InteractiveFly: GeneBrief

Thor: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The messenger RNA 5' cap-binding protein eIF4E is regulated by its binding protein (4E-BP), a downstream target of phosphatidylinositol-3-OH kinase [PI(3)K] signaling. Drosophila 4E-BP (d4E-BP) activity becomes critical for survival under dietary restriction and oxidative stress, and is linked to life span. The Drosophila forkhead transcription factor (dFOXO) activates d4E-BP transcription. Ectopic expression of d4E-BP in dFOXO-null flies restores oxidative stress resistance to control levels. Thus, d4E-BP is an important downstream effector of a dFOXO phenotype, and regulation of translation by eIF4E is vital during environmental stress (Tettweiler, 2005).

4E-BP has been studied extensively in cell culture; however, to date the biological role of 4E-BP in developing organisms is unclear. Since TOR (see Drosophila Tor) has been shown to control tissue growth during animal development, 4E-BP has also been assumed to serve as a growth regulator. The relevance of 4E-BP function for organismal development has been studied, and evidence is presented for an alternate view of 4E-BP function. 4E-BP strongly affects fat metabolism in Drosophila. It is suggested 4E-BP works as a metabolic brake that is activated under conditions of environmental stress to control fat metabolism. 4E-BP mutants lack this regulation, reducing their ability to survive under unfavorable conditions (Teleman, 2005).

A rapid response is a crucial early line of defense in preventing cellular death in situations of stress. Translational regulation allows an organism to generate quick responses to environmental cues by controlling the expression of protein from existing cellular mRNAs. Translation initiation of most eukaryotic mRNAs requires binding of eIF4F, a protein complex made up of eIF4A, eIF4G, and eIF4E, to the 5' cap structure. eIF4E activity is highly regulated both by Mnk1/Mnk2-dependent phosphorylation and by repressor proteins termed eIF4E-binding proteins (4E-BPs), which compete with eIF4G for the same binding site on eIF4E (Haghighat, 1995; Mader, 1995). 4E-BPs themselves are negatively regulated by phosphorylation, and are downstream effectors of the PI3K/TOR pathway (Miron, 2001). Under nutritionally favorable conditions, the evolutionarily conserved TOR pathway is active and results in 4E-BP phosphorylation. This prevents 4E-BP binding to eIF4E, thus upregulating translation (Schmelzle, 2000; Hay, 2004). Conversely, poor nutrition causes inhibition of the TOR pathway, such that unphosphorylated 4E-BP represses translation through eIF4E binding (Tettweiler, 2005).

Activation of the PI3K pathway stimulates Akt activity. Akt directly phosphorylates the forkhead transcription factor dFOXO, which is a transcriptional activator of d4E-BP in flies, resulting in reduced d4E-BP transcription (Jünger, 2003; Puig, 2003). Akt also activates TOR through tuberous sclerosis complex 2 (TSC2/Gigas), which functions as a GTPase-activating protein (GAP) for the GTPase protein Rheb that activates TOR. Thus, activation of the PI3K pathway in Drosophila represses both the expression of the d4E-BP gene and the activity of d4E-BP protein (Tettweiler, 2005).

Whether d4E-BP is essential under starvation and oxidative stress conditions was investigated, because dFOXO activates the transcription of d4E-BP and d4E-BP mRNA levels increase upon starvation (Zinke, 2002). Evidence is provided that d4E-BP activity is linked to life span, since overexpression of dFOXO is linked to increased longevity. This work establishes that d4E-BP is the critical effector of the dFOXO-induced stress-sensitive phenotype (Tettweiler, 2005).

To investigate whether 4E-BP activity influences life span in Drosophila, the median life span of d4E-BP^null flies was determined in comparison to revertant control flies that were produced by a precise excision of the P{lacW} insertion of Thor¹ (which disrupts the gene encoding d4E-BP) but which otherwise have the identical genetic background (Rodriguez, 1996; Bernal, 2000; Bernal, 2004). These will be referred to as revertant flies. A null mutation in d4E-BP caused a significant decrease in longevity. The median life span of mutant males was 19.8 d, ~25% shorter than that of control males, median life span of 26.6 d). The life span of females was longer than for males, but a comparable relative effect of the null mutation in d4E-BP on life span was observed in both sexes. These results show that d4E-BP, a target of the conserved PI3K/TOR signaling pathway that has been strongly implicated in longevity, has a significant impact itself on life span (Tettweiler, 2005).

In Drosophila larvae, protein-rich nutrition is critical for survival, while adults can survive up to 3 wk without protein. Strikingly, it was observed that larval d4E-BP protein levels rise rapidly during nutritional stress. A dramatic increase of ~10-fold is observed after 8 h of starvation. To determine whether this increase in d4E-BP level is important for survival following starvation, eggs were collected from Oregon-R, d4E-BP^null, and revertant flies, transferred to starvation medium: mortality was scored at 12-h intervals. Drosophila 4E-BP^null larvae die substantially faster than their control counterparts under starvation, (median life span of 20.8 h for starved d4E-BP^null larvae, 26.4 h for revertant, and 26.2 h for Oregon-R larvae). To examine whether this effect can be overcome by induced expression of a d4E-BP(wt) transgene in a d4E-BP^null background, the UAS-GAL4 system was used with the heat-shock inducible Hsp70 promoter. Induction of d4E-BP expression in this manner fully rescues the increased sensitivity of d4E-BP^null larvae to starvation (median life span of 27 h). Importantly, transgenic flies expressing a mutant version of d4E-BP [d4E-BP(Y54A,M59A)] that does not bind to eIF4E (Miron, 2001) are susceptible to starvation similar to the d4E-BP^null animals (median life-span 22.6 h). While the d4E-BP(Y54A,M59A) larvae resist nutritional stress somewhat better in the first 12 h, after 36 h of complete starvation their survival was as poor as that of d4E-BP^null larvae (survival rates of 8.5% and 9.6%, respectively) and far lower than that of control animals, or animals expressing d4E-BP from a transgene [survival rates of 26.8% for revertant larvae, 27.6% for Oregon-R larvae, and 30.3% for transgenic d4E-BP(wt) larvae]. These results demonstrate clearly that the protective role of d4E-BP during starvation requires binding to eIF4E (Tettweiler, 2005).

dFOXO is a transcriptional activator of d4E-BP (Jünger, 2003; Puig, 2003; Wang, 2005). dFOXO loss-of-function mutants exhibit increased sensitivity to oxidative stress (Jünger, 2003). Since d4E-BP is a downstream target of dFOXO, the sensitivity of EP-dFOXO²¹/EP-dFOXO²⁵ flies and d4E-BP^null flies to oxidative stress was compared. EP-dFOXO²¹/EP-dFOXO²⁵ is null for dFOXO function (Jünger, 2003) and is subsequently referred to here as dFOXO-null for brevity. Indeed, on medium containing 5% hydrogen peroxide, a reduced median life span was observed of dFOXO-null and d4E-BP^null flies (median life spans of 34.6 h and 23.2 h, respectively). The survival rate at 60 h after exposure to oxidative stress was 0% for d4E-BP^null animals, and only 2% for dFOXO-null flies, compared with 66.1% for wild-type controls. Thus, an intriguing possibility is that d4E-BP acts as the downstream mediator of the dFOXO-null phenotype. As is the case for d4E-BP-mediated protection against starvation, this effect can be rescued by ectopic expression of d4E-BP (median life span of 55.4 h). The resistance to oxidative stress was also dependent on eIF4E binding, since d4E-BP(Y54A, M59A) was unable to rescue stress sensitivity (median life span of 24.1 h, 0.4% survival rate at 60 h). The heat shock itself did not significantly affect life span. To examine whether reduced levels of d4E-BP protein could account for the oxidative stress sensitivity of dFOXO-null mutants, d4E-BP was ectopically expressed in a dFOXO-independent manner in these animals. Remarkably, ectopic expression of d4E-BP completely rescues the sensitivity of dFOXO-null animals to oxidative stress (median life span of 56.8 h, 39.7% survival rate after 60 h exposure to 5% H₂O₂). Taken together, these results demonstrate that d4E-BP is a critical downstream mediator of dFOXO in oxidative stress resistance (Tettweiler, 2005).

Normal growth and development are suspended during stress in order to concentrate resources on an appropriate stress response, and repressing translation does, in fact, slow growth. d4E-BP is strongly up-regulated during starvation, and its absence leads to compromised survival under outright starvation. The importance of d4E-BP in nutrient stress response is underscored by the fact that it is one of a small set of ~14 genes whose expression is up-regulated as a consequence of starvation (Zinke, 2002; Tettweiler, 2005). The data support the current models to explain how oxidative stress activates transcription of d4E-BP. Oxidative stress promotes dephosphorylation of dFOXO, which causes its transport into the nucleus and activation of d4E-BP transcription (Jünger, 2003). Amino acid starvation activates the dTOR pathway in the larval fat body. This triggers a starvation signal that suppresses the Inr/PI3K pathway in peripheral tissues (Colombani, 2003). Suppression of this pathway results in inactivation of dAkt, which in turn can no longer phosphorylate and inactivate dFOXO, causing enhanced transcription of d4E-BP (Tettweiler, 2005).

Since the function of d4E-BP in stress resistance requires its eIF4E-binding activity, it is proposed that d4E-BP involves repression of cap-dependent translation, concomitant with the stimulation of cap-independent translation. The up-regulation of d4E-BP could result in preferential translation of mRNAs, which translate in a cap-independent manner, via an internal ribosome entry site (IRES) element. Such stimulation has been documented under stress conditions including irradiation, hypoxia, and nutrient deprivation. It is also possible that d4E-BP exerts its effects by inhibiting the translation of key mRNA targets as was documented in mammals (Gingras, 1999). Mammalian eIF4E preferentially stimulates the translation of mRNAs with a high degree of secondary structure in their mRNA 5 'UTRs (Gingras, 1999). These mRNAs, which are inefficiently translated, mostly encode proteins that play important roles in cell growth and proliferation (Tettweiler, 2005).

Recent studies show that overexpression of dFOXO in adult fat bodies can extend the Drosophila life span. Further, overexpression of dTSC1, dTSC2, or dominant-negative forms of dTOR or dS6K all extend the life span of Drosophila. Interestingly, TOR also regulates life span in Caenorhabditis elegans. The data support a role for d4E-BP in mediating life span, most likely as an effector of dFOXO. Taken together, these results add to the increasing body of evidence supporting a key role for 4E-BP-mediated regulation of eIF4E in controlling cell growth, proliferation, and survival (Miron, 2001; Fingar, 2002; Li, 2002; Avdulov, 2004; Bjornsti, 2004; Tettweiler, 2005 and references therein).

Drosophila miR2 primarily targets the m⁷GpppN cap structure for translational repression

Understanding the molecular mechanism(s) of how miRNAs repress mRNA translation is a fundamental challenge in RNA biology. This study used a validated cell-free system from Drosophila embryos to investigate how miR2 inhibits translation initiation. By screening a library of chemical m⁷GpppN cap structure analogs, defined modifications of the triphosphate backbone were identified that augment miRNA-mediated inhibition of translation initiation but are 'neutral' toward general cap-dependent translation. Interestingly, these caps also augment inhibition by 4E-BP. Kinetic dissection of translational repression and miR2-induced deadenylation shows that both processes proceed largely independently, with establishment of the repressed state involving a slow step. These data demonstrate a primary role for the m⁷GpppN cap structure in miRNA-mediated translational inhibition, implicate structural determinants outside the core eIF4E-binding region in this process, and suggest that miRNAs may target cap-dependent translation through a mechanism related to the 4E-BP class of translational regulators (Zdanowicz, 2009).

MicroRNAs (miRNAs) are posttranscriptional regulators of a wide range of biological processes including development, growth control, cellular differentiation, and apoptosis. With few exceptions, miRNAs fulfill their regulatory function by imperfect base pairing with the 3′UTR of target mRNAs inhibiting translation and/or triggering mRNA destabilization. Despite active investigation, the molecular mechanism(s) of how miRNAs and their associated proteins mediate their repressive or destabilizing effects remains controversial (Zdanowicz, 2009).

Much evidence now suggests that miRNAs can regulate translation initiation, although perhaps not exclusively. Since miRNAs induce mRNA deadenylation in vivo and in vitro, this effect could theoretically suffice for inhibition of translation initiation. However, multiple lines of evidence suggest that neither a poly(A) tail nor its removal by deadenylation is required for miRNA regulation, implying that the primary mechanism of inhibition must be sought elsewhere (Zdanowicz, 2009).

Both cap-dependent small ribosomal subunit recruitment and 60S subunit joining at the translation initiation codon have been reported to be regulated by miRNAs. Binding of the eIF4F complex to the cap promotes recruitment of the small ribosomal subunit to mRNAs and was implicated as a primary target of miRNA regulation by early reports investigating miRNA-mediated control; subsequent work in cultured cells and in vitro systems further supported this notion. However, the strength of conclusions from all of these studies has been questioned on the grounds that they rely on experimental approaches that alter the mode (internal ribosome entry sequences) and/or rate of nonregulated translation. Kinetic modeling studies frame this concern in quantitative terms and actually favored a late step in translation initiation as the likely target for miRNAs (Zdanowicz, 2009).

To overcome these inherent limitations, a strategy was devised that probes miRNA-mediated regulation without affecting general (nonregulated) cap-dependent translation. This approach, based on modified cap structural analogs, circumvents by its very design the major caveats limiting other experimental approaches used to date. Advantage was also taken of the properties of an in vitro system to probe more deeply the relationship between translational inhibition and mRNA deadenylation. The data unambiguously demonstrate the importance of the cap structure as a primary target for miRNA-mediated translational control and show that deadenylation is an independent, rapid process that can contribute to repression. Moreover, the approach reveals that miR2's cap targeting mechanism bears similarities to repression by 4E-BP, highlighting interactions between cap-bound eIF4E and eIF4G as potential molecular targets of miR-RISC function (Zdanowicz, 2009).

The discovery of two cap structure analogs that specifically augment miRNA-mediated repression without affecting overall mRNA translation provides a uniquely powerful argument that the cap structure serves as the primary functional target of miR2 translational inhibition. Both modifications to the cap structure also yield stronger repression of translation by 4E-BP. How do these cap modifications selectively sensitize translation to specific inhibitory pathways, and what does this say about how miRNAs target the cap? Structural details of eIF4E binding to the m⁷GpppN cap structure readily explain why the modifications of cap analogs cap 16 and cap 21 do not interfere with general translation: the modifications affect the end of the triphosphate linker, outside the 'core' m⁷G nucleotide recognition region that features critical contacts for high-affinity cap binding. Nevertheless, the observation that these modifications result in sensitivity to both miR2-RISC and 4E-BP suggests that changes to this region of the cap subtly affect the way eIF4F interacts with the cap and perhaps also downstream portions of the 5' UTR via eIF4G. Since the effects of these changes are effectively nonconsequential at the level of general translation initiation, and are only revealed in the presence of specific inhibitors, they appear to introduce an Achilles' heel in the translation initiation pathway. Enhanced sensitivity is observed with both 4E-BP and miR2-RISC, but not with m⁷GpppG cap analog itself, suggesting that miR2-RISC may use a mechanism similar to 4E-BP to target cap function. Since 4E-BP directly interferes with interaction between eIF4E and eIF4G, the results highlight physical interaction between eIF4E and eIF4G–or a related functional step–as a potential target of miRNA action (Zdanowicz, 2009).

As seen in multiple experimental in vivo and in vitro settings, specific, miR-dependent deadenylation of the reporter mRNA was observed. The data using cap 18 support earlier work suggesting that a poly(A) tail and mRNA deadenylation are not required for miRNA-mediated translational inhibition but can quantitatively contribute to it. At least in vitro, deadenylation is a kinetically rapid process that occurs even under conditions where the mRNA fails to be translationally repressed (lack of preincubation or A capping). In this sense, deadenylation and repression are separable processes that are both specifically triggered by miR2. The data also challenge the recent conclusion that miR/Ago1-mediated translational repression primarily occurs via deadenylation, as m⁷GpppN-capped mRNA is fully deadenylated but essentially unrepressed if it has not undergone preincubation. Thus, deadenylation cannot be the primary cause of repression (Zdanowicz, 2009).

Based on published work and the new data, a 'two-hit model' is proposed; the miR-RISC complex affects both ends of mRNAs to which it is bound. Repression primarily targets the cap structure, preventing recruitment of the small ribosomal subunit. This process is normally facilitated and reinforced by the independent action of miR-RISC removal of the poly(A) tail. Both hits converge on the inhibition of cap-dependent small ribosomal subunit recruitment via the eIF4F complex. While this model can explain much of the published work, it does not exclude the existence of additional mechanisms that could target later steps in translation initiation or postinitiation steps (Zdanowicz, 2009).

Acute fasting regulates retrograde synaptic enhancement through a 4E-BP-dependent mechanism

While beneficial effects of fasting on organismal function and health are well appreciated, little is known about the molecular details of how fasting influences synaptic function and plasticity. Genetic and electrophysiological experiments demonstrate that acute fasting blocks retrograde synaptic enhancement that is normally triggered as a result of reduction in postsynaptic receptor function at the Drosophila larval neuromuscular junction (NMJ). This negative regulation critically depends on transcriptional enhancement of eukaryotic initiation factor 4E binding protein (4E-BP) under the control of the transcription factor Forkhead box O (Foxo). Furthermore, these findings indicate that postsynaptic 4E-BP exerts a constitutive negative input, which is counteracted by a positive regulatory input from the Target of Rapamycin (TOR). This combinatorial retrograde signaling plays a key role in regulating synaptic strength. These results provide a mechanistic insight into how cellular stress and nutritional scarcity could acutely influence synaptic homeostasis and functional stability in neural circuits (Kauwe, 2016).

Many forms of dietary restriction can reduce cellular stress, improve organismal health, and in many instances extend lifespan in a number of model organisms. A major cellular function that is highly sensitive to nutrient intake from yeast to mammals is cap-dependent translation under the regulation of the target of rapamycin (TOR). TOR promotes cap-dependent translation primarily through phosphorylation of 4E-BPs (eukaryotic initiation factor 4E binding proteins) and p70 S6Ks (S6 ribosomal protein kinases). Phosphorylation of 4E-BP suppresses its ability to bind and inhibit the interaction between eIF4E (eukaryotic initiation factor 4E) and the initiation factor eIF4G, a critical step for translation initiation. In addition to the regulation by TOR, 4E-BP undergoes upregulation in response to dietary restriction and starvation. Together these two responses result in a strong inhibition of protein synthesis and act as a metabolic brake. Multiple lines of evidence suggest that fasting-induced increase in ketone bodies influences neuronal excitability and aspects of neurotransmitter release; however, little is known about how different forms of dietary restriction, by influencing protein translation, can exert an effect on the regulation of synaptic function and plasticity (Kauwe, 2016).

At the Drosophila larval neuromuscular junction (NMJ), the genetic removal of GluRIIA, one of five glutamate receptor subunits, reduces the postsynaptic response to unitary release of neurotransmitter. As a result of this reduced response to neurotransmitter, a retrograde signal is triggered in the postsynaptic muscle that ultimately leads to a compensatory enhancement in presynaptic release from the motor neuron, a process that is conserved at the vertebrate NMJs. The maintenance of this homeostatic synaptic compensation or retrograde synaptic enhancement is highly sensitive to postsynaptic cap-dependent translation in Drosophila; mutations in either Target of Rapamycin (TOR) or eIF4E can dominantly suppress the synaptic compensation in GluRIIA mutants (Penney, 2012). Interestingly, postsynaptic overexpression of TOR or S6K, in an otherwise wild-type muscle, is also sufficient to trigger a retrograde enhancement in presynaptic neurotransmitter release, suggesting that normal synaptic strength may be affected by a postsynaptic signal from the muscle (Penney, 2012; Kauwe, 2016 and references therein).

Previous findings have demonstrated that postsynaptic translation plays a critical role in the regulation of retrograde synaptic enhancement at the NMJ. Therefore, in light of the effect of dietary restriction on TOR-dependent translation, this study set out to investigate the consequence of nutrient restriction on retrograde synaptic compensation in GluRIIA mutants. Electrophysiological analysis indicates that acute fasting, but not amino acid restriction, blocks this retrograde synaptic compensation. This block is not merely due to reduced TOR activity, but rather a result of transcriptional upregulation of postsynaptic 4E-BP under the control of the transcription factor Foxo. These results indicate that the retrograde regulation of synaptic strength at the NMJ depends on the balance between 4E-BP and TOR (Kauwe, 2016).

A few hours of fasting can have a strong impact on retrograde synaptic enhancement at the Drosophila larval NMJ. Removal of food source acutely activates 4E-BP transcription in postsynaptic muscles in a Foxo-dependent manner, thereby leading to the inhibition of retrograde synaptic enhancement at the NMJ. The results indicate that Foxo and 4E-BP act cell autonomously in postsynaptic muscles to exert a retrograde negative regulation on presynaptic neurotransmitter release. Future studies are needed to test whether fasting-induced alterations in insulin signaling underlie the transcriptional upregulation of 4E-BP via its effect on Foxo in postsynaptic muscles. While 4E-BP-mediated suppression of synaptic enhancement as a result of fasting could be considered undesirable during development, it can be beneficial under conditions of abnormally high synaptic activity. As such, 4E-BP-mediated inhibition of retrograde synaptic enhancement and the subsequent dampening of circuit activity might provide an explanation for the beneficial effects of fasting in reducing seizures in some cases. Similarly, in cases where dysregulation of TOR activity is thought to underlie abnormal circuit activity, such as in TSC models, intermittent fasting could potentially dampen the increase in synaptic release through a 4E-BP-dependent inhibition, thereby stabilizing neuronal circuits (Kauwe, 2016).

In addition to its role as a molecular responder to stress, 4E-BP exerts a constitutive negative regulation on presynaptic neurotransmitter release at the NMJ. Electrophysiological analysis of loss-of-function mutant larvae indicates that 4E-BP functions in postsynaptic muscles to constitutively provide a retrograde negative influence on synaptic strength. In light of these findings, a two-pronged scheme is proposed for the retrograde regulation of synaptic strength at the NMJ. On the one hand, a positive input from TOR is mediated through S6K/eIF4A and eIF4E to enhance postsynaptic translation. Synaptic compensation in GluRIIA mutant larva appears to rely mostly on this axis as evidenced by strong sensitivity to S6K heterozygosity and no change in the proportion of phosphorylated 4E-BP versus non-phosphorylated 4E-BP levels. Opposing this positive input, 4E-BP inhibits translation by sequestering eIF4E and adjusting the degree of retrograde compensation. Indeed, loss of 4E-BP leads to a strong increase in quantal content that is highly sensitive to eIF4E heterozygosity but not sensitive to S6K heterozygosity. The balance between these two forces reveals itself also when 4E-BP loss-of-function mutants are rescued by a non-phosphorylatable 4E-BP transgene. In this combination TOR can no longer inhibit 4E-BP, and this study finds that the presynaptic neurotransmitter release is lower than wild-type, similarly to what is observed in TOR hypomorphic mutants (Penney, 2012). A working model is proposed in which the negative force of 4E-BP is under constant check via phosphorylation by TOR, and the positive input from TOR/S6K is constitutively countered by 4E-BP’s ability to sequester eIF4E, a dynamic duel that ensures a tight regulation of synaptic strength (Kauwe, 2016).

The GCN2-ATF4 signaling pathway induces 4E-BP to bias translation and boost antimicrobial peptide synthesis in response to bacterial infection

Bacterial infection often leads to suppression of mRNA translation, but hosts are nonetheless able to express immune response genes through as yet unknown mechanisms. This study used a Drosophila model to demonstrate that antimicrobial peptide (AMP) production during infection is paradoxically stimulated by the inhibitor of cap-dependent translation, 4E-BP (eIF4E-binding protein; encoded by the Thor gene). 4E-BP was found to be induced upon infection with pathogenic bacteria by the stress-response transcription factor ATF4 and its upstream kinase, GCN2. Loss of gcn2, atf4, or 4e-bp compromised immunity. While AMP transcription is unaffected in 4e-bp mutants, AMP protein levels are substantially reduced. The 5' UTRs of AMPs score positive in cap-independent translation assays, and this cap-independent activity is enhanced by 4E-BP. These results are corroborated in vivo using transgenic 5' UTR reporters. These observations indicate that ATF4-induced 4e-bp contributes to innate immunity by biasing mRNA translation toward cap-independent mechanisms, thus enhancing AMP synthesis (Vasudevan, 2017).

Much research has focused on the transcriptional response to infection mediated by the innate immune response pathways. Although translational inhibitors such as GCN2 and 4E-BP have been implicated in the antibacterial response, the incongruence of translation inhibition and the need for AMP synthesis had not been addressed experimentally. This study provides results that resolve this discrepancy and establish that AMPs have evolved mechanisms to bypass translation inhibition imposed by pathogenic bacteria via 4E-BP activation. This study has shown that the ISR pathway promotes innate immunity against pathogenic bacteria by mediating 4e-bp induction, which in turn biases cellular translation toward cap-independent mechanisms that favor AMP synthesis. The newly identified role of the ISR pathway in the innate immune response is likely coordinated with the established roles of other innate immune response pathways, such as those mediated by FOXO, IMD, and Dorsal/Dif (Vasudevan, 2017).

In addition to regulation by GCN2/ATF4 and FOXO, 4E-BP is also famously regulated post-translationally by another amino acid-sensitive kinase, TOR. While under steady-state conditions, TOR phospho-inactivates 4E-BP, and TOR itself is inactivated in response to amino acid deprivation. Thus, 4E-BP newly synthesized in response to infection-mediated amino acid deprivation will likely not be subject to inactivation by TOR (Vasudevan, 2017).

These observations are consistent with the positive effects of 4e-bp induction against a non-lethal pathogen such as Ecc15. However, infection by a severe pathogen such as Pseudomonas entomophila results in a GCN2-mediated translation block in the gut and is detrimental to the host immune response (Chakrabarti, 2012). Why GCN2 mediates such different outcomes remains unresolved, but it could be because of factors other than ATF4 that are downstream of GCN2 (Vasudevan, 2017).

It is worth noting that GCN2 engages two different translation inhibition mechanisms: (1) phospho-eIF2α, which induces ATF4; and (2) 4e-bp, which is transcriptionally induced by ATF4. This study primarily focused on 4E-BP because the effect of phospho-eIF2α on translation may be temporary because ATF4 induces the expression of an eIF2α phosphatase subunit, GADD34, thereby stimulating eIF2α dephosphorylation. It is speculated that eIF2α phosphorylation may not serve as a long-term deterrent for AMP translation, while the second translational block by 4E-BP may persist longer. In addition, the data show that 4e-bp itself is synthesized favorably by cap-independent translation conferred by its 5' UTR, suggesting that this inhibition mechanism is self-sustaining (Vasudevan, 2017).

Intriguingly, the 5' UTRs of Drosomycin and Attacin A, which confer cap-independent translation capabilities to their respective mRNA, are relatively small at 63 and 29 bases, respectively. These are the smallest characterized cap-independent translation elements known. Although there are no known consensus sequences for IRESs, scoring positively in biochemical assays such as bicistronic assays and cap competition assays has been recognized to be a reliable indicator for them. Although both Drosomycin and Attacin A are likely translated cap-independently via IRES-like elements in their 5' UTRs, they may utilize slightly different mechanisms. Although both 5' UTRs confer cap-independence, which is enhanced by 4E-BP, the Attacin A 5' UTR reporter shows basal translation of the reporter even in the absence of 4E-BP, whereas the Drosomycin 5' UTR relies heavily on 4E-BP. Additionally, eliminating competing transcripts in the presence of excess m7GpppG also had a small but significant effect on Attacin A 5' UTR reporter mRNA, suggesting that there is an additional layer of regulatory mechanisms acting on these 5' UTRs (Vasudevan, 2017).

Because suppression of cap-dependent translation by 4E-BP appears to stimulate the synthesis of AMPs, it is speculated that the AMP 5' UTRs do not compete well with other mRNAs for ribosomes and initiation factors. Thus, in addition to the established role of 4E-BP in the inhibition of cap-dependent translation, the data show a more nuanced role for 4E-BP as a promoter of cap-independent translation required for driving the synthesis of essential immune response proteins. Interestingly, it had been previously suggested that eIF2α-kinases such as GCN2 could promote cap-independent translation, although the effectors of such regulation were unknown. Based on the curren work showing that 4E-BP downstream of GCN2/ATF4 signaling regulates AMP translation, it can now be surmised that these previously observed instances of cap-independent translation downstream of phospho-eIF2α are also likely mediated by ATF4-induced 4E-BP. Additionally, given the conservation of translation inhibition during infection in mammals, it would be interesting to examine whether the mammalian innate immune response is aided by any of the three known mammalian 4E-BPs and if first-response cytokines are synthesized cap-independently (Vasudevan, 2017).

Bacterial recognition by PGRP-SA and downstream signalling by Toll/DIF sustain commensal gut bacteria in Drosophila

The gut sets the immune and metabolic parameters for the survival of commensal bacteria. This study reports that in Drosophila, deficiency in bacterial recognition upstream of Toll/NF-κB signalling resulted in reduced density and diversity of gut bacteria. Translational regulation factor 4E-BP (Thor), a transcriptional target of Toll/NF-κB, mediated this host-bacteriome interaction. In healthy flies, Toll activated 4E-BP, which enabled fat catabolism, which resulted in sustaining of the bacteriome. The presence of gut bacteria kept Toll signalling activity thus ensuring the feedback loop of their own preservation. When Toll activity was absent, TOR-mediated suppression of 4E-BP made fat resources inaccessible and this correlated with loss of intestinal bacterial density. This could be overcome by genetic or pharmacological inhibition of TOR, which restored bacterial density. These results give insights into how an animal integrates immune sensing and metabolism to maintain indigenous bacteria in a healthy gut (Bahuguna, 2022).

The animal gut accommodates a diverse array of bacteria, which assist in regulation of digestion, supply of nutrients and metabolites as well as in immune development. To reap benefits from these microbes, the host provides a symbiotic environment for sustaining them in the gut. The Drosophila gut and its bacteriome is used as a simpler model to study such host-microbe interactions. Although much less diverse compared to humans, the fly bacteriome is equally dynamic and changes with age and environmental conditions connected to reinfections during fly culture (Bahuguna, 2022).

