InteractiveFly: GeneBrief

Rheb: Biological Overview | Regulation | Developmental Biology | Effects of Mutation and Overexpression | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

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

Studies in Drosophila have greatly enhanced understanding of growth regulation. From these efforts, two highly conserved signalling pathways dedicated to the control of growth have emerged: the insulin receptor (InR)/phosphatidylinositol-3-OH kinase (PI(3)K) and TOR pathways. Recent studies have also shown that these two pathways interact, although the mechanisms by which they communicate are the subject of controversy. In addition, each pathway seems to be modulated by distinct tumor suppressor genes: PTEN (phosphatase and tensin homolog deleted in chromosome 10) and TSC1-TSC2, respectively. Whereas it is clear that PTEN constrains PI(3)K signalling by dephosphorylation of phosphatidylinositol-3,4,5-triphosphate (PtdInsP₃), an understanding of the mechanism by which TSC1 and TSC2 counteract TOR signalling remains elusive. Importantly, TSC2 possesses a putative GTPase-activating protein (GAP) domain, which has been shown to increase the intrinsic GTPase activity of the small GTPases Rap1 and Rab5. Genetic and biochemical data from Drosophila suggest a novel role for the small GTPase Rheb in the TOR/S6K signalling pathway (Stocker, 2003 and references therein).

To identify growth-regulating genes, two complementary screens were performed for loss- and gain-of-function mutations, respectively. In the loss-of-function screen, a novel complementation group of ten alleles was discovered that impairs cell and organ growth. The ethylmethane sulphonate (EMS)-induced mutations were identified on the basis of reduced head size of mosaic animals, consisting of heads largely made up of homozygous mutant cells and bodies containing heterozygous cells. This phenotype is reminiscent of mutations in InR signalling components. Genetic mapping of two representative alleles and subsequent testing of candidate open reading frames identified alterations in the gene CG1081 in seven alleles. CG1081 encodes a small GTPase most closely related to mammalian Rheb. Therefore, this complementation group was named Rheb (Stocker, 2003).

The gain-of-function screen for genes that stimulate growth when overexpressed resulted in the identification of an EP element in the Rheb locus (EP 50.084). EP-mediated overexpression of Rheb in the developing eye substantially increases eye size. Six additional Rheb loss-of-function alleles were generated by imprecise excision of EP 50.084. Whereas all combinations of the EMS-induced Rheb alleles are lethal, some hetero-allelic combinations of EMS-induced alleles and EP excision alleles are viable and result in flies of reduced size. The size reduction is caused by a decrease in cell number (3%-11%), as well as in cell size (9%-14% in wing cells -- more than 20% in eye cells as judged by ommatidial size). In addition, the small flies eclose with a delay of at least one day and the females have rudimentary ovaries and are sterile. Thus, the surviving Rheb mutant flies display all the hallmarks of impaired InR signalling activity, resembling flies lacking the insulin-receptor substrate (IRS) protein Chico (Stocker, 2003).

A more severe reduction in Rheb function (in heteroallelic combinations of Rheb mutations) is lethal at late larval or early pupal stages. Mutant larvae and pupae are consistently smaller, although the phenotype is variable. Interestingly, the size reduction is more pronounced in the endoreplicative larval tissue than in the imaginal discs, similar to the larval phenotype of TOR mutants (Oldham, 2000; Zhang, 2000). Staining of DNA in salivary glands and fat body cells demonstrate a severe deficit in endoreplication (Stocker, 2003).

The behavior of Rheb mutant cells was studied during development by means of mitotic recombination. Clones of cells homozygous for EMS-induced Rheb alleles grow poorly and are consistently smaller than their corresponding sister clones. When provided with a proliferative advantage (by means of the Minute technique), Rheb mutant cells still fail to cover large regions of the imaginal discs. Instead, the resulting clones typically display elongated shapes with thin extensions. A possible explanation for this unusual phenotype may reside in the attempt of mutant cells to minimize contact with other mutant cells. This phenomenon has not been previously described in the context of growth-regulating genes. Despite this abnormal behavior, Rheb mutant cells differentiate properly into adult structures. For example, analysis of clones in the adult eye reveals the presence of extremely small photoreceptor cells of otherwise normal structure and arrangement in the mutant tissue. The size reduction phenotype is strictly cell-autonomous. Taken together, the characterization of the mutant phenotypes demonstrates that Rheb is required for proper growth regulation in a cell-autonomous manner (Stocker, 2003).

