosphorylates and activates Akt (Manning, 2002).

The regulation of S6K1 is much more complex, with both PI3K-dependent and -independent signaling pathways involved in its activation. Several PI3K-regulated effectors are known to participate in the activation of S6K1, including PDK1, PKCzeta/lambda, Cdc42, Rac1, and Akt. However, the molecular mechanism of how these contribute to S6K1 activation remains unclear. In addition to mitogen-regulated signaling to S6K1, the metabolic state of the cell and the availability of nutrients control S6K1 activation through the mammalian target of rapamycin (mTOR, also known as FRAP, RAFT, and RAPT). Recent studies suggest that mTOR is also regulated by mitogenic signals. Interestingly, it has been suggested that the point of convergence of the mitogenic and nutrient-sensing signals in the regulation of S6K1 may be at the level of Akt directly phosphorylating mTOR. However, this phosphorylation does not appear to affect mTOR activity or S6K1 activation. Thus, of the PI3K-regulated effectors thought to participate in S6K1 activation, the molecular basis of how Akt regulates S6K1 remains the least well understood (Manning, 2002).

Akt itself has been implicated in many of the PI3K-regulated cellular events, and several substrates have been shown to be phosphorylated in vitro and/or in vivo by Akt. Therefore, the total cellular effect of PI3K activation and subsequent activation of Akt is mediated through a variety of different targets. However, it seems unlikely that the large array of processes controlled by the PI3K-Akt pathway can be accounted for by current knowledge of downstream targets (Manning, 2002).

An approach has been developed to screen for substrates of PI3K-dependent S/T kinases, such as Akt. This approach uses phospho-specific antibodies generated against a phosphorylated protein kinase consensus recognition motif in combination with a protein database motif scanning program called Scansite (http://scansite.mit.edu). Scansite is a web-based program that searches protein databases for optimal substrates of specific protein kinases and for optimal binding motifs for specific protein domains with data generated by peptide library screens. The phospho-motif antibody is used to recognize proteins phosphorylated specifically under conditions in which the kinase of interest is active. Scansite is then used to identify candidate substrates of this protein kinase that have the predicted molecular mass of the proteins recognized by the phospho-motif antibody. This approach successfully identifies known substrates of Akt. The tuberous sclerosis complex-2 (TSC2) tumor suppressor gene product, tuberin, is also identified and characterized as an Akt substrate. Furthermore, it is found that overexpression of a tuberin mutant lacking the major Akt phosphorylation sites can inhibit growth factor-induced activation of S6K1. These results provide a biochemical link between the PI3K-Akt pathway and regulation of S6K1 and also indicate a biochemical basis for the disease tuberous sclerosis complex (TSC) (Manning, 2002).

Expression in human cells of the tuberinS939A/T1462A mutant, which lacks the major PI3K-dependent phosphorylation sites, at levels comparable to endogenous tuberin leads to a decrease in growth factor-induced S6K1 phosphorylation and activity. This phosphorylation and subsequent activation of S6K1 has been previously demonstrated to be dependent on PI3K. These results, along with those from genetic studies in other systems, are consistent with a model in which growth factors activate PI3K leading to the phosphorylation of tuberin by Akt. This phosphorylation inhibits the tuberin-hamartin complex, thereby relieving its inhibition of S6K1. In this model, expression of the tuberinS939A/T1462A mutant, which would not be phosphorylated and inhibited, would have a dominant effect over endogenous tuberin and block growth factor-induced S6K1 activation. It will be of great interest to determine the molecular nature of S6K1 inhibition by the tuberin-hamartin complex in the absence of mitogenic stimuli. It is possible that the complex does so upstream of mTOR, because the constitutive activation of S6K1 in TSC1-/- MEFs is sensitive to rapamycin. Alternatively, mTOR might regulate S6K1 in a nutrient-sensitive pathway parallel to the mitogen-sensitive PI3K-Akt-tuberin pathway (Manning, 2002).

Recent studies have suggested that S6K1 activation can occur independent of PI3K and Akt. These studies demonstrate the existence of multiple pathways regulating S6K1 and that the tuberin-hamartin complex might integrate signals from many different inputs. The identification of tuberin as a direct downstream target of the PI3K-Akt pathway provides the missing link between this signaling cascade and control of S6K1 activity (Manning, 2002).

The identification of this biochemical relationship between the mammalian TSC tumor suppressor gene products and the oncogenic PI3K-Akt pathway could have important implications in human diseases. For instance, in approximately 10%-15% of patients diagnosed with TSC, mutations in TSC1 or TSC2 have not been detected. Based on the findings of this study, it is possible that mutations leading to aberrant activation of the PI3K-Akt pathway, such as PTEN mutations, could inhibit the function of the tuberin-hamartin complex by causing constitutive phosphorylation of tuberin. It will be interesting to examine the phosphorylation state of tuberin within hamartomas from such TSC patients. Indeed, in PTEN-/- cell lines derived from both glioblastoma and prostate tumors, growth factor-independent phosphorylation of tuberin on the PI3K-dependent T1462 site is detected (Manning, 2002).