The Drosophila intestinal epithelium is immunocompetent and upon enteric infection initiates innate immune responses via the NF-κB pathway IMD, mediating the production of antimicrobial peptides (AMPs) as well as the pathway centered on Dual Oxidase, the enzyme needed for the generation of Reactive Oxygen Species (ROS). However, it also preserves commensal bacteria, since transcription of AMPs is suppressed by Caudal and Nubbin, while bacterial-derived uracil is important for distinguishing pathogens from commensals (Bahuguna, 2022).

The evolutionary conserved Target of Rapamycin (TOR) pathway is a major pathway controlling cellular metabolism and growth. TOR balances lipid and glucose anabolism and catabolism in the cell through the activity of the TORC1 protein complex. TORC1 promotes protein synthesis primarily through phosphorylation of the eIF4E Binding Protein (4E-BP/Thor) and p70S6 Kinase 1 (S6K1). 4E-BP is a translational inhibitor, which binds and inhibits the activity of eIF4E an eukaryotic translation initiation factor responsible for the recruitment of 40s ribosomal subunit at the 5'-cap of mRNA. Phosphorylation of 4E-BP lowers its affinity towards eIF4E. This frees eIF4E, enabling it to promote cap-dependent translation. In the case of S6K1, activated S6K1 promotes protein synthesis by activating inducers of mRNA translation initiation whilst degrading inhibitors (Bahuguna, 2022).

Drosophila TOR has been extensively studied for its role in growth and development, using fly mutants or by treating flies with rapamycin. Rapamycin treatment in stress conditions, led to upregulation of 4E-BP activity resulting in an increase of whole-fly lipid reserves that could be used for the long-term survival of these stress conditions. In contrast, 4E-BP mutants were unable to preserve lipid stores and had thus compromised survival following starvation or oxidative stress. More broadly, the consensus is that in both Drosophila and mice, 4E-BP regulates fat levels in stress conditions like starvation and oxidative stress. During larval stages and when in food with poor nutritional value, the presence of the commensal Lactobacillus plantarum is important to sustain development through TOR, which is in turn crucial for sustaining this mutualistic relationship. The metabolic state of the gut is also influenced by dietary conditions and in its turn influences the bacteriome. Diet-dependent adaptations of the microbiota require NF-κB-dependent control of the translational regulator 4E-BP and this where TOR and NF-κB 'meet' (Bahuguna, 2022).

Drosophila has three NF-κB proteins namely, Relish, Dorsal and the Dorsal-related Immunity Factor (DIF). DIF is downstream of the Toll signalling pathway. Toll and Toll-like receptor (TLR) signalling is one of the most important evolutionary conserved mechanisms by which the innate immune system senses the invasion of pathogenic microorganisms. Unlike its mammalian counterparts however, Drosophila Toll is activated by an endogenous cytokine-like ligand, the Nerve Growth Factor homologue, Spz. Spz is processed to its active form by the Spz-Processing Enzyme (SPE). Two serine protease cascades converge on SPE: one triggered by bacterial or fungal serine proteases through the host serine protease Persephone and a second activated by host receptors that recognise bacterial or fungal cell wall. Prominent among these host receptors is the Peptidoglycan Recognition Protein-SA or PGRP-SA. PGRP-SA binds to peptidoglycan on the bacterial cell wall without structural preference but depending on accessibility and generates the downstream signal (Bahuguna, 2022).

When the recognition signal reaches the cell surface, it is communicated intracellularly via the Toll receptor and a membrane-bound receptor-adaptor complex including dMyd88, Tube (as an IRAK4 functional equivalent) and the Pelle kinase (as an IRAK1 functional homologue). Transduction of the signal culminates in the phosphorylation of the IκB homologue, Cactus probably by Pelle, leaving the NF-κB homologue DIF to move to the nucleus and regulate hundreds of target genes including antimicrobial peptides (AMPs) (Bahuguna, 2022).

In this study, evidence is presented that PGRP-SA is important for the preservation of commensal intestinal bacterial density. The results reveal that larvae and adults that are deficient in PGRP-SA or DIF have a significantly reduced commensal gut bacterial density. Inhibition of the activity of TOR by Rapamycin or TOR RNAi in enterocytes, restores bacterial density (but not diversity) in PGRP-SA or DIF mutant guts. However, flies mutants for PGRP-SA and deficient for 4EBP in enterocytes were unable to restore bacterial density upon Rapamycin treatment or TOR RNAi, demonstrating the important role of 4EBP. PGRP-SA mutants had increased intestinal fat stores that were restored to normal levels through Rapamycin or TOR-RNAi treatment in enterocytes. This restoration failed in PGRP-SA;4EBP double mutants indicating that 4EBP was crucial in regulating fat stores in the gut. Fat catabolism was important for gut bacterial restoration as flies deficient for PGRP-SA and treated with rapamycin were unable to restore bacterial density if the triglyceride lipase Brummer was knocked down in enterocytes. This mechanism gives an insight into how the host integrates immunity and metabolism to maintain commensal bacteria at the intestinal epithelium (Bahuguna, 2022).

In the absence of the bacterial receptor PGRP-SA from enterocytes, a reduction was observed in intestinal bacterial density. It was restored with the use of rapamycin, which targets TORC1 or by knocking-down TOR in enterocytes. This suggested that loss of the immune receptor PGRP-SA, generated a metabolic environment unfavourable for intestinal bacterial growth. The results indicated that at the centre of this relationship was 4E-BP, which is activated by Toll and suppressed by TORC1. In keeping with this, PGRP-SA^seml; NP1GAL4>4E-BPRNAi flies treated with rapamycin were unable to restore gut bacterial density. Intestinal lipid catabolism downstream of 4EBP was paramount for the maintenance of cultivable bacterial density because the loss of the lipase Bmm blocked restoration of gut bacteria after rapamycin treatment. Silencing of bmm in enterocytes caused intestinal lipid accumulation and prevented any restoration via rapamycin in PGRP-SA^seml flies (Bahuguna, 2022).

These results indicate that downstream of Toll, intestinal triglyceride levels were under 4E-BP control in enterocytes. Although the phenomenon of cultivable bacteriome reduction in PGRP-SA^seml flies was readily manifested in larvae and young flies, the results indicated that it was also there in older flies. Conventional fly rearing techniques ensure a steady stream of defaecation and re-introduction of bacteria over time. However, when food vials were changed rapidly re-infection was reduced and CFUs in 30-day old flies were significantly lower in PGRP-SA^seml than yw flies. Preservation of the bacteriome was dependent on PG recognition as the rescue of enteric CFUs in PGRP-SA^seml flies was only possible with PGRP-SA transgenes that had an intact PG binding ability. This indicated that bacterial sensing was the initial trigger point to activate the process (Bahuguna, 2022).

A working model is depicted A schematic model outlining the role of PGRP-SA/Toll/Dif in the retention of the gut bacteriome. PGRP-SA recognises components of the intestinal bacteriome and activates the Toll pathway in enterocytes. In turn, this keeps increased 4E-BP transcription/4E-BP protein phosphorylation in enterocytes, preserving a steady rate of intestinal lipid catabolism. The latter is important for maintaining normal density of commensal bacteria. It is hypothesised that Bmm-mediated lipid catabolism is regulated by 4E-BP and released triglycerides act as fuel for the maintenance of commensal bacteria. In keeping with this, stopping lipid catabolism by silencing the Bmm lipase in ECs resulted in accumulation of lipids and reduction of enteric CFUs. According to the model, bacteria should trigger lipid catabolism and 5-day old axenic flies showed a clear trend for lipid accumulation in their gut, but this was marginally not statistically significant. Studies with Vibrio cholera, have shown that intestinal acetate leads to deactivation of host insulin signalling and lipid accumulation in enterocytes, resulting in host lethality. Loss of PGRP-SA/Dif leads to a decrease in lifespan. Whether this is due to the long-term accumulation of lipids is an open question (Bahuguna, 2022).

PGRP-SA recognises components of the intestinal bacteriome and activates the Toll pathway in enterocytes. This increases 4E-BP transcription/4E-BP protein phosphorylation in enterocytes. 4EBP is important for maintaining normal density of commensal bacteria. It is hypothesized that Bmm-mediated lipid catabolism is regulated by 4E-BP and released triglycerides act as fuel for the maintenance of commensal bacteria (Bahuguna, 2022).

More work is needed to understand whether/how stored intestinal lipids maybe released to circulation, how commensal bacteria receive them and which component(s) of the bacteriome are recognised by PGRP-SA (Bahuguna, 2022).

REGULATION

Promoter Structure

An important question with regard to the possibilities of Thor induction being involved in cell growth regulation is whether or not eIF4E also is induced by infection. In mammalian cells, overexpression of eIF4E is associated with malignant transformation, and concomitant overexpression of 4E-BP has been shown to negate this overgrowth (for review see Clemens, 1999). In the Drosophila immune response, large quantities of antimicrobial proteins are produced and one possibility is that eIF4E could be up-regulated to increase translation. In this scenario, Thor up-regulation would have the homeostatic function of producing more 4E-BP to keep growth regulation in balance. The hypothesis that eIF4E also is up-regulated by bacterial infection was tested. This is not the case; Thor is up-regulated upon infection while the levels of eIF4E mRNA remain the same. Therefore, if these two components are coordinately regulated, it is not at the level of transcription (Bernal, 2000).

All of the promoter regions studied for Drosophila antimicrobial genes induced by infection have been found to have elements similar to those of immune inducible genes in mammals. Sequence analysis of the 5'-flanking region has determined that Thor has an array of these types of elements. The Thor promoter has the canonical NFkappaB recognition sequence that has been shown to be essential for immune induction. Furthermore, Thor has the GATA sequence associated with NFkappaB elements that has been also shown to be important for immune induction and conserved in other Drosophila species. Additional sequences found upstream of Drosophila immune response genes have been also identified, in particular those involved in vertebrate cytokine regulation and liver specific expression. TRANSFAC analysis has identified more sequences related to liver-specific regulation, such as hepatocyte nuclear factor/forkhead, and also interferon-related regulatory sequences (Bernal, 2000).

Transcriptional Regulation

Foxo regulates cell cycle arrest possibly by transcriptionally activating genes implicated in cell division or in cell growth. As an initial attempt to identify target genes of Foxo, DNA microarrays were used to assess gene expression profiles in S2 cells stably transfected with mutant Foxo and grown in the presence of insulin. Cells expressing wild-type Foxo or untransfected S2 cells subjected to the same treatment were assayed as controls (Puig, 2003).

Two-hundered and seventy-seven genes were found to be up-regulated in Foxoa3-expressing cells when compared with Foxo-expressing cells or untransfected S2 cells. Interestingly, two genes that were consistently and specifically up-regulated in these conditions were the Drosophlia InR gene (13.5-fold) and the Drosophila 4EBP gene (25-fold). Both genes have been implicated in the regulation of cell growth by insulin. To confirm that InR and 4EBP are bona fide transcriptional targets of Foxo, the same experiment described above was performed but in the presence of cycloheximide to inhibit translation. As expected, both InR and 4EBP continue to be transcriptionally activated (2.5- and 3.1-fold, respectively) by FOXOA3 but not Foxo in the insulin-repressed state. This result suggests that Foxo, when released from control by the insulin/dAkt cascade, is involved in transcription from the InR and 4EBP promoters (Puig, 2003).

To confirm these microarray results and to independently quantitate the increase in mRNA transcription, RNase protection assays were performed with mRNAs extracted from cells stably transfected with either Foxo or FoxoA3. Indeed, FoxoA3 stimulates transcription of Drosophila 4EBP and InR by 16.3- and 11-fold, respectively. A time-course experiment confirmed that Drosophila InR mRNA increases rapidly upon FoxoA3 expression: 3 h after CuSO₄ addition, there is already an 8-fold increase, reaching 20-fold after 9 h of CuSO₄ induction. Similar results were obtained for Drosophila 4EBP. These experiments suggest that Foxo expression specifically activates both Drosophila InR and 4EBP transcription, thus unmasking an important feedback control mechanism in this pathway involving Foxo and InR (Puig, 2003).

Having obtained evidence that exogenously transfected Foxo responds to insulin and regulates both the downstream target gene 4EBP and the feedback control target InR, it was of interest to know if endogenous Foxo would also activate transcription of these genes. The PI3K inhibitor LY294002 was used to activate endogenous Foxo or insulin to deactivate it. S2 cells grown in the absence of serum for 48 h were treated either with LY294002 or insulin. Total RNA was extracted and RNase protection was performed to detect Drosophila InR and 4EBP mRNAs. Both mRNA levels are significantly increased after LY294002 treatment (5.3-fold for dInR and 4-fold for d4EBP) when compared with insulin treatment. This result provides further evidence indicating that the PI3K–Akt pathway regulates InR and 4EBP transcription via Foxo (Puig, 2003).

It was of interest to determine whether Foxo directly binds to the promoters of Drosophila 4EBP and InR. To identify the DNA region recognized by Foxo in these two promoters, a 1708-bp fragment of the 4EBP promoter and a 1562-bp fragment of the InR promoter were inserted into a luciferase reporter vector. When transfected into S2 cells, these fragments responded to Foxo activation (3-fold for 4EBP, >200-fold for InR. A series of deletions lacking upstream sequences still responded to Foxo activation, albeit more weakly, suggesting that Foxo can bind the DNA in a region close to the start of transcription (485 bp for the d4EBP promoter and 194 bp for the dInR promoter). In contrast, Foxo completely fails to activate a reporter construct in which upstream activating sequences (UAS) for the transcription factor GAL4 are fused to the luciferase gene, confirming that transcription activation is specific for both 4EBP and InR promoters (Puig, 2003).

Interestingly, 125 bp upstream of the transcription start site of the d4EBP promoter there are three tandem copies of a putative FOXO4 recognition element (FRE). These elements are reminiscent of the ones present in the human glucose-6-phosphatase promoter, previously shown to bind FOXO4 (Yang, 2002). This was reassuring because Foxo and FOXO4 share 85% identity in the core of the forkhead DNA-binding domain. Similarly, several putative FRE sequences appear in the InR promoter in the region comprising nucleotides -1434 to -70 (Puig, 2003).

To determine whether Foxo binds these putative FREs, band shift experiments were performed with a 113-bp DNA probe encompassing the 4EBP FRE motifs and with 12 separate DNA probes (ranging from 100 to 150 bp) spanning a region of 1.4 kb from the InR promoter. Purified recombinant Foxo expressed in Escherichia coli efficiently binds the 113-bp FRE-containing fragment from the 4EBP promoter compared with control DNA fragments. Furthermore, Foxo binding to the 4EBP promoter fragment can be efficiently competed with an unlabeled 113-bp 4EBP promoter fragment but not with nonspecific DNA. Similarly, purified recombinant Foxo binds efficiently to 5 out of 12 of the DNA fragments located within the InR promoter. As expected, each of the five DNA fragments bound by Foxo contains putative FREs. Thus, Foxo can specifically bind to both promoters in vitro. To determine whether Foxo also binds these same DNA regions in vivo, chromatin immunoprecipitation (ChIP) experiments were performed with S2 cells expressing either Foxo or dFoxoA3. Cells were incubated with serum, and Foxo expression was induced with the addition of CuSO₄. After 6 h, cells were cross-linked with formaldehyde, and extracts were prepared and immunoprecipitated. After reversal of cross-links, DNA was recovered, and PCR was performed with primers encompassing regions containing putative FREs in both promoters. The results indicate that Foxo can directly bind to both the 4EBP and InR promoters in vivo. These results establish that Foxo can specifically bind the 4EBP and InR promoters both in vitro and in vivo (Puig, 2003).

To demonstrate that Foxo can directly activate transcription of these promoters in vitro, the constructs were used that contain 485 bp of the 4EBP promoter region and 514 bp of the InR promoter region, respectively. Addition of purified recombinant Foxo to in vitro reactions activates transcription of these promoters by at least 3-fold (4EBP) and 5.5-fold (InR), which is comparable to the activation observed in vivo. Under in vitro transcription conditions, activation of the 4EBP promoter by Foxo becomes rapidly saturated with increasing amounts of Foxo. As expected, Foxo also activates (up to sixfold) a synthetic promoter bearing four FOXO4-binding sites placed upstream of the alcohol dehydrogenase distal promoter. Together these results show that transcription of 4EBP and InR can be directly activated by Foxo in vitro (Puig, 2003).

Drosophila embryonic Kc167 cells respond to insulin stimulation with upregulated activities of PKB and S6K. mRNA profiling experiments were performed using the Affymetrix GeneChip system to measure on a genome-wide scale the transcriptional changes induced by insulin in these cells. On the basis of the currently held model that FOXO transcription factors are transcriptional activators that are negatively regulated by insulin, potential Foxo target genes were expected to be repressed in Kc167 cells upon insulin stimulation. Foxo target gene candidates were selected that are transcriptionally downregulated by a factor of two or more upon insulin stimulation and whose promoter regions contain one or more conserved forkhead-response elements (FHREs) with the consensus sequence (G/A)TAAACAA. Three of these candidate gene products are each involved in one of two biological processes known to be negatively regulated by insulin, namely gluconeogenesis (PEPCK) and lipid catabolism (CPTI and long-chain-fatty-acid-CoA-ligase). The remaining candidates are involved in stress responses (cytochrome P450 enzymes), DNA repair (DNA polymerase iota), transcription and translation control (4E-BP and CDK8), and cell-cycle control (centaurin gamma and CG3799). Several of the insulin-repressed genes have been reported to be transcriptionally induced in Drosophila larvae under conditions of complete starvation (4E-BP and PEPCK) or sugar-only diet (CPTI and long-chain-fatty-acid-CoA-ligase) (Jünger, 2003).

4E-BP was chosen for further investigation, because it has previously been reported to be insulin-regulated at the level of protein phosphorylation, but not at the level of gene expression. The 4E-BP gene encodes a translational repressor and was initially identified as the immune-compromised Thor mutant in a genetic screen for genes involved in the innate immune response to bacterial infection. There are several FHREs in the genomic region around the 4E-BP locus. The 4E-BP protein is negatively regulated by insulin through LY294002- and rapamycin-sensitive phosphorylation, suggesting involvement of the Dp110 and TOR signaling pathways. Phosphorylation of 4E-BP leads to the dissociation of 4E-BP from its binding partner, the translation initiation factor eIF4E, which then participates in the formation of a functional initiation complex. Positive transcriptional regulation of 4E-BP by Foxo, which corresponds to negative transcriptional regulation by insulin, would be a complementary mechanism of regulation (Jünger, 2003).

Whether overexpression of endogenous foxo can induce transcriptional upregulation of the 4E-BP gene was investigated. On the basis of overexpression results, the Dp110DN-Foxo coexpression was used to efficiently activate Foxo. Eye imaginal discs from Dp110DN-expressing third instar larvae display a low level of basal 4E-BP transcription throughout the disc, which is not induced by the driver construct alone. Coexpression of foxo elicits a dramatic upregulation of 4E-BP transcription posterior to the morphogenetic furrow. Consistent with this observation, it was possible to induce expression of the 4E-BP enhancer trap line Thor¹ with human FOXO3a-TM . It remains unclear, however, whether regulation of d4E-BP expression by Foxo is of physiological relevance (Jünger, 2003).

Overexpression of 4E-BP partially suppresses the PKB overexpression phenotype, but since ectopic expression experiments have to be interpreted with some caution, whether loss of 4E-BP function suppresses the cell-number reduction in insulin-signaling mutants as does loss of Foxo function was investigated. Double-mutant flies were generated for PKB and 4E-BP and it was observed that the Thor¹ mutation slightly but significantly suppressed the reduced cell-number phenotype in a dose-dependent manner. The Thor¹ mutation itself had no effect on ommatidial number compared to wild-type flies, so additive effects of d4E-BP and dPKB can be ruled out. These observations strongly argue that under conditions of reduced insulin-signaling activity, the Foxo-dependent reduction in cell number is in part mediated by the transcriptional upregulation of its target 4E-BP. Microarray studies in both mammalian and Drosophila cells imply that FOXO transcription factors exert their physiological functions by modulating expression of large sets of target genes (Jünger, 2003).

All animals coordinate growth and maturation to reach their final size and shape. In insects, insulin family molecules control growth and metabolism, whereas pulses of the steroid 20-hydroxyecdysone (20E) initiate major developmental transitions. 20E signaling also negatively controls animal growth rates by impeding general insulin signaling involving localization of the transcription factor dFOXO and transcription of the translation inhibitor 4E-BP. The larval fat body, equivalent to the vertebrate liver, is a key relay element for ecdysone-dependent growth inhibition. Hence, ecdysone counteracts the growth-promoting action of insulins, thus forming a humoral regulatory loop that determines organismal size (Colombani, 2005).

FOXO-regulated transcription restricts overgrowth of Tsc mutant organs

FOXO is thought to function as a repressor of growth that is, in turn, inhibited by insulin signaling. However, inactivating mutations in Drosophila melanogaster FOXO result in viable flies of normal size, which raises a question over the involvement of FOXO in growth regulation. Previously, a growth-suppressive role for FOXO under conditions of increased target of rapamycin (TOR) pathway activity was described. This study further characterizes this phenomenon. Tuberous sclerosis complex 1 mutations cause increased FOXO levels, resulting in elevated expression of FOXO-regulated genes, some of which are known to antagonize growth-promoting pathways. Analogous transcriptional changes are observed in mammalian cells, which implies that FOXO attenuates TOR-driven growth in diverse species (Harvey, 2008).

To investigate mechanisms by which the TOR pathway controls tissue growth, transcriptional profiles were analyzed of tissue lacking Tsc1, which leads to hyperactivation of the TOR pathway and excessive growth. Eye-antennal imaginal discs from third instar Drosophila larvae were generated that were composed almost entirely of tissue derived from one of two different genotypes: Tsc1 or wild-type isogenic control. Three biologically independent first strand cDNA samples from each genotype were hybridized to microarray chips. Expression levels of 157 genes were elevated 1.5-fold or more, whereas 211 genes were repressed 1.5-fold or more when compared with control tissue. These genes have been implicated in diverse cellular functions including metabolism, membrane transport, stress response, cell growth, and cell structure (Harvey, 2008).

Observed transcriptional changes were validated for several genes using Drosophila gene-enhancer trap lines. The UAS-Gal4 system was used to activate the TOR pathway in a specific tissue domain by driving expression of Rheb under the control of the glass multiple reporter (GMR) promoter. Induction of astray (aay) and 4E-BP (both of which were found to be elevated in Tsc1 tissue by microarray analysis) were observed in the GMR expression domain (posterior to the morphogenetic furrow) when Rheb was misexpressed but were not induced when the negative control Gal4 gene was misexpressed. QPCR was also used to confirm expression changes observed in Tsc1 tissue for charybdis (chrb), scylla (scy), phosphoenolpyruvate carboxy kinase, 4E-BP, and aay (Harvey, 2008).

Intriguingly, several gene products whose expression was elevated in Tsc1 tissue have been implicated in tissue growth controlled by the insulin and TOR pathways, including 4E-BP, Chrb, and Scy. 4E-BP is a repressor of cap-dependent translation. Upon phosphorylation by TOR, 4E-BP dissociates from eIF4e, allowing assembly of the initiation complex at the mRNA cap structure, ribosome recruitment, and subsequent translation. Scy and Chrb, and their mammalian orthologues REDD1 and REDD2, inhibit insulin and TOR signaling in response to hypoxia and energy stress and restrict growth during Drosophila development. The finding that inhibitors of growth are highly expressed in Tsc1 tissue led to the hypothesis that such genes are transcriptionally induced as part of a feedback loop that restricts tissue growth under conditions of excessive TOR activity. Feedback loops are an important activity-modulating feature of many signaling pathways, including the TOR and insulin pathways (Harvey, 2008).

To examine the mechanism whereby transcription of growth inhibitors is induced in response to TOR hyperactivation, attempts were made to determine which transcription factors were responsible for their expression. One obvious candidate was FOXO, a member of the forkhead transcription factor family, which has a well-established role as an effector of insulin signaling. If FOXO has a role in inducing expression of negative regulators of growth in Tsc tissue, then expression of some of those genes should be elevated under conditions of increased FOXO activity. To investigate this hypothesis, the expression profiles were examined of Tsc1 LOF tissue and Drosophila S2 cells expressing FOXOA3, a mutant version of FOXO that is insensitive to phosphorylation-dependent inhibition by Akt. This analysis revealed that 25 genes were up-regulated 1.5-fold or greater in both Tsc1 LOF and FOXO GOF expression profiles, which represents a highly significant degree of overlap as determined by calculation of the hypergeometric distribution. A highly statistically significant P value strongly suggests that there is a functional overlap between these two datasets that cannot be explained by random variation (Harvey, 2008).

Interestingly, two genes previously implicated in tissue growth regulated by the insulin and TOR pathways 4E-BP and scy were elevated in both microarray experiments, whereas the chrb growth-inhibiting gene was not. Thus, a subset of genes elevated in Tsc1 tissue appears to respond to FOXO activity and was investigated further (Harvey, 2008).

4E-BP is a well-characterized FOXO target gene. To determine whether FOXO could directly activate transcription of genes that were elevated in Tsc1 tissue other than 4E-BP, focus was placed on scy and the phosphoserine phosphatase aay (one of the most highly elevated transcripts in each microarray experiment). scy and aay both possess consensus FOXO recognition elements (FREs) in their promoters comparable to those found in dInR and 4E-BP promoters. Therefore, whether these genes are bona fide FOXO targets was examined by measuring their expression in Drosophila S2 cells misexpressing FOXOA3 in the presence of insulin. aay and scy mRNAs were up-regulated 19.4- and 4.3-fold, respectively, relative to a control gene, actin, as determined by QPCR (Harvey, 2008).

Luciferase reporter assays in S2 cells were used to determine whether the aay promoter region containing putative FREs was sensitive to FOXO activity. Luciferase activity dependent on the aay promoter was strongly induced by FOXOA3. In addition, using in vitro band shift assays, it was demonstrated that FOXO directly binds to the aay promoter, indicating that FOXO likely activates expression of aay by directly binding to the FRE. Surprisingly, in parallel luciferase reporter assays, activation of the scy promoter by FOXO could not be demonstrated, despite the fact that strong binding of FOXO to the putative scy FRE was observed using in vitro band-shift assays. A possible explanation is that the scy-promoter construct lacked the minimal promoter elements required for transcription of luciferase (Harvey, 2008).

TOR pathway hyperactivation caused by Tsc deficiency has been shown to strongly repress activity of Akt. FOXO is normally inactivated by Akt-dependent phosphorylation, which restricts nuclear entry of FOXO and leads to its ubiquitin-dependent destruction. Therefore, in response to TOR pathway hyperactivation, it was predicted that reduced Akt activity would cause FOXO protein to accumulate. To examine this hypothesis, expression of FOXO protein was analyzed in mosaic Tsc1 imaginal discs. It was found that FOXO protein was markedly increased in Tsc1 clones when compared with neighboring wild-type tissue (Harvey, 2008).

In addition, FOXO protein appeared to be mostly nuclear in Tsc1 tissue and cytoplasmic in wild-type tissue. Consistent with this observation, nuclear localization of the mouse FOXO orthologue FOXO1 is observed in endothelial cells of Tsc2 mutant hemangiomas, whereas FOXO1 is mostly cytoplasmic in normal cells. FOXO mRNA levels are unchanged in Tsc1 tissue as determined by microarray analysis, which suggests that changes in translation or stability of FOXO protein account for its accumulation in Tsc1 tissue. The presence of increased FOXO protein in the nuclei of Tsc1 cells is consistent with the hypothesis that FOXO is responsible for increased expression of some of the growth inhibitors that are up-regulated in Tsc1 cells (Harvey, 2008).

To determine whether FOXO was necessary for transcriptional induction of genes that were elevated in Tsc1 tissue, QPCR analysis was used to measure 4E-BP, aay, and scy expression in Tsc1 and Tsc1-FOXO double mutant eye-antennal imaginal discs. Consistent with microarray analysis, increased expression of 4E-BP, aay, and scy was observed in Tsc1 tissue. In Tsc1-FOXO tissue, however, 4E-BP was expressed at approximately equivalent amounts as in wild-type tissue, whereas aay and scy expression was only partially reduced. This demonstrates that elevated expression of 4E-BP in Tsc1 tissue is dependent on the FOXO transcription factor and provides evidence that FOXO activity increases when the TOR pathway is hyperactivated. Expression of aay and scy appear to be partially dependent on FOXO but are likely stimulated by additional transcription factors in Tsc1 tissue (Harvey, 2008).

Next, attempts were made to determine whether FOXO is required to limit growth of tissues with increased TOR pathway activity. In addition, a potential role was examined for another transcription factor, HIF-1, for retardation of TOR-driven growth. HIF-1 is a dual-subunit transcription factor consisting of α and β subunits that functions in response to insulin/TOR signaling and drives transcription of the growth-inhibiting genes scy and chrb, both of which are elevated in Tsc1 tissue (Harvey, 2008).

Drosophila possesses several HIF-1α subunits and a sole HIF-1β subunit, tango (tgo), which partners with each HIF-1α subunit. If FOXO and/or HIF-1 are required to induce expression of genes that limit tissue growth when the TOR pathway is hyperactivated, one might predict that Tsc1-FOXO and/or Tsc1-tgo double mutant tissue would possess a greater capacity to grow than Tsc1 tissue alone. To test this hypothesis, the size was examined of Drosophila eyes comprised almost entirely of the following genotypes: control, tgo, FOXO, Tsc1, Tsc1-tgo, and Tsc1-FOXO. Mutant eyes were created by driving mitotic recombination of chromosomes bearing flipase recognition target (FRT) sites and the appropriate gene mutations, specifically in developing Drosophila eye-antennal imaginal discs. Eyes lacking either tgo or FOXO were approximately the same size as control eyes, whereas Tsc1 eyes were considerably larger. Tsc1-tgo double mutant eyes did not exhibit a further increase in size, which suggests that HIF-1 is not required to inhibit tissue growth in response to Tsc1 loss. In contrast, Tsc1-FOXO double mutant eyes were substantially larger than Tsc1 eyes. This finding is particularly significant in light of the finding that eyes lacking FOXO were indistinguishable in size from wild-type eyes. Thus, it appears that FOXO is normally dispensable for control of eye size, but when growth control is altered by virtue of increased TOR activity, FOXO partially offsets the increased tissue growth. These findings are consistent with observations that FOXO protein accumulates in Tsc1 tissue and that transcriptional profiles of FOXO GOF and Tsc1 LOF cells overlap significantly (Harvey, 2008).