Does overexpression of Rheb promote growth? The effect of overexpressing Rheb during development through the use of the EP 50.084 line and two independent UAS-Rheb lines was monitored in marked clones in imaginal discs and in the adult eye. All the lines yielded qualitatively similar results, with the EP line consistently showing the strongest effects. Clones overexpressing Rheb in the wing imaginal disc attained a substantially larger size when compared with control clones. This enlargement is caused by a significant increase in cell size (a 48% increase in area covered per cell). In contrast, the cell doubling time remained unchanged in cells expressing Rheb versus control cells. Consistent with the size effect in the imaginal discs, cells expressing Rheb in differentiating cells posterior to the morphogenetic furrow (under the control of GMR regulatory sequences) resulted in enlarged but fully differentiated photoreceptor cells (a 66% size increase of the rhabdomeres). As in the case of the loss-of-function clones, the size alteration was cell-autonomous. Thus, Rheb is sufficient to promote cellular growth (Stocker, 2003).

Since both InR and TOR signalling have been implicated in the response to nutrient availability, it was asked whether overexpression of Rheb would promote growth even under starvation conditions. It has been shown that depriving larvae of amino acids blocks endoreplication of the larval tissues, but that this can be overcome by expression of Dp110/PI(3)K. Rheb is expressed in small clones of cells in the salivary glands and in the fat body. Under normal food conditions, only a very subtle increase in cell size is observed. In larvae starved of amino acids, however, Rheb expression has a pronounced effect on both DNA content (as visualized by DAPI staining) and cell size. Despite the lack of amino acids, larval cells expressing Rheb reach a normal size in the fat body, and the size and endoreplication deficits are significantly alleviated in the salivary glands. It is concluded that Rheb is sufficient to counteract the effects of amino-acid deprivation and thus may function in amino-acid sensing (Stocker, 2003).

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

Complete loss of Tsc1 function results in larval lethality. Importantly, it was found that a simultaneous reduction of Rheb function is sufficient to restore viability. The emerging double-mutant flies display a weak Rheb hypomorphic phenotype (a moderate size reduction). These findings suggest that the major consequence of a lack of Tsc1 is overactivation of Rheb (Stocker, 2003).

Thus genetic analysis indicates that Rheb regulates S6K through TOR. Therefore, whether S6K activity is dependent on Rheb function was tested. Larval extracts of various heteroallelic Rheb combinations were subjected to S6K and PKB kinase assays. Indeed, S6K activity is significantly reduced in all combinations without any apparent effect on S6K protein levels. PKB activity, however, is consistently increased. This is in agreement with the hypothesis that S6K is an essential component of a negative feedback loop regulating InR signalling (Radimerski, 2002; Haruta, 2000). Conversely, ubiquitous expression of Rheb results in an increase in S6K activity and a concomitant decrease in PKB activity. The stimulation of S6K activity by Rheb was also observed after amino-acid deprivation. Thus, Rheb is both necessary and sufficient for S6K activation (Stocker, 2003).

Although Rheb is essential for S6K activity, and the overgrowth phenotype elicited by Rheb overexpression depends on S6K, regulation of S6K is clearly not the only effect of Rheb activity. Whereas flies lacking S6K function are semi-viable (exhibiting a severe delay in development and a reduced body size (Montagne, 1999), loss of Rheb is lethal. Moreover, reduction of Rheb activity results in a decrease in cell number and cell size (as opposed to S6K mutants, where only cell size is affected). Finally, the characteristic shape of Rheb mutant cell clones suggests that Rheb has other functions in addition to growth control (Stocker, 2003).

Two models of Rheb activity can be envisaged: (1) Rheb could function in the TOR signalling pathway directly downstream of and negatively regulated by Tsc1-Tsc2; (2) Rheb might be a component of an independent pathway that impinges on S6K. In the latter model, the TOR signalling pathway and the putative parallel pathway would both be necessary for the full activation of S6K. This could explain why impairing the activity of one pathway interferes with the consequences of overactivating the other. Nevertheless, the former model is favored because Rheb mutants show striking similarities with TOR signalling defects and because of the intimate genetic interactions of Rheb with Tsc1-Tsc2. A particularly attractive hypothesis implicates Tsc2 as the GAP of Rheb. Indeed, Zhang (2003) provides evidence that Tsc2 is the GAP for Rheb in Drosophila, and the same conclusion (Garami, 2003) has been derived from studies on the mammalian homologs of Drosophila Tsc2 and Rheb (Stocker, 2003).

Interestingly, loss of rhb1 function in the fission yeast Schizosaccharomyces pombe results in a growth arrest phenotype that is very similar to that of nitrogen-starved cells (Mach, 2000). Thus, the function of Rheb in growth regulation in response to nutrients (amino acids) may have been conserved during evolution. Furthermore, the fact that impaired Rheb function is sufficient to suppress the phenotypic consequences of loss of PTEN and TSC1-TSC2 suggests that Rheb might be a suitable target for therapeutic intervention in a wide range of tumors (Stocker, 2003 and references therein).

eIF4A inactivates TORC1 in response to amino acid starvation

Amino acids regulate TOR complex 1 (TORC1) via two counteracting mechanisms, one activating and one inactivating. The presence of amino acids causes TORC1 recruitment to lysosomes where TORC1 is activated by binding Rheb. How the absence of amino acids inactivates TORC1 is less well understood. Amino acid starvation recruits the TSC1/TSC2 complex to the vicinity of TORC1 to inhibit Rheb; however, the upstream mechanisms regulating TSC2 are not known. This study identified the the eIF4A-containing eIF4F translation initiation complex (composed of three subunits: eIF4E, eIF4A and eIF4G) as an upstream regulator of TSC2 in response to amino acid withdrawal in Drosophila. TORC1 and translation preinitiation complexes bind each other. Cells lacking eIF4F components retain elevated TORC1 activity upon amino acid removal. This effect is specific for eIF4F and not a general consequence of blocked translation. This study identifies specific components of the translation machinery as important mediators of TORC1 inactivation upon amino acid removal (Tsokanos, 2016).