Germline mutations in either PTEN or the TSC genes cause autosomal dominant diseases that are characterized by the occurrence of widespread hamartomas due to loss of heterozygosity at these loci. However, the tissue distribution of these benign tumors varies between patients with loss of PTEN and those with TSC. These differences might be explained by a model in which the tuberin-hamartin complex is the primary growth-inhibiting target of the PI3K-Akt pathway in tissues affected in TSC patients. In other tissues, such as those affected in patients with PTEN mutations, this complex might be one of many targets of the PI3K-Akt pathway. Interestingly, though, recent studies have suggested that mTOR activity is essential for oncogenic transformation of cells by activated PI3K or Akt and for growth of PTEN-/- tumors. Therefore, aberrant phosphorylation and inhibition of the tuberin-hamartin complex, and subsequent increased activity of mTOR and/or S6K1, would likely contribute to tumorigenesis caused by mutations that activate the PI3K-Akt pathway. Future studies using crosses between PTEN and TSC knockout mice should help determine the contribution of the tuberin-hamartin complex in prevention of the variety of tumors caused by uncontrolled signaling through the PI3K-Akt pathway. Finally, the elucidation of a PI3K-Akt-tuberin pathway controlling S6K1 activity will have important implications in the understanding and treatment of the prevalent TSC disease (Manning, 2002).

The mammalian target of rapamycin (mTOR) regulates cell growth and proliferation via the downstream targets ribosomal S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). Phosphatidic acid (PA) has been identified as a mediator of mitogenic activation of mTOR signaling. This study tests the hypotheses that phospholipase D 1 (PLD1) is an upstream regulator of mTOR and that the previously reported S6K1 activation by Cdc42 is mediated by PLD1. Overexpression of wild-type PLD1 is found to increase S6K1 activity in serum-stimulated cells, whereas a catalytically inactive PLD1 exerts a dominant-negative effect on S6K1. More importantly, eliminating endogenous PLD1 by RNAi leads to drastic inhibition of serum-stimulated S6K1 activation and 4E-BP1 hyperphosphorylation in both HEK293 and COS-7 cells. Knockdown of PLD1 also results in reduced cell size, suggesting a critical role for PLD1 in cell growth control. Using a rapamycin-resistant S6K1 mutant, Cdc42's action has been demonstrated to be through the mTOR pathway. When Cdc42 is mutated in a region specifically required for PLD1 activation, its ability to activate S6K1 in the presence of serum is hindered. However, when exogenous PA is used as a stimulus, the PLD1-inactive Cdc42 mutant behaves similarly to the wild-type protein. These observations reveal the involvement of PLD1 in mTOR signaling and cell size control, and provide a molecular mechanism for Cdc42 activation of S6K1. A new cascade is proposed to connect mitogenic signals to mTOR through Cdc42, PLD1, and PA (Fang, 2003).

The tuberous sclerosis tumor suppressors TSC1 and TSC2 regulate the mTOR pathway to control translation and cell growth in response to nutrient and growth factor stimuli. The stress response REDD1 gene has been identified as a mediator of tuberous sclerosis complex (TSC)-dependent mTOR regulation by hypoxia. REDD1 (Drosophila homologs Scylla and Charybde) inhibits mTOR function to control cell growth in response to energy stress. Endogenous REDD1 is induced following energy stress, and REDD1^-/- cells are highly defective in dephosphorylation of the key mTOR substrates S6K and 4E-BP1 following either ATP depletion or direct activation of the AMP-activated protein kinase (AMPK). REDD1 likely acts on the TSC1/2 complex, because regulation of mTOR substrate phosphorylation by REDD1 requires TSC2 and is blocked by overexpression of the TSC1/2 downstream target Rheb but is not blocked by inhibition of AMPK. Tetracycline-inducible expression of REDD1 triggers rapid dephosphorylation of S6K and 4E-BP1 and significantly decreases cellular size. Conversely, inhibition of endogenous REDD1 by short interfering RNA increases cell size in a rapamycin-sensitive manner, and REDD1^-/- cells are defective in cell growth regulation following ATP depletion. These results define REDD1 as a critical transducer of the cellular response to energy depletion through the TSC-mTOR pathway (Sofer, 2005).

Rheb is targeted by TSC1/TSC2 in the TOR pathway upstream of S6K

Tumor suppressor genes evolved as negative effectors of mitogen and nutrient signaling pathways, such that mutations in these genes can lead to pathological states of growth. Tuberous sclerosis (TSC) is a potentially devastating disease associated with mutations in two tumor suppressor genes, TSC1 and 2 (See Drosophila Gigas), that function as a complex to suppress signaling in the mTOR/S6K/4E-BP pathway. However, the inhibitory target of TSC1/2 and the mechanism by which it acts are unknown. Evidence is provided that TSC1/2 is a GAP for the small GTPase Rheb (see Drosophila Rheb) and that insulin-mediated Rheb activation is PI3K dependent. Moreover, Rheb overexpression induces S6K1 phosphorylation and inhibits PKB phosphorylation, as do loss-of-function mutations in TSC1/2, but contrary to earlier reports Rheb has no effect on MAPK phosphorylation. Finally, coexpression of a human TSC2 cDNA harboring a disease-associated point mutation in the GAP domain, failed to stimulate Rheb GTPase activity or block Rheb activation of S6K1 (Garami, 2003).