Because individual components of the insulin and TOR pathways are highly conserved among eukaryotes, important regulatory mechanisms that control tissue growth via these pathways are also likely to be conserved. To investigate this idea, transcriptional control was analyzed of mouse orthologues of genes that were elevated in D. melanogaster Tsc1 tissue. Initially, Northern blotting analysis was performed on Tsc2 primary mouse embryonic fibroblasts (MEFs; derived on a p53 background to overcome premature senescence induced by Tsc2 loss). It is reasonable to predict that transcriptional changes that occur because of loss of either Tsc1 or Tsc2 should be very similar because TSC1 and TSC2 function together in an obligate fashion, and mutation of either gene leads to almost indistinguishable phenotypes. It was found that several gene expression changes observed in Drosophila Tsc1 tissue are conserved in Tsc2 MEFs (Harvey, 2008).

The homologues of aay, heat shock protein (hsp) 23, scy, and chrb (PSPH, hsp 27, REDD1, and REDD2, respectively) were all significantly up-regulated in Tsc2 MEFs when compared with control MEFs and expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control. To demonstrate that these expression changes were a specific consequence of Tsc2 loss, Tsc2 expression was reconstituted in Tsc2 null cells, which substantially suppressed mammalian TOR activity and expression of these genes. Interestingly, expression of phosphoenolpyruvate carboxy kinase and 4E-BP1/2 was not altered between wild-type and Tsc2 cells, which might reflect tissue- or species-specific differences in the transcriptome of Drosophila epithelial cells and MEFs (Harvey, 2008).

To determine whether the mode of transcription of these genes was also conserved in mammals, expression was analyzed of the scy homologue REDD1. Like scy, mammalian REDD1 orthologues possess a putative consensus FRE within their proximal promoters. Cotransfection of a version of FOXO that is insensitive to phosphorylation-dependent inhibition by Akt (TM-FKHRL-1) induced robust activation of a mouse REDD1 reporter construct in primary MEFs. To determine whether induction was mediated through the identified FRE, a mutant reporter was created lacking this sequence. Deletion of the REDD1 FRE consistently reduced FOXO-mediated induction of the REDD1 promoter. Finally, to directly assess whether FOXO-dependent transcription was activated in mammalian cells lacking Tsc2, activity of the REDD1 promoter reporter or the corresponding mutant FRE reporter was examined in wild-type and Tsc2 MEFs. As predicted, the wild-type REDD1 promoter exhibited robust activation in Tsc2 cells compared with wild-type cells, and this activation was substantially reduced by deletion of the FRE. Together, these findings provide evidence that transcriptional changes resulting from Tsc1/Tsc2 deficiency are conserved in diverse species (Harvey, 2008).

This study has identified of an evolutionary conserved transcriptional program important for restricting tissue overgrowth driven by excessive activation of the TOR pathway. The FOXO transcription factor plays a key role in this transcriptional response, likely by stimulating expression of several growth inhibitory genes. Thus, although the requirement for FOXO in restricting growth under normal development conditions appears dispensable, this is no longer the case under conditions of excessive TOR activation. These findings have important implications for cancer syndromes that arise because of inappropriate TOR pathway activation, such as the human hamartomatous syndrome, tuberous sclerosis. TOR-dependent feedback inhibition is thought to contribute to the benign nature of Tsc1 and Tsc2 tumors (Ma, 2005; Manning, 2005). Conceivably, inactivating mutations in FOXO family transcription factors and/or FOXO target genes that possess growth-inhibiting properties could promote further growth in normally benign Tsc1 and Tsc2 tumors (Harvey, 2008).

Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila

Animals use the insulin/TOR signaling pathway to mediate their response to fluctuations in nutrient availability. Energy and amino acids are monitored at the single-cell level via the TOR branch of the pathway and systemically via insulin signaling to regulate cellular growth and metabolism. Using a combination of genetics, expression profiling, and chromatin immunoprecipitation, this study examined nutritional control of gene expression and identified the transcription factor Myc as an important mediator of TOR-dependent regulation of ribosome biogenesis. myc was also identified as a direct target of FOXO, and genetic evidence is provided that Myc has a key role in mediating the effects of TOR and FOXO on growth and metabolism. FOXO and TOR also converge to regulate protein synthesis, acting via 4E-BP and Lk6, regulators of the translation factor eIF4E. This study uncovers a network of convergent regulation of protein biosynthesis by the FOXO and TOR branches of the nutrient-sensing pathway (Teleman, 2008).

The global transcriptional analysis reported in this study has revealed a surprising degree of interconnectedness between the two branches of the nutrient-sensing pathway. Insulin, acting through PI3K and Akt, feeds into the FOXO and TORC1 branches of the pathway, whereas energy levels (AMP/ATP) and amino acids act directly on the TORC1 branch. How are these inputs integrated to maintain energy balance? It was previously known that 4E-BP is transcriptionally regulated by FOXO and posttranslationally regulated by TOR. This study has identified the protein kinase Lk6 as a second direct FOXO target. Thus, there appear to be two parallel, independent mechanisms by which the TOR and FOXO branches of the insulin signaling pathway converge to regulate eIF4E activity and hence cellular protein translation. This 'belt and suspenders' approach to translational control might be important to make the system robust (Teleman, 2008).

A key finding of this study is the identification of Myc as a point of convergent regulation by the FOXO and TOR branches of the pathway. myc mRNA levels are controlled by FOXO in a tissue-specific manner. In addition, Myc protein levels are dependent on TORC1. Why use two independent means to control Myc levels? Transcription alone would limit the speed with which the system can respond to changing nutritional conditions. This might be detrimental, particularly as conditions worsen. Regulation of Myc activity by TORC1 permits a rapid response to changes in energy levels or amino acid availability and could serve to fine tune the nutritional response in the cell by controlling translational outputs. This parallels the situation with 4E-BP, albeit with a slightly different logic. Reduced insulin signaling allows FOXO to enter the nucleus and increase 4E-BP expression and at the same time alleviates TORC1-mediated inhibition of the existing pool of 4E-BP. A subsequent increase in energy or amino acid levels would permit rapid reinhibition of 4E-BP and thus allow a flexible response during the time needed for the pool of protein elevated in response to reduced insulin levels to decay (Teleman, 2008).

In yeast, TORC1 is known to regulate ribosome biogenesis through different nuclear RNA polymerases. It has been shown that yeast TORC1 can bind DNA directly at the 35S rDNA promoter and activate Pol I-mediated transcription in a rapamycin-sensitive manner. Moreover, yeast TORC1 is known regulate Pol II-dependent RP gene expression by controlling the nuclear localization of the transcription factor SFP1 and CRF1, a corepressor of the forkhead transcription factor FHL1. In Drosophila, TORC1 has recently been reported to regulate a set of protein-coding genes involved in ribosome assembly. This study has identified Myc as the missing link mediating TORC1-dependent regulation of this set of genes. Indeed, the fact that more than 90% of TORC1-activated genes contain E boxes suggests that Myc might be the main mediator of this transcriptional program. This connection suggests that expression of Myc targets as a whole should be responsive to nutrient conditions. Indeed, this study found that 33% of direct Myc targets -- defined as genes reported to be bound by Myc when assayed by DNA adenine methyltransferase ID (DamID) in Kc cells and to be regulated by myc overexpression in larvae -- are downregulated upon nutrient deprivation. This is a significant enrichment of 4-fold relative to all genes in the genome, despite the comparison being based on correlating data from different tissue types (Teleman, 2008).

It seems reasonable that cellular translation rates need to be dampened if the TOR branch of the pathway senses low amino acid levels. As ribosome biogenesis is energetically expensive, it may be advantageous to link ribosome biogenesis and translational control via TORC1. This dual regulation is well reflected in tissue growth, since this study observed that Myc, the regulator of ribosome biogenesis, is essential for tissue growth driven by the TOR pathway but not sufficient to drive growth in the absence of TOR activity. The FOXO branch of the pathway senses reduced insulin or mitogen levels. FOXO is also highly responsive to oxidative and other stresses and would integrate this information into the cellular control of translation. The data support the notion of a network in which TOR and FOXO regulate protein biosynthesis by converging on Myc to regulate ribosome biogenesis and on eIF4E activity via 4E-BP and Lk6 to regulate translation initiation (Teleman, 2008).

The work presented in this study complements a previous study in which larvae were either starved completely or starved for amino acids only, while having a supply of energy in the form of sugar. A significant and positive correlation (~0.4) indicates general agreement between the two data sets, but they differ in two ways. The current goal was to explore the regulatory network by which insulin controls cellular transcription. Individual tissues were isolated rather than assaying the whole animal. Genes found to be regulated in a previous but not in the current assays may be regulated in tissues other than muscle or adipose tissue. Conversely, genes identified only by the current study might be regulated oppositely in different tissues or might only be regulated in a subset of tissues and so be missed in a whole-animal analysis.

Is Myc also involved in nutritional signaling networks in mammals? No similar rapid downregulation of c-myc was seen in response to rapamycin in human cell lines, suggesting that the mechanism by which TOR signaling controls gene expression may differ between phyla. This is further supported by the fact that the sets of genes reported to be rapamycin regulated also appear to be largely distinct in Drosophila and mammalian cells, with the caveat that different cell types were used in the two analyses. Although the mechanism does not appear to be identical in mammals, there are several suggestions in the literature of a connection between c-Myc and nutritional signaling. For example, dMyc and c-Myc share the ability to regulate ribosome biogenesis, although the specific target genes through which they do so are different. There is also evidence that mammalian c-myc expression in liver is regulated by nutrition and that transgenic expression of c-myc in liver affects metabolism, i.e., glucose uptake and gluconeogenesis. Furthermore, it has been reported that FOXO3 represses Myc activity in colon cancer cells by inducing members of the Mad/Mxi family, which are known to antagonize Myc. The current data suggest that Max and Mnt are not transcriptionally regulated by insulin or FOXO in Drosophila, whereas myc is. This is similar to what has been reported in murine lymphoid cells, in which c-myc expression is regulated by the FOXO homolog FKHRL1. These parallels between the fly and mammalian systems suggest a broader connection between insulin signaling and activity of the Myc/Mnt/Max network. Although some features may be different in the two systems, the similarities merit further investigation (Teleman, 2008).

Finally, this work has revealed a surprising amount of tissue specificity in the transcriptional response to insulin signaling. Roughly half of the genes regulated by insulin in adipose tissue or in muscle were not significantly regulated in the other tissue. Furthermore, 155 genes were differentially regulated in the two tissues (i.e., upregulated in one tissue and downregulated in the other). This likely reflects the roles of the different tissues in the organism's response to nutrient deprivation. Further work will elucidate the underlying molecular mechanisms (Teleman, 2008).

4E-BP extends lifespan upon dietary restriction by enhancing mitochondrial activity in Drosophila

Dietary restriction (DR) extends lifespan in multiple species. To examine the mechanisms of lifespan extension upon DR, genome-wide translational changes were assayed in Drosophila. A number of nuclear encoded mitochondrial genes, including those in Complex I and IV of the electron transport chain, showed increased ribosomal loading and enhanced overall activity upon DR. Various mitochondrial genes were found to possess shorter and less structured 5'UTRs, which were important for their enhanced mRNA translation. The translational repressor 4E-BP, the eukaryotic translation initiation factor 4E binding protein, was upregulated upon DR and mediated DR dependent changes in mitochondrial activity and lifespan extension. Inhibition of individual mitochondrial subunits from Complex I and IV diminish the lifespan extension obtained upon DR, reflecting the importance of enhanced mitochondrial function during DR. These results imply that translational regulation of nuclear-encoded mitochondrial gene expression by 4E-BP plays an important role in lifespan extension upon DR (Zid, 2009).

The Drosophila FoxA ortholog Fork head regulates growth and gene expression downstream of Target of rapamycin

Forkhead transcription factors of the FoxO subfamily regulate gene expression programs downstream of the insulin signaling network. It is less clear which proteins mediate transcriptional control exerted by Target of rapamycin (TOR) signaling, but recent studies in nematodes suggest a role for FoxA transcription factors downstream of TOR. This study presents evidence that outlines a similar connection in Drosophila, in which the FoxA protein Fork head (FKH) regulates cellular and organismal size downstream of TOR. Ectopic expression and targeted knockdown of FKH in larval tissues elicits different size phenotypes depending on nutrient state and TOR signaling levels. FKH overexpression has a negative effect on growth under fed conditions, and this phenotype is not further exacerbated by inhibition of TOR via rapamycin feeding. Under conditions of starvation or low TOR signaling levels, knockdown of FKH attenuates the size reduction associated with these conditions. Subcellular localization of endogenous FKH protein is shifted from predominantly cytoplasmic on a high-protein diet to a pronounced nuclear accumulation in animals with reduced levels of TOR or fed with rapamycin. Two putative FKH target genes, CG6770, a nuclear DNA binding phosphoprotein, and cabut, are transcriptionally induced by rapamycin or FKH expression, and silenced by FKH knockdown. Induction of both target genes in heterozygous TOR mutant animals is suppressed by mutations in fkh. Furthermore, TOR signaling levels and FKH impact on transcription of the dFOXO target gene d4E-BP (Thor), implying a point of crosstalk with the insulin pathway. In summary, these observations show that an alteration of FKH levels has an effect on cellular and organismal size, and that FKH function is required for the growth inhibition and target gene induction caused by low TOR signaling levels (Bulow, 2010).

Modulation of gurken translation by insulin and TOR signaling in Drosophila

Localized Gurken (Grk) translation specifies the anterior-posterior and dorsal-ventral axes of the developing Drosophila oocyte; spindle-class females lay ventralized eggs resulting from inefficient grk translation. This phenotype is thought to result from inhibition of the Vasa RNA helicase. In a screen for modifiers of the eggshell phenotype in spn-B flies, a mutation was identified in the lnk gene. lnk mutations restore Grk expression but do not suppress the persistence of double-strand breaks nor other spn-B phenotypes. This suppression does not affect Egfr directly, but rather overcomes the translational block of grk messages seen in spindle mutants. Lnk was recently identified as a component of the insulin/insulin-like growth factor signaling (IIS) and TOR pathway. Interestingly, direct inhibition of TOR with rapamycin in spn-B or vas mutant mothers can also suppress the ventralized eggshell phenotype. When dietary protein is inadequate, reduced IIS-TOR activity inhibits cap-dependent translation by promoting the activity of the translation inhibitor eIF4E-binding protein (4EBP). It is hypothesized that reduced TOR activity promotes grk translation independent of the canonical Vasa- and cap-dependent mechanism. This model might explain how flies can maintain the translation of developmentally important transcripts during periods of nutrient limitation when bulk cap-dependent translation is repressed (Ferguson, 2012).

Reproduction represents a substantial energy investment for an organism. Many studies have shown that ovarian physiology is exquisitely sensitive to nutritional status. Limitation of dietary protein intake results in a dramatic slowing of egg chamber maturation via developmental arrest, programmed cell death, or loss of germline stem cells. Several signaling pathways are integrated to bring about this response including 20-hydroxyecdysone, Juvenile Hormone (JH), and insulin/insulin-like signaling (IIS). IIS is stimulated by protein feeding and is required for oogenesis to progress. The IIS pathway integrates nutritional signals at two distinct points during oogenesis. The first is in region 2A of the germarium where developing germline cysts undergo apoptosis in the absence of a source of maternal dietary protein. The second point of nutritional control is at stage 8 of oogenesis during the onset of vitellogenesis. In the absence of food, egg chambers develop to stage 8, where they are arrested until a favorable food source is located. These two checkpoints represent points at which the energetically expensive process of oogenesis can be halted if insufficient resources are available (Ferguson, 2012 and references therein).

The IIS pathway elicits its effect on Drosophila physiology through several effector pathways, namely the dFOXO transcription factor and the Target of Rapamycin kinase (TOR). IIS inhibits dFOXO activity by promoting its phosphorylation by PKB/Akt and subsequent exclusion from the nucleus. Starvation or mutations in the insulin pathway allow dFOXO to translocate to the nucleus where it directs the transcription of genes that promote longevity, stress resistance, fat storage, and growth attenuation. TOR activity is stimulated by both IIS through the dRheb GTPase and by amino acids via Rag GTPases. When nutrients are plentiful, high TOR activity stimulates the translation of mRNA by phosphorylating S6K which in turn phosphorylates eIF4B and promotes its interaction with eIF3. These steps are critical for recruiting the translation preinitiation complex (PIC) to the m7G cap at the 5’ end of the mRNA. Once bound, the PIC recruits the small ribosomal subunit and proceeds to scan the transcript for an initiating AUG codon. This process requires the activity of the eIF4A RNA helicase. TOR also phosphorylates and inactivates the inhibitory eIF4E binding protein, 4EBP. Starvation inhibits cap- dependent translation through reduced TOR activity. When nutrients are limiting and TOR activity is low, eIF4B is not phosphorylated and can no longer participate in PIC assembly, furthermore 4EBP inhibition is lifted and it proceeds to inhibit cap-recognition by eIF4E. Both activities have the effect of strongly blocking cap-dependent translation initiation when nutrients are scarce. A select few transcripts escape this translational block by upregulating the utilization of an alternative mechanism that relies on an Internal Ribosomal Entry Site (IRES) that obviates the requirement for cap recognition and start codon scanning. The list of transcripts that contain IRES sequences is growing and includes numerous growth factors such as VEGF-A , PDGF2, and IGF-II. A prominent example of IRES-mediated nutritional adaptation is the Drosophila insulin receptor dInR, the translation of which is upregulated in response to starvation as a way to sensitize the cell to insulin when nutrients become available (Ferguson, 2012 and references therein).

Control of translation is vitally important to developmental patterning. The transcripts of many morphogens, including nanos, oskar, and gurken, are co-transcriptionally packaged into silencing particles and transported in a translationally quiescent form. Once localized, this repression is alleviated and translation proceeds in the developmentally appropriate locale. Gurken (Grk) is a TGF-α related ligand for the Drosophila Egfr. Localized translation of the spatially restricted grk transcript results in signaling by germline-derived Grk to the Egfr in the overlying follicle cells. This signal is required to specify the posterior fate in early oogenesis and the dorsal fate during mid oogenesis. Mutations that reduce grk translation are female sterile due to an inability to correctly pattern the developing oocyte and result in concomitant patterning defects in the embryo. grk translation requires the eIF4A-related DEAD-box helicase Vasa (Vas). Mutations in vas are female sterile owing to a failure to specify dorsal structures in the egg shell or posterior structures in the embryo (Ferguson, 2012 and references therein).

Spindle class genes are responsible for repairing DNA double strand breaks (DSBs) that are induced during homologous recombination in Drosophila oogenesis. In wild type females, DSBs are induced in germ line cells entering pachytene in region 2A of the germarium. This process is initiated by the Spo11 homologue Mei-W68 and Mei-P22, a protein that aids in break site selection. These breaks are then repaired by homologous recombination, a process that requires the RAD-51 homologue spindle-B (spn-B). Mutations in spn-B result in an accumulation of unrepaired DSBs that lead to activation of a meiotic checkpoint. The checkpoint is comprised of the ATR homologue mei-41 and the downstream kinase chk-2. Persistent DSBs in spn-B^BU females activate the checkpoint that requires the Mei-41 and Chk2 kinases and leads to inefficient grk translation and ventralized eggshell phenotypes. Checkpoint activation also results in phosphorylation of Vasa, a modification that is thought to inhibit its function. Early in oogenesis, the oocyte nucleus becomes arrested in pachytene and forms a compact structure called the karyosome. The formation of the karyosome is disrupted in spindle-class mutants where the chromatin appears fractured or ellipsoid. Weak grk translation and an inability to properly form the karyosome are both spindle phenotypes that are consistent with reduced Vasa activity (Ferguson, 2012).

This study has identified the SH2B family adaptor gene lnk in a genetic screen for modifiers of the ventralized eggshell phenotype seen in spn-B^BU mutant flies. SH2B proteins are known to regulate intracellular signaling by membrane bound receptor tyrosine kinases (RTKs). SH2Bs can promote signaling by scaffolding downstream effectors to the RTK or mediate proteosomal receptor destruction by recruiting the Cbl ubiquitin ligase. Lnk was recently identified as a positive regulator of the Insulin/Insulin-like Signaling (IIS) pathway that functions at the level of the insulin receptor substrate Chico. This study shows that lnk mutations can promote grk translation and suppress the ventralized eggshell phenotype in a spn-B^BU mutant background. This suppression occurs independent of Vasa activity and does not suppress the karyosome phenotype. No genetic interactions were found with a weak grk allele nor downstream targets of Egfr suggesting that lnk-mediated suppression of spindle phenotypes does not occur by directly modulating Egfr activity. The data suggest that lnk mutations promote grk translation by inhibiting TOR activity as Rapamycin feeding experiments can also suppress the eggshell phenotype of spn-B and vas mutant flies. A model is proposed in which reduced IIS/TOR signaling inhibits cap-dependent translation and promotes utilization of an alternative translation initiation mechanism of the grk mRNA. This mechanism enables flies to faithfully pattern their oocytes when nutrients are scarce (Ferguson, 2012).

This study demonstrates a novel interaction between a meiotic checkpoint, the insulin/insulin- like signaling pathway, and translation of gurken mRNA in Drosophila oogenesis. Mutations in meiotic DNA repair enzymes such as spn-B result in persistent DSBs in early oogenesis that activate an ATR- Chk2-dependent meiotic checkpoint. Checkpoint activation results in phosphorylation of the eIF4A-like RNA helicase Vasa, the activity of which is important for grk translation. In these mutants, low levels of Grk protein are synthesized which is insufficient to pattern the eggshell correctly and results in ventralized eggs. Using forward genetics, an allele was isolated of the insulin receptor adapter, lnk. This mutation can suppress the weak grk translation phenotype and restore normal patterning to eggs laid by spn-B^BU flies. Clonal analysis has shown that lnk mutations reduce IIS in a cell-autonomous manner in the ovary. As in mammals, Drosophila IIS controls the rate of cap-dependent translation initiation in the cell by regulating the activity of the TOR kinase. Rapamycin inhibits TOR activity, and feeding rapamycin can suppress the ventralized eggshell phenotype not only in spn-B^BU females, but also in vasa^PH165 / vas^aRG53 flies. These data suggest an alternative translation initiation mechanism for the grk mRNA by which flies can maintain D/V axis patterning in times of moderate nutrient limitation (Ferguson, 2012).

The discovery that mutations in lnk, a positive regulator of IIS, can suppress the patterning defects in spn-B flies was initially surprising. The eggshell phenotypes of the different genotypes were assessed after keeping the flies on apple or grape juice agar plates on which abundant amounts of yeast paste had been added thus allowing the females to eat a very protein rich diet. A protein rich diet stimulates the activity of the TOR kinase via two mechanisms. Insulin-like peptides (dilps) are secreted into the hemolymph by neuroendocrine cells in response to nutrient availability. This in turn activates the IIS cascade comprised of Chico/Lnk, PI3K, Akt, Tsc1/2, and Rheb which promotes TOR-C1 activity. The second mechanism acts more directly through the levels of intracellular amino acids that are imported in part by the slimfast and pathetic transporters. Both of these mechanisms stimulate TOR-C1 activity which has been shown to promote cap-dependent translation by inhibiting 4EBP sequestration of eIF4E. Therefore, reducing TOR activity either by a mutation in lnk or by addition of rapamycin, would be expected to interfere with cap-dependent translation and therefore further enhance the mutant phenotype. However, in spn-B mutant flies, cap-dependent translation is already inhibited by the activity of the checkpoint, presumably acting via Vasa modification. The fact that a suppression of the ventralized phenotype was observed in lnk mutants indicates that reduction in TOR signaling must activate a second mode of translation that allows Gurken protein to be produced independently of the block in cap-dependent translation (Ferguson, 2012).

Several ovarian phenotypes are shared between mutations in spindle genes and vas mutants, including failure to form a compact karyosome, very weak grk translation, and ventralized eggs. Combined with the reproducible phosphorylation of Vas protein in spindle-class mutants, these phenotypes are consistent with a defect in Vas activity. While the specific effect of this phosphorylation is unknown, Vas serves several functions in cap-dependent translation initiation of grk mRNA. Vasa has been shown to interact with eIF5B and mutations that interfere with this interaction inhibit grk translation. This interaction is thought to facilitate assembly of the 60S ribosomal subunit at the AUG start codon. Furthermore, as a DEAD-box RNA helicase, Vasa may permit the pre-initiation complex to scan the 5’ UTR of grk and negotiate secondary structures that may impede the progress of this complex. IRES sequences adopt strong secondary structures in the 5’ UTR of RNAs that they regulate. If it can be demonstrated in the future that grk possess an IRES sequence, this may explain the requirement for Vasa helicase activity to unwind this structure when translation is initiated from the 5'cap during conditions of adequate nutrient availability. Whether the checkpoint dependent phosphorylation of Vas affects its stability, RNA helicase activity, or its eIF5B interaction, the expected result is a block in cap-dependent translation initiation of grk mRNA and concomitant D/V patterning defects. The observation that grk translation can be induced to occur in spn-B^BU and in vasa^PH165 / vas^aRG53 flies indicates that an alternative mechanism for supporting translation initiation is taking place. Because reduced IIS and TOR activity both block bulk cap-dependent translation initiation through sequestration of eIF4E by 4EBP, yet stimulate IRES activity, it is proposed that the latter may provide an explanation for the results (Ferguson, 2012).

Grk plays a central role in shaping the development of the egg and subsequent embryo. Mutations that disrupt Grk / Egfr signaling during oogenesis result in female sterility. Blocking the translation of this essential morphogen in spindle class mutants that are unable to repair DNA damage is an effective mechanism to prevent the transmission of mutations to the progeny. This reproductive checkpoint is effective when nutrients are abundant, however as this study has demonstrated, the strategy breaks down when IIS/TOR activity is low. Under these conditions, grk can be translated and result in eggs that are patterned correctly, even though the DNA damage and karyosome malformation phenotypes persist. It is proposed that this difference occurs because the DNA-damage checkpoint can only impinge on one of the two mechanisms by which grk translation can be initiated (Ferguson, 2012).

One mechanism by which suppression of the D/V patterning defects of spn-B^BU may occur is through the effects of the additional time that lnk^CR642 egg chambers spend completing oogenesis. While Grk production is reduced in spn-B^BU flies, it is not completely blocked and some Grk protein is made. If the reduced rate of Grk production is integrated over the extended time spent during mid oogenesis, sufficient Grk levels could accumulate and support normal D/V patterning. However, this model is inconsistent with the inability of lnk^CR642 to suppress the ventralized eggs laid by grk^ED22 females. These flies do retain some Grk activity as is evident by the single appendage that is specified, however if the mechanism of suppression were via accumulation, then grk^ED22 should be suppressed by lnk mutations. Therefore, the IRES-dependent model proposed in this study is favored (Ferguson, 2012).

The selective pressure that may have driven the evolution of this bi-modal translation mechanism for grk can be best understood by considering that in wild populations of Drosophila, females feed and oviposit at locations where yeast is abundant. This behavior ensures adequate nutrition to support oogenesis in the female as well as for the developing larvae. If however nutrients become scarce, females adjust the rate of oogenesis to match nutrient availability. In response to complete starvation, egg chambers undergo apoptosis and are reabsorbed, however moderate reductions in IIS slow the rate of oogenesis until an abundant protein source is found. The conserved response to dietary restriction is to repress cap- dependent translation of most cellular transcripts while a select population of RNAs that are essential for survival escape this repression by utilizing a cap-independent IRES mechanism. It is posited that grk may be one such transcript. Oocytes that are in mid development when nutrients are scarce must still be patterned appropriately so that the resulting eggs are fertile. IRES activity may facilitate Grk expression to maintain normal D/V patterning in times of lean whereas when nutrients are abundant, cap-dependent translation predominates (Ferguson, 2012).

AMPK modulates tissue and organismal aging in a non-cell-autonomous manner

AMPK exerts prolongevity effects in diverse species; however, the tissue-specific mechanisms involved are poorly understood. This study shows that upregulation of AMPK in the adult Drosophila nervous system induces autophagy both in the brain and also in the intestinal epithelium. Induction of autophagy is linked to improved intestinal homeostasis during aging and extended lifespan. Neuronal upregulation of the autophagy-specific protein kinase Atg1 is both necessary and sufficient to induce these intertissue effects during aging and to prolong the lifespan. Furthermore, upregulation of AMPK in the adult intestine induces autophagy both cell autonomously and non-cell-autonomously in the brain, slows systemic aging, and prolongs the lifespan. The organism-wide response to tissue-specific AMPK/Atg1 activation is linked to reduced insulin-like peptide levels in the brain and a systemic increase in 4E-BP expression. Together, these results reveal that localized activation of AMPK and/or Atg1 in key tissues can slow aging in a non-cell-autonomous manner (Ulgherait, 2014. PubMed ID: 25199830).

Pelle modulates dFoxO-mediated cell death in Drosophila

Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals. Recent studies also suggest a critical role of IRAKs in tumor development, though the underlying mechanism remains elusive. Pelle is the sole Drosophila IRAK homolog implicated in the conserved Toll pathway that regulates Dorsal/Ventral patterning, innate immune response, muscle development and axon guidance. This study reports a novel function of pll in modulating apoptotic cell death, which is independent of the Toll pathway. It was found that loss of pll results in reduced size in wing tissue, which is caused by a reduction in cell number but not cell size. Depletion of pll up-regulates the transcription of pro-apoptotic genes, and triggers caspase activation and cell death. The transcription factor dFoxO is required for loss-of-pll induced cell death. Furthermore, loss of pll activates dFoxO, promotes its translocation from cytoplasm to nucleus, and up-regulates the transcription of its target gene Thor/4E-BP. Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly. This study not only identifies a previously unknown physiological function of pll in cell death, but also sheds light on the mechanism of IRAKs in cell survival/death during tumorigenesis (Wu, 2015).