To maintain homeostasis, biological systems frequently use a combination of two distinct mechanisms that converge and counteract each other. For instance, the level of phosphorylation of a target protein depends not only on the rate of phosphorylation by the upstream kinase, but also on the rate of dephosphorylation by the phosphatase. Both the activating kinase and the inactivating phosphatase can be regulated separately. Likewise, the activity of TORC1 in response to amino acid levels appears to reflect a balance between activating and inactivating mechanisms that converge on Rheb. When amino acids are re-added to cells, TORC1 is activated via Rag or Arf1 GTPase-dependent recruitment to the lysosome where TORC1 binds Rheb. In contrast, when amino acids are removed from cells, TORC1 activity drops in part by blocking this activation mechanism and in part via a distinct inactivation mechanism whereby TSC2 is recruited to the vicinity of TORC1 to act on Rheb (Demetriades, 2014). The existence of this distinct and counteracting mechanism is highlighted by the fact that in the absence of TSC2, both Drosophila and mammalian cells do not appropriately inactivate TORC1 in response to amino acid removal. The upstream mechanisms regulating TSC2 in response to amino acid withdrawal, however, are not known. This study has identified the translational machinery, and in particular components of the eIF4F complex, as one upstream regulatory mechanism working via TSC2 to inactivate TORC1 upon amino acid withdrawal (Tsokanos, 2016).

The subcellular localization of TORC1 plays an important role in its regulation. A significant body of evidence shows that TORC1 needs to translocate to the lysosome or Golgi to become reactivated following amino acid starvation and re-addition. Whether active TORC1 then remains on the lysosome, or whether it can move elsewhere in the cell to phosphorylate target proteins, is less clear. Several findings in the literature, as well as the data presented in this study, indicate that active TORC1 can leave the lysosome, yet remain active: (1) Upon amino acid re-addition in starved cells, the Rag GTPases are necessary for mTORC1 lysosomal localization and reactivation. In contrast, Rag depletion in cells growing under basal conditions, replete of serum and amino acids, does not cause a strong drop in mTORC1 activity, although it causes a similar delocalization of mTORC1 away from lysosomes. Hence, under these conditions, mTORC1 is non-lysosomal, but still active to a large extent. (2) Similarly, particular stresses such as arsenite treatment can cause TORC1 to localize away from the lysosome, yet remain active. (3) The Rag GTPases tether TORC1 to the LAMTOR complex present on the lysosome. Amino acid restimulation, which activates TORC1, actually decreases binding between Rag GTPases and LAMTOR, suggesting that active Rag-bound TORC1 complexes can leave the lysosome and reside elsewhere in the cell. Additional mechanisms also contribute to the delocalization of the Rag GTPases away from lysosomes (4) Active TORC1 phosphorylates target proteins such as 4E-BP and S6K, which are physically associated with translation preinitiation complexes. Indeed, this study reports physical interactions between the TORC1 complex and translation preinitiation complexes, in agreement with what has also been observed by others. Therefore, either translation preinitiation complexes need to translocate to lysosomes to meet TORC1, or TORC1 needs to come off the lysosome to meet translation preinitiation complexes in the cytoplasm. (5) Using proximity ligation assay, an interaction was observed between Raptor and eIF4A, which does not colocalize with either lysosomes or endoplasmic reticulum, suggesting that it takes place in the cytoplasm. (6) In agreement with these PLA data, antibody staining of cells in the presence of amino acids with anti-TOR antibody reveals an accumulation of TOR on lysosomes, as well as a more diffuse, non-lysosomal TORC1 localization throughout the cytoplasm. (7) A recent report employing a FRET-based probe detects mTORC1 activity at lysosomes as well as in the cytoplasm and nucleus. Taken together, these data suggest that although TORC1 is activated on the lysosome, it then in part translocates to other sites in the cell including the cytoplasm to phosphorylate target proteins (Tsokanos, 2016).