The tuberous sclerosis complex 2 (TSC2) tumor suppressor gene product is a negative regulator of protein synthesis upstream of the mTOR and ribosomal S6 kinases. Because of the homology of TSC2 with GTPase-activating proteins for Rap1, whether a Ras/Rap-related GTPase might be involved in this process was examined. TSC2 was found to bind to Rheb-GTP in vitro and to reduce Rheb GTP levels in vivo. Over-expression of Rheb but not Rap1 promotes the activation of S6 kinase in a rapamycin-dependent manner, suggesting that Rheb acts upstream of mTOR. The ability of Rheb to induce S6 phosphorylation is also inhibited by a farnesyl transferase inhibitor, suggesting that Rheb may be responsible for the Ras-independent anti-neoplastic properties of this drug (Castro, 2003).

Tuberous sclerosis complex is a genetic disease caused by mutation in either TSC1 or TSC2. The TSC1 and TSC2 gene products form a functional complex and inhibit phosphorylation of S6K and 4EBP1. These functions of TSC1/TSC2 are likely mediated by mTOR. TSC2 is a GTPase-activating protein (GAP) toward Rheb, a Ras family GTPase. Rheb stimulates phosphorylation of S6K and 4EBP1. This function of Rheb is blocked by rapamycin and dominant-negative mTOR. Rheb stimulates the phosphorylation of mTOR and plays an essential role in regulation of S6K and 4EBP1 in response to nutrients and cellular energy status. These data demonstrate that Rheb acts downstream of TSC1/TSC2 and upstream of mTOR to regulate cell growth (Inoki, 2003).

The small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, it is shown that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently. It is concluded that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells (Tee, 2003).

To examine whether Rheb overexpression could modulate S6K1 activity, S6K1 was coexpressed with Rheb at two different expression levels in HEK293E cells and kinase activity was assayed by using GST-S6 as a substrate. Coexpression of Rheb significantly increased the basal and insulin-stimulated activity of S6K1. Higher levels of Rheb expression enhanced the basal and insulin-stimulated activity of S6K1 by 5.6- and 1.7-fold, respectively, and this activity level is more potent than the S6K1 activity observed in the presence of lower levels of Rheb protein. These results indicate that Rheb activates signaling cascades that result in S6K1 activation (Tee, 2003).

Given that the activity of S6K1 is enhanced upon cell signaling through mTOR, PI3K, and mitogen-activated protein kinase (MAPK) and protein kinase C (PKC)-mediated pathways, it was determined which signaling pathway was activated upon Rheb overexpression. To investigate PI3K-mediated signaling, Rheb, along with Akt, a downstream target of PI3K, was expressed within HEK293E cells and kinase activity was assayed. Whereas EGF stimulation led to a 4-fold increase in Akt activity, Rheb overexpression did not enhance basal or EGF-stimulated Akt activity. In contrast, Rheb potently activated S6K1 by 11-fold when assayed in parallel. Wortmannin was used as a control to show that PI3K-mediated activation of Akt upon stimulation with EGF was specifically measured. To examine whether Rheb enhanced MAPK-mediated signaling, Rheb was coexpressed with RSK1, a known downstream signaling component of MAPK, within HEK293E cells. Although EGF stimulation led to a 12-fold increase in RSK1 activity, Rheb did not augment the basal or EGF-induced activation of RSK1 assayed with GST-S6 as a substrate. In contrast, Rheb overexpression drastically increased S6K1 activity when assayed in parallel. To inhibit activation of ERK (extracellular signal-regulated kinase) through MEK (MAPK/ERK-kinase)-mediated signaling, cells were treated with the U0126 compound to specifically inhibit MEK. These findings suggest that Rheb does not function upstream of either PI3K/Akt or ERK/RSK1 signaling pathways (Tee, 2003).