Drosophila melanogaster has emerged as an excellent model organism to study apoptotic cell death and has made significant contribution to understand cell death regulation and its role in development. While mammalian IRAKs function as the mediator of IL-1Rs/TLRs signal transduction in the immune and inflammatory responses, Pll, the sole Drosophila orthologue of IRAKs, has been implicated as a central regulator of Toll pathway involved in embryonic dorsal/ventral patterning, innate immune response, muscle development and axon guidance. This work identified a Toll pathway independent function of Pll in modulating caspase-mediated cell death in animal development (Wu, 2015).

Previous studies have suggested that Drosophila wing vein formation is a result of cell fate specification regulated by multiple signaling pathways including Notch, Hedgehog, EGF (epidermal growth factor) and BMP (bone morphogenetic proteins) pathways. The present study found that knock-down pll along the A/P compartment boundary of the developing wing (ptc>pll-IR) resulted in extensive cell death in the wing disc and a loss-of-ACV phenotype in the adult wing, implying a potential role of cell death in vein patterning. Consistent with this notion, the loss-of-ACV phenotype is rescued by blocking apoptotic cell death, suggesting cell death is responsible for the loss of ACV. To investigate whether cell death is able to impede vein patterning, apoptosis was initiated by expressing the pro-apoptotic protein Grim under the control of ptc-Gal4. ptc>Grim caused extensive cell death in tissue ablation between L3 and L4 in the adult wing. In most cases, L3 and L4 were fused in the proximal area where ACV is located. To adjust Grim expression and cell death, Tub-Gal80^ts that represses Gal4 activity was added in a temperature sensitive manner. At 25°C, Tub-Gal80^ts partially blocks ptc-Gal4 activity and allows limited Grim expression and therefore, cell death, between L3 and L4. Intriguingly, under this condition, the loss-of-ACV phenotype was observed to be accompanied by a slight reduction of area between L3 and L4, suggesting that both reduced area and loss-of-ACV phenotypes are consequences of cell death. The loss-of-ACV phenotype is more sensitive to cell death, since weak cell death is sufficient to generate the phenotype, whereas stronger cell death is required to delete tissue between L3 and L4. Consistent with this notion, while ptc>pll-IR flies reared at 25°C only displayed the loss-of-ACV phenotype , those raised at 29°C also showed reduced area between L3 and L4, which was caused by a reduction in cell number, but not cell size (Wu, 2015).

Pll regulates caspase activation and cell death through dFoxO. Mechanistically, loss of pll promotes the nuclear translocation of dFoxO, which otherwise is retained in the cytoplasm by phosphorylation. A number of kinases, including AKT, IκK and JNK, have been reported to phosphorylate FoxO and regulate its nuclear-cytoplasmic trafficking. This study provides evidence that Pll is another dFoxO kinase that phosphorylates dFoxO and inhibits its nuclear localization. Thus, it would be very interesting to check whether a similar interaction is conserved between IRAKs and FoxOs in mammal (Wu, 2015).

The FoxO family proteins have been implicated in multiple important biological processes, including cell death and tumor suppression. It has been reported that conditional deletion of FoxO1, FoxO3 and FoxO4 simultaneously results in the development of hemangiomas and thymic lymphomas, and IκB kinase represses FoxO3a activity to promote human breast tumorigenesis and acute myeloid leukemia (AML). IRAKs also show altered expression level in tumors and surrounding stroma, and participate in tumor initiation and progression, yet the underlying mechanisms remain poorly understood. Thus, the inhibitory effect of Pll on dFoxO activity in Drosophila provides a beneficial framework for a better understanding of mammalian IRAKs’ crucial roles in tumor development (Wu, 2015).

As the Toll/NF-κB pathway is not implicated in loss-of-pll triggered dFoxO-dependent cell death, it was of interest to learn about what are the pathways or factors act upstream of Pll to regulate its role in cell death. Since dFoxO has been reported as a downstream transcription factor in the JNK and Insulin pathways in Drosophila, it was asked whether Pll is also involved in these pathways. Activation of JNK signaling between L3 and L4 by expressing Egr (Drosophila TNF) or Hep (Drosophila JNK Kinase), or depleting puc (encoding a JNK inhibitor), produced similar loss-of-ACV and reduced area phenotypes as that of pll depletion, yet the phenotypes were not affected by gain or loss of pll. Inactivation of the Insulin pathway by expressing a dominant negative form of PI3K, or knocking-down PI3K or Akt, resulted in diminished area between L3 and L4, which remained unaffected by gain or loss of pll. The Hippo pathway, known to play a crucial role in regulating cell death and organ size, was also examined. Up-regulation of Hippo pathway in distinct wing areas by different Gal4 drivers led to various small wing or wing tissue ablation phenotypes, which were not altered by changing Pll level. Finally, dMyc, the fly homolog of c-Myc that regulates cell growth and cell death in Drosophila, and the cell polarity gene scribble (scrib), whose depletion promotes cell death were also examined. Depletion of dMyc triggered wing phenotype and loss of scrib induced cell death are both independent of Pll. Thus, while Pll directly regulates dFoxO-mediated caspase-dependent cell death in development, the upstream factors modulating Pll activity remain unknown, which deserve further investigation (Wu, 2015)

Mitochondrial retrograde signaling regulates neuronal function

Mitochondria are key regulators of cellular homeostasis, and mitochondrial dysfunction is strongly linked to neurodegenerative diseases, including Alzheimer's and Parkinson's. Mitochondria communicate their bioenergetic status to the cell via mitochondrial retrograde signaling. To investigate the role of mitochondrial retrograde signaling in neurons, mitochondrial dysfunction was induced in the Drosophila nervous system. Neuronal mitochondrial dysfunction causes reduced viability, defects in neuronal function, decreased redox potential, and reduced numbers of presynaptic mitochondria and active zones. Neuronal mitochondrial dysfunction stimulates a retrograde signaling response that controls the expression of several hundred nuclear genes. Drosophila hypoxia inducible factor alpha (HIFalpha) ortholog Similar (Sima) regulates the expression of several of these retrograde genes, suggesting that Sima mediates mitochondrial retrograde signaling. Remarkably, knockdown of Sima restores neuronal function without affecting the primary mitochondrial defect, demonstrating that mitochondrial retrograde signaling is partly responsible for neuronal dysfunction. Sima knockdown also restores function in a Drosophila model of the mitochondrial disease Leigh syndrome and in a Drosophila model of familial Parkinson's disease. Thus, mitochondrial retrograde signaling regulates neuronal activity and can be manipulated to enhance neuronal function, despite mitochondrial impairment (Cagin, 2015).

The human brain constitutes approximately 2% of body weight but consumes 20% of available oxygen because of its high energy demand. Mitochondria are abundant in neurons and generate the majority of cellular ATP through the action of the mitochondrial ATP synthase complex. Mitochondrial disorders are one of the most common inherited disorders of metabolism and have diverse symptoms, but tissues with a high metabolic demand, such as the nervous system, are frequently affected. The primary insult in all mitochondrial diseases is to mitochondrial function, but the etiology of these diseases is highly pleiotropic. This phenomenon is poorly understood, but suggests that the cellular response to mitochondrial dysfunction may be complex and vary between cell types and tissues (Cagin, 2015).

Mitochondrial retrograde signaling is defined as the cellular response to changes in the functional state of mitochondria. Mitochondrial retrograde signaling enables communication of information about changes in processes such as mitochondrial bioenergetic state and redox potential to the rest of the cell and is thus a key mechanism in cellular homeostasis. The best characterized retrograde responses involve mitochondrial dysfunction eliciting changes in nuclear gene transcription. In yeast, mitochondrial dysfunction causes changes in the expression of genes involved in supplying mitochondria with oxaloacetate and acetyl CoA, the precursors of α-ketoglutarate and glutamate, to compensate for failure of the tricarboxylic acid (TCA) cycle (Cagin, 2015).

In proliferating mammalian cell models, mitochondrial retrograde signaling is more diverse and involves increases in cytosolic-free Ca2+, leading to activation of Ca2+-responsive calcineurin, causing the up-regulation of genes controlling Ca2+ storage and transport. In addition to mitochondrial diseases, alterations in mitochondrial function are also associated with late onset neurodegenerative diseases such as Alzheimer's and Parkinson's. Thus, the neuronal response to mitochondrial function may be altered in these diseases and contribute to disease progression. However, neuronal-specific mitochondrial retrograde signaling is poorly understood and its role in neuronal homeostasis is completely unknown (Cagin, 2015).

This study has developed a neuronal-specific model of mitochondrial dysfunction in Drosophila and used this to characterize mitochondrial retrograde signaling in vivo. Retrograde signaling is shown to regulate neuronal function and can be manipulated to alleviate the effects of mitochondrial dysfunction in neurons (Cagin, 2015).

This study shows that the Drosophila HIFα ortholog Sima is potentially a key regulator of the mitochondrial retrograde response in the nervous system and that knockdown of Sima dramatically improves neuronal function in this and other models of mitochondrial dysfunction. Surprisingly, Sima activity in part causes the dysfunction of neurons containing defective mitochondria. Previous studies of Drosophila mutants in the regulatory and catalytic subunits of the mitochondrial DNA polymerase Polγ have demonstrated that loss of mtDNA replication in Drosophila causes mtDNA loss, reduced neuronal stem cell proliferation, and developmental lethality. To avoid the pleiotropic effects of using homozygous mutant animals, this study developed a neuronal-specific model of mitochondrial dysfunction. The phenotypes resulting from TFAM overexpression and expression of a mitochondrially targeted restriction enzyme were characterized, and both of these tools were used to model neuronal-specific mitochondrial dysfunction (Cagin, 2015).

Overexpression of mitochondrial transcription factor A (TFAM) results in mitochondrial dysfunction caused by inhibition of mitochondrial gene expression, rather than an alteration in mtDNA copy number. Overexpression of TFAM has been shown to have different effects depending on the cell type, model system, or ratio of TFAM protein to mtDNA copy number. The current results are consistent with in vitro studies and overexpression of human TFAM in mice and human cells, which have shown that excess TFAM results in the suppression of mitochondrial gene transcription. Ubiquitous expression of mitoXhoI causes early developmental lethality and that, although there was no significant mtDNA loss, the majority of mtDNA was linearized. Given that mtDNA is transcribed as two polycistronic mRNAs, a double-stranded break in coxI would block the transcription of the majority of mitochondrially encoded genes, resulting in severe mitochondrial dysfunction (Cagin, 2015).

Using a Drosophila motor neuron model, mitochondrial dysfunction was found to cause a reduction in the number of active zones, loss of synaptic mitochondria, and locomotor defects. Mitochondrial dysfunction caused by overexpression of PINK1 or Parkin decreases the rate of mitochondrial transport in vitro and in vivo. Furthermore, a recent study using KillerRed demonstrated that local mitochondrial damage results in mitophagy in axons. Therefore, the acute loss of synaptic mitochondria in the current model may result from defects in mitochondrial transport and/or mitophagy (Cagin, 2015).

Previous studies in mice have examined the effects of neuronal mitochondrial dysfunction by using mitoPstI expression, or targeted knockout of TFAM. Knockout of TFAM specifically in mouse dopaminergic neurons (the 'MitoPark' mouse model) causes progressive loss of motor function, intraneuronal inclusions, and eventual neuronal cell death. Interestingly, cell body mitochondria are enlarged and fragmented and striatal mitochondria are reduced in number and size in MitoPark dopaminergic neurons, suggesting that the effects of neuronal mitochondrial dysfunction are conserved in Drosophila and mammals. Larvae mutant for the mitochondrial fission gene drp1 have fused axonal mitochondria and almost completely lack mitochondria at the NMJ, similar to motor neurons overexpressing TFAM or expressing mitoXhoI (Cagin, 2015).

Adult drp1 mutant flies also have severe behavioral defects. Synaptic reserve pool vesicle mobilization is inhibited in drp1 mutant larvae because of the lack of ATP to power the myosin ATPase required for reserve pool tethering and release. Reserve pool vesicle mobilization is likely to be similarly affected in TFAM overexpressing or mitoXhoI-expressing motor neurons, which would result in locomotor defects in these animals (Cagin, 2015).

Interestingly, expression of the Arctic form of β-amyloid1-42 (Aβ) in Drosophila giant fiber neurons also leads to the depletion of synaptic mitochondria and decreased synaptic vesicles. Synaptic loss and alterations in neuronal mitochondrial morphology have also been observed in postmortem tissue from Alzheimer's disease patients. The parallels between these phenotypes and those in the current model suggest a common underlying mechanism (Cagin, 2015).

Using microarray analysis, this study found that mitochondrial dysfunction in neurons regulates the expression of hundreds of nuclear genes. The Drosophila CNS contains different neuronal subtypes, and glial cells, so the results of the microarray are heterogeneous, representing the pooled response to mitochondrial dysfunction throughout the CNS. Mitochondrial dysfunction was phenotypically characterized in motor neurons, but not all of the genes identified from the microarrays are expressed in motor neurons, e.g., Ilp3. The specific genes that are regulated differ depending on whether mitochondrial dysfunction results from TFAM overexpression or knockdown of ATPsynCF6. However, a core group of approximately 140 genes are similarly regulated in both conditions (Cagin, 2015).

Yeast mutants in different components of the TCA cycle result in differing retrograde responses and comparison of somatic cell hybrids (cybrids) carrying the A3243G mtDNA mutation with cybrids completely lacking mtDNA (ρ0 cells) showed overlapping but distinct gene expression profiles. Moreover, another study comparing cybrids with increasing levels of the A3243G mtDNA mutation showed markedly different alterations in nuclear gene expression, depending on the severity of mitochondrial dysfunction (Cagin, 2015).

Taken together, these data suggest that the cellular response to mitochondrial dysfunction is not uniform and adapts to the specific defect and severity of the phenotype. Therapeutic strategies targeting mitochondrial dysfunction in human disease may therefore need to be tailored to the specific mitochondrial insult. Concomitant with the current findings, previous studies have shown that in yeast, Drosophila, and mammalian-proliferating cells, retrograde signaling activates the expression of hypoxic/glycolytic genes and the insulin-like growth factor-1 receptor pathway to compensate for mitochondrial dysfunction. Rtg1 and Rtg3, the transcription factors that coordinate the mitochondrial retrograde response in yeast, are not conserved in metazoans. In mammalian proliferating cellular models, the retrograde response activates the transcription factors nuclear factor of activated T cells (NFAT), CAAT/enhancer binding protein δ (C/EBPδ), cAMP-responsive element binding protein (CREB), and an IκBβ-dependent nuclear factor κB (NFκB) c-Rel/p50. Whether these transcription factors regulate mitochondrial retrograde signaling in the mammalian nervous system is not known (Cagin, 2015).

HIFα/Sima is a direct regulator of LDH expression in flies and mammals, and this study found that Sima also regulates the expression of two other retrograde response genes, Thor and Ilp3, in the Drosophila nervous system. Importantly, Sima is required for the increase in Thor expression in response to mitochondrial dysfunction. Sima has been strongly implicated as a key regulator of mitochondrial retrograde signaling in Drosophila S2 cells knocked down for the gene encoding subunit Va of complex IV. sima, Impl3, and Thor expression were all increased in this model, and there is a significant overlap with the genes regulated in the current model (Cagin, 2015).

These data support the possibility that the Drosophila HIFα ortholog Sima is a key transcriptional regulator of neuronal mitochondrial retrograde signaling. HIFα is stabilized in hypoxia through the action of prolyl hydroxylases and this mechanism was thought to require ROS, but HIFα stabilization may in fact be ROS independent. In mammalian cells carrying the mtDNA A1555G mutation in the 12S rRNA gene, mitochondrial retrograde signaling has been shown to be activated by increased ROS, acting through AMPK and the transcription factor E2F1 to regulate nuclear gene expression. In the Drosophila eye, loss of the complex IV subunit cytochrome c oxidase Va (CoVa) causes decreased ROS. However, retrograde signaling upon loss of CoVa was not mediated by decreased ROS, but by increased AMP activating AMPK. Similarly, the small decrease in redox potential in neurons in response to mitochondrial dysfunction in the current model makes it unlikely that ROS are the mediator of the retrograde signal. Moreover, HIFα physically interacts with several transcriptional regulators including the Drosophila and mammalian estrogen-related receptor and Smad3, as well as its heterodimeric binding partner HIFβ, to regulate gene expression. Mitochondrial retrograde signaling may modulate these or other unidentified HIFα interactors and, thus, control HIF target gene expression without directly regulating HIFα (Cagin, 2015).

In cancer cell models, mitochondrial dysfunction promotes cell proliferation, increased tumourigenicity, invasiveness, and the epithelial-to-mesenchymal transition via retrograde signaling. In these models, inhibition of retrograde signaling prevents these tumourigenic phenotypes. Neuronal mitochondrial dysfunction in the current model causes a cellular response, resulting in a severe deficit in neuronal function. This response may have evolved to protect neurons, through decreased translation and increased glycolysis, from the short-term loss of mitochondrial function. Over longer periods, however, this response may be counterproductive because it results in decreased neuronal activity and locomotor function. Inhibition of neuronal mitochondrial retrograde signaling, through knockdown of Sima, dramatically improves neuronal function. Thus, mitochondrial retrograde signaling contributes to neuronal pathology and can be modified to improve the functional state of the neuron (Cagin, 2015).

Importantly, this intervention works without altering the primary mitochondrial defect. Knockdown of Sima not only abrogates the acute defects in neuronal function, but also suppresses the reduced lifespan caused by neuronal mitochondrial damage. The benefits of reduced Sima expression therefore extend throughout life. In addition to TFAM overexpression, this study also shows that Sima knockdown in neurons rescues a Drosophila model of the mitochondrial disease Leigh syndrome. However, Sima knockdown does not rescue the lethality caused by a temperature-sensitive mutation in coxI (Cagin, 2015).

Mitochondrial diseases are complex, and mutations in different COX assembly factors cause varying levels of COX deficiency in different tissues. The increasing number of Drosophila models of mitochondrial dysfunction will help to unravel the mechanisms underlying the varied pathology of mitochondrial diseases. Ubiquitous knockdown of Sima also partially restores the climbing ability of parkin mutant flies. The ability of reduced Sima expression to rescue both mitochondrial dysfunction and Parkinson's disease models reinforces the link between mitochondrial deficiency and Parkinson's and suggests that retrograde signaling may be a therapeutic target in Parkinson's disease. HIF1α inhibitors are in clinical trials for lymphoma and so, if the current findings can be replicated in mammalian models, HIF1α inhibitors may be candidates for repurposing to treat mitochondrial diseases and neurodegenerative diseases associated with mitochondrial dysfunction, such as Parkinson's disease (Cagin, 2015).

Bunched and Madm function downstream of Tuberous Sclerosis Complex to regulate the growth of intestinal stem cells in Drosophila

The Drosophila adult midgut contains intestinal stem cells that support homeostasis and repair. This study shows that the leucine zipper protein Bunched and the adaptor protein MLF1-adaptor molecule (Madm) are novel regulators of intestinal stem cells. MARCM mutant clonal analysis and cell type specific RNAi revealed that Bunched and Madm were required within intestinal stem cells for proliferation. Transgenic expression of a tagged Bunched showed a cytoplasmic localization in midgut precursors, and the addition of a nuclear localization signal to Bunched reduced its function to cooperate with Madm to increase intestinal stem cell proliferation. Furthermore, the elevated cell growth and 4EBP phosphorylation phenotypes induced by loss of Tuberous Sclerosis Complex or overexpression of Rheb were suppressed by the loss of Bunched or Madm. Therefore, while the mammalian homolog of Bunched, TSC-22, is able to regulate transcription and suppress cancer cell proliferation, these data suggest the model that Bunched and Madm functionally interact with the TOR pathway in the cytoplasm to regulate the growth and subsequent division of intestinal stem cells (Nie, 2015).

Homeostasis and regeneration of an adult tissue is normally supported by resident stem cells. Elucidation of the mechanisms that regulate stem cell-mediated homeostasis is important for the development of therapeutics for various diseases. The intestine with fast cell turnover rate supported by actively proliferating stem cells is a robust system to study tissue homeostasis. In the mouse intestine, two interconverting intestinal stem cell (ISC) populations marked by Bmi1 and Lgr5 located near the crypt base can replenish cells of various lineages along the crypt-villus axis Furthermore, recent data suggest that Lgr5+ cells are the main stem cell population and that immediate progeny destined for the secretory lineage can revert to Lgr5+ stem cells under certain conditions [6, 7]. Together, the results suggest previously unexpected plasticity in stem cell maintenance and differentiation in the adult mammalian intestine (Nie, 2015).

In the adult Drosophila midgut, which is equivalent to the mammalian stomach and small intestine, ISCs are distributed evenly along the basal side of the monolayered epithelium to support repair. The maintenance and regulation of Drosophila midgut ISCs depend on both intrinsic and extrinsic factors. When a midgut ISC divides, it generates a renewed ISC and an enteroblast (EB) that ceases to divide and starts to differentiate. The ISC-EB asymmetry is established by the Delta-Notch signaling, with Delta in the renewed ISC activating Notch signaling in the newly formed neighboring EB . Growth factors such as Wingless/ Wnt, insulin-like peptides, Decapentaplegic/BMP, Hedgehog and ligands for the EGF receptor and JAK-STAT pathways are secreted from surrounding cells and constitute the niche signals that regulate both ISC division and EB differentiation. ISC-intrinsic factors including Myc, Target of Rapamycin (TOR) and Tuberous Sclerosis Complex act to coordinate the growth and division of ISCs. Furthermore, chromatin modifiers such as Osa, Brahma and Scrawny function within ISCs to regulate Delta expression or ISC proliferation (Nie, 2015).

This study reports the identification of the leucine zipper protein Bunched (Bun) and the adaptor protein myeloid leukemia factor 1 adaptor molecule (Madm) as intrinsic factors for ISC proliferation. A single bun genomic locus generates multiple predicted transcripts that encode 4 long isoforms, BunA, F, G and P, and 5 short isoforms, BunB, C, D, E, H and O. The first identified mammalian homolog of Bun is TGF-β1 stimulated clone-22 (TSC-22). In the mouse genome four different TSC- 22 domain genes also encode multiple short and long isoforms. All isoforms of Bun and TSC-22 contain an approximately 200 amino acids C-terminal domain where the conserved TSC-box and leucine zippers are located. The originally identified TSC-22 is a short isoform and various assays suggest that it suppresses cancer cell proliferation and may function as a transcriptional regulator. Meanwhile, in Drosophila, the long Bun isoforms positively regulate growth, while the short isoforms may antagonize the function of long isoforms. Transgenic fly assays also demonstrate that the long TSC-22 can rescue the bun mutant phenotypes, whereas short isoforms cannot. These results suggest an alternative model that the long Bun isoforms positively regulate proliferation, while the short isoforms may dimerize with and inhibit the functions of long isoforms (Nie, 2015).

Madm also can promote growth. The long isoform BunA binds to Madm via a conserved motif located in the N- terminus that is not present in the short Bun isoforms. The molecular function of this novel BunA- Madm complex, nonetheless, remains to be elucidated. The results in this report demonstrate that Bun and Madm modulate the Tuberous Sclerosis Complex-target of Rapamycin (TOR)-eIF4E binding protein (4EBP) pathway to regulate the growth and division of ISCs in the adult midgut (Nie, 2015).

This report shows that Bun and Madm are intrinsically required for ISC growth and division. The results suggest a model that Bun and Madm form a complex in the cytoplasm to promote cellular growth and proliferation. The evidence that support this model includes the observation that transgenic expressed Bun localizes in the cytoplasm of midgut precursor cells, similar to the results from transfection in S2 cells and immune-staining in eye discs. Bun physically and functionally interacts with Madm, which has also been proposed as a cytoplasmic adaptor protein. Adding a nuclear localization signal to Bun reduced the growth promoting ability of Bun. Although there is a possibility this signal peptide changes the functionality in an unpredicted way, the interpretation is favored that Bun normally acts in the cytoplasm and with Madm to regulate the proliferation of ISCs. This is in contrast to mammalian TSC-22, which was reported to function in the nucleus (Nie, 2015).

The results seem to contradict a previous publication reporting that TSC-22 arrests proliferation during human colon epithelial cell differentiation. However, this apparent contradiction is resolved when the growing evidence for distinct functions for large and small Bun/ TSC-22 isoforms is considered. The Bun/TSC-22 proteins have short and long isoforms that contain the conserved TSC-box and leucine zippers in the C-terminal domain. The prototypical TSC-22 protein, TSC22D1-001, may act as a transcriptional regulator and repress cancer cell proliferation, particularly for blood lineages. Another recent model suggests that in Drosophila the long Bun isoforms interact with Madm and have a growth promoting activity, which is inhibited by the short Bun isoforms. Similarly, the long isoform, TSC22D1-002, enhances proliferation in mouse mammary glands, whereas the short isoform promotes apoptosis. Unpublished result that transgenic expression of BunB also has lower function than BunA in fly intestinal progenitor cells is consistent with this model where large isoforms have a distinct function, namely in growth promotion (Nie, 2015).

Loss of either Bun or Madm can potently suppress all the growth stimulation by multiple pathways in the midgut as shown in this report. These results are intrepeted to indicate that Bun and Madm do not act specifically in one of the signaling pathways tested but instead function in a fundamental process required for cell growth, such as protein synthesis or protein turnover. It is therefore speculated that Bun and Madm may regulate the TOR pathway. In support of this idea, it was shown that bunRNAi or MadmRNAi efficiently suppresses the Tuberous Sclerosis Complex 2RNAi-induced cell growth and p4EBP phenotypes. A recent study of genetic suppression of TOR complex 1-S6K function in S2 cells also suggests that Bun and Madm can interact with this pathway. Furthermore, proteomic analyses of Bun and Madm interacting proteins in S2 cells have shown interactions with ribosomal proteins and translation initiation factors. Therefore, a model is proposed that Bun and Madm function in the Tuberous Sclerosis Complex-TOR- 4EBP pathway to regulate protein synthesis in ISCs for their growth, which is a prerequisite for ISC proliferation. Suppression of Tuberous Sclerosis Complex mutant cell growth phenotype by bun or Madm RNAi was substantial but not complete. Earlier papers demonstrated that Bun also interacts with Notch and EGF pathway in ovary follicle cells. Therefore by definition Bun and Madm are neither 100% essential nor restricted to the TOR pathway. The genetic data suggest that Bun and Madm work downstream of Tuberous Sclerosis Complex and upstream of 4EBP, but they could also work in parallel to the TOR pathway components (Nie, 2015).

ISCs with loss of Tuberous Sclerosis Complex function have substantial cell size increase. Meanwhile, the Bun/ Madm overexpression caused increased ISC division but not cell hypertrophy. Both loss of Tuberous Sclerosis Complex and overexpression of Bun/Madm should promote cell growth but the phenotypes at the end are different. It is speculated that the reason is the Bun/Madm overexpressing ISCs are still capable of mitosis, while the Tuberous Sclerosis Complex mutant ISCs do not divide anymore thereby resulting in the very big cells. In Bun and Madm overexpressing mid- guts, the p-H3+ and GFP+ cell count showed a significant increase, indicating increased mitosis. Therefore, an explanation is that Bun and Madm overexpression may increase cell size/cell growth, but when they grow to certain size they divide, resulting in rather normal cell size (Nie, 2015). The knockout of the Madm mammalian homolog, NRBP1, can cause accumulation of the short isoform TSC22D2. Up-regulation of Madm/NRBP1 has been associated with poor clinical outcome and increased growth of prostate cancer. Further analysis based on this model may reveal whether high ratio of long Bun/TSC22 isoforms over short isoforms may associate with high Madm activity and poor clinical outcomes (Nie, 2015).

4E-BP is a target of the GCN2-ATF4 pathway during Drosophila development and aging

Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation-activated kinase GCN2 mediates this response in part by phosphorylating eIF2α. In addition, the cap-dependent translational inhibitor 4E-BP (Thor) is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. This study shows that GCN2 and its downstream transcription factor, ATF4 (Cryptocephal), mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging (Kang, 2016).

Previous studies had established the importance of 4E-BP transcription by FOXO in several distinct biological contexts, including the regulation of cell number, metabolism, response to oxidative stress, and cardiac function. Alternative transcriptional regulatory mechanisms for 4E-BP and their biological significance have remained poorly characterized. This study shows evidence that another pathway, mediated by GCN2 and ATF4, mediates the induction of 4E-BP transcription in response to the restriction of amino acids in the diet and during the development of specific tissues. The specific data presented in this study include examination of 4E-BP protein through Western blot from starved larval extracts and examination of transcripts through quantitative PCR in cultured S2 cells, larvae, and adult tissues. A new 4E-BP intron reporter, which responds to ATF4 activation, is widely expressed in Drosophila, indicating that ATF4 is a major mediator of 4E-BP induction during normal development as well as in response to dietary restriction of amino acids (Kang, 2016).

The results also show that Drosophila gcn2 mutants have a shorter lifespan than wild-type controls when reared in food with low yeast content. These results are similar to what had been observed with mutants of C. elegans gcn2 and yeast GCN4, an ATF4 equivalent gene in that organism. The Drosophila gcn2 mutant phenotype is also similar to the reported phenotype of 4E-BP mutant flies. However, this study did not examine through double-mutant analysis whether the two genes have a strictly linear genetic relationship in regulating lifespan. Based on current understanding, the two genes do not have a strictly linear relationship: GCN2-ATF4 has other transcriptional targets that also contribute to their phenotypes, and ATF4-independent regulatory inputs into 4E-BP exist, such as those mediated by FOXO and TOR. Thus, it is speculated that the similar reported phenotypes of gcn2 and 4E-BP mutants on lifespan may be due to a broad effect of 4E-BP on other GCN2-ATF4 target gene expression, as 4E-BP's target, eIF-4E, is thought to be involved in the expression of most eukaryotic genes (Kang, 2016).

Emerging evidence indicates that 4E-BP is not indiscriminate in the inhibition of general translation. For example, ribosome profiling studies in mammalian cultured cells have found that 4E-BP1's effect on translation is highly selective, with some transcripts being highly sensitive to 4E-BP1 and others indifferent. Accordingly, it appears that 4E-BP activation would have cells shift their overall protein synthesis profile. The data in this study are consistent with that view. Specifically, it was found that BiP and other stress-responsive transcripts score positive in the Internal Ribosomal Entry Site (IRES) assay and are resistant to suppression by 4E-BP. It is noted that mammalian BiP also reportedly has an IRES element in its 5' UTR. The finding that 4E-BP is a target of the UPR helps make sense of such an observation; IRES would help transcripts evade suppression by 4E-BP, whose expression level is high in stressed cells, allowing BiP to be expressed and help resolve stress. As 4E-BP activation results in a specific biological phenotype of enhanced stress resistance and lifespan extension, it appears that the proteome shift brought on by 4E-BP favors stress-responsive gene expression (Kang, 2016).