Upon amino acid withdrawal, both cytoplasmic and lysosomal fractions of active TORC1 need to be inactivated. The data presented in this study suggest that upon amino acid removal, inactivation of TORC1 happens in part via an eIF4A-dependent mechanism acting on TSC2 to inactivate Rheb in the cytosol. In agreement with this, TORC1 inactivation upon amino acid removal can be rescued by supplying cells with dominantly active, but not wild-type Rheb. It has been previously reported that a pool of TSC2 is also recruited to lysosomes upon amino acid removal. This study shows in Drosophila cells, upon amino acid removal, some TSC2 accumulates in lysosomes, whereas some remains in the cytosol. Therefore, TSC2 is likely recruited to all subcellular sites where active TORC1 is located to inactivate it. Indeed, Rheb and TSC2 have been observed at several subcellular compartments. Since Rheb localizes to many endomembranes in the cell, Rheb that is not bound to TORC1 could potentially remain active, to provide a pool for subsequent TORC1 reactivation (Tsokanos, 2016).

Upon inactivation, the data indicate that TORC1 remains bound to preinitiation complexes, in agreement with previous reports. This finding is reminiscent of the fact that Raptor is also recruited to stress granules, which are essentially stalled preinitiation complexes, in response to another stress-oxidative stress. Whether the Rag GTPases also remain bound to preinitiation complexes upon amino acid removal is unclear because some experiments showed a decrease in binding between Rag GTPases and initiation factors, and some did not (Tsokanos, 2016).

How could eIF4A affect TORC1 activity? The data indicate that the effects of eIF4A knockdown cannot be explained as a consequence of generally impaired translation, since other means of blocking translation do not have the same effects on TORC1 activity upon amino acid starvation. Instead, knockdown of any of the three members of the eIF4F complex gives this elevated TORC1 phenotype, indicating that it is specific for the eIF4F complex. The data are consistent with two interpretations: One option is that the eIF4F complex is specifically required to translate a protein that promotes TSC2 function. An alternate option is that the eIF4F complex acts directly on TSC2, regulating its activity. The latter is supported by the fact that eIF4A and TSC2 proteins are seen interacting with each other. Interestingly, eIF4A has been reported to have additional functions that are not translation-related (Tsokanos, 2016).

Some differences were noted between Drosophila cells and mammalian cells. The first is that overexpression of wild-type Rheb is sufficient to activate TORC1 upon amino acid removal in mammalian cells, whereas this is not the case in Drosophila cells. This could be due to a difference in the biology of the two cell types, or simply to a technical difference having to do with levels of Rheb overexpression. A second difference is that cycloheximide treatment is sufficient to maintain elevated TORC1 levels in HeLa or HEK293 cells upon amino acid removal, whereas this is not the case in Drosophila cells. This could be due to differences in rates of amino acid efflux and levels of autophagy in mammalian compared to S2 and Kc167 cells, causing intracellular amino acid levels to remain elevated in mammalian cells when both amino acid import from the medium and amino acid expenditure via translation are simultaneously blocked (Tsokanos, 2016).

A number of studies have looked at the involvement of Rheb in the cellular response to amino acids, with some disagreement on whether amino acids affect Rheb GTP-loading or Rheb-mTOR binding. The current data fit with previous reports that Rheb GTP-loading is affected by amino acids and with the conclusion that amino acids affect TORC1 activity via both a Rheb-dependent and a Rheb-independent mechanism (Tsokanos, 2016).

The data indicate a close physical relationship between TORC1 and the translational machinery. This is in part mediated by a direct interaction between the major scaffolding subunit of the initiation complex, eIF4G, and RagC and in part likely mediated by additional interactions between TORC1 and preinitiation supercomplexes as previously reported. Interestingly, TORC2 is also physically associated with the ribosome and requires ribosomes, but not translation, for its activation. Hence, both TORC1 and TORC2 have close physical connections to the translational machinery (Tsokanos, 2016).

Some side observations in this study are interesting and could constitute a starting point for further studies. For instance, eIF4A-knockdown cells inactivate TORC1 more robustly than control cells upon serum removal. Also, eIF2b knockdown causes S6K phosphorylation to decrease significantly in S2 cells. It is not known why this occurs. The latter might suggest that there are additional points of cross-talk between TORC1 and the translation machinery (Tsokanos, 2016).

How cells sense the presence or the absence of amino acids has been an open question in the field. The data presented in this study indicate that the translational machinery itself might sense the absence of amino acids. Indeed, the relevant parameter for a cell is likely not the absolute levels of intracellular amino acids, but rather whether the available amino acid levels are sufficient to support the amount of translation that a cell requires. Hence, the translation machinery itself might be best poised to make this assessment. Binding is observed between eIF4A and NAT1 that is strong in the presence of amino acids, and is reduced upon amino acid withdrawal, independently of TORC1 signaling. These epistasis experiments are consistent with NAT1 acting as the upstream mediator of the amino acid signal, binding and inhibiting eIF4A in the presence of amino acids, but not in the absence of amino acids. Hence, NAT1 might play a role in this sensing process (Tsokanos, 2016).

In sum, these data identify the eIF4F complex as an important upstream regulator of TORC1, which acts via TSC2 to inactivate TORC1 upon withdrawal of amino acids (Tsokanos, 2016).