The above data implies that Rheb may modulate mTOR signaling. Given that the loss of Rheb in yeast mimics nutrient starvation, Rheb overexpression may promote mTOR signaling through a nutrient-regulated signaling pathway. To address this possibility, whether Rheb promotes S6K1 activation was investigated in the absence of amino acids. During conditions of amino acid withdrawal, Rheb overexpression potently activated S6K1, which was completely blocked by rapamycin but only partially inhibited by wortmannin. Importantly, insulin stimulation of these amino acid-deprived cells potently activated Akt (as observed by Akt phosphorylation on Ser473) but only weakly activated S6K1. Therefore, unlike Rheb-mediated signaling, acute stimulation of PI3K and Akt is not sufficient to fully activate S6K1 during nutrient insufficiency. The modest insulin-induced activation of S6K1 during amino acid insufficiency was blocked by wortmannin, revealing that this activation is completely dependent on PI3K. Activation of S6K1 upon readdition of amino acids was enhanced when Rheb was overexpressed or when cells were stimulated with insulin). Interestingly, Rheb-induced S6K1 activation upon readdition of amino acids was completely inhibited by rapamycin but only partially inhibited by wortmannin. In contrast, insulin-induced S6K1 activity was markedly impaired by both rapamycin and wortmannin. These data convincingly reveal that Rheb potently activates S6K in the absence of nutrients through mTOR rather than PI3K-mediated signaling. Therefore, it is likely that Rheb enhances nutrient-mediated signaling through mTOR (Tee, 2003).

To decisively determine whether Rheb positively activates mTOR signaling, use was made of a rapamycin-resistant mutant of S6K1 (S6K1-F5A-DeltaCT). Unlike treatment with wortmannin, rapamycin treatment was unable to prevent insulin-induced activation of S6K1-F5A-DeltaCT, demonstrating that this mutant is responsive to PI3K signaling but not mTOR signaling. As a positive control, PDK1 and PKCζ, which are known to activate S6K1 through a PI3K-dependent input, were coexpressed. Overexpression of Rheb potently activates wild-type S6K1 basally (by 11-fold) and during insulin stimulation (by 17-fold) but does not enhance the activity of the S6K1-F5A-DeltaCT mutant. In contrast, increased PI3K-mediated signaling toward S6K1 by coexpression of PDK1 and PKCζ results in significantly enhanced activation of both wild-type S6K1 and S6K1-F5A-DeltaCT. These findings strongly suggest that Rheb induces S6K1 activation via a signaling input that is upstream of mTOR but not PI3K (Tee, 2003).

Overexpression of wild-type Rheb has been shown to lead to a significant increase in its activity and implies that the majority of the overexpressed Rheb must exist in the active GTP bound form. If this is true, then the RhebGAP activity must be a limiting factor. If Tuberin possesses RhebGAP activity, overexpression of Tuberin should switch Rheb from an active GTP bound state to an inactive GDP bound state. To indirectly measure Rheb activity, Rheb-induced S6K1 activation was analyzed within nutrient-deprived HEK293E cells. Coexpression of Hamartin and Tuberin completely block Rheb's ability to activate S6K1, implying that Tuberin may function as a RhebGAP (Tee, 2003).

If the GAP domain of Tuberin is essential for Rheb inactivation, then patient-derived TSC2 GAP domain point mutants should not block Rheb-induced S6K1 activation. To address this, three Tuberin mutants were generated that mimic patient-derived TSC2 mutations that occur within the GAP domain and these Tuberin mutants were coexpressed with Hamartin and S6K1. Under serum-starved conditions, Rheb potently activates S6K1, which was fully blocked by coexpression of wild-type Tuberin with Hamartin. In contrast, the three TSC2 GAP domain point mutants were unable to repress Rheb-induced S6K1 activation, revealing that the GAP domain of Tuberin is critical for Tuberin's ability to repress Rheb-mediated signaling (Tee, 2003).

Thus, Rheb functions upstream of mTOR within the nutrient signaling pathway. Rheb specifically activates mTOR-mediated signaling rather than cell signaling through MEK/ERK and PI3K, as shown by Rheb-mediated activation of S6K1 but not Akt or RSK1. Therefore, it is unlikely that Rheb activates PI3K and Raf, two downstream effectors of Ras. Rheb has previously been shown to interact with Raf in vitro, but the current data suggest that Raf is not an effector of Rheb in vivo. Additionally, Rheb overexpression does not increase the activity of the rapamycin-resistant S6K1 mutant that is unresponsive to mTOR signaling inputs but is activated in response to PI3K signaling. S6K1 activation is regulated by multiple signaling inputs, one of which is directed by PI3K. Therefore, these findings are important and confirm that Rheb overexpression specifically promotes mTOR rather than PI3K signaling. Furthermore, Rheb-induced 4E-BP1 phosphorylation is completely sensitive to rapamycin but not to wortmannin, which further strengthens the notion that Rheb acts upstream of mTOR rather than PI3K. 4E-BP1 dissociates from eIF4E upon Rheb overexpression, revealing that Rheb-mediated signaling through mTOR promotes cap-dependent translation (Tee, 2003).