In Drosophila, 4E-BP is widely understood as a transcriptional target of FOXO. However, the role of FOXO in mediating the effects of dietary restriction of amino acids has been disputed. The experiments presented in this paper show that the loss of foxo does not impair 4E-BP transcription, at least under conditions of amino acid restriction. Notably, a foxo mutant allele was used that is different from those used in the earlier studies on 4E-BP. Although the earlier studies had used foxo21/25 alleles with premature stop codons, recent studies indicate that full-length FOXO protein is still expressed in the foxo25 mutants. Thus, it is possible that these alleles have neomorphic properties that may have led to results different from the current work. On the other hand, the foxo mutant allele used in this study has been validated to be a null allele. The negative result with the foxo mutant is mostly related to the amino acid deprivation response and does not contradict FOXO's known role in the induction of 4E-BP in other contexts (Kang, 2016).

In regards to the cellular response to amino acid deprivation, much focus had been placed on the TOR signaling pathway. It is interesting that the other amino acid-response pathway mediated by GCN2 leads to the transcriptional regulation of this TOR phosphorylation substrate. The current observation suggests that the two amino acid-responsive pathways work cooperatively (Kang, 2016).

Targets of Activity

It is generally accepted that the growth rate of an organism is modulated by the availability of nutrients. One common mechanism to control cellular growth is through the global down-regulation of cap-dependent translation by eIF4E-binding proteins (4E-BPs). Evidence is reported for a novel mechanism that allows eukaryotes to coordinate and selectively couple transcription and translation of target genes in response to a nutrient and growth signaling cascade. The Drosophila insulin-like receptor (dINR) pathway incorporates 4E-BP resistant cellular internal ribosome entry site (IRES) containing mRNAs, to functionally couple transcriptional activation with differential translational control in a cell that is otherwise translationally repressed by 4E-BP. Although examples of cellular IRESs have been previously reported, their critical role mediating a key physiological response has not been well documented. These studies reveal an integrated transcriptional and translational response mechanism specifically dependent on a cellular IRES that coordinates an essential physiological signal responsible for monitoring nutrient and cell growth conditions (Marr, 2007).

Coupled transcription and protein synthesis is a hallmark of prokaryotic gene expression. The advantages of such a linked system are well recognized as it provides smooth coordination to ensure that cells respond appropriately to signals such as nutrient availability. A rapid response to such environmental signals also allows for multiple points of regulation and a fine-tuning mechanism for controlling gene expression. In eukaryotic organisms, the compartmentalization of the cell nucleus makes the direct coupling of transcription and translation problematic. Nevertheless, like prokaryotes, the metazoan cell must respond to many external as well as internal signals, and a coupled response would be highly advantageous. However, there is currently little evidence for such a direct linkage, either physical or functional, in metazoans. In attempts to dissect the transcriptional regulatory circuitry of the insulin-like signaling cascade in Drosophila, a potentially new mechanism that functionally links transcription and translation has been identified (Marr, 2007).

Metazoan organisms must strictly control both body and organ size during development. Thus, cell size and cell number are tightly controlled to determine the final size of an animal. One of the cues used in determining growth regulation is nutrient availability. The insulin receptor (INR) and insulin-like growth factor (IGF) receptor pathways have evolved as key sensors of nutrient availability and play an important role in both cell-autonomous and nonautonomous decisions controlling cellular proliferation, cell size determination, and the response to nutrient availability. In Drosophila, this pathway is critical for determining body and organ size as well as metabolic homeostasis and life span. Perhaps most notably, misregulation of this pathway in humans can lead to type 2 diabetes and all of its associated pathologies, which is becoming a rapidly escalating worldwide epidemic (Marr, 2007).

The INR/IGF pathway is highly conserved, with homologs of the key molecular players present in metazoan organisms from flies to humans. The downstream targets of this signaling cascade are thought to separately modulate both transcription and translation to potentiate signals for either growth or stasis. In the presence of insulin or insulin-like peptides, the signaling cascade activates the oncogenic protein kinase Akt. To control RNA synthesis, Akt phosphorylates the Forkhead-box-binding protein (dFOXO) family of transcription factors, sequestering them in the cytoplasm and thus effectively inactivating them. This in turn prevents activated transcription of the dFOXO target genes. In addition, Akt stimulates the modification of the target of rapamycin (TOR) protein, which in turn phosphorylates and inactivates the translation initiation inhibitor eIF4E-binding protein (d4E-BP). In its unphosphorylated and active state, d4E-BP binds to the 7-methyl-guanosine (m7G) cap-binding protein eIF4E. This prevents formation of the translation initiation complex eIF4F, thereby inhibiting cap-dependent translation. This combination of inactivated dFOXO and inactive d4E-BP efficiently drives the cell toward growth and proliferation. Conversely, active dFOXO and d4E-BP conspire to arrest cell growth until the cell receives favorable nutrient and physiological signals to continue proliferation (Marr, 2007).

Drosophila melanogaster has proven to be a valuable model organism for working out the molecular details of this conserved pathway. In the absence of insulin or insulin-like peptides, dFOXO activates the transcription of both the insulin-like receptor (dINR) gene and the gene for Drosophila 4E-BP, establishing a transcriptional signaling loop that sensitizes the cell to receive further nutrient-dependent signals while preventing the cell from proliferating. In order to investigate this intriguing transcriptional feedback control, the start site of transcription for the dINR gene was precisely mapped using a modification of the cap-trapping cDNA synthesis method. This method, which depends on an intact m7G cap for capture of the mRNA, when combined with rapid amplification of five prime (5') cDNA ends (5' RACE) maximizes the yield of full-length 5' untranslated regions (UTRs). The use of this methodology allowed detection of critical UTRs associated with the mRNA that had previously gone undocumented. The dINR gene is actually controlled by a complex set of three distinct promoters (P1, P2, and P3) spread over 38 kb of the Drosophila genome. These combined promoters and associated introns and exons encompass the entire region between the Drosophila E2F gene and the currently annotated dINR gene. This complex control region fills a gap in the genome annotation that contains no other annotated genes or gene predictions (Marr, 2007).

Each of the dINR promoters produces a transcript with a unique and unusually long 5'UTR spliced to a short common exon that is in turn spliced to the first coding exon. The UTR originating from P1 is 1118 bases, the UTR originating from P2 is 419 bases, and the UTR originating from P3 is 485 bases. In contrast, the average 5'UTR in Drosophila is only 256 bases. All three UTRs contain multiple AUG initiator codons upstream of the legitimate INR initiator codon. In the case of the transcript that originates from P1, there are 12 AUGs before the legitimate translational start signal (Marr, 2007).

The DNA sequences immediately upstream of the mapped transcript start sites contain easily recognizable sequences similar to the computationally and biochemically determined common core promoter elements. P1 contains a TATA box, an Initiator element, and a downstream promoter element (DPE). P2 contains a TATA-like box and a DPE but no recognizable Initiator. P3 contains a recognizable Initiator but no recognizable TATA box or DPE. Importantly, a constitutively active form of dFOXO (dFOXO-A3) activates all three promoters in Drosophila Schneider line 2 (S2) cells, and this increased RNA synthesis can produce dINR protein even in the presence of insulin. The transcript originating at P1 is by far the most abundant transcript under both unactivated and activated conditions. P2 is present at an intermediate level, and P3 is a low-abundance transcript. Interestingly, the level of transcription correlates with the number of recognized core promoter elements, illustrating the important role these different elements play in determining the total level of transcription from a gene in both activated and unactivated states (Marr, 2007).

In the animal, all three transcripts are detectable in multiple developmental stages. They are present in whole animal extracts in the same relative order of abundance that is detected in S2 cells (P1 >> P2 > P3). When compared with the Rp49 transcript, a common control transcript that changes little over the stages tested, all three transcripts fluctuate in abundance. Notably, all three transcripts diminish significantly in the L3 larva, a time when the animal is voraciously eating. In contrast, these dINR transcripts peak in the pupae, a time when the animal is fasting and expending much of the energy gained during the larval stage. This observation is consistent with a previous finding that dINR expression is linked to nutrient availability (Marr, 2007).

Strikingly, dINR is not only transcriptionally up-regulated but also robustly translated. Growing S2 cells in the absence of serum and insulin causes a marked decrease in the rate of incorporation of radiolabeled cysteine and methionine consistent with a global decrease in the rate of translation. Despite this slowing of overall translation, dINR protein accumulates in S2 cells. This is detectable by immunoblot of whole cell extracts with antisera raised against the dINR protein. The increase in dINR protein levels is at least partially due to the absence of insulin itself and not another component of serum because the accumulation of dINR protein is inhibited by addition of insulin to media containing insulin-depleted serum. In addition, the increased dINR protein level is most likely due to increased synthesis since serum starved cells contain more radiolabeled receptor that binds to insulin-agarose. This raises the intriguing question of how translation of dINR can proceed in the presence of a quantitatively dephosphorylated, potently active, and up-regulated inhibitor of protein synthesis, d4E-BP. This paradoxical finding that the dINR pathway transcriptionally up-regulates both dINR and d4E-BP combined with the newly discovered unusually long 5'UTRs of these transcripts suggest that perhaps the INR gene engages the translation machinery in an unconventional manner that bypasses the need for eIF4E. A potential d4E-BP resistant internal ribosome entry site (IRES) exists in these Drosophila genes that contain long UTRs, as has been seen in other instances. For example, both the Antennapedia and Ultrabithorax long 5'UTRs contain IRESs, although their physiological role has remained undetermined (Marr, 2007).

As a first test of whether the dINR 5'UTRs also contain an IRES activity, a bicistronic construct, commonly used to assess IRES activity, was generated. The various 5'UTRs of dINR were inserted in both the forward and reverse orientations between the Renilla and firefly luciferase genes. The reverse orientation was used as a spacer length control equivalent. The ratio of Renilla luciferase expression to firefly luciferase expression should provide an indication of the cap-independent translational potential of the various 5'UTRs. Since resistance to d4E-BP is most relevant to this pathway, these experiments were carried out in the presence and absence of a constitutively active form of d4E-BP. Because the Renilla luciferase ORF is the first in the mRNA, it should be uniquely sensitive to inhibition of cap-dependent translation, while the firefly gene expression, if any, should be dependent on internal ribosome entry. The data are expressed as a ratio of the activity in the presence of d4E-BP to the activity in the absence of d4E-BP. Therefore, a number close to 1 indicates that there is no resistance to d4E-BP. In these cell-based assays, the 5'UTR from both P1 and P2 showed significant resistance to d4E-BP (about fourfold better than the reverse orientation in both cases), but only when inserted in the forward direction. Curiously, the 5'UTR from P3 showed unusual resistance to d4E-BP in either orientation. Indeed, the P3 UTR showed a perplexing increase in expression of the firefly ORF in the presence of d4E-BP compared with no UTR in both orientations. This finding reveals a potential limitation of using the bicistronic assays since interfering effects from cryptic promoters, cryptic splicing, or secondary effects of expression of d4E-BP cannot be ruled out with this assay (Marr, 2007).

To circumvent some of the inherent idiosyncrasies of the bicistronic constructs, monocistronic constructs were used that more closely mimic the situation of the endogenous dINR gene. Potential IRES activity esd measured in two complementary ways. First, in a DNA-based transient transfection, either the constitutively active form of d4E-BP or a control protein, green fluorescent protein (GFP), was expressed and resistance to d4E-BP was measure as the ratio of luciferase activity (provided by a second plasmid) in the presence of d4E-BP to the activity in the presence GFP. In this set of experiments, the minimal Antennapedia IRES, a Drosophila 5'UTR known to support cap-independent initiation of translation, was included as a positive control. Under these cell-based assay conditions, the P1 and P2 UTRs again displayed robust resistance to d4E-BP, while P3 and the common exons showed little resistance. Notably, the P2 5'UTR is as efficient as the minimal Antennapedia IRES, and the P1 5'UTR is actually significantly more efficient than the control IRES. Taken together, these two cell-based assays suggest that the 5'UTRs of at least the P1 and P2 transcripts can direct substantial IRES activity, while the P3 UTR appears to have much less if any such activity in S2 cells. Second, to complement these plasmid-based assays and directly investigate the contribution of the UTRs to translation, an RNA-based transfection assay was used. The RNAs contained either a m7G cap or an ApppG cap mimic. Only the 7mG cap allows cap-dependent translation. The ApppG cap stabilizes the transcript but does not allow cap-dependent translation, so it is a direct measure of the contribution of IRES activity. In this assay, the UTRs again showed significant IRES activity. The P1 UTR confers the same activity with or without a m7G cap, indicating a strong IRES activity. The P2 and P3 UTRs also confer cap-independent translation activity, although the level of activity is not equal to UTR plus cap. In contrast, the common exon or nonspecific UTR retains only 20% of their translation potential without the m7G cap. Taken together, these cell-based assays provide encouraging evidence for IRES activity of the dINR 5'UTRs (Marr, 2007).

However, given the well-recognized limitations inherent with using cell-based assays to establish IRES activity, a Drosophila embryo-derived cap-dependant in vitro translation system was used to test more directly the putative IRES activity and more specifically the potential d4E-BP resistance of the INR UTRs. The translation extracts were treated with micrococcal nuclease to destroy the bulk of competing endogenous transcripts so that translation would be largely dependent on exogenously added RNA. As expected, addition of normal capped transcripts results in robust translation from all of the UTR-containing RNAs as well as the common UTR and a short nonspecific UTR control RNA. To test the dependence of translation on eIF4E, exogenous m7G cap analog was added as a competitor. This excess free cap efficiently binds and sequesters the available eIF4E, preventing this essential initiation factor from binding capped RNA, thus effectively blocking the nucleation of the eIF4F complex and cap-dependent initiation. Remarkably, only the transcripts containing the P1, P2, and P3 UTRs are resistant to exogenously added competitor cap analog, whereas the common UTR fragment and the short nonspecific leader are effectively inhibited. This finding strongly suggests that the various dINR-specific UTRs, indeed, provide a cap-independent mechanism of translation initiation. To directly test the resistance of these transcripts to d4E-BP-mediated translation inhibition, recombinant d4E-BP was added to the reactions. Whereas the common exon and control RNAs are efficiently inhibited by this blocker of eIF4E-mediated translation initiation, the P1, P2, and P3 UTR-containing transcripts are highly resistant to d4E-BP. These findings taken together with cell-based assays suggest that, indeed, dINR protein synthesis can proceed via an IRES-mediated eIF4E-independent mechanism of initiation both in vitro and in vivo (Marr, 2007).

What purpose might a cap-independent translation activity serve beyond simple resistance to the active d4E-BP in the absence of insulin? Perhaps by functionally coupling transcription and translation, such a mechanism could serve to amplify the signal received from the insulin receptor pathway. To test this idea, in vitro translation experiments were used. In the absence of miccrococal nuclease treatment, the endogenous transcripts present in the translation extract should effectively compete with the experimental dINR transcripts for limiting amounts of the translation machinery. Advantage was taken of this inevitable competition for translation machinery to test the response of the various UTRs in a situation that may more closely reflect the cellular environment, where multiple variable abundant transcripts must compete for a limited supply of the translational apparatus. Under these competitive conditions, addition of either m7G or d4E-BP actually results in an even more robust increase in translation of the dINR UTR-containing RNAs relative to the unchallenged state. This finding suggests that these RNAs that contain dINR UTRs, and presumably IRES activity, are highly effective at out-competing other transcripts for access to the translational machinery when m7G cap-dependent initiation is inhibited. While the molecular mechanism of 4E-BP resistance of the dINR transcripts have not been unequivocally defined, it is clear that the UTRs allow significant translation in conditions when cap-dependent translation is inhibited (Marr, 2007).

These data allowed formulation of a new model to explain the effects of nutrients and insulin levels on dINR feedback regulation. In times of high nutrients and therefore high insulin-like peptides, both dFOXO and d4E-BP are phosphorylated and inactive. Under these 'rich' conditions, dFOXO is sequestered in the cytoplasm and phosphorylated d4E-BP is unable to interact with eIF4E. This situation allows efficient translation of most cellular transcripts regardless of the mechanism of initiation (cap-dependent vs. cap-independent). In contrast, in low nutrient conditions or in the absence of insulin or insulin-like peptides, both dFOXO and d4E-BP become dephosphorylated and active. Activated dFOXO directs a robust increase in the transcription of both dINR and d4E-BP (among other genes). Additionally, the active and up-regulated d4E-BP effectively inhibits cap-dependent translation, freeing up the protein synthesis machinery to selectively translate IRES-containing transcripts like dINR. These two coordinated mechanisms consequently orchestrate the integration of a specific transcriptional response and simultaneously a translational response that greatly amplifies the signal and sensitizes the cell for detection of small changes in nutrient availability as well as, possibly, developmental and environmental cues (Marr, 2007).

Interestingly, the dFOXO-responsive dINR promoters produce three distinct transcripts. Why such a complex regulatory network? A hint may be that the P3 UTR does not seem to have detectable IRES activity in the S2 cells but shows substantial activity in vitro with extracts derived from whole Drosophila embryos. It is likely that the three transcripts are produced in a tissue- or temporal-specific manner during development, and it is speculated that each may depend on cell-specific IRES trans-acting factors (ITAFs) that are required for activity. This would direct tissues to respond differentially to dINR signaling. In tissues lacking specific ITAFs, the IRES activity would be diminished and the tissue may produce only a moderate level of dINR protein (Marr, 2007).

An interesting parallel was found between mechanisms for reprogramming the gene expression machinery in a cell to respond to physiological cues and the more commonly observed viral takeover of the cellular macromolecular synthesis machinery. When some viruses, such as polio, infect a cell, they target the translation initiation machinery (either eIF4G or 4E-BP) so that there is a switch from cap-dependent synthesis to IRES-dependent synthesis. This leads to a robust and specific stimulation of viral protein synthesis at the expense of most cellular protein synthesis. By the evolution of cellular mechanisms that activate 4E-BP and simultaneously produce transcripts containing cellular IRESs, a critical physiological signaling cascade can evidently adopt a similar mechanism to effectively usurp the macromolecular synthesis machinery to drive cellular physiology in a very specific direction. Indeed, viruses may have merely co-opted the mechanism from cells in the eternal battle between host and virus (Marr, 2007).

Although the initial characterization of the INR transcriptional feedback loop was carried out in Drosophila, a similar regulatory circuit has been found in vertebrates. It is interesting to note that the transcripts for human insulin receptor and IGF-2 receptor remain associated with polysomes when cap-dependant translation is inhibited by poliovirus infection. Although the level of INR mRNA up-regulation by FOXO in mouse muscle cells is only twofold, the levels of INR protein increase much more dramatically (six- to eight-fold), consistent with a coupled transcription/translation mechanism of the signal in vertebrates. It seems likely, given the findings report in this study, that the same type of coupling between the transcriptional program of FOXO proteins and translational control by IRES activity is also occurring in vertebrate systems. Understanding this novel mechanism that couples transcription and translation may provide new insight into disease states such as insulin-resistant type 2 diabetes (Marr, 2007).

Protein Interactions

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 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).

Insulin treatment caused a strong increase in the immunoreactivity of d4E-BP to antibodies directed against human phospho-4E-BP1(Thr37/46). Since the residues in the region of Thr46, but not Thr37, are perfectly conserved between 4E-BP1 and d4E-BP, it is probably Thr46 that is hyperphosphorylated in d4E-BP after insulin treatment. Regulation of d4E-BP phosphorylation thus appears to differ from that of mammalian 4E-BP1. Phosphorylation of Thr37 and Thr46 of 4E-BP1 is only modestly induced after serum stimulation in serum-starved HEK293 cells (Gingras, 1999). In serum-starved HEK293 cells treated with rapamycin, the phosphorylation of 4E-BP1 at Thr37 and Thr46 is reduced, but upon serum addition, it is restored to its original state, whereas phosphorylation of Ser65 and Thr70 remains blocked (Gingras, 1999; Gingras, 2001). Thus, Ser65 and Thr70 are the rapamycin-sensitive sites of 4E-BP1. In contrast, in S2 cells, phosphorylation of d4E-BP at Thr46 is robustly induced after insulin treatment, and rapamycin completely blocks its phosphorylation. Therefore, unlike 4E-BP1, phosphorylation at Thr46 is a major insulin-stimulated and rapamycin-sensitive event involved in d4E-BP regulation. A dependency between Thr37 and Thr46 that is analogous to that of 4E-BP1 is important for d4E-BP phosphorylation. For 4E-BP1, phosphorylations at Thr37 and Thr46 are intimately linked; these two phosphorylation events are regulated coordinately by mTOR (Gingras, 1999). The link between Thr37 and Thr46 in d4E-BP is conserved, but it is not known if Thr37 acts as a priming event for the subsequent phosphorylation of Thr46 or if Thr37 and Thr46 are regulated coordinately, similar to 4E-BP1. Hence, phosphorylation of d4E-BP may be explained by three possible models: (1) In the primed model, d4E-BP is already phosphorylated at Thr37 and is subsequently phosphorylated on one additional site, Thr46, after insulin stimulation. (2) In the sequential model, d4E-BP is phosphorylated on Thr37 and then Thr46 (or vice versa). (3) In the coordinated model, d4E-BP is phosphorylated coordinately on Thr37 and Thr46 (Miron, 2003).

The results suggest a simpler mode of regulation of d4E-BP by phosphorylation compared with the hierarchical phosphorylation of mammalian 4E-BP1. The phosphorylation of 4E-BP1 is the best understood, but not all mammalian 4E-BPs are regulated in a similar manner. 4E-BP2 is phosphorylated on fewer residues (Lin, 1996) and is dephosphorylated more slowly than 4E-BP1. Also, 4E-BP3 is weakly stimulated by insulin treatment, causing poor release from eIF4E. This has been attributed to the lack of the four-residue RAIP motif found in the N terminus of 4E-BP1 and -2 but not in 4E-BP3 Tee, 2003). The RAIP motif seems to be required for the efficient overall phosphorylation of 4E-BP1, and intriguingly, this motif is also lacking in d4E-BP. Another important motif for 4E-BP1 regulation is the TOR signaling (TOS) motif (Schalm, 2002), which is conserved (FQLDL, at the C terminus) in d4E-BP. Hence, a consequence of the lack of the RAIP motif may be the simpler regulation of d4E-BP to improve its release from eIF4E when d4E-BP is recruited to the dTOR/dRaptor complex through the TOS motif (Nojima, 2003, Schalm, 2002). Moreover, because of a divergent eIF4E-binding site, d4E-BP does not interact as strongly with deIF4E as 4E-BP1 (Miron, 2001). It is conceivable that because of the poorer interaction of d4E-BP with deIF4E, phosphorylation at Thr37 and Thr46 is sufficient to bring about its release from eIF4E. IEF-SDS-PAGE results indicate that in serum-starved S2 cells, some d4E-BP is already phosphorylated. Although these additional sites do not prevent the interaction between deIF4E and d4E-BP, it is possible that they contribute to the release from deIF4E once Thr46 becomes phosphorylated (Miron, 2003).

A large body of work has established the paramount importance of the InR-IRS-PI3K-PTEN signaling module in the control of cell growth. This module coordinates cellular metabolism with the nutritional state. The primary outcome of its activation is the modulation of the PI3K/PTEN cycle and consequent PIP₃ production. The increase in PIP₃ facilitates the recruitment of pleckstrin homology domain-containing proteins, such as dAkt and dPDK1, to the plasma membrane. dPTEN mutant flies die because of increased membrane translocation and activation of dAkt. Phosphorylation of the Tsc2 subunit of TSC by Akt results in inhibition of the complex by causing its dissociation or by blocking its interaction with other proteins. TSC inhibits S6K and 4E-BP1 by repressing the GTPase Rheb, preventing it from activating mTOR through an unknown mechanism. Thus, mTOR, is clearly a critical regulator of S6K and 4E-BP1 in mammals and Drosophila (Miron, 2003 and references therein).

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).

Interaction of Drosophila 4E-BP with eIF4E isoforms

The Drosophila genome-sequencing project has revealed a total of seven genes encoding eight eukaryotic initiation factor 4E (eIF4E) isoforms. Four of them (eIF4E-1,2, eIF4E-3, eIF4E-4 and eIF4E-5) share exon/intron structure in their carboxy-terminal part and form a cluster in the genome. All eIF4E isoforms bind to the cap (m7GpppN) structure. All of them, except eIF4E-6 and eIF4E-8 are able to interact with Drosophila eIF4G or eIF4E-binding protein (4E-BP). eIF4E-1, eIF4E-2, eIF4E-3, eIF4E-4 and eIF4E-7 rescue a yeast eIF4E-deficient mutant in vivo. Only eIF4E-1 mRNAs and, at a significantly lower level, eIF4E3 and eIF4E-8 are expressed in embryos and throughout the life cycle of the fly. The transcripts of the remaining isoforms are detected from the third instar larvae onwards. This indicates the cap-binding activity relies mostly on eIF4E-1 during embryogenesis. This agrees with the proteomic analysis of the eIF4F complex purified from embryos and with the rescue of l(3)67Af, an embryonic lethal mutant for the eIF4E-1,2 gene, by transgenic expression of eIF4E-1. Overexpression of eIF4E-1 in wild-type embryos and eye imaginal discs results in phenotypic defects in a dose-dependent manner (Hernandez, 2005).

Phosphorylation of 4E-BP by LRRK2 affects the maintenance of dopaminergic neurons in Drosophila

Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent molecular lesions so far found in Parkinson's disease (PD), an age-dependent neurodegenerative disorder affecting dopaminergic (DA) neuron. The molecular mechanisms by which mutations in LRRK2 cause DA degeneration in PD are not understood. This study shows that both human LRRK2 and the Drosophila orthologue of LRRK2 phosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP), a negative regulator of eIF4E-mediated protein translation and a key mediator of various stress responses. Although modulation of the eIF4E/4E-BP pathway by LRRK2 stimulates eIF4E-mediated protein translation both in vivo and in vitro, it attenuates resistance to oxidative stress and survival of DA neuron in Drosophila. These results suggest that chronic inactivation of 4E-BP by LRRK2 with pathogenic mutations deregulates protein translation, eventually resulting in age-dependent loss of DA neurons (Imai, 2008).

This study used Drosophila as a model system to understand the normal physiological function of LRRK2 and how its dysfunction leads to DA neurodegeneration. Genetic and biochemical evidence is provided that dLRRK modulates the maintenance of DA neuron by regulating protein synthesis. LRRK2 primes phosphorylation of 4E-BP, and this event has an important function in mediating the pathogenic effects of mutant dLRRK. These results link deregulation of the eIF4E/4E-BP pathway of protein translation with DA degeneration in PD (Imai, 2008).

eIF4E is a key component of the eIF4F complex that initiates cap-dependent protein synthesis. It has long been recognized that a key mechanism regulating eIF4E function is through phosphorylation-induced release of 4E-BP from eIF4E. A number of candidate kinases, including mTOR, have been implicated on the basis of in vitro or cell culture studies, but the physiological kinases remain to be identified. This study shows that LRRK2 is one of the physiological kinases for 4E-BP. LRRK2 exerts an effect on 4E-BP primarily at the T37/T46 sites. Phosphorylation at T37/T46 by LRRK2 likely facilitates subsequent phosphorylation at T70 and S65 in vivo by other kinase or LRRK2 itself. 4E-BP phosphorylation by LRRK2, therefore, could serve as an initiating event in an ordered, multisite phosphorylation process to generate hyperphosphorylated 4E-BP, similar to the phosphorylation of the Alzheimer's disease-associated tau. These results show that LRRK2 is not the only kinase that phosphorylates 4E-BP T37/T46 sites. Similarly, 4E-BP is unlikely the only substrate of LRRK2. A recent study showed that human LRRK2 phosphorylates moesin, the physiological relevance of which remains to be determined (Imai, 2008).

The role of 4E-BP in regulating eIF4E function has been well established in vitro. Recent studies in Drosophila, however, have revealed the complexity of the in vivo function of 4E-BP. Loss of the only d4E-BP gene does not affect cell size or animal viability, suggesting that it is dispensable for cell growth or survival under normal conditions. However, d4E-BP mutant flies are defective in responses to various stress stimuli. d4E-BP has also been proposed to exert an effect as a metabolic brake for fat metabolism under stress condition. Whether this role of 4E-BP is relevant to dLRRK function in stress resistance and DA neuron maintenance remains to be tested. eIF4E, the target of 4E-BP, functions primarily in regulating general protein translation in vitro. It has been suggested that overactivation of eIF4E is linked to the aging process and lifespan regulation. This study observed that overexpression of eIF4E as well as dLRRK leads to an aging-related phenotype in DA neurons, which strongly suggested that chronic attenuation of 4E-BP activity promotes oxidative stress and consequent aging in DA neurons. This is consistent with the finding of similar patterns of gene expression under oxidative stress and aging conditions, and the fact that PD caused by LRRK2 mutations is of late onset, with aging being a major risk factor (Imai, 2008).

This study analysed effects of removing dLRRK activity using a transposon insertion allele (dLRRK−), a chromosomal deletion allele (dLRRK Df) and gene knockdown (dLRRK RNAi). dLRRK(−/−), dLRRK (Df/−) and dLRRK RNAi flies are all resistant to oxidative stress treatments and show reduced endogenous ROS damages. In the paraquat treatment assay, dLRRK (Df/−) appeared more resistant than dLRRK(−/−). It is possible that dLRRK(−/−), which contains a transposon insertion in the COR domain of dLRRK, is not a null allele, although it has not been possible to detect a truncated protein product using an antibody against the N-terminus of the protein. Alternatively, the chromosomal deletion in dLRRK (Df) may include other gene(s) relevant to stress sensitivity. One candidate is the gene for PI3K Dp110 subunit. A recent study reported that dLRRK(−/−) animals are slightly sensitive to hydrogen peroxide but are comparable to control animals in response to paraquat. It is possible that the different genetic backgrounds and the nutrient conditions may account for the divergent results. In the current studies, the mutant chromosome was backcrossed to w⁻ WT background for six generations in an effort to eliminate potential background mutations. A consistent finding from this study and two other studies of dLRRK(−/−) animals is that dLRRK is dispensable for the maintenance of DA neurons, although in one study it was reported that dLRRK(−/−) animals show reduced TH immunoreactivity and shrunken morphology of DA neurons. In contrast, overexpression of hLRRK2 containing a pathogenic G2019S mutation, or overexpression of mutant dLRRK as reported in this study, caused DA neuron degeneration, supporting the fact that the pathogenic mutations cause disease by a GOF mechanism (Imai, 2008).