RagC phosphorylation autoregulates mTOR complex 1

The mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) controls cell growth, proliferation, and metabolism in response to diverse stimuli. Two major parallel pathways are implicated in mTORC1 regulation including a growth factor-responsive pathway mediated via TSC2/Rheb and an amino acid-responsive pathway mediated via the Rag GTPases. This study identified and characterize three highly conserved growth factor-responsive phosphorylation sites on RagC, a component of the Rag heterodimer, implicating cross talk between amino acid and growth factor-mediated regulation of mTORC1. RagC phosphorylation is associated with destabilization of mTORC1 and is essential for both growth factor and amino acid-induced mTORC1 activation. Functionally, RagC phosphorylation suppresses starvation-induced autophagy, and genetic studies in Drosophila reveal that RagC phosphorylation plays an essential role in regulation of cell growth. Finally, mTORC1 was identified as the upstream kinase of RagC on S21. These data highlight the importance of RagC phosphorylation in its function and identify a previously unappreciated auto-regulatory mechanism of mTORC1 activity (Yang, 2018).

mTOR is an evolutionarily conserved atypical serine/threonine kinase belonging to the phosphoinositide 3 kinase (PI3K)-related kinase family. mTOR is found in two structurally and functionally distinct complexes-mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2)-defined by their unique components, in particular raptor (mTORC1) and rictor (mTORC2). Through the coordinated phosphorylation of its downstream effectors, mTORC1 integrates extra- and intra-cellular signal inputs such as amino acids, growth factors (GF), stress, and energy status, to regulate major cellular processes including growth, proliferation, and survival. Underlining its crucial role in cellular and organismal homeostasis, mTORC1 dysregulation occurs in numerous human diseases including cancer, metabolic disorders, and neurodegeneration. Growth factors and amino acids both acutely enhance mTORC1 activity, and two different types of small GTPases-Ras-homolog enriched in brain (Rheb) and the Rag GTPases-cooperatively regulate mTORC1 activity via these two parallel activation mechanisms. Rheb is activated under conditions of high cellular ATP and upstream growth factor signals. Once activated, Rheb interacts with and activates mTORC1 and is required for mTORC1 activation by all signals, including amino acids. Rag GTPases are considered amino acid-specific regulators of the mTORC1 pathway. Mammals have four Rag proteins-RagA to RagD-which form obligate heterodimers comprising RagA or RagB together with RagC or RagD. Amino acids cause Rag GTPases to switch to an active conformation, in which RagA/B is GTP-loaded and Rag C/D is GDP-loaded. The active Rag heterodimer physically interacts with raptor, recruiting mTORC1 to the lysosome where its activator Rheb resides. Extensive work has revealed several mechanisms implicated in the regulation of Rag activity that enables them to function as nutrient sensors. A common feature among these is the control of Rag nucleotide status, particularly through the activation of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). These include the Ragulator (GEF for Rag A/B; Bar-Peled, 2012), the GAP Activity Towards Rags complex 1 (GATOR1) complex (GAP for RagA/B; Bar-Peled, 2013), folliculin (FLCN, GAP for RagC/D; Petit, 2013; Tsun, 2013), and leucyl-tRNA synthetase (LeuRS, GAP for RagD; Han, 2012). Ubiquitination has also recently emerged as a post-translational modification (PTM) capable of inhibiting Rag GTPase signaling by recruiting GATOR1 to RagA. Importantly, these pathways regulating Rag activity are all amino acid-dependent, and much less is known about the control of growth factor-mediated Rag GTPase signaling (Yang, 2018).

In a recent global mass spectrometry-based phosphoproteomics study in adipocytes, insulin-dependent phosphorylation was observed of several highly conserved residues on RagC including S2, S21, and T394. These data highlight a possible role for the Rag GTPases in mTORC1 growth factor sensing. This study demonstrates that both growth factors and amino acids trigger RagC phosphorylation and that phosphorylated RagC potentiates mTORC1 activity and affects mTORC1-dependent cell growth and autophagy. Moreover, the phosphorylation of RagC at S21 (and likely T394) was shown to be catalyzed directly by mTORC1, revealing a novel auto-regulatory feedback loop within the mTORC1 signaling pathway (Yang, 2018).

This study identified a new auto-regulatory branch of mTORC1 signaling, involving phosphorylation of the Rag GTPase RagC. This is the first report that Rag GTPase phosphorylation can regulate mTORC1 activity. More importantly, the results confirm that Rag GTPases are not only involved in the amino acid-sensing mTORC1 pathway, but could also participate in growth factor sensing in the mTORC1 pathway. Although previous studies show that in Rag heterodimers, the GTP/GDP loading of Rag heterodimers plays a dominant role in the interaction between Rag heterodimers and mTORC1, the data indicate that RagC is also a positive regulator of mTORC1 through post-translational modification. Interestingly, phosphoproteomics data suggest that most phosphorylation is concentrated on RagC compared with other Rag GTPases, and S21 is not conserved between RagC and RagD, suggesting that RagC is not functionally redundant and potentially has distinct biological functions to RagD (Yang, 2018).