Evidence has also been provided that Rheb functions within the nutrient signaling cascade upstream of mTOR, as shown by Rheb's ability to potently stimulate S6K1 activity during amino acid insufficiency. During amino acid withdrawal, acute insulin stimulation is still able to elicit high levels of Akt phosphorylation but poorly activates S6K1, showing that the nutrient-mediated mTOR signaling input is essential for optimal S6K1 activation. Therefore, Rheb overexpression supersedes the dependency of the nutrient input to mTOR, suggesting that Rheb is an activator of mTOR within the nutrient-signaling pathway. Interestingly, resupplying cells with amino acids further enhances the activity of S6K1 when Rheb is overexpressed, suggesting that amino acids may promote the activation of Rheb. This research has revealed that the Tuberin-Hamartin heterodimer functions as an inhibitor of nutrient signaling through mTOR. The Tuberin-Hamartin heterodimer inhibits Rheb-induced S6K1 activation during conditions of amino acid withdrawal. This work, therefore, extends these earlier studies, revealing that inhibition of Rheb is the mechanism by which the Tuberin-Hamartin heterodimer inhibits nutrient-mediated signaling. Importantly, the Rheb-inhibitory function of Tuberin-Hamartin heterodimers depends on an intact Tuberin GAP domain; patient-derived point mutations within the GAP domain of TSC2 prevented the Tuberin-Hamartin heterodimer from blocking Rheb-induced S6K1 activation. These data indicate that the GAP activity of Tuberin promotes inactivation of Rheb in vivo, presumably through increasing the intrinsic GTPase activity of Rheb. Confirming this hypothesis, in vitro Rheb GTPase activity assays have revealed that Tuberin enhances the intrinsic GTPase activity of Rheb. Interestingly, coexpression of Hamartin with Tuberin markedly enhances the GTPase activity of Rheb, implying that Hamartin promotes the GAP function of Tuberin toward Rheb. A model is proposed whereby Rheb promotes mTOR signaling when it is in an active GTP bound form, whereas the Tuberin-Hamartin heterodimer inhibits Rheb by converting it to an inactive GDP bound state. These findings reveal that the Tuberin-Hamartin heterodimer and Rheb respectively inhibit and activate the nutrient-signaling input to mTOR. Small G proteins are additionally regulated by guanine nucleotide exchange factors (GEFs). In this model it is proposed that a RhebGEF becomes activated during conditions of nutrient sufficiency, and its activation switches Rheb to an active GTP bound form. Therefore, identifying this Rheb-GEF will be of great importance and may provide new insights into how mTOR senses intracellular amino acids. However, at this point the possibility that Rheb- and nutrient-mediated signaling may function in parallel pathways upstream of mTOR cannot be ruled out. Further experiments will be carried out to investigate this possibility (Tee, 2003).

Dominant negative mutants of S. pombe Rheb (SpRheb) are reported. Screens of a randomly mutagenized SpRheb library yielded a mutant, SpRhebD60V, whose expression in S. pombe results in growth inhibition, G1 arrest, and induction of fnx1+, a gene whose expression is induced by the disruption of Rheb. Alteration of the Asp-60 residue to all possible amino acids by site-directed mutagenesis led to the identification of two particularly strong dominant negative mutants, D60I and D60K. Characterization of these dominant negative mutant proteins revealed that D60V and D60I exhibit preferential binding of GDP, while D60K lost the ability to bind both GTP and GDP. A possible use of the dominant negative mutants in the study of mammalian Rheb was explored by introducing dominant negative mutations into human Rheb. Transient expression of the wild type Rheb1 or Rheb2 causes activation of p70S6K, while expression of Rheb1D60K mutant results in inhibition of basal level activity of p70S6K. In addition, Rheb1D60K and Rheb1D60V mutants block nutrient- or serum-induced activation of p70S6K. This provides critical evidence that Rheb plays a role in the mTOR/S6K pathway in mammalian cells (Tabancay, 2003).

The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity

The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner. This study demonstrates that the p90 ribosomal protein S6 kinase (RSK; see Drosophila) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3 (Shahbazian, 2006).

Separation of the gluconeogenic and mitochondrial functions of PGC-1α through S6 kinase

PGC-1α is a transcriptional coactivator that powerfully regulates many pathways linked to energy homeostasis. Specifically, PGC-1α controls mitochondrial biogenesis in most tissues but also initiates important tissue-specific functions, including fiber type switching in skeletal muscle and gluconeogenesis and fatty acid oxidation in the liver. S6 kinase, activated in the liver upon feeding, can phosphorylate PGC-1α directly on two sites within its arginine/serine-rich (RS) domain. This phosphorylation significantly attenuates the ability of PGC-1α to turn on genes of gluconeogenesis in cultured hepatocytes and in vivo, while leaving the functions of PGC-1α as an activator of mitochondrial and fatty acid oxidation genes completely intact. These phosphorylations interfere with the ability of PGC-1α to bind to HNF4α, a transcription factor required for gluconeogenesis, while leaving undisturbed the interactions of PGC-1α with ERRα and PPARα, factors important for mitochondrial biogenesis and fatty acid oxidation. These data illustrate that S6 kinase can modify PGC-1α and thus allow molecular dissection of its functions, providing metabolic flexibility needed for dietary adaptation (Lustig, 2011).