The pathogenesis caused by mutations in LRRK2 could be partially explained by their higher kinase activity. Indeed, some pathogenic mutants of both hLRRK2 and dLRRK show elevated kinase activity towards 4E-BP. However, other mutants (e.g., hLRRK2 Y1699C and dLRRK Y1383C) did not show elevated kinase activity in vivo. Therefore, these pathogenic hLRRK2 mutations might confer cellular toxicity through mechanisms other than protein translation. For example, some hLRRK2 mutants are prone to aggregation in cultured cells. Consistently, dLRRK Y1383C mutant appeared as more prominent vesicular aggregates in fly DA neurons. Nevertheless, the facts that overexpression of eIF4E is sufficient to confer hypersensitivity to oxidative stress and DA neuron loss and that co-expression of 4E-BP suppresses the dopaminergic toxicity caused by more than one pathogenic dLRRK mutants provide compelling evidence that the eIF4E-4E-BP axis has an important function in mediating the pathogenic effects of overactivated LRRK2. The more downstream events that lead to DA neurotoxicity remain to be elucidated. So far, no clear evidence has been found of altered autophagy, caspase activation or DNA fragmentation (Imai, 2008).

There are several possibilities of how elevated protein translation could contribute to PD pathogenesis. First, given that protein synthesis is a highly energy-demanding process, stimulation of protein translation by LRRK2 could perturb cellular energy and redox homoeostasis. This could be especially detrimental in aged cells or stressed post-mitotic cells such as DA neurons. Second, increased protein synthesis could lead to the accumulation of misfolded or aberrant proteins, overwhelming the already compromised ubiquitin proteasome and molecular chaperone systems in aged or stressed cells. Third, altered LRRK2 kinase activity may affect synapse structure and function, which is known to involve local protein synthesis. Deregulation of this process could lead to synaptic dysfunction and eventual neurodegeneration (Imai, 2008).

LRRK2 kinase regulates synaptic morphology through distinct substrates at the presynaptic and postsynaptic compartments of the Drosophila neuromuscular junction

Mutations in leucine-rich repeat kinase 2 (LRRK2) are linked to familial as well as sporadic forms of Parkinson's disease (PD), a neurodegenerative disease characterized by dysfunction and degeneration of dopaminergic and other types of neurons. The molecular and cellular mechanisms underlying LRRK2 action remain poorly defined. This study shows that LRRK2 controls synaptic morphogenesis at the Drosophila neuromuscular junction. Loss of Drosophila LRRK2 results in synaptic overgrowth, whereas overexpression of Drosophila LRRK or human LRRK2 has opposite effects. Alteration of LRRK2 activity also affects neurotransmission. LRRK2 exerts its effects on synaptic morphology by interacting with distinct downstream effectors at the presynaptic and postsynaptic compartments. At the postsynapse, LRRK2 interacts with the previously characterized substrate 4E-BP (Imai, 2008), an inhibitor of protein synthesis. At the presynapse, LRRK2 phosphorylates and negatively regulates the microtubule (MT)-binding protein Futsch. These results implicate synaptic dysfunction caused by deregulated protein synthesis and aberrant MT dynamics in LRRK2 pathogenesis and offer a new paradigm for understanding and ultimately treating PD (Lee, 2010).

Parkinson's disease (PD) is one of the most common neurodegenerative diseases and is characterized by locomotor abnormalities as a result of the dysfunction and eventual loss of dopaminergic (DA) neurons. Most PD cases are sporadic with no known cause. Recent advances in PD genetics have led to the identification of familial PD (FPD) genes. It is anticipated that understanding the disease mechanisms of the FPD cases will provide insights into PD pathogenesis in general. Despite intensive studies of the FPD gene products at the biochemical and cell biological levels, understanding of their physiological function and the molecular and cellular pathways underlying disease pathogenesis is still fragmentary. Of all FPD genes, leucine-rich repeat kinase 2 (LRRK2) is the most frequently mutated. LRRK2 encodes a large serine/threonine kinase with multiple other domains. Some pathogenic mutations in LRRK2, such as the I2020T and G2019S substitutions in the kinase domain and R1441C substitution in the ROC domain, appear to augment kinase activity. In Drosophila and mouse models, pathogenic human (hLRRK2) or Drosophila (dLRRK) LRRK2 induce parkinsonian phenotypes in an age-dependent manner. A number of LRRK2-interacting proteins and substrates have been identified through in vitro studies, which implicate diverse biological functions for LRRK2 in translational control, vesicular trafficking, and cytoskeletal regulation. The physiological relevance of these interacting proteins and substrates remain to be established (Lee, 2010).

Actin and microtubule (MT) cytoskeleton dynamics play a crucial role in the formation of the nervous system, regulating such fundamental processes as axonal guidance and synaptogenesis. Dynamic modulation of synaptic structure and function is fundamental to neural network formation during development and is the molecular basis of learning and memory. Synaptic dysfunction is tightly linked to the pathogenesis of major neurodegenerative diseases such as Alzheimer's disease, and its role in PD is beginning to be appreciated. In Drosophila, the MT-associated protein 1B (MAP1B) homolog Futsch is required for axonal and dendritic growth during embryogenesis and for synaptic morphogenesis during larval neuromuscular junction (NMJ) development. This study shows that dLRRK phosphorylates and negatively regulates Futsch function at the presynapse. The previously characterized dLRRK substrate 4E-BP functionally interacts with LRRK2 at the postsynapse. These results implicate defects in presynaptic MT cytoskeleton dynamics and postsynaptic protein synthesis in LRRK2 pathogenesis (Lee, 2010).

Genetic mutations in LRRK2 are frequently found in familial and sporadic PD cases. Understanding the physiological function of LRRK2 will therefore offer insights into PD pathogenesis in general. This study reveals a new physiological function of LRRK2 and offers molecular mechanisms underlying such function. The key findings from this study are that LRRK2 plays an important role in regulating synaptic morphogenesis and that it does so through distinct substrate proteins at the presynaptic and postsynaptic compartments. The results also show that the precise level of LRRK2 activity is important for synaptic morphogenesis and neurotransmission, but the regulation of these two synaptic processes likely involve different mechanisms and players. Given the similarity of Drosophila NMJ synapse to mammalian excitatory glutamatergic synapses, it is possible that the findings reported here are relevant to mammalian systems (Lee, 2010).

Synaptic loss is a major neurobiological substrate of cognitive dysfunction in a number of neurological diseases. Extensive studies in patients and animal models have documented that synaptic failure is one of the earliest events in the pathogenesis of Alzheimer's disease. Interestingly, neurotransmission defects have been repeatedly observed in rodent FPD models, including the LRRK2 model, although no obvious signs of neurodegeneration accompany the electrophysiological defects. These results suggest that synaptic dysfunction is a primary effect of FPD gene mutations and that synaptic failure is intimately involved in PD pathogenesis. The molecular mechanisms underlying these synaptic transmission defects, however, remain elusive. This study of the LOF and GOF models of LRRK2 in Drosophila provides mechanistic insights into the possible cause of synaptic dysfunction in LRRK2-associated PD. It was found that LRRK2 regulates synaptic morphogenesis at the presynaptic and postsynaptic compartments through distinct substrates (Lee, 2010).

In the presynaptic side, LRRK2 forms a complex with tubulin and the MT-binding protein Futsch. Furthermore, LRRK2 phosphorylates Futsch and negatively regulates the presynaptic function of Futsch in controlling MT dynamics. MT cytoskeleton is critical for the generation and maintenance of neuronal axons and dendrites, transport of synaptic vesicles and organelles along the processes, and the initiation and maintenance of synaptic transmission. Disrupted MT dynamics in neuronal synapses has been implicated as an underlying cause for several neurological diseases, including hereditary spastic paraplegia and fragile X syndrome. LRRK2-associated PD may share this feature with the aforementioned diseases. Disrupted MT dynamics could be responsible for the presynaptic effects observed in LRRK2 LOF and GOF mutants, such as aberrant mitochondria distribution. The synaptic vesicle transport phenotypes seen in Caenorhabditis elegans LRK-1 mutant could also be attributable to altered MT dynamics. These could all contribute to synaptic dysfunction. Futsch/MAP1B proteins are large multidomain proteins that are phosphorylated by multiple kinases, including Sgg/GSK-3β, PAR-1/MARK, and Cdk5, which also phosphorylate tau and are implicated in tau pathology. Tau-related pathology has been observed in LRRK2 transgenic animals. It would be interesting to test for possible interplay between LRRK2 and these other kinases in regulating MT-binding proteins and MT dynamics. In mammalian hippocampal neurons, overexpression of pathogenic hLRRK2 led to reduced neurite length and branching, whereas deficiency of LRRK2 had opposite effects. Whether MT dynamics regulated by Futsch/MAP1B is contributing to this LRRK2 effect in mammals will await additional investigation (Lee, 2010).

This study also showed that LRRK2 interacts with 4E-BP at the postsynapse. 4E-BP acts as a negative regulator of the translational initiation factor eIF4E through direct binding and sequestration. Phosphorylation of 4E-BP by LRRK2 weakens 4E-BP binding to eIF4E (Imai, 2008), therefore releasing the inhibitory effect of 4E-BP on eIF4E. Previous studies have demonstrated an important postsynaptic role for eIF4E-mediated protein synthesis in activity-dependent synaptic growth at the Drosophila NMJ (Sigrist, 2000). Genetic interaction studies demonstrate a functional role for 4E-BP in mediating the synaptic effects of LRRK2. However, the exact roles of 4E-BP and LRRK2 in this process appear complex. For example, (1) despite the prominent effects of 4E-BP overexpression on NMJ development, its loss of function has no obvious effect. One would expect a gain of eIF4E function in the absence of 4E-BP and therefore a synaptic-overgrowth phenotype. (2) 4E-BP activity is predicted to be high in dLRRK mutant and low in LRRK2 overexpression condition, but this study observed synapse phenotypes opposite of what is predicted based on the presumed roles of eIF4E and 4E-BP on Drosophila NMJ morphogenesis. One possible explanation of these seemingly disparate results is that phospho-4E-BP, the product of LRRK2-mediated phosphorylation of 4E-BP, instead of being inactive and inert, may actually perform some new synaptic function at the NMJ. In this scenario, loss of 4E-BP function in d4E-BP mutant would not show the same phenotype as LRRK2 overexpression, which produces more phospho-4E-BP. Recent studies in Drosophila dopaminergic neurons suggest a functional role for phospho-4E-BP in vivo. Alternately, effectors other than 4E-BP may also mediate the effects of LRRK2 on NMJ development and neurotransmission. Although 4E-BP overexpression might have masked the effects of these other effectors, in dLRRK mutant background, the functional roles of these other effectors might manifest themselves. A similar situation was observed in pumillo mutant, in which a synapse-loss phenotype was observed despite the upregulation of eIF4E activity in this mutant attributable to the derepression of translational inhibition of eIF4E, which would have resulted in a predicted synapse-overgrowth phenotype. Involvement of other effectors in mediating the effects of LRRK2 on NMJ development and neurotransmission, and possibly different effectors for NMJ development versus neurotransmission, could also explain the complex electrophysiological phenotypes of dLRRK mutant and LRRK2 overexpression animals, as well as the uncoupling of the effects on synaptic differentiation and neurotransmission by the various genetic manipulations. Future studies will test these possibilities as well as the relevance of the NMJ studies to dopaminergic neuron synapses (Lee, 2010).

In addition to LRRK2, the TOR pathway also regulates 4E-BP function through phosphorylation. This pathway primarily regulates cell and organism growth through diverse outputs, including protein synthesis, cytoskeletal change, autophagy, and cell survival. This study found that treatment of flies with rapamycin, an inhibitor of TOR, has the same effects as 4E-BP overexpression in wild type as well as LRRK2 overexpression backgrounds. Although rapamycin has been extensively studied in the context of autophagy induction and neurodegeneration models, its effect on Drosophila NMJ development is unlikely attributable to autophagy, because the current results show that presynaptic or postsynaptic induction of autophagy through Atg1 overexpression had no obvious effect on synapse number. The similar effects of rapamycin and 4E-BP overexpression on NMJ development support that rapamycin acts via the 4E-BP translational control pathway to impact NMJ development. A recent report showed that either 4E-BP overexpression or 4E-BP activation by rapamycin could suppress the muscle and dopaminergic neurodegeneration phenotypes seen in Drosophila Pink1 and Parkin models of PD. These results suggest that deregulation of protein synthesis could be generally involved in PD pathogenesis and that rapamycin or its analogs could be developed into effective PD therapeutics (Lee, 2010).

4E-BPs require non-canonical 4E-binding motifs and a lateral surface of eIF4E to repress translation

eIF4E-binding proteins are a widespread class of translational regulators that share a canonical (C) eIF4E-binding motif (4E-BM) with eIF4G. Consequently, 4E-BPs compete with eIF4G for binding to the dorsal surface on eIF4E to inhibit translation initiation. Some 4E-BPs contain non-canonical 4E-BMs (NC 4E-BMs), but the contribution of these motifs to the repressive mechanism-and whether these motifs are present in all 4E-BPs-remains unknown. This study shows that the three annotated Drosophila melanogaster 4E-BPs contain NC 4E-BMs. These motifs bind to a lateral surface on eIF4E that is not used by eIF4G. This distinct molecular recognition mode is exploited by 4E-BPs to dock onto eIF4E-eIF4G complexes and effectively displace eIF4G from the dorsal surface of eIF4E. These data reveal a hitherto unrecognized role for the NC4E-BMs and the lateral surface of eIF4E in 4E-BP-mediated translational repression, and suggest that bipartite 4E-BP mimics might represent efficient therapeutic tools to dampen translation during oncogenic transformation (Igreja, 2014).

Cyclin G functions as a positive regulator of growth and metabolism in Drosophila

In multicellular organisms, growth and proliferation is adjusted to nutritional conditions by a complex signaling network. The Insulin receptor/target of rapamycin (InR/TOR) signaling cascade plays a pivotal role in nutrient dependent growth regulation in Drosophila and mammals alike. This study identifies Cyclin G (CycG) as a regulator of growth and metabolism in during larval development in Drosophila. CycG mutants have a reduced body size and weight and show signs of starvation accompanied by a disturbed fat metabolism. InR/TOR signaling activity is impaired in cycG mutants, combined with a reduced phosphorylation status of the kinase Akt1 and the downstream factors S6-kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP). Moreover, the expression and accumulation of Drosophila insulin like peptides (dILPs) is disturbed in cycG mutant brains. Using a reporter assay, it was shown that the activity of one of the first effectors of InR signaling, Phosphoinositide 3-kinase (PI3K92E), is unaffected in cycG mutants. However, the metabolic defects and weight loss in cycG mutants are rescued by overexpression of Akt1 specifically in the fat body and by mutants in widerborst (wdb), the B'-subunit of the phosphatase PP2A, known to downregulate Akt1 by dephosphorylation. Together, these data suggest that CycG acts at the level of Akt1 to regulate growth and metabolism via PP2A in Drosophila (Fischer, 2015).

This study analyzed the role of Cyclin G in growth regulation and metabolism of Drosophila. Two different cycG null mutant alleles were used, thereby allowing the following of the developmental consequences resulting from the absence of cycG gene activity instead of drawing conclusions from overexpression or RNAi experiments. Misexpression studies initially raised the assumption that CycG negatively regulated cell growth and cell proliferation in Drosophila. The current results now indicate that CycG is required for normal growth, affecting both cell size and cell number. In fact, clonal analysis revealed a cell autonomous requirement of CycG not only in the wing but also the eye anlagen. In addition, the cycG null mutants show signs of metabolic disorder. Evidence is provided that CycG facilitates InR/TORC1 mediated growth regulation via PP2A, thereby helping to sustain nutrient dependent growth in Drosophila (Fischer, 2015).

Drosophila CycG appears to have extraordinarily diverse roles. It has been involved in epigenetic regulation of homeotic gene activity, in cell cycle regulation, developmental stability and in DNA repair, and now also in metabolic homeostasis. The current work confirmed molecular interactions between CycG and Wdb proteins in vivo that had been predicted from genome-wide proteome analyses in vitro. Interestingly, similar molecular interactions have been described before for mammalian CycG1 and CycG2: both proteins interact with several B' subunits, thereby mediating the recruitment of PP2A to its different substrates. In contrast to mammals, the genetic relationship between CycG and PP2A is antagonistic in Drosophila as a reduction of PP2A activity ameliorates the consequences of CycG loss. The cycG mutation could be formally explained by a gain of PP2A activity. It is tempting to speculate that the diversity of CycG functions results from a regulation of PP2A by CycG. PP2A affects a plethora of developmental and cellular processes, hence, pleiotropy is expected in case of its misregulation. Most likely, this hypothesis is too simplified. For example, loss of cycG in the female germ line results in an increase of phosphorylated H2Av (gamma-H2Av), a known target of PP2A activity. One might have expected a reduced amount of gamma-H2Av if loss of CycG equated with a gain in PP2A activity. Instead, this study has shown that CycG is found in a protein complex together with Rad9 and BRCA2 that primarily acts in the sensing of DNA double strand breaks. The importance of Drosophila CycG in DNA double strand break repair is reminiscent of functions described for mammalian CycG proteins: albeit CycG1 and CycG2 mutant mice are viable and healthy, they are both sensitive to DNA damaging reagents. Moreover, upregulation of CycG2 was involved in the activation of Chk2 and in damage induced G2/M cell cycle arrest, i.e. in DNA damage response in mammals as well. Whether the other phenotypes and interactions reported for Drosophila CycG are linked to the regulation of PP2A remains to be addressed in more detail (Fischer, 2015).

The cycG mutants display several phenotypic characteristics of a diminished TORC1 signaling activity, including weight reduction, a reduced egg laying rate, impaired endoreplication and a general increase in lipid mobilization. Moreover, CycG activity promotes phosphorylation of the primary TORC1 targets, i.e. S6K and 4E-BP. In contrast to TOR mutants, however, cycG mutants are viable, implying that CycG facilitates InR/TOR signaling rather than being an essential factor. Overall, cycG mutant flies show typical signs of nutritional starvation distress even under normal food conditions, suggesting a problem in their capacity to take up food and/or to sense and utilize the food. This defect is not due to a general inability of the animal to grasp the feed, but instead reflects a defect in coordinating the energy status with the regulation of systemic growth. As dILP accumulation in the brain is altered in cycG mutants, it is known that the signals transmitted from the nutritional sensor fat body must be disturbed. The fact, that the growth defects of cycG mutants can be strongly ameliorated by an induction of Akt1 specifically in the fat body rules out a function of CycG in the endocrine signal emanating from the fat body. Instead, all of the data indicate that CycG acts genetically at the level of Akt1, thereby controlling TOR signaling activity (Fischer, 2015).

Akt1 is negatively regulated by PP2A, supporting a model whereby CycG exerts its positive input on Akt1 via an inhibition of PP2A. In accordance, mutations in wdb efficiently rescue the growth and metabolic defects observed in cycG mutants. Likewise a downregulation of Wdb ameliorates the weight deficits resulting from a loss of Akt1 activity. In Drosophila, Wdb acts as a tissue-specific negative regulator of Akt1: it modulates lipid metabolism in the ovary as a result of a direct interaction with Akt1, whereas no such influence was seen in eye tissue. This study has shown that Wdb-Akt1 binding in the adult head is favored in the absence of CycG, i.e. CycG is able to influence the interaction between Wdb and Akt1 presumably by its direct binding to Wdb. A consequence of CycG loss may be the enhanced binding of PP2A to Akt1 and an enforced dephosphorylation of Akt1, resulting in the inhibition of downstream TOR signaling activity and affecting lipid metabolism and growth. Moreover, the second B'-subunit of Drosophila PP2A (also called Well rounded, Wrd) is involved in the negative regulation of the S6K. Assuming a molecular interaction of Wrd and CycG, a likewise regulatory input of CycG on PP2A containing the Wrd B'-subunit is conceivable. In this case, CycG might influence S6K activity as well, having a regulatory input on InR/TOR signaling also downstream of TORC1. This scenario is complicated by the negative feed back regulation of InR signaling by S6K and of Akt1 by TORC1. Circular regulation of InR/TOR signaling has been described at several levels, implementing a tight control of dietary signals and growth but complicating genetic analyses (Fischer, 2015).

In conclusion, the identification of CycG as a novel regulator of InR/TOR signaling in Drosophila highlights the importance of studying the regulatory network at the Akt1—PP2A nexus. Based on the high conservation of the InR/TOR signaling pathway and its regulation by PP2A, mammalian fat homeostasis is likely to involve similar regulatory control mechanisms to those that have been uncovered in Drosophila. This work raises the possibility of an involvement of CycG in InR/TOR-associated diseases that might be modulated by PP2A. A better understanding of the underlying mechanisms could therefore open up avenues for new strategies to fight InR/TOR-associated disorders in the future (Fischer, 2015).

Diminished MTORC1-dependent JNK activation underlies the neurodevelopmental defects associated with lysosomal dysfunction

This study evaluated the mechanisms underlying the neurodevelopmental deficits in Drosophila and mouse models of lysosomal storage diseases (LSDs). Lysosomes promote the growth of neuromuscular junctions (NMJs) via Rag GTPases and mechanistic target of rapamycin complex 1 (MTORC1). However, rather than employing S6K/4E-BP1, MTORC1 stimulates NMJ growth via JNK, a determinant of axonal growth in Drosophila and mammals. This role of lysosomal function in regulating JNK phosphorylation is conserved in mammals. Despite requiring the amino-acid-responsive kinase MTORC1, NMJ development is insensitive to dietary protein. This paradox is attributed to anaplastic lymphoma kinase (ALK), which restricts neuronal amino acid uptake, and the administration of an ALK inhibitor couples NMJ development to dietary protein. These findings provide an explanation for the neurodevelopmental deficits in LSDs and suggest an actionable target for treatment (Wong, 2015).

Mucolipidosis type IV (MLIV) and Batten disease are untreatable lysosomal storage diseases (LSDs) that cause childhood neurodegeneration. MLIV arises from loss-of-function mutations in the gene encoding TRPML1, an endolysosomal cation channel belonging to the TRP superfamily. The absence of TRPML1 leads to defective lysosomal storage and autophagy, mitochondrial damage, and macromolecular aggregation, which together initiate the protracted neurodegeneration observed in MLIV). Batten disease arises from the absence of a lysosomal protein, CLN3), and results in psychomotor retardation. Both diseases cause early alterations in neuronal function. For instance, brain imaging studies revealed that MLIV and Batten patients display diminished axonal development in the cortex and corpus callosum, the causes of which remain unknown (Wong, 2015).

To better understand the etiology of MLIV in a genetically tractable model, flies were generated lacking the TRPML1 ortholog. The trpml-deficient (trpml¹) flies have led to insight into the mechanisms of neurodegeneration and lysosomal storage (Wong, 2015).

This study reports that trpml¹ larvae exhibit diminished synaptic growth at the NMJ, a well-studied model synapse. Lysosomal function supports Rag GTPases and MTORC1 activation, and this is essential for JNK phosphorylation and synapse development (Wong, 2015).

Drosophila larvae and mice lacking CLN3 also exhibit diminished Rag/ MTORC1 and JNK activation, suggesting that alterations in neuronal signaling are similar in different LSDs and are evolution- arily conserved. Interestingly, the NMJ defects in the two fly LSD models were suppressed by the administration of a high-protein diet and a drug that is currently in clinical trials to treat certain forms of cancer. These findings inform a pharmacotherapeutic strategy that may suppress the neurodevelopmental defects observed in LSD patients (Wong, 2015).

This study shows that lysosomal dysfunction in Drosophila MNs results in diminished bouton numbers at the larval NMJ. Evidence is presented that lysosomal dysfunction results in decreased activation of the amino-acid-responsive cascade involving Rag/MTORC1, which are critical for normal NMJ development (Wong, 2015).

Despite the requirement for MTORC1 in NMJ synapse development, previous studies and the current findings show that bouton numbers are independent of S6K and 4E-BP1. Rather, MTORC1 promotes NMJ growth via a MAP kinase cascade culminating in JNK activation. Therefore, decreasing lysosomal function or Rag/MTORC1 activation in hiw^ND8 suppressed the associated synaptic overgrowth. However, the 'small-bouton' phenotype of hiw^ND8 was independent of MTORC1. Thus, MTORC1 is required for JNK-dependent regulation of bouton numbers, whereas bouton morphology is independent of MTORC1. Furthermore, although both rheb expression and hiw loss result in Wnd-dependent elevation in bouton numbers, the supernumerary boutons in each case show distinct morphological features. Additional studies are needed for deciphering the complex interplay between MTORC1-JNK in regulating the NMJ morphology (Wong, 2015).

Biochemical analyses revealed that both JNK phosphorylation and its transcriptional output correlated with the activity of MTORC1, which are consistent with prior observations that cln³ overexpression promotes JNK activation and that tsc1/tsc2 deletion in flies result in increased JNK-dependent transcription. These findings point to the remarkable versatility of MTORC1 in controlling both protein trans lation and gene transcription (Wong, 2015).

Using an in vitro kinase assay, this study demonstrates that Wnd is a target of MTORC1. Because axonal injury activates both MTORC1 and DLK/JNK, these findings imply a functional connection between these two pathways. Interestingly, the data also suggest that MTORC1 contains additional kinases besides MTOR that can phosphorylate Wnd. One possibility is that ULK1/Atg1, which associates with MTORC1, could be the kinase that phosphorylates Wnd. Consistent with this notion, overexpression of Atg1 in the Drosophila neurons has been shown to promote JNK signaling and NMJ synapse overgrowth via Wnd) (Wong, 2015).

This study also found that developmental JNK activation in axonal tracts of the CC and pJNK levels in cortical neurons were compromised in a mouse model of Batten disease. Thus, the signaling deficits identified in Drosophila are also conserved in mammals. The activity of DLK (the mouse homolog of Wnd) and JNK signaling are critical for axonal development in the mouse CNS. Therefore, decreased neuronal JNK activation during development might underlie the thinning of the axonal tracts observed in many LSDs (Wong, 2015).

Although the findings of this study demonstrate a role for an amino-acid- responsive cascade in the synaptic defects associated with lysosomal dysfunction, simply elevating the dietary protein content was not sufficient to rescue these defects. These findings were reminiscent of an elegant study that showed that the growth of Drosophila neuroblasts is uncoupled from dietary amino acids owing to the function of ALK, which suppresses the uptake of amino acids into the neuroblasts (Cheng, 2011). Indeed, simultaneous administration of an ALK inhibitor and a high-protein diet partially rescued the synaptic growth defects associated with the lysosomal dysfunction, and improved the rescue of pupal lethality associated with trpml¹. Although these studies do not causally link the defects in synapse development with pupal lethality, they do raise the intriguing possibility that multiple phenotypes associated with LSDs could be targeted using ALK inhibitors along with a protein-rich diet (Wong, 2015).

Although LSDs result in lysosomal dysfunction throughout the body, neurons are exceptionally sensitive to these alterations. The cause for this sensitivity remains incompletely understood. Given the findings of this study that mature neurons do not efficiently take up amino acids from the extracellular medium, lysosomal degradation of proteins serves as a major source of free amino acids in these cells. Therefore, disruption of lysosomal degradation leads to severe shortage of free amino acids in neurons, regardless of the quantity of dietary proteins, thus explaining the exquisite sensitivity of neurons to lysosomal dysfunction (Wong, 2015).

DEVELOPMENTAL BIOLOGY

The developmental profile of wild-type Thor expression shows transcripts present throughout all stages of development, with a noticeable increase in the larval stages, especially the third instar. beta-galactosidase in the P{lacW} Thor strain localizes to the embryonic nervous system and to many tissues in larvae and adults (Rodriguez, 1996). The same results were found by tissue in situ hybridizations with Thor probes, and the analysis has been extended to the reproductive system, Thor expression is found in the testes of third instar males and in the ovaries of adult females. This expression in the testes and ovarian nurse cells is part of a more complex pattern of expression in both the female and male reproductive systems. The general expression of Thor and in particular the expression in the ovaries, in which there are intricate systems of translational regulation, are consistent with the role of Thor as a 4E-BP, but the role of a 4E-BP in the immune response is not consistent with or predicted by current views in immunity (Bernal, 2000).

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, 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, 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, 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, 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, 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, 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, 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, 2011).

EFFECTS OF MUTATION

The initiation factor 4E for eukaryotic translation (eIF4E) binds the messenger RNA 5'-cap structure and is important in the regulation of protein synthesis. Mammalian eIF4E activity is inhibited when the initiation factor binds to the translational repressors, the 4E-binding proteins (4E-BPS). The Drosophila 4E-BP (d4E-BP) is a downstream target of the phosphatidylinositol-3-OH kinase [PI(3)K] signal-transduction cascade, which affects the interaction of d4E-BP with eIF4E. Ectopic expression of a highly active d4E-BP mutant in wing-imaginal discs causes a reduction of wing size, brought about by a decrease in cell size and number. A marked reduction in cell size is also observed in post-mitotic cells. Expression of d4E-BP in the eye and wing together with PI(3)K or dAkt1, the serine/threonine kinase downstream of PI(3)K, results in suppression of the growth phenotype elicited by these kinases. These results support a role for d4E-BP as an effector of cell growth (Miron, 2001).

Drosophila 4E-BP (d4E-BP) was isolated by interaction cloning from a complementary DNA expression library using ³²P-labelled deIF4EI. d4E-BP is identical to Drosophila Thor (Bernal, 2000) and homologous to 4E-BPs from other species. Phosphorylation sites in mammalian 4E-BP1 are conserved in d4E-BP, but the predicted eIF4E-binding motif in d4E-BP (YERAFMK) diverges from the canonical consensus sequence (Miron, 2001).