One of the RagC phosphorylation sites, S21, was established as a novel rapamycin-insensitive mTORC1 substrate in vitro and in cells, and the T394 is phosphorylated by mTOR in vitro. The S2 and T394 sites may also be mTORC1 substrates in vivo, because the kinetics of their phosphorylation resembles that of other bona fide mTORC1 substrates and they also have surrounding sequence features matching the preferred sequence motif of mTORC1. These findings indicate the presence of a positive feedback loop between mTORC1 and RagC, which may contribute to the fine-tuning of mTORC1 activity (Yang, 2018).

There is evidence that the stability of the raptor-mTOR complex is related to mTORC activity, and the current data implicate RagC phosphorylation in the destabilization of mTORC1. This is likely to be a direct effect of RagC phosphorylation, because RagC 3E still destabilized mTOR-raptor complex under serum starvation. This is consistent with the observation that RagC 3E causes hyper-phosphorylation of ULK1 and inhibits autophagy under serum starvation. The next major question is what is the underlying cause of this instability. One possibility is that RagC phosphorylation influences the interaction with other regulators, resulting in 'locking' or 'opening' of the mTOR-raptor complex. Interestingly, it was observed that RagC 3A binds more FLCN, which is a GAP for RagC/D, and RagC 3E can bind more raptor under both steady and amino acid starvation/re-fed condition. One possibility is that RagC phosphorylation regulates its nucleotide binding status by modulating the interaction with FLCN. However, no substantial difference was observed in FLCN binding between wild-type RagC and RagC 3E, or raptor binding between wild-type RagC and 3A. The temporal change in mTOR/raptor stability upon amino acid re-feeding is similar to that of FLCN/RagC stability, but not with raptor/RagC interaction. A possible explanation is that FLCN has two functions: serving as a GAP for RagC/D and a 'lock' for mTORC1. This model could help explain why FLCN releases from Rag GTPases in the presence of amino acids if it is a GAP for RagC/D, which is a positive regulator for mTORC1 activity: After activating RagC/D, FLCN needs to be disassociated from the lysosome to unlock mTORC1, and RagC phosphorylation may affect this process. Further studies will be needed to investigate these possibilities (Yang, 2018).

Other explanations cannot be ruled out for the impact of RagC phosphorylation on impaired mTORC1 activity. For example, it is well established that raptor recruits substrate proteins such as S6K and 4E-BP1 to mTORC1 so that they can be phosphorylated by mTOR. Therefore, RagC phosphorylation may affect the recruitment of mTORC1 substrates by raptor. Recently, two elegant studies showed that under amino acid or growth factor starvation, the Rag heterodimer binds and recruits TSC2 to lysosomes to inhibit Rheb, resulting in mTORC1 inactivation. Therefore, a final possibility is that RagC phosphorylation may mediate its effects by acting through TSC2. Future studies into the underlying mechanics of how RagC phosphorylation exerts its effects on mTORC1 signaling are therefore likely to shed light on this newly identified mechanism that sits at the intersection between amino acid sensing and growth factor signaling (Yang, 2018).

Phosphatidic acid drives mTORC lysosomal translocation in the absence of amino acids

mTOR Complex (mTORC1) promotes cell growth and proliferation in response to nutrients and growth factors. Amino acids induce lysosomal translocation of mTORC via the Rag GTPases. Growth factors activate Ras homolog enriched in brain (Rheb), which in turn, activates mTORC at the lysosome. Amino acids and growth factors also induce the phospholipase D (PLD)-phosphatidic acid (PA) pathway, required for mTORC signaling through mechanisms that are not fully understood. Using human and murine cell lines, along with immunofluorescence, confocal microscopy, endocytosis, PLD activity, and cell viability assays, this study shows that exogenously supplied PA vesicles deliver mTORC to the lysosome in the absence of amino acids, Rag GTPases, growth factors, and Rheb. Of note, pharmacological or genetic inhibition of endogenous PLD prevented mTORC lysosomal translocation. This study observed that precancerous cells with constitutive Rheb activation through loss of TSC complex subunit (TSC2) exploit the PLD-PA pathway and thereby sustain mTORC activation at the lysosome in the absence of amino acids. These findings indicate that sequential inputs from amino acids and growth factors trigger PA production required for mTORC translocation and activation at the lysosome (Frias, 2019).

mTORC18 is a conserved serine/threonine catalytic complex that integrates signals from nutrients and growth factors to regulate cell growth, proliferation, survival, and metabolism. Activation of mTORC1 is a two-step process whereby amino acids induce Rag-dependent translocation of mTORC1 from the cytoplasm to the lysosome, followed by mTOR kinase activation by the lysosomal small GTPase Rheb upon growth factor stimulation (Frias, 2019).