The mTORC1/S6K1 pathway regulates glutamine metabolism through the eIF4B-dependent control of c-Myc translation

Growth-promoting signaling molecules, including the mammalian target of rapamycin complex 1 (mTORC1; see Drosophila Tor), drive the metabolic reprogramming of cancer cells required to support their biosynthetic needs for rapid growth and proliferation. Glutamine is catabolyzed to alpha-ketoglutarate (alphaKG), a tricarboxylic acid (TCA) cycle intermediate, through two deamination reactions, the first requiring glutaminase (GLS) to generate glutamate and the second occurring via glutamate dehydrogenase (GDH) or transaminases. Activation of the mTORC1 pathway has been shown previously to promote the anaplerotic entry of glutamine to the TCA cycle via GDH. Moreover, mTORC1 activation also stimulates the uptake of glutamine, but the mechanism is unknown. It is generally thought that rates of glutamine utilization are limited by mitochondrial uptake via GLS, suggesting that, in addition to GDH, mTORC1 could regulate GLS. This study demonstrates that mTORC1 positively regulates GLS and glutamine flux through this enzyme. mTORC1 controls GLS levels through the S6K1-dependent regulation of c-Myc (Myc; see Drosophila Myc). Molecularly, S6K1 enhances Myc translation efficiency by modulating the phosphorylation of eukaryotic initiation factor eIF4B (see Drosophila eIF4B), which is critical to unwind its structured 5' untranslated region (5'UTR). Finally, these data show that the pharmacological inhibition of GLS is a promising target in pancreatic cancers expressing low levels of PTEN (Csibi, 2014).

p70S6K controls cell growth and cell cycle progression

Phosphoinositide 3-kinase (PI3K) has been shown to regulate cell and organ size in Drosophila, but the role of PI3K in vertebrates in vivo is not well understood. To examine the role of PI3K in intact mammalian tissue, transgenic mice expressing constitutively active or dominant-negative mutants of PI3K in the heart have been created and characterized. Cardiac-specific expression of constitutively active PI3K results in mice with larger hearts, while dominant-negative PI3K results in mice with smaller hearts. The increase or decrease in heart size is associated with comparable increase or decrease in myocyte size. Cardiomyopathic changes, such as myocyte necrosis, apoptosis, interstitial fibrosis or contractile dysfunction, are not observed in either type of transgenic mouse. Thus, the PI3K pathway is necessary and sufficient to promote organ growth in mammals (Shioi, 2000).

What are the downstream targets of PI3K that are involved in the regulation of organ size? The available information does not provide a definitive answer to this question. However, Akt, a well characterized downstream target of PI3K, is likely to be one of the major mediators of this process. Akt is necessary and sufficient for phosphorylation and subsequent inactivation of 4E-BP1, a repressor of mRNA translation. Akt can also activate p70S6K in some contexts, although activation of p70S6K might not be solely dependent on Akt. p70S6K is upregulated in constitutively active PI3K hearts and downregulated in dominant negative PI3K hearts. The amount of phosphorylated S6 protein correlates with the activation of p70S6K. The inhibition of the p70S6K pathway by rapamycin at nanomolar concentrations selectively suppresses an increase in protein synthesis of cultured neonatal myocytes in response to growth factors. Interestingly, rapamycin does not inhibit other phenotypic changes associated with myocyte hypertrophy, such as re-activation of fetal genes and sarcomere organization. This raises the possibility that p70S6K may selectively regulate cell size via controlling the rate of protein synthesis. Gene disruption of p70S6K is known to result in smaller body size in mice. In Drosophila, deficiency of the S6K gene is associated with a reduction in body size associated with smaller cells (Shioi, 2000 and references therein).

The role of the PI3K-p70 S6 kinase (S6K) signaling cascade in the stimulation of endothelial cell proliferation has been examined. Inhibitors of the p42/p44 MAPK pathway (PD98059) and the PI3K-p70 S6K pathway (wortmannin, Ly294002, and rapamycin) all block thymidine incorporation stimulated by fetal calf serum in the resting mouse endothelial cell line 1G11. The action of rapamycin can be generalized, since it completely inhibits the mitogenic effect of fetal calf serum in primary endothelial cell cultures (human umbilical vein endothelial cells) and another established capillary endothelial cell line (LIBE cells). The inhibitory effect of rapamycin is only observed when the inhibitor is added at the early stages of G(0)-G(1) progression, suggesting an inhibitory action early in G(1). Rapamycin completely inhibits growth factor stimulation of protein synthesis, which perfectly correlates with the inhibition of cell proliferation. In accordance with its inhibitory action on protein synthesis, activation of cyclin D1 and p21 proteins by growth factors is also blocked by preincubation with rapamycin. Expression of a p70 S6K mutant partially resistant to rapamycin reverses the inhibitory effect of the drug on DNA synthesis, indicating that rapamycin action is via p70 S6K. Thus, in vascular endothelial cells, activation of protein synthesis via p70 S6K is an essential step for cell cycle progression in response to growth factors (Vinals, 1999).