To examine the binding of d4E-BP to deIF4E, residues within the consensus eIF4E-binding site were mutated. Recombinant proteins were expressed in Escherichia coli, and far Western blotting was performed using ³²P-labelled deIF4EI. Mutation of Tyr 54 to Ala (Y54A) or Phe (Y54F), and Met 59 to Ala (M59A) abrogates the interaction of d4E-BP with deIF4E. Mutation of Lys 60 to Ala (K60A) decreases deIF4E binding by 87%, indicating that Lys 60 contributes to deIF4E binding. However, when either Met 59 or Lys 60 are mutated to the consensus Leu, the interaction of d4E-BP with deIF4EI is 2.5-fold higher than with the wild type, and when both Met 59 and Lys 60 are so changed, deIF4E binding increases by 3.4-fold. These results indicate that d4E-BP interacts with deIF4E, albeit more weakly than previously characterized 4E-BPs, owing to its divergent eIF4E-binding motif (Miron, 2001).

4E-BP1 is hyperphosphorylated in response to insulin in many cell types. To test whether this response operates in Drosophila, Schneider-2 (S2) cells were deprived of serum and treated with insulin. Increasing levels of a slower migrating form of d4E-BP (d4E-BP) were observed consequent to insulin treatment. To determine whether the ß-form corresponds to phosphorylated d4E-BP, extracts from insulin-stimulated S2 cells were treated with either calf intestine alkaline phosphatase (CIP) or protein phosphatase 2A (PP2A). Untreated extracts (or extracts kept on ice) contain both the faster migrating alpha- and the slower migrating ß-forms. In contrast, phosphatase-treated extracts contained only the alpha-form (Miron, 2001).

LY294002 and rapamycin inhibit PI(3)K and target of rapamycin (TOR) activity, respectively, and block the insulin-induced hyperphosphorylation of 4E-BP1. Similarly, exposure of serum-deprived S2 cells to either drug before treatment with insulin, results in a dose-dependent decrease in d4E-BP phosphorylation. To determine whether phosphorylation of d4E-BP prevents its binding to deIF4E, m⁷GDP-agarose precipitation was performed. The alpha form is present primarily in the bound fraction, whereas the ß-form is found exclusively in the unbound fraction. These results show that d4E-BP is a downstream target of the PI(3)K pathway, and that the binding of d4E-BP to deIF4E is modulated by its phosphorylation state (Miron, 2001).

Assembly of eIF4F is essential for translational control, and overexpression of eIF4E in mammalian cells results in malignant transformation. To investigate whether eIF4F is also linked to growth control, eIF4F assembly was perturbed in Drosophila. UAS transgenic fly lines were generated that express wild-type d4E-BP or the mutant d4E-BP that binds deIF4E most strongly, d4E-BP(LL). Expression of d4E-BP was targeted to the wing-imaginal disc using MS1096-GAL4. The size and cell number of wings from males were measured. Expression of wild-type d4E-BP has no effect on wing size or pattern. However, expression of d4E-BP(LL) from one line [d4E-BP(LL)^w] causes a marked reduction of wing size without affecting cell number. Another line, [d4E-BP(LL)^s], which expresses d4E-BP(LL) more strongly, causes a larger reduction, which is partly due to a decrease in cell number. Since direct inhibition of cellular proliferation increases, rather than decreases, cell size, it is possible that d4E-BP(LL) also affects cell size directly, and cell proliferation as a consequence. This is supported by analysis of the effects of d4E-BP(LL) expression in larval-wing discs. Although discs from the d4E-BP(wt) and d4E-BP(LL)^w lines are indistinguishable from control discs, d4E-BP(LL)^s discs are 52% smaller. d4E-BP(LL)^s males also required 1-2 days longer to eclose, which would account for the smaller decrease in adult wings (Miron, 2001).

Acridine-orange staining shows that d4E-BP(LL)^s discs contain many apoptotic cells. Co-expression of p35, the baculovirus inhibitor of apoptosis, with d4E-BP(LL)^s partially rescues the size of adult wings. To distinguish between apoptosis and decreased proliferation, cell clones expressing d4E-BP(LL), with or without p35, and co-expressing green fluorescent protein (GFP), were induced 72 h after egg deposition in developing wing discs using the flip-out technique. Clones expressing d4E-BP(LL)^w contain fewer cells than wild-type clones, but co-expression of p35 with d4E-BP(LL)^w does not affect the number of cells per clone, indicating that decreased proliferation, but not increased apoptosis, is the cause of reduction. Few clones expressing d4E-BP(LL)^s are recovered, and they usually contain 1-2 cells. Co-expression of p35 greatly increases the number of clones recovered, but only marginally increases the number of cells per clone (1-4 cells) (Miron, 2001).

Direct interference with cell proliferation using string mutants results in increased cell size. To help distinguish effects on size from effects on proliferation, cell size was evaluated by flow cytometry (FACS). Mean forward-light scatter values for GFP-positive cells that expressed d4E-BP(LL) were reduced by 6%-8%. Because cells that expressed d4E-BP(LL) are smaller and proliferate more slowly than their wild-type counterparts, it is conceivable that d4E-BP(LL) directly affects cell growth and consequently retards proliferation, which would lead to reduced viability and ultimately apoptosis. Similar results were observed in dTOR mutants, and interpreted as a primary defect in cellular growth coupled with a consequent decrease in cell proliferation. The possibility that growth and proliferation are affected independently by d4E-BP(LL) expression cannot be excluded (Miron, 2001).

To exclude proliferation effects, the growth and viability of d4E-BP(LL) cells were examined in a post-mitotic tissue. Polyploid fat-body cells undergo successive rounds of DNA synthesis without mitoses. Cells that express d4E-BP(LL)^s, induced 48 h after egg deposition in the fat body, are 45%-70% smaller than neighboring wild-type cells, but their frequency is much higher than in mitotically active tissues, such as the wing-imaginal disc. Thus, viability of cells that express d4E-BP(LL) is maintained in the absence of mitogenic signals, indicating that proliferation of wild-type neighboring cells is necessary to cause the disappearance of cells expressing d4E-BP(LL). In support of this notion is the finding that when d4E-BP(LL)^s clones are induced during development of eye-imaginal discs, only the clones that are generated posterior to the morphogenetic furrow survive; the clones generated anterior to the furrow (that is, in mitotically active cells), are eliminated (Miron, 2001).

To study the possible role of d4E-BP as an effector of cell growth through the PI(3)K signaling pathway, potential interactions between d4E-BP and relevant signaling genes of this pathway were examined. Expression of wild-type d4E-BP in eye-imaginal discs, using GMR-GAL4, does not engender any discernible phenotype, whereas expression of dAkt1 results in an enlarged eye. However, co-expression of wild-type d4E-BP and dAkt1 partially suppresses the enlarged-eye phenotype, and fully suppresses the roughness induced by expression of dAkt1. Since d4E-BP by itself has no effect on eye size but is able to suppress the dAkt1 phenotype, there is a genuine epistatic relationship between d4E-BP and dAkt1 (Miron, 2001).

Other components of the PI(3)K pathway were also examined for potential epistatic interactions with d4E-BP in the wing, using dpp-GAL4 and 4E-BP(LL)^s. Ectopic expression of Dp110 and dAkt1 causes an enlargement of the region encompassed by the third and fourth longitudinal veins, the anterior crossvein and wing margin. In contrast, expression of a dominant-negative mutant form of PI(3)K (Dp110^D954A) or d4E-BP(LL)^s results in reduction of the size of this region. Co-expression of d4E-BP(LL)^s with Dp110 or dAkt1 suppresses the growth enhancement engendered by expression of these kinases, whereas co-expression of d4E-BP(LL)^s with Dp110^D954A results in further size reduction. Flies that lacked a copy of the gene encoding the adaptor protein p60 [the Drosophila homolog of mammalian PI(3)K subunit p85] are also reduced in size when d4E-BP(LL)^s is co-expressed. These results provide genetic evidence that d4E-BP is a downstream effector of the PI(3)K pathway (Miron, 2001).

Null mutants of d4E-BP are viable and although their immune response is compromised (Bernal, 2000), they do not exhibit increased growth. Furthermore, ectopic expression of Drosophila eIF4E in a wild-type or d4E-BP null background fails to produce a growth-related phenotype. Therefore, an increase in eIF4E activity alone is not sufficient to promote cell growth in Drosophila imaginal discs. This is consistent with data in primary mouse-embryo fibroblasts, in which eIF4E overexpression leads only to oncogenic transformation when co-expressed with c-myc or E1A. Attempts were made to rescue the d4E-BP(LL)-induced growth defects in imaginal discs by co-expressing deIF4E. Unexpectedly, growth is further reduced. Thus, endogenous eIF4E expression levels are optimal for cell growth and proliferation, and in the absence of activation of the PI(3)K pathway, a further increase in eIF4E expression is either without effect or deleterious (Miron, 2001).

Many studies have shown that PI(3)K and TOR-mediated signaling is important for normal cell growth and proliferation. However, one downstream target of this pathway, S6K, regulates cell size but not proliferation. Constitutive expression of dS6K in dTOR mutants only partially suppresses the dTOR phenotype, indicating that S6K-independent targets operate downstream of dTOR. Regulation of eIF4E activity, independent of S6K, contributes to the control of cell size. In Drosophila, the activity of eIF4E is modulated through 4E-BP. Phosphorylation of eIF4E is correlated with increased translation rates. Mutation of the phosphorylation site in Drosophila eIF4E causes a cell size reduction. In summary, the results presented here show that d4E-BP acts as an important downstream effector of PI(3)K in the regulation of cell proliferation and growth, independent of S6K, and further underline the importance of translation initiation in the latter process (Miron, 2001).

Response of mutant Thor flies to bacterial infection.

Thor has been identified as a new type of gene involved in Drosophila host immune defense. Thor is a member of the 4E-binding protein (4E-BP) family, which in mammals has been defined as comprising critical regulators in a pathway that controls initiation of translation through binding eukaryotic initiation factor 4E (eIF4E). Without an infection, Thor is expressed during all developmental stages and transcripts localize to a wide variety of tissues, including the reproductive system. In response to bacterial infection and, to a lesser extent, by wounding, Thor is up-regulated. The Thor promoter has the canonical NFkappaB and associated GATA recognition sequences that have been shown to be essential for immune induction, as well as other sequences commonly found for Drosophila immune response genes, including interferon-related regulatory sequences. In survival tests, Thor mutants show symptoms of being immune compromised, indicating that Thor may be critical in host defense. In contrast to Thor, Drosophila eIF4E is not induced by bacterial infection. These findings for Thor provide the first evidence that a 4E-BP family member has a role in immune induction in any organism. Further, no gene in the translation initiation pathway that includes 4E-BP has been previously found to be immune induced. These results suggest either a role for translational regulation in humoral immunity or a new, nontranslational function for 4E-BP type genes (Bernal, 2000).

The most critical test of immune response is for survival after infection. To determine the effect of Thor mutation on immune response, Thor mutations were identified and then both control and Thor mutant flies were subjected to different types of bacterial infection and their survival was observed for 4 days (Bernal, 2000).

The P{lacW} insertion that led to identifying Thor also resulted in the Thor¹ mutation. P{lacW} inserted adjacent to the TATA box, separating the coding region from the promoter region, and Northern analysis of flies with this insertion reveals that only a small amount of the 0.85-kb Thor transcript is present, and it does not increase after infection. The P-element insertion has thus resulted in the production of the Thor¹ mutation, a noninducible, very weakly expressing hypomorph. To control for background effects, a wild-type revertant of the Thor¹ mutation, Thor^1rv1, was used, that was produced by precise excision of the P{lacW} insertion. To confirm that any differences between Thor¹ and Thor^1rv1 flies are the result of mutation of Thor, a second Thor mutation, Thor², was used. In Thor², P{lacW} also is excised, but leaving Thor still mutant because of imprecise excision that deleted bases between P{lacW} and the first B site. Thor¹ and Thor² mutants are homozygous viable and fertile, and in noninfected and sterile wounding controls, survival is similar to wild-type Oregon R flies, Thor^1rv1 and imd. Thor^1rv1 survives like Oregon R and is referred to as the designated wild-type control. As a control for susceptibility to infection, imd mutants were used. In similar experiments, flies with the imd mutation have been shown to be highly susceptible to bacterial infection, and imd clearly operates in controlling induction of antibacterial genes (Bernal, 2000).

Survival of mutant Thor adults is similar to wild-type adults when infected by E. coli and E. cloacae B₁₂. Both bacterial types affected imd flies as expected, with on average 11% surviving 4 days after infection with E. cloacae B₁₂ and 1% surviving with E. coli infection. After infection (4 days) with S. epidermidis, however, on average only 46% of Thor¹ and 47% of Thor² flies survived. Surprisingly, 66% of imd flies survived, which is much higher than Thor mutants and also the previously reported survival for imd flies, which was approximately 8% after E. coli infection. Tests with M. roseus showed that even wild-type flies are susceptible, with 47% surviving on average. Thor mutants and imd flies are both more susceptible, with 11% for Thor¹, 16% for Thor², and 2% for imd surviving on average 4 days after infection. The antibacterial response provides a strong defense, and failure of survival by mutants indicates that a critical facet of the response has been disrupted. These results show that Thor mutants are immune compromised, and when subjected to some types of bacteria, the disruption of the immune response leads to failure of survival (Bernal, 2000).

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).

FOXO/4E-BP signaling in Drosophila muscles regulates organism-wide proteostasis during aging

The progressive loss of muscle strength during aging is a common degenerative event of unclear pathogenesis. Although muscle functional decline precedes age-related changes in other tissues, its contribution to systemic aging is unknown. This study shows that muscle aging is characterized in Drosophila by the progressive accumulation of protein aggregates that associate with impaired muscle function. The transcription factor FOXO and its target 4E-BP remove damaged proteins at least in part via the autophagy/lysosome system, whereas foxo mutants have dysfunctional proteostasis. Both FOXO and 4E-BP delay muscle functional decay and extend life span. Moreover, FOXO/4E-BP signaling in muscles decreases feeding behavior and the release of insulin from producing cells, which in turn delays the age-related accumulation of protein aggregates in other tissues. These findings reveal an organism-wide regulation of proteostasis in response to muscle aging and a key role of FOXO/4E-BP signaling in the coordination of organismal and tissue aging (Demontis, 2010).

By using a number of behavioral, genetic and molecular assays, this study has described a mechanism in the pathogenesis of muscle aging that is based on the loss of protein homeostasis (proteostasis) and the resulting decrease in muscle strength (see FOXO/4E-BP Signaling in Muscles Controls Proteostasis and Systemic Aging). Increased activity of Pten and the transcription factor FOXO is sufficient to delay this process, while foxo null animals experience accelerated loss of proteostasis during muscle aging. Pten and FOXO induce multiple protective responses, including the expression of folding chaperones, and the regulator of protein translation 4E-BP that has a pivotal role in preserving proteostasis. FOXO and 4E-BP preserve muscle function at least in part by sustaining the basal activity of the autophagy/lysosome system, which removes aggregates of damaged proteins. However, additional mechanisms may be involved. For example, the proteasome system may degrade damaged proteins and thus avoid their accumulation in aggregates. Thus, perturbation in proteasome assembly and subunit composition may contribute to muscle aging in response to FOXO activity. In addition, while overexpression of a single chaperone had limited effects, interventions to effectively limit the extent of protein damage are likely to delay the decay in proteostasis by decreasing the workload for the proteasome and autophagy systems (Demontis, 2010).

By comparing the accumulation of poly-Ubiquitinated proteins in aggregates of aging muscles, retinas, brains, and adipose tissue, this study has found that reduced protein homeostasis is a general feature of tissue aging that is particularly prominent in muscles. The observation that muscle aging is characterized by loss of proteostasis further suggests some similarity between muscle aging and neurodegenerative diseases, many of which are characterized by the accumulation of protein aggregates (Demontis, 2010).

Mechanical, thermal, and oxidative stressors occur during muscle contraction and therefore muscle proteins may be particularly susceptible to damage in comparison with other tissues. While the current findings refer to the loss of proteostasis in the context of normal aging, it is likely that a better understanding of this process will likely help cure muscle pathologies associated with aging, as some of the underlying mechanisms of etiology may be shared. For example, most cases of inclusion body myositis (IBM) arise over the age of 50 years, defining aging as a major risk factor for the pathogenesis of this disease. Interestingly, muscle weakness in patients with IBM is characterized by the accumulation of protein aggregates, which we have now described to occur in the context of regular muscle aging in Drosophila. Thus, FOXO may interfere with the pathogenesis of muscle degenerative diseases in addition to muscle aging. Studies in animal disease models of IBM will be needed to test this hypothesis (Demontis, 2010).

There is an apparent contradiction between the current findings and data describing the FOXO-dependent induction of muscle atrophy in mice, a serious form of age-related muscle degeneration that results in decreased muscle strength. The observation that different degrees of FOXO activation can promote stress resistance or rather cell death could explain why FOXO activity can be protective or rather detrimental during muscle aging. In particular, while physiologic FOXO activation can preserve protein homeostasis and muscle function, its excessive activation may lead to decreased muscle function due to hyper-activation of protein turnover pathways. Consistent with this view, the macroautophagy pathway has also been involved in both muscle atrophy as well as in the preservation of muscle sarcomere organization, highlighting the importance of fine tuning the degree of activation of stress resistance pathways to maintain muscle homeostasis. In addition, the output of FOXO activity may radically differ in growing versus pre-existing myofibers. In particular, the current study indicates that FOXO protects pre-existing myofibers against age-dependent changes in proteostasis, while it also blunts developmental muscle growth in flies (Demontis, 2009), as observed in mammals. Thus, deleterious effects of FOXO activation as observed in mammalian muscles may result from the inhibition of growth of novel myofibers in post-natal development and adulthood, a process which is thought to be limited to development in Drosophila (Demontis, 2010).

An interesting observation of this study is that interventions that decrease muscle aging also extend the lifespan of the organism. In particular, this work raises the prospect that the extent of muscle aging may be a key determinant of systemic aging. Reduced muscle proteostasis may be detrimental per se for life expectancy, presumably due to the involvement of muscles in a number of key physiological functions. Consistent with this view, overexpression in muscles of aggregation-prone human Huntington’s disease proteins is sufficient to decrease lifespan. Moreover, FOXO signaling in muscles regulates proteostasis in other tissues, via inhibition of feeding behavior and decreased release of Insulin from producing cells, that in turn promote 4E-BP activity systemically. Thus, it is proposed that FOXO/4E-BP signaling in muscles regulates lifespan and remotely controls aging events in other tissues by bringing about some of the protection associated with decreased food intake (Demontis, 2010).

In mammals, muscles produce a number of cytokines involved in the control of systemic metabolism. For example, Interleukin-6 (IL-6) is produced by muscles and has been proposed to control glucose homeostasis and feeding behavior through peripheral and brain mechanisms. Thus, a muscle-based network of systemic aging as observed in flies may occur in humans (Demontis, 2010).

This study supports the common belief that preserving muscle function is beneficial for overall aging and the notion that muscles are central tissues to coordinate organism-wide processes, including aging and metabolic homeostasis. Moreover, the observation that FOXO signaling in muscles influences aging events in other tissues suggests that the systemic regulation of aging relies on tissue-to-tissue communication, which may provide the basis for interventions to extend healthy lifespan (Demontis, 2010).

Regulation of cell growth by Notch signaling and its differential requirement in normal vs. tumor-forming stem cells in Drosophila

Cancer stem cells (CSCs) are postulated to be a small subset of tumor cells with tumor-initiating ability that shares features with normal tissue-specific stem cells. The origin of CSCs and the mechanisms underlying their genesis are poorly understood, and it is uncertain whether it is possible to obliterate CSCs without inadvertently damaging normal stem cells. This study shows that a functional reduction of eukaryotic translation initiation factor 4E (eIF4E) in Drosophila specifically eliminates CSC-like cells in the brain and ovary without having discernable effects on normal stem cells. Brain CSC-like cells can arise from dedifferentiation of transit-amplifying progenitors upon Notch hyperactivation. eIF4E is up-regulated in these dedifferentiating progenitors, where it forms a feedback regulatory loop with the growth regulator dMyc to promote cell growth, particularly nucleolar growth, and subsequent ectopic neural stem cell (NSC) formation. Cell growth regulation is also a critical component of the mechanism by which Notch signaling regulates the self-renewal of normal NSCs. These findings highlight the importance of Notch-regulated cell growth in stem cell maintenance and reveal a stronger dependence on eIF4E function and cell growth by CSCs, which might be exploited therapeutically (Song, 2011).

The differential cell growth rates observed between ectopic NBs and normal or primary NBs and the correlation between cell growth defects and NB fate loss prompted a test of whether slowing down cell growth might selectively affect the formation of ectopic NBs. Attenuation of TOR signaling, a primary mechanism of cell growth regulation, through NB-specific overexpression of TSC1/2, a strong allele of eIF4E antagonist 4EBP [4EBP(LL)s], or a dominant-negative form of TOR (TOR.TED) all partially suppressed ectopic NB formation in α-adaptin (ada) mutants without affecting normal or primary NBs. Interestingly, RNAi-mediated knockdown of eIF4E, a stimulator of oncogenic transformation and a downstream effector of TOR signaling, showed a better suppression than manipulating other TOR pathway components, suggesting that eIF4E might play a more important role in ectopic NB formation. Strikingly, the brain tumor phenotypes caused by overactivation of N signaling - as in lethal giant larvae (lgl) mutant, aPKC^CAAX overexpression, or N overexpression conditions - were also fully suppressed by eIF4E knockdown. Furthermore, the brain tumor phenotypes of brat mutants were also completely rescued by eIF4E RNAi (Song, 2011).

In contrast, normal NB formation or maintenance was not affected by eIF4E knockdown. NBs with eIF4E knockdown remained highly proliferative, as evidenced by the mitotic figures, and displayed relatively normal apical basal cell polarity. There are several other eIF4E-like genes in the fly genome (Hernandez, 2005), which may play partially redundant roles in normal NB maintenance. eIF4E knockdown appeared to specifically block ectopic NB formation caused by the dedifferentiation of IPs in type II NB lineages, since it did not affect ectopic type I NB formation in cnn or polo mutants that are presumably caused by symmetric divisions of type I NBs. In addition, cell fate transformation induced by N overactivation in the SOP lineage was not affected by eIF4E RNAi, supporting the idea that eIF4E is particularly required for type II NB homeostasis. Supporting the specificity of the observed eIF4E RNAi effect, another eIF4E RNAi transgene (eIF4E-RNAi-s) also prevented ectopic NB formation. Moreover, a strong loss-of-function mutation of eIF4E also selectively eliminated ectopic NBs induced by N overactivation without affecting normal NBs, reinforcing the hypothesis that ectopic NBs exhibit higher dependence on eIF4E (Song, 2011).

To further support the notion that the ectopic NBs are particularly vulnerable to eIF4E depletion, a conditional expression experiment was carried out in which eIF4E-RNAi-s was turned on in brat mutants using the 1407^ts system, after ectopic NBs had been generated. Whereas the brain tumor phenotype exacerbated over time in the brat mutants, 1407-GAL4-driven eIF4E-RNAi-s expression in brat mutants effectively eliminated ectopic NBs, leaving normal NBs largely unaffected (Song, 2011).

In normal type II NB lineage, eIF4E protein was enriched in the NBs. Ectopic NBs induced by N overactivation in ada mutants also expressed eIF4E at high levels, whereas spdo mutant NBs exhibited reduced eIF4E expression. Thus, eIF4E up-regulation correlates with N-induced ectopic NB formation in a dedifferentiation process that likely involves elevated cell growth (Song, 2011).

Given the coincidence of nucleolar size change with ectopic NB formation, the involvement of the growth regulator dMyc was tested. dMyc protein levels were up-regulated in normal or N overactivation-induced ectopic NBs, but were down-regulated in spdo mutant NBs. Furthermore, dMyc transcription, as detected with a dMyc-lacZ transcriptional fusion reporter, was also up-regulated in both normal and ectopic NBs in ada mutants. A previous study in Drosophila S2 cells identified dMyc as a putative N target. In vivo chromatin immunoprecipitation (ChIP) experiments were carried out to assess whether dmyc transcription is directly regulated by N signaling in NBs. Using chromatin isolated from wild-type larval brains and a ChIP-quality antibody against the N coactivator Suppressor of Hairless [Su(H)], specific binding was demonstrated of Su(H) to its putative binding sites within the second intron of dmyc (dmyc-A). No binding to an internal negative control region proximal to the first exon of dmyc (dmyc-B) or to the promoter region of the rp49 gene was detected. N signaling thus directly activates dMyc transcription in the NBs. Similar to eIF4E RNAi, knockdown of dMyc strongly suppressed ectopic NB formation induced by Brat or Ada inactivation or N overactivation. Intriguingly, the strong tumor suppression effect of eIF4E knockdown was partially abolished by dMyc overexpression. Furthermore, dMyc function, as reflected by its promotion of nucleolar growth in IPs, was attenuated by eIF4E RNAi, although eIF4E RNAi alone had no obvious effect. Different from the reported eIF4E regulation of Myc expression in mammalian cells (Lin, 2008), dMyc promoter activity or protein levels remained unaltered under eIF4E RNAi conditions, suggesting that eIF4E may modulate dMyc activity without altering its expression. One possibility is that eIF4E may enter the nucleus to interact with Myc and promote its transcriptional activity. To test this hypothesis, HEK293T cells were transfected with Flag-tagged human eIF4E alone or in combination with HA-tagged dMyc. Indeed, both Drosophila dMyc and endogenous human c-Myc specifically coimmunoprecipitated with human eIF4E from nuclear extracts, indicating a conserved interaction between eIF4E and Myc within the nuclei of proliferating cells. Consistent with these biochemical data, dMyc transcriptional activity within NBs, which could be monitored with an eIF4E-lacZ reporter, was drastically reduced upon eIF4E knockdown (Song, 2011).

In contrast, eIF4E transcription, as detected with an eIF4E-lacZ transcriptional fusion reporter, as well as eIF4E protein levels detected by immunostaining were up-regulated upon dMyc overexpression and down-regulated by dMyc RNAi. It is unlikely that the changes in eIF4E-lacZ activity were due to global increases or decreases in β-galactosidase (β-gal) translation caused by altered dMyc levels, since lacZ expression from a dMyc-lacZ reporter was unaffected under similar conditions. Furthermore, like dMyc protein, eIF4E-lacZ reporter expression was up-regulated in normal NBs or ectopic NBs in ada mutants, further supporting the notion that dMyc may up-regulate eIF4E transcription. Moreover, ChIP experiments using chromatins isolated from wild-type larval brains and a ChIP-quality antibody against dMyc demonstrated specific binding of dMyc to an eIF4E promoter region harboring a cluster of adjacent noncanonical E boxes, supporting a direct regulation of eIF4E transcription by dMyc. dMyc and eIF4E thus appeared to form a regulatory feedback loop that promoted NB growth and renewal. Consistent with this model, while knocking down either dMyc or eIF4E had no noticeable effect on type II NB maintenance and only a mild effect on NB nucleolar size in the case of dMyc RNAi, their simultaneous knockdown led to a significant reduction in nucleolar size, premature neuronal differentiation, and loss of NBs (Song, 2011).

If the dMyc-eIF4E axis of cell growth control is a crucial downstream effector of N signaling in regulating NB maintenance, its up-regulation might be able to rescue the type II NB depletion phenotype resulting from reduced N signaling. Indeed, the loss of NBs associated with reduced Notch signaling was preventable when cell growth was boosted by dMyc overexpression. Thus, while N-IR directed by 1407-GAL4 led to complete elimination of type II NBs, the coexpression of dMyc, but not CD8-GFP or Rheb, an upstream component of the TOR pathway, resulted in the preservation of approximately half of type II NBs with apparently normal cell sizes, cell fate marker expression, and lineage composition. A similar effect was observed when dMyc was coexpressed with N-IR using the conditional 1407^ts system, with transgene expression induced at the larval stage. While both dMyc and Rheb promote cell growth, they do so through distinct mechanisms, with the former increasing nucleolar size and the latter expanding cytoplasmic volume. These results thus provide compelling evidence that control of cell growth, particularly nucleolar growth, is a critical component in the maintenance of NB identity by N signaling (Song, 2011).

The differential responses of normal and tumor-initiating stem cells to functional reduction of eIF4E prompted a test of whether chemicals that specifically inhibit eIF4E function might have therapeutic potential in preventing CSC-induced tumorigenesis. Indeed, the brain tumor phenotypes induced by N overactivation or ada loss of function were effectively suppressed by feeding animals with fly food containing Ribavirin, an eIF4E inhibitor that interferes with eIF4E binding to mRNA 5' caps and promotes the relocalization of eIF4E from the nucleus to the cytoplasm (Kentsis, 2004; Assouline, 2009) (Song, 2011).

The CSC hypothesis was initially developed based on studies in mammalian systems. Various studies have supported the notion that CSCs share many functional features with normal stem cells, such as signaling molecules, pathways, and mechanisms governing their self-renewal versus differentiation choice. However, the cellular origin of CSCs and the molecular and cellular mechanisms underlying their development or genesis remain poorly understood. It has been proposed that CSCs could arise from (1) an expansion of normal stem cell niches, (2) normal stem cells adapting to different niches, (3) normal stem cells becoming niche-independent, or (4) differentiated progenitor cells gaining stem cell properties. This study has showen that in the Drosophila larval brain, CSCs can arise from the dedifferentiation of transit-amplifying progenitor cells back to a stem cell-like state. Importantly, eIF4E was identified as a critical factor involved in this dedifferentiation process. More significantly, it was shown that reduction of eIF4E function can effectively prevent the formation of CSCs without affecting the development or maintenance of normal stem cells. This particular dependence on eIF4E function by CSCs appears to be a general theme, as reduction of eIF4E function also effectively prevented the formation of CSCs, but not normal GSCs, in the fly ovary. These findings may have important implications for stem cell biology and cancer biology, in terms of both mechanistic understanding and therapeutic intervention (Song, 2011).