Phospholipase D (PLD) and its product, the signaling lipid phosphatidic acid (PA) play a role in mTORC1 activation in response to amino acids and growth factors. Amino acids induce lysosomal translocation of PLD1. Once on the lysosome, PLD1 binds to Rheb, which activates PLD1 in response to growth factors. PLD1 is widely expressed in mammals and converts the most abundant membrane phospholipid phosphatidylcholine to choline and PA. Conserved basic amino acids in the FKBP12-rapamycin binding (FRB) domain of mTOR lead to proton dissociation to generate PA with two negative charges. This locks mTOR onto deprotonated PA, promoting mTORC1 assembly and stability (Frias, 2019).

This study reports that PA with an unsaturated fatty acid stimulates lysosomal translocation and activation of mTORC1 in the absence of amino acids, Rag GTPases, growth factors, or Rheb. This work provides a unifying model showing that PA is critical for translocation and full activation of mTORC1 at the lysosome in response to sequential signals provided by amino acids and growth factors (Frias, 2019).

The data support a model where PLD1, similar to mTORC1, acts like a coincidence detector and effector of both amino acids and growth factors. Amino acids induce PLD activity and production of PA. Exogenously supplied PA vesicles enter the cell through endocytosis and drive mTOR to the lysosome, suggesting that amino acids induce production of PA-containing endosomes that carry mTOR to the lysosome. In agreement, inhibition of endogenous PLD prevented mTOR translocation to the lysosome in response to amino acids. Amino acid-induced RagA/B-GTP RagC/D-GDP heterodimers provide a parallel pathway that locks mTOR on the lysosome. Amino acids also induce the translocation of PLD1 from cytoplasmic puncta to the lysosome. Once on the lysosome, PLD1 binds to Rheb. Growth factors activate Rheb, which then activates PLD1. Lysosomal PA production promotes further binding of PA to mTOR to allow complex stability and activation (Frias, 2019).

Previous studies showed that exogenously supplied PA induced mTORC1 activation in the presence but not in the absence of amino acids. This study was able to induce mTORC1 translocation to the lysosome and mTORC1 activity with exogenously supplied PA-18:1 vesicles in the absence of amino acids. The main difference between the two studies is that this study performed amino acid and serum deprivation (to prevent contamination of amino acids present in serum) for 1 h, followed by amino acid stimulation for 10 min. In contrast, the previous study performed amino acid starvation for 2 h after overnight serum starvation, followed by amino acid stimulation for 30 min (Frias, 2019).

Previous findings suggest that mTORC1 assembles before reaching the lysosome because binding of mTOR to the Rag GTPases requires the mTORC1 component raptor. PA promotes mTORC1 assembly and stability. PA-containing endosomes carrying mTORC1 to the lysosome is therefore an attractive model in which PA would allow mTORC1 formation and stability. This study showed that exogenously supplied PA can drive mTOR to the lysosome and induce mTORC1 activity in TSC2-null MEFs where RagC and D were genetically ablated. Therefore, it is proposed that delivery of mTORC1 to the lysosome does not require the Rags. However, this study found that residual retention of mTOR at the lysosome in TSC2-null MEFs was lost upon RagC and D knockdown. This suggests that the Rags operate in parallel to PA to lock mTORC1 on the lysosome, after mTORC1 delivery by PA. This study showed that genetic ablation of PLD1 induced lysosomal scattering. This favors the idea that PA-containing endosomes carry mTORC1 to the lysosome, fuse with the lysosome, and increase its size (Frias, 2019).

Exogenously supplied PA vesicles induce mTORC1 translocation and activation in the absence of Rheb, indicating that the key step in Rheb activation of mTORC1 is increased PLD1 activity and PA production. Consistent with this observation, the Rheb association with mTOR is independent of GTP loading, whereas the Rheb association with PLD1 depends on GTP loading. Additionally, this study found that PLD1/2 inhibitors, in combination, abolished mTORC1 activity in RagAGTP/GTP MEFs, indicating that PA production is required for complex assembly and stability. If mTORC1 is not intact, then constitutive Rag activation is lost. Thus, the effect of PA on mTORC1 is downstream of Rheb and parallel to Rag GTPases. Genetic deletion of mTOR in mice is embryonic lethal. Unlike mTOR, mice with genetic deletion of PLD1, PLD2, or both are viable, suggesting that PLD and PA may not be required for steady state but rather acute activation of mTOR. PLD inhibitors preferentially killed TSC-null MEFs, whereas rapamycin did not. This suggests an advantage in terms of cancer therapeutics because targeting PLD may selectively target cancer cells, with minimal side effects. PLD inhibitors were developed from halopemide, a psychotropic drug extensively used in humans without toxicities. Thus, PLD inhibition might be a viable alternative to current therapies in cancers with mTORC1 hyperactivation that requires PLD-generated PA (Frias, 2019).