In T lymphocytes, the hematopoietic cytokine interleukin-2 (IL-2) uses phosphatidylinositol 3-kinase (PI 3-kinase)-induced signaling pathways to regulate E2F transcriptional activity, a critical cell cycle checkpoint. PI 3-kinase also regulates the activity of p70(s6k), the 40S ribosomal protein S6 kinase, a response that is abrogated by the macrolide rapamycin. This immunosuppressive drug is known to prevent T-cell proliferation, but the precise point at which rapamycin regulates T-cell cycle progression has yet to be elucidated. Moreover, the effects of rapamycin on, and the role of p70(s6k) in, IL-2 and PI 3-kinase activation of E2Fs have not been characterized. The present results show that IL-2- and PI 3-kinase-induced pathways for the regulation of E2F transcriptional activity include both rapamycin-resistant and rapamycin-sensitive components. Expression of a rapamycin-resistant mutant of p70(s6k) in T cells can restore rapamycin-suppressed E2F responses. Thus, the rapamycin-controlled processes involved in E2F regulation appear to be mediated by p70(s6k). However, the rapamycin-resistant p70s6k could not rescue rapamycin inhibition of T-cell cycle entry, consistent with the involvement of additional, rapamycin-sensitive pathways in the control of T-cell cycle progression. The present results thus show that p70s6k is able to regulate E2F transcriptional activity and provide direct evidence for the first time for a link between IL-2 receptors, PI 3-kinase, and p70s6k that regulates a crucial G1 checkpoint in T lymphocytes (Brennan, 1999).

High-resistance exercise training results in an increase in muscle wet mass and protein content. To begin to address the acute changes following a single bout of high-resistance exercise, a new model has been developed. Training rats twice a week for 6 wk resulted in 13.9% and 14.4% hypertrophy in the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles, respectively. Polysome profiles after high-resistance lengthening contractions suggest that the rate of initiation is increased. The activity of the 70-kDa S6 protein kinase (p70S6k), a regulator of translation initiation, is also increased following high-resistance lengthening contractions. Furthermore, the increase in p70S6k activity 6 h after exercise correlates with the percent change in muscle mass after 6 wk of training. The tight correlation between the activation of p70S6k and the long-term increase in muscle mass suggests that p70S6k phosphorylation may be a good marker for the phenotypic changes that characterize muscle hypertrophy and may play a role in load-induced skeletal muscle growth (Baar, 1999).

The mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K) signaling pathways promote cell growth and cell cycle progression in response to nutritional, energy, and mitogenic cues. In mammalian cells, the ribosomal protein S6 kinases, S6K1 and S6K2, lie downstream of mTOR and PI3K, suggesting that translational control through the phosphorylation of S6 regulates cell growth. Interestingly, genetic experiments predict that a substrate that is specific to S6K1 but not S6K2 regulates cell growth. SKAR has been identified as a novel and specific binding partner and substrate of S6K1 but not S6K2. Serines 383 and 385 of human SKAR are insulin-stimulated and rapamycin-sensitive S6K1 phosphorylation sites. Quantitative mass spectrometry reveals that serine 383/385 phosphorylation is sensitive to RNA interference (RNAi)-mediated S6K1 reduction, but not S6K2 reduction. Furthermore, RNAi-mediated reduction of SKAR decreases cell size. SKAR is nuclear protein with homology to the Aly/REF family of RNA binding proteins, which has been proposed to couple transcription with pre-mRNA splicing and mRNA export. Database searches reveal SKAR homologs in mouse and rat with 91% amino acid identity and a potential homolog in Drosophila but not in lower eukaryotes. This study has identified a novel and specific target of S6K1, SKAR, which regulates cell growth. The homology of SKAR to the Aly/REF family links S6K1 with mRNA biogenesis in the control of cell growth (Richardson, 2004).

S6 phosphorylation is a determinant of cell size and glucose homeostasis

The regulated phosphorylation of ribosomal protein (rp) S6 has attracted much attention since its discovery in 1974, yet its physiological role has remained obscure. To directly address this issue, viable and fertile knock-in mice have been established, whose rpS6 contains alanine substitutions at all five phosphorylatable serine residues (rpS6^P-/-). Contrary to the widely accepted model, this mutation does not affect the translational control of mRNAs characterized by an oligopyrimidine tract at their 5' terminus (TOP mRNAs) rpS6^P-/- mouse embryo fibroblasts (MEFs) display an increased rate of protein synthesis and accelerated cell division, and they are significantly smaller than rpS6^P+/+ MEFs. This small size reflects a growth defect, rather than a by-product of their faster cell division. Moreover, the size of rpS6^P-/- MEFs, unlike wild-type MEFs, is not further decreased upon rapamycin treatment, implying that the rpS6 is a critical downstream effector of mTOR in regulation of cell size. The small cell phenotype is not confined to embryonal cells, since it also selectively characterizes pancreatic beta-cells in adult rpS6^P-/- mice. These mice suffer from diminished levels of pancreatic insulin, hypoinsulinemia, and impaired glucose tolerance (Ruvinsky, 2005).