This study also offers mechanistic insights into the cellular processes leading to the dedifferentiation of progenitors back to stem cells. In Drosophila type II NB clones with overactivated N signaling, ribosome biogenesis within ectopic NBs appears to be faster than in normal NBs, as shown by the fact that the ratio of nucleolar to cellular volume of the ectopic NBs is approximately fivefold higher than that of normal NBs. The faster growth rate is accompanied by the up-regulation of dMyc and eIF4E and appears to be essential for transit-amplifying progenitors to undergo complete dedifferentiation back to a stem cell-like state. When the function of cell growth-promoting factors such as eIF4E is attenuated, the faster cell growth of ectopic NBs can no longer be sustained and the dedifferentiation process stalls. As a result, brain tumor formation caused by uncontrolled production of ectopic NBs is suppressed. In contrast, normal NBs, which presumably have relatively lower requirements for cell growth and hence eIF4E function, maintain their stem cell fate and development under similar conditions. Therefore, a potential key to a successful elimination of CSC-induced tumors would be to find the right level of functional reduction in eIF4E, which causes minimal effects on normal stem cells but effectively obliterates CSCs. An ongoing clinical trial with Ribavirin in treating acute myeloid leukemia (AML) (Assouline, 2009), a well-characterized CSC-based cancer, demonstrated exciting proof of principle that such a strategy is feasible. The current version of Ribavirin, however, has certain limitations, such as its poor specificity and the high dosage (micromolar range) required for effective treatment. Thus, more specific and effective eIF4E inhibitors are urgently needed. The drug treatment experiments with Ribavirin validated Drosophila NBs as an excellent CSC model for searching further improved drugs. More importantly, the nuclear interaction between eIF4E and Myc unraveled by this biochemical analysis not only provides a new mechanistic explanation for the synergistic effects of eIF4E and Myc in tumorigenesis (Ruggero, 2004; Wendel, 2007), but also sheds new light on how to rationally optimize drug design and therapy for treating CSC-based cancer (Song, 2011).

The results offer new information on how N signaling helps specify and maintain NSC fate. N signaling regulates stem cell behavior in various tissues of diverse species. However, it remains unclear how differential N signaling determines distinct cell fate within the stem cell hierarchy. This study demonstrates that N signaling maintains Drosophila NSC fate at least in part through promoting cell growth. The following evidence supports that cell growth, but not cell fate, change is the early and primary effect of N signaling inhibition in type II NBs: (1) Pros expression is not immediately turned on in spdo mutant NBs with reduced cell sizes. Instead, it gradually increases during the course of spdo mutant NB divisions. (2) Up-regulation of Pros is not the cause of stem cell fate loss in spdo mutant NBs, as shown by spdo pros double-mutant analysis. (3) Cell growth defects precede the up-regulation of Ase expression in aph-1 mutant NBs. (4) Promotion of cell growth, and particularly nucleolar growth, by dMyc is sufficient to prevent NB loss caused by N inhibition. At the molecular level, N signaling appears to regulate the transcription of dMyc, which in turn up-regulates the transcription of eIF4E. Such a transcriptional cascade and feedback regulation of dMyc activity by eIF4E may help to sustain and amplify the activity of the Notch-dMyc-eIF4E molecular circuitry. Hence, differential N signaling within the lineage can lead to different cell growth rates, which partially determine differential cell fates. Consistent with this notion, knockdown of both eIF4E and dMyc results in defects of NB cell growth and loss of stem cell fate (Song, 2011).

While many signaling pathways and molecules have been implicated in the maintenance of stem cell identity, the question of how a stem cell loses its 'stemness' at the cellular level remains poorly understood. A stem cell may lose its stem cell fate by undergoing a symmetric division to yield two daughter cells that are both committed to differentiation or through cell death. Earlier studies provided intriguing hints that cell growth and translational regulation could influence stem cell maintenance in the Drosophila ovary. This study usded detailed clonal analyses of NSCs over multiple time points to provide direct evidence that a NSC with impaired N signaling will gradually lose its identity due to a gradual slowing down of cell growth and loss of cell mass. Remarkably, such loss of stem cell fate can be prevented when cell growth is restored by dMyc, but not Rheb, overexpression, demonstrating the functional significance of regulated cell growth, particularly nucleolar growth, in stem cell maintenance. More importantly, this information offers clues on how to specifically eliminate tumor-initiating stem cells. These studies suggest that a stem cell, normal or malignant, has to reach a certain growth rate in order to acquire and maintain its stemness, presumably because when the stem cell grows below such a threshold, its proliferative capacity becomes too low, whereas the concentration of differentiation-promoting factors becomes too high to be compatible with the maintenance of stem cell fate. Consistent with this notion are the strong correlation between the expression of ribosomal proteins and cellular proliferation (van Riggelen, 2010) as well as the correlation between the reduction of NB sizes and the up-regulation of differentiation-promoting factor Pros or Ase in different developmental contexts (Song, 2011).

The results also provide new insights into how the evolutionarily conserved tripartite motif and Ncl-1, HT2A, and Lin-41 (TRIM-NHL) domain proteins regulate stem cell homeostasis. The TRIM-NHL protein family, to which Brat and Mei-P26 belong, include evolutionarily conserved stem cell regulators that prevent ectopic stem cell self-renewal by inhibiting Myc. However, the downstream effectors of the TRIM-NHL proteins remain largely unknown. This study identified eIF4E as such a factor. NB-specific knockdown of eIF4E completely suppresses the drastic brain tumor phenotype caused by loss of Brat. Interestingly, eIF4E knockdown is even more effective than dMyc knockdown in this regard. N signaling and Brat have been proposed to act in parallel in regulating Drosophila type II NB homeostasis. However, at the molecular level, how deregulation of these two rather distinct pathways causes similar brain tumor phenotypes remain largely unknown. The current results suggest that these two pathways eventually converge on the dMyc-eIF4E regulatory loop to promote cell growth and stem cell fate. N overactivation and loss of Brat both result in up-regulation of eIF4E and dMyc in transit-amplifying progenitors, accelerating their growth rates and helping them acquire stem cell fate. Consistent with a general role of eIF4E and dMyc in stem cell regulation, it was shown that partial reduction of eIF4E or dMyc function in the Drosophila ovary effectively rescues the ovarian tumor phenotype due to the loss of Mei-P26. The vertebrate member of the TRIM-NHL family, TRIM32, is shown to suppress the stem cell fate of mouse neural progenitor cells, partially through degrading Myc. Whether eIF4E acts as a downstream effector of TRIM32 in balancing stem cell self-renewal versus differentiation in mammalian tissues awaits future investigation (Song, 2011).

EVOLUTIONARY HOMOLOGS

Interaction of 4E-BP with eIF-4E

Eukaryotic translation initiation factor 4E (eIF-4E), which possesses cap-binding activity, functions in the recruitment of mRNA to polysomes as part of a three-subunit complex, eIF-4F (cap-binding complex). eIF-4E is the least abundant of all translation initiation factors and a target of growth regulatory pathways. Recently, two human cDNAs encoding novel eIF-4E-binding proteins (4E-BPs), which function as repressors of cap-dependent translation, have been cloned. Their interaction with eIF-4E is negatively regulated by phosphorylation in response to cell treatment with insulin or growth factors. The present study aimed to characterize the molecular interactions between eIF-4E and the other subunits of eIF-4F and to similarly characterize the molecular interactions between eIF-4E and the 4E-BPs. A 49-amino-acid region of eIF-4 gamma, located in the N-terminal side of the site of cleavage by Picornaviridae protease 2A, was found to be sufficient for interacting with eIF-4E. Analysis of deletion mutants in this region led to the identification of a 12-amino-acid sequence conserved between mammals and Saccharomyces cerevisiae that is critical for the interaction with eIF-4E. A similar motif is found in the amino acid sequence of the 4E-BPs, and point mutations in this motif abolish the interaction with eIF-4E. These results shed light on the mechanisms of eIF-4F assembly and on the translational regulation by insulin and growth factors (Mader, 1995).

Translation initiation in eukaryotes is mediated by the cap structure (m⁷GpppN, where N is any nucleotide) present at the 5' end of all cellular mRNAs, except organellar. The cap is recognized by eukaryotic initiation factor 4F (eIF4F), which consists of three polypeptides, including eIF4E, the cap-binding protein subunit. The interaction of the cap with eIF4E facilitates the binding of the ribosome to the mRNA. eIF4E activity is regulated in part by two translational repressors, 4E-BP1 and 4E-BP2, which bind to it and prevent its assembly into eIF4F. 4E-BP3, a new member of the 4E-BP family, is homologous to 4E-BP1 and 4E-BP2, exhibiting 57%Êand 59% identity, respectively. The homology is most striking in the middle region of the protein, which contains the eIF4E binding motif and residues that are phosphorylated in 4E-BP1. 4E-BP3 is a heat stable protein that binds to eIF4E in vitro as well as in vivo. Further, 4E-BP3 overexpression specifically reduces eIF4E-dependent translation. The overlapping function and expression of the different 4E-BP family members imply that there is redundancy in this translational control mechanism, underscoring its importance (Poulin, 1998).

eIF4G uses a conserved Tyr-X-X-X-X-Leu-phi segment (where X is variable and phi is hydrophobic) to recognize eIF4E during cap-dependent translation initiation in eukaryotes. High-resolution X-ray crystallography and complementary biophysical methods have revealed that this eIF4E recognition motif undergoes a disorder-to-order transition, adopting an L-shaped, extended chain/alpha-helical conformation when it interacts with a phylogenetically invariant portion of the convex surface of eIF4E. Inhibitors of translation initiation known as eIF4E-binding proteins (4E-BPs) contain similar eIF4E recognition motifs. These molecules are molecular mimics of eIF4G, which act by occupying the same binding site on the convex dorsum of eIF4E and blocking assembly of the translation machinery. The implications of these results for translation initiation are discussed in detail, and a molecular mechanism for relief of translation inhibition following phosphorylation of the 4E-BPs is proposed (Marcotrigianno, 1999).

Phosphorylation of 4E-BP

The effects of insulin and rapamycin on the phosphorylation of the translation regulator, initiation factor 4E-binding protein 1 (4E-BP1) have been studied in rat fat cells by following changes in the incorporation of 32P from [32P]Pi under steady-state conditions. Both unbound 4E-BP1 and 4E-BP1 bound to eukaryotic initiation factor 4E (eIF4E) were isolated from the cells and then digested with trypsin and other proteases; the radiolabelled phosphopeptides were then separated by two-dimensional thin- layer analysis and HPLC. The results provide confirmation of the conclusion of Fadden, Haystead and Lawrence [J. Biol. Chem. (1997) 272, 10240-10247] that insulin increases the phosphorylation of four sites that fit a Ser/Thr-Pro motif (Thr-36, Thr-45, Ser-64 and Thr-69) and that taken together these phosphorylations result in the dissociation of 4E-BP1 from eIF4E. The effects of insulin on the phosphorylation of these sites, and hence dissociation from eIF4E, are blocked by rapamycin. However, the present study also provides evidence that insulin increases the phosphorylation of 4E-BP1 bound to eIF4E on a further site (Ser-111) and that this is by a rapamycin-insensitive mechanism. Extraction of rat epididymal fat cells followed by chromatography on Mono-S and Superose 12 columns results in the separation of both an insulin-stimulated eIF4E kinase and an apparently novel kinase that is highly specific for Ser-111 of 4E-BP1. The 4E-BP1 kinase is activated more than 10-fold by incubation of the cells with insulin and is markedly more active towards 4E-BP1 bound to eIF4E than towards unbound 4E-BP1. The effects of insulin are blocked by wortmannin, but not by rapamycin. A 14-mer peptide based on the sequence surrounding Ser-111 of 4E-BP1 is also a substrate for the kinase, but peptide substrates for other known protein kinases were not. The kinase is quite distinct from casein kinase 2, which also phosphorylates Ser-111 of 4E-BP1. The possible importance of these kinases in the phosphorylation of 4E-BP1 in fat cells is discussed. It is suggested that the phosphorylation of Ser-111 might be a priming event that facilitates the subsequent phosphorylation of Thr-36, Thr-45, Ser-64 and Thr69 by a rapamycin-sensitive process that initiates the dissociation of 4E-BP1 from eIF4E and hence the formation of the eIF4F complex (Heesom, 1998).

The multisubunit eukaryotic translation initiation factor (eIF) 4F recruits 40S ribosomal subunits to the 5' end of mRNA. The eIF4F subunit eIF4E interacts directly with the mRNA 5' cap structure. Assembly of the eIF4F complex is inhibited by a family of repressor polypeptides, the eIF4E-binding proteins (4E-BPs). Binding of the 4E-BPs to eIF4E is regulated by phosphorylation: Hypophosphorylated 4E-BP isoforms interact strongly with eIF4E, whereas hyperphosphorylated isoforms do not. 4E-BP1 is hypophosphorylated in quiescent cells, but is hyperphosphorylated on multiple sites following exposure to a variety of extracellular stimuli. The PI3-kinase/Akt pathway and the kinase FRAP/mTOR signal to 4E-BP1. FRAP/mTOR has been reported to phosphorylate 4E-BP1 directly in vitro. However, it is not known if FRAP/mTOR is responsible for the phosphorylation of all 4E-BP1 sites, nor which sites must be phosphorylated to release 4E-BP1 from eIF4E. To address these questions, a recombinant FRAP/mTOR protein and a FRAP/mTOR immunoprecipitate were utilized in in vitro kinase assays to phosphorylate 4E-BP1. Phosphopeptide mapping of the in vitro-labeled protein yielded two 4E-BP1 phosphopeptides that comigrated with phosphopeptides produced in vivo. Mass spectrometry analysis indicates that these peptides contain phosphorylated Thr-37 and Thr-46. Thr-37 and Thr-46 are efficiently phosphorylated in vitro by FRAP/mTOR when 4E-BP1 is bound to eIF4E. However, phosphorylation at these sites was not associated with a loss of eIF4E binding. Phosphorylated Thr-37 and Thr-46 are detected in all phosphorylated in vivo 4E-BP1 isoforms, including those that interact with eIF4E. Finally, mutational analysis has demonstrated that phosphorylation of Thr-37/Thr-46 is required for subsequent phosphorylation of several carboxy-terminal serum-sensitive sites. Taken together, these results suggest that 4E-BP1 phosphorylation by FRAP/mTOR on Thr-37 and Thr-46 is a priming event for subsequent phosphorylation of the carboxy-terminal serum-sensitive sites (Gingras, 1999).

In most instances, translation is regulated at the initiation phase, when a ribosome is recruited to the 5' end of an mRNA. The eIF4E-binding proteins (4E-BPs) interdict translation initiation by binding to the translation factor eIF4E, and preventing recruitment of the translation machinery to mRNA. The 4E-BPs inhibit translation in a reversible manner. Hypophosphorylated 4E-BPs interact avidly with eIF4E, whereas 4E-BP hyperphosphorylation, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E-binding activity. Phosphorylation of 4E-BP1 on Thr-37 and Thr-46 is relatively insensitive to serum deprivation and rapamycin treatment, and phosphorylation of these residues is required for the subsequent phosphorylation of a set of unidentified serum-responsive sites. Using mass spectrometry, the serum-responsive, rapamycin-sensitive sites have been identified as Ser 65 and Thr 70. Utilizing a novel combination of two-dimensional isoelectric focusing/SDS-PAGE and Western blotting with phosphospecific antibodies, the order of 4E-BP1 phosphorylation has been established in vivo; phosphorylation of Thr 37/Thr 46 is followed by Thr 70 phosphorylation, and Ser 65 is phosphorylated last. Finally, phosphorylation of Ser 65 and Thr 70 alone is shown to be insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF4E (Gingras, 2001).

Eukaryotic initiation factor 4E (eIF4E) binding proteins (4E-BPs) regulate the assembly of initiation complexes required for cap-dependent mRNA translation. 4E-BP1 undergoes insulin-stimulated phosphorylation, resulting in its release from eIF4E, allowing initiation complex assembly. 4E-BP1 undergoes caspase-dependent cleavage in cells undergoing apoptosis. Cleavage occurs after Asp24, giving rise to the N-terminally truncated polypeptide Delta4E-BP1, which possesses the eIF4E-binding site and all the known phosphorylation sites. Delta4E-BP1 binds to eIF4E and fails to become sufficiently phosphorylated upon insulin stimulation to bring about its release from eIF4E. Therefore, Delta4E-BP1 acts as a potent inhibitor of cap-dependent translation. Using a mutagenesis approach, a novel regulatory motif of four amino acids (RAIP) has been identified that lies within the first 24 residues of 4E-BP1 and that is necessary for efficient phosphorylation of 4E-BP1. This motif is conserved among sequences of 4E-BP1 and 4E-BP2 but is absent from 4E-BP3. Insulin increases the phosphorylation of 4E-BP3 but not sufficiently to cause its release from eIF4E. However, a chimeric protein that was generated by replacing the N terminus of 4E-BP3 with the N-terminal sequence of 4E-BP1 (containing this RAIP motif) undergoes a higher degree of phosphorylation and is released from eIF4E. This suggests that the N-terminal sequence of 4E-BP1 is required for optimal regulation of 4E-BPs by insulin (Tee, 2002).

Domain structure or of 4E-BP

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).

Eukaryotic initiation factor 4E (eIF4E) binds the mRNA cap structure and forms eIF4F complexes that recruit 40S subunits to the mRNA. Formation of eIF4F is blocked by eIF4E-binding proteins such as 4E-BP1, which interacts with eIF4E via a motif in the center of its 118-residue sequence. 4E-BP1 plays key roles in cell proliferation, growth, and survival. Binding of 4E-BP1 to eIF4E is regulated by hierarchical multisite phosphorylation. Three different features in the C terminus of 4E-BP1 play distinct roles in regulating its phosphorylation and function. (1) A new phosphorylation site in its C terminus (S101) has been identifed. A serine or glutamate at this position is required for efficient phosphorylation at Ser65. (2) A second C-terminal site, S112, directly affects binding of 4E-BP1 to eIF4E without influencing phosphorylation of other sites. (3) A conserved C-terminal motif influences phosphorylation of multiple residues, including rapamycin-insensitive sites. These relatively long-range effects are surprising given the reportedly unstructured nature of 4E-BP1 and may imply that phosphorylation of 4E-BP1 and/or binding to eIF4E induces a more-ordered structure. 4E-BP2 and -3 lack phosphorylatable residues corresponding to both S101 and S112. However, in 4E-BP3, replacement of the alanine at the position corresponding to S112 by serine or glutamate does not confer the ability to be released from eIF4E in response to insulin (Wang, 2003).

The TOS motif of 4E-BP functions as a docking site for the mTOR/raptor complex, which is required for multisite phosphorylation of 4E-BP1, eIF4E release from 4E-BP1, and cell growth

The mammalian target of rapamycin (mTOR) controls multiple cellular functions in response to amino acids and growth factors, in part by regulating the phosphorylation of p70 S6 kinase (p70S6k) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Raptor (regulatory associated protein of mTOR) is a recently identified mTOR binding partner that also binds p70S6k and 4E-BP1 and is essential for TOR signaling in vivo. Raptor binds to p70S6k and 4E-BP1 through their respective TOS (conserved TOR signaling) motifs which are shown to be required for amino acid- and mTOR-dependent regulation of these mTOR substrates in vivo. A point mutation of the TOS motif also eliminates all in vitro mTOR-catalyzed 4E-BP1 phosphorylation and abolishes the raptor-dependent component of mTOR-catalyzed p70S6k phosphorylation in vitro. Raptor appears to serve as an mTOR scaffold protein, the binding of which to the TOS motif of mTOR substrates is necessary for effective mTOR-catalyzed phosphorylation in vivo and perhaps for conferring their sensitivity to rapamycin and amino acid sufficiency (Nojima, 2003).

The mammalian target of rapamycin, mTOR, is a serine/threonine kinase that controls cell growth and proliferation via the translation regulators eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). A TOR signaling (TOS) motif has been identified in the N terminus of S6K1 and the C terminus of 4E-BP1; in S6K1, the TOS motif is necessary to facilitate mTOR signaling to phosphorylate and activate S6K1. However, it is unclear how the TOS motif in S6K1 and 4E-BP1 mediates mTOR signaling. This study shows that a functional TOS motif is required for 4E-BP1 to bind to raptor (a recently identified mTOR-interacting protein), for 4E-BP1 to be efficiently phosphorylated in vitro by the mTOR/raptor complex, and for 4E-BP1 to be phosphorylated in vivo at all identified mTOR-regulated sites. mTOR/raptor-regulated phosphorylation is necessary for 4E-BP's efficient release from the translational initiation factor eIF4E. Consistently, overexpression of a mutant of 4E-BP1 containing a single amino acid change in the TOS motif (F114A) reduces cell size, demonstrating that mTOR-dependent regulation of cell growth by 4E-BP1 is dependent on a functional TOS motif. These data demonstrate that the TOS motif functions as a docking site for the mTOR/raptor complex, which is required for multisite phosphorylation of 4E-BP1, eIF4E release from 4E-BP1, and cell growth (Schalm, 2003).

Signaling upstream of 4E-BP

The eukaryotic initiation factor 4E (eIF-4E)-binding proteins PHAS-I and PHAS-II have overlapping but different patterns of expression in tissues. Both PHAS proteins are expressed in 3T3-L1 adipocytes, in which insulin stimulates their phosphorylation, promotes dissociation of PHAS-IF-4E complexes, and decreases the ability of both to bind exogenous eIF-4E. The effects of insulin are attenuated by rapamycin and wortmannin, two agents that block activation of p70S6K. Unlike PHAS-I, PHAS-II is readily phosphorylated by cAMP-dependent protein kinase in vitro; however, the effects of insulin on both PHAS proteins are attenuated by agents that increase intracellular cAMP, by cAMP derivatives, and by phosphodiesterase inhibitors. These agents also markedly inhibit the activation of p70S6K. In summary, the results indicate that PHAS-I and -II are controlled by the mammalian target of rapamycin and p70S6K signaling pathway and that in 3T3-L1 adipocytes, this pathway is inhibited by increased cAMP (Lin, 1996).

Recent studies indicate that phosphatidylinositide-3OH kinase (PI3K)-induced S6 kinase (S6K1) activation is mediated by protein kinase B (PKB). Support for this hypothesis has largely relied on results obtained with highly active, constitutively membrane-localized alleles of wild-type PKB, whose activity is independent of PI3K. This study examines the importance of PKB signaling in S6K1 activation. In parallel, glycogen synthase kinase 3beta (GSK-3beta) inactivation and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation were monitored as markers of the rapamycin-insensitive and -sensitive branches of the PI3K signaling pathway, respectively. The results demonstrate that two activated PKBalpha mutants, whose basal activity is equivalent to that of insulin-induced wild-type PKB, inhibit GSK-3beta to the same extent as a highly active, constitutively membrane-targeted wild-type PKB allele. However, of these two mutants, only the constitutively membrane-targeted allele of PKB induces S6K1 activation. Furthermore, an interfering mutant of PKB, which blocks insulin-induced PKB activation and GSK-3beta inactivation, has no effect on S6K1 activation. Surprisingly, all the activated PKB mutants, regardless of constitutive membrane localization, induce 4E-BP1 phosphorylation and the interfering PKB mutant blocks insulin-induced 4E-BP1 phosphorylation. The results demonstrate that PKB mediates S6K1 activation only as a function of constitutive membrane localization, whereas the activation of PKB appears both necessary and sufficient to induce 4E-BP1 phosphorylation independently of its intracellular location (Dufner, 1999).

Hormones and growth factors induce protein translation in part by phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). The rapamycin and FK506-binding protein (FKBP)-target 1 (RAFT1, also known as FRAP) is a mammalian homolog of the Saccharomyces cerevisiae target of rapamycin proteins (mTOR) that regulates 4E-BP1. However, the molecular mechanisms involved in growth factor-initiated phosphorylation of 4E-BP1 are not well understood. Protein kinase Cdelta (PKCdelta) associates with RAFT1 and PKCdelta is required for the phosphorylation and inactivation of 4E-BP1. PKCdelta-mediated phosphorylation of 4E-BP1 is wortmannin resistant but rapamycin sensitive. As shown for serum, phosphorylation of 4E-BP1 by PKCdelta inhibits the interaction between 4E-BP1 and eIF4E and stimulates cap-dependent translation. Moreover, a dominant-negative mutant of PKCdelta inhibits serum-induced phosphorylation of 4E-BP1. These findings demonstrate that PKCdelta associates with RAFT1 and thereby regulates phosphorylation of 4E-BP1 and cap-dependent initiation of protein translation (Kumar, 2000a).

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 [p70(S6k)] 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 p70(S6k). 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).

Cell size is controlled by mTOR and its downstream target 4EBP1/eIF4E

The coordinated action of cell cycle progression and cell growth (an increase in cell size and cell mass) is critical for sustained cellular proliferation, yet the biochemical signals that control cell growth are poorly defined, particularly in mammalian systems. This study demonstrates that cell growth and cell cycle progression are separable processes in mammalian cells and that growth to appropriate cell size requires mTOR- and PI3K-dependent signals. Expression of a rapamycin-resistant mutant of mTOR rescues the reduced cell size phenotype induced by rapamycin in a kinase-dependent manner, showing the evolutionarily conserved role of mTOR in control of cell growth. Expression of S6K1 mutants that possess partial rapamycin-resistant activity or overexpression of eIF4E individually and additively partially rescues the rapamycin-induced decrease in cell size. In the absence of rapamycin, overexpression of S6K1 or eIF4E increases cell size, and, when coexpressed, they cooperate to increase cell size further. Expression of a phosphorylation site-defective mutant of 4EBP1 that constitutively binds the eIF4E-Cap complex to inhibit translation initiation reduces cell size and blocks eIF4E effects on cell size. These data show that mTOR signals downstream to at least two independent targets, S6K1 and 4EBP1/eIF4E, that function in translational control to regulate mammalian cell size (Fingar, 2002).

4E-BP and the initiation of development in sea urchin embryos

The eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) inhibit translation initiation by binding eIF4E and preventing recruitment of the translation machinery to mRNA. Fertilization of sea urchin eggs triggers eIF4E-4E-BP complex dissociation and 4E-BP degradation. Microinjection of eIF4E-binding motif peptide into unfertilized eggs delays the onset of the first mitosis triggered by fertilization, demonstrating that dissociation of the eIF4E-4E-BP complex is functionally important for the first mitotic division in sea urchin embryos. eIF4E is present in unfertilized eggs as an 80 kDa molecular mass complex containing 4E-BP and a new 4E-BP of 40 kDa. Fertilization triggers the dissociation of eIF4E from these two 4E-BPs and triggers the rapid recruitment of eIF4E into a high-molecular-mass complex. Release of eIF4E from the two 4E-BPs is correlated with a decrease in the total level of both 4E-BPs following fertilization. Abundance of the two 4E-BPs has been monitored during embryonic development. The level of the two proteins remains very low during the rapid cleavage stage of early development and increases 8 hours after fertilization. These results demonstrate that these two 4E-BPs are down- and up-regulated during the embryonic development of sea urchins. Consequently, these data suggest that eIF4E availability to other partners represents an important determinant of the early development of sea urchin embryos (Salaun, 2005).

The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs

Adult stem cells must limit their rate of protein synthesis, but the underlying mechanisms remain largely unexplored. Differences in protein synthesis among hematopoietic stem cells (HSCs) and progenitor cells did not correlate with differences in proteasome activity, total RNA content, mRNA content, or cell division rate. However, adult HSCs had more hypophosphorylated eukaryotic initiation factor 4E-binding protein 1 (4E-BP1; see Drosophila Thor) and 4E-BP2 as compared with most other hematopoietic progenitors. Deficiency for 4E-BP1 and 4E-BP2 significantly increased global protein synthesis in HSCs, but not in other hematopoietic progenitors, and impaired their reconstituting activity, identifying a mechanism that promotes HSC maintenance by attenuating protein synthesis (Signer, 2016).

4E-BP and apoptosis

Translational control has been recently added to well-recognized genomic, transcriptional, and posttranslational mechanisms regulating apoptosis. Overexpressed eukaryotic initiation factor 4E (eIF4E) rescues cells from apoptosis, while ectopic expression of wild-type eIF4E-binding protein 1 (4E-BP1), the most abundant member of the 4E-BP family of eIF4E repressor proteins, activates apoptosis, but only in transformed cells. To test the possibility that nontransformed cells require less cap-dependent translation to suppress apoptosis than do their transformed counterparts, the level of translational repression was intensified in nontransformed fibroblasts. Inhibition of 4E-BP1 phosphorylation by rapamycin triggers apoptosis in cells ectopically expressing wild-type 4E-BP1 and expression of 4E-BP1 phosphorylation site mutants potently activates apoptosis in a phosphorylation site-specific manner. In general, proapoptotic potency paralleled repression of cap-dependent translation. However, this relationship is not a simple monotone. As repression of cap-dependent translation intensifies, apoptosis increases to a maximum value. Further repression results in less apoptosis -- a state associated with activation of translation through internal ribosomal entry sites. These findings show: (1) that phosphorylation events govern the proapoptotic potency of 4E-BP1, that 4E-BP1 is proapoptotic in normal as well as transformed fibroblasts, and (2) that malignant transformation is associated with a higher requirement for cap-dependent translation to inhibit apoptosis. These results suggest that 4E-BP1-mediated control of apoptosis occurs through qualitative rather than quantitative changes in protein synthesis, mediated by a dynamic interplay between cap-dependent and cap-independent processes (Li, 2002).

4E-BP and cancer

Common human malignancies acquire derangements of the translation initiation complex, eIF4F, but their functional significance is unknown. Hypophosphorylated 4E-BP proteins negatively regulate eIF4F assembly by sequestering its mRNA cap binding component eIF4E, whereas hyperphosphorylation abrogates this function. Breast carcinoma cells harbor increases in the eIF4F constituent eIF4GI and hyperphosphorylation of 4E-BP1 which are two alterations that activate eIF4F assembly. Ectopic expression of eIF4E in human mammary epithelial cells enabled clonal expansion and anchorage-independent growth. Transfer of 4E-BP1 phosphorylation site mutants into breast carcinoma cells suppresses their tumorigenicity, whereas loss of these 4E-BP1 phosphorylation site mutants accompanies spontaneous reversion to a malignant phenotype. Thus, eIF4F activation is an essential component of the malignant phenotype in breast carcinoma (Avdulov, 2004).

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date revised: 1 March 2024

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