REGULATION

Protein Interactions

Mutations in the TSC1 or TSC2 genes cause tuberous sclerosis, a benign tumor syndrome in humans. Tsc2 possesses a domain that shares homology with the GTPase-activating protein (GAP) domain of Rap1-GAP2, suggesting that a GTPase might be the physiological target of Tsc2. The small GTPase Rheb (Ras homolog enriched in brain) has been shown to be a direct target of Tsc2 GAP activity both in vivo and in vitro. Point mutations in the GAP domain of Tsc2 disrupt its ability to regulate Rheb without affecting the ability of Tsc2 to form a complex with Tsc1. These studies identify Rheb as a molecular target of the TSC tumor suppressor genes (Zhang, 2003).

TSC1 and TSC2 were initially discovered as tumor suppressor genes mutated in tuberous sclerosis, a human syndrome characterized by the widespread development of benign tumors termed harmatomas. TSC2 encodes a putative GAP protein, whereas TSC1 encodes a novel protein containing two coiled-coil domains. Studies of Drosophila TSC1 and TSC2 homologs have identified a specific function for TSC1-TSC2 in the control of cell growth, with loss of TSC1-TSC2 resulting in increases in cell size. Recent studies further suggest that Tsc1-Tsc2 antagonizes the amino-acid-TOR signalling pathway, which normally couples amino-acid availability to S6 Kinase (S6K) activation, translation initiation and cell growth. Strikingly, loss of Drosophila TSC1-TSC2 results in a TOR-dependent increase of S6K activity that is resistant to amino-acid starvation (Zhang, 2003 and references therein).

Despite these new advances, the biochemical activity of the Tsc1-Tsc2 complex remains unknown. Tsc2 possesses a domain homologous to Rap1-GAP. The GAP homology domain of Tsc2 is important for its function, and mis-sense mutations of this domain have been identified in a high proportion of TSC patients. These observations suggest that an unknown small GTPase might be the direct target of Tsc2. This study set out to determine the target GTPase of Tsc2-GAP using an RNAi-based screen in Drosophila S2 cells. It was reasoned that this putative GTPase should be expressed in S2 cells and that RNAi of this GTPase should result in downregulation of S6K-Thr 398 phosphorylation, a phenotype opposite that caused by Tsc2 RNAi. During the course of the RNAi screen, genetic studies have implicated the small GTPase Rheb as a potential target of Tsc2. In S2 cells, RNAi inhibition of Rheb, but not any of the other 17 GTPases tested so far, abolished S6K-Thr 398 phosphorylation, as predicted for a Tsc2 GAP substrate. Among the 17 GTPases screened were Rab5 and Rap1, two proteins previously implicated as TSC2 GAP substrates from in vitro studies, suggesting that Rab5 and Rap1 are improbable physiological substrates of Tsc2. The highly specific effect of Rheb RNAi on S6K phosphorylation suggests that Rheb might be the physiological substrate of TSC2 GAP activity (Zhang, 2003).

Rheb is an evolutionarily conserved small GTPase found from yeast to mammals. Unlike Ras and most other Ras superfamily GTPases, Rheb has an arginine at the third residue of the G1 box (residue 15 of mammalian Rheb) instead of glycine. Rheb is unique, compared with many small GTPases, in that it exists in a highly activated state in mammalian cells. Studies of mammalian Rheb further implicated the existence of a Rheb-GAP that is normally present at relatively limiting concentrations, since overexpression of Rheb results in a progressive increase in the proportion of Rheb in the active GTP-bound state. Genetic analyses in Drosophila support a model in which Tsc2 functions as a Rheb-GAP. These studies also suggest that similarly to mammalian cells, Tsc2, the putative Rheb-GAP, is normally present in limiting concentrations in Drosophila, because overexpression of wild-type Rheb results in an activated phenotype and overexpression of Tsc2 (together with Tsc1) results in the opposite phenotype (Zhang, 2003).

To test directly whether Rheb is a physiological substrate of Tsc2 GAP activity, it was asked if Tsc2 could regulate Rheb in vivo. Rheb, similar to other small GTPases, cycles between an active GTP-bound form and an inactive GDP-bound form. Thus, the steady state GTP/GDP-loading status of Rheb can be used as a measurement of its in vivo activity. An in vivo labelling procedure was adapted to analyse the steady-state GTP/GDP-binding status of Rheb. Drosophila S2 cells expressing Myc-tagged Rheb were labelled with ³²P-orthophosphate. Rheb protein was then purified by immunoprecipitation and Rheb-associated GTP/GDP was analysed by thin-layer chromatography (TLC) on polyethyleneimine (PEI) cellulose plates. In wild-type S2 cells, Rheb binds preferentially to GTP, in agreement with studies of mammalian Rheb. In addition, co-overexpression of Tsc1 and Tsc2 results in a marked decrease (approximately eightfold) in the ratio of GTP to GDP bound on Rheb. Interestingly, overexpression of Tsc2 alone has much weaker effect on GTP:GDP