Translational control by MAPK signaling in long-term synaptic plasticity and memory: Phosphorylation of S6K

Enduring forms of synaptic plasticity and memory require new protein synthesis, but little is known about the underlying regulatory mechanisms. The role of MAPK signaling in these processes has been investigated. Conditional expression of a dominant-negative form of MEK1 in the postnatal murine forebrain inhibits ERK activation and causes selective deficits in hippocampal memory retention and the translation-dependent, transcription-independent phase of hippocampal L-LTP. In hippocampal neurons, ERK inhibition blocks neuronal activity-induced translation as well as phosphorylation of the translation factors eIF4E, 4EBP1, and ribosomal protein S6. Correspondingly, protein synthesis and translation factor phosphorylation induced in control hippocampal slices by L-LTP-generating tetanization are significantly reduced in mutant slices. Translation factor phosphorylation induced in the control hippocampus by memory formation is similarly diminished in the mutant hippocampus. These results suggest a crucial role for translational control by MAPK signaling in long-lasting forms of synaptic plasticity and memory (Kelleher, 2004).

Translation of eukaryotic mRNAs is primarily regulated at the level of initiation. Studies in mitotic cells have defined 5' cap recognition and ribosomal recruitment by translation initiation factors as key events in this multistep process. Cap recognition is accomplished by eukaryotic initiation factor 4E (eIF4E), and the eIF4E-associated factor eIF4G then recruits the 40S ribosomal subunit. Cap-dependent translation accounts for the synthesis of the vast majority of cellular proteins, since all mRNAs transcribed in the nucleus bear a 5' cap. The synthesis of the translation machinery itself is additionally regulated by an inhibitory cis-acting element, termed a 5' terminal oligopyrimidine tract (5' TOP), which occurs adjacent to the cap in mRNAs encoding ribosomal proteins and translation factors (Meyuhas, 2000).

Prior work in mitotic cells has identified the ERK-dependent kinase Mnk1 as the major eIF4E kinase, indicating a dominant role for ERK signaling in eIF4E phosphorylation. The complex pattern of inducible 4E-BP1 hyperphosphorylation appears to be mediated primarily by rapamycin-sensitive mTOR-dependent pathways, while some evidence has suggested ERK-dependent modulation of Ser65 phosphorylation. Studies on the mitogen-induced hyperphosphorylation of S6 have delineated a central role for mTOR-dependent activation of S6 kinase. The current findings demonstrate a major role for the ERK pathway in the neuronal activity-induced phosphorylation of S6, eIF4E, and 4E-BP1, with consistently greater effects on eIF4E relative to S6 across all levels of analysis. Interestingly, the phosphorylation of all three factors was also found to be highly sensitive to rapamycin, with the greatest effect on S6. These observations suggest that the ERK and mTOR pathways cooperate in the coordinate regulation of cap-dependent and 5' TOP-dependent translation. Hippocampal L-LTP and serotonin-induced LTF in Aplysia have been shown to be sensitive to rapamycin, implicating mTOR-dependent translation in these processes. Translational efficiency during the establishment of long-term synaptic plasticity and memory may therefore be determined through the functional interplay of ERK- and mTOR-dependent signaling mechanisms. General translational induction of a broad range of neuronal mRNAs by such activity-dependent mechanisms may provide the protein products required for the input-specific 'capture' of long-term synaptic plasticity by 'tagged' synapses (Meyuhas, 2000).

Pharmacologic targeting of S6K1 in PTEN-deficient neoplasia

Genetic S6K1 (see Drosophila S6k) inactivation can induce apoptosis in PTEN-deficient cells (see Drosophila Pten). This study analyzed the therapeutic potential of S6K1 inhibitors in PTEN-deficient T cell leukemia and glioblastoma. Results revealed that the S6K1 inhibitor LY-2779964 was relatively ineffective as a single agent, while S6K1-targeting AD80 induced cytotoxicity selectively in PTEN-deficient cells. In vivo, AD80 rescued 50% of mice transplanted with PTEN-deficient leukemia cells. Cells surviving LY-2779964 treatment exhibited inhibitor-induced S6K1 phosphorylation due to increased mTOR-S6K1 (see Drosophila Tor) co-association, which primed the rapid recovery of S6K1 signaling. In contrast, AD80 avoided S6K1 phosphorylation and mTOR co-association, resulting in durable suppression of S6K1-induced signaling and protein synthesis. Kinome analysis revealed that AD80 coordinately inhibits S6K1 together with the TAM family tyrosine kinase AXL. TAM suppression by BMS-777607 or genetic knockdown potentiated cytotoxic responses to LY-2779964 in PTEN-deficient glioblastoma cells. These results reveal that combination targeting of S6K1 and TAMs is a potential strategy for treatment of PTEN-deficient malignancy (Liu, 2017).

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Search PubMed for articles about Drosophila Ribosomal protein S6 kinase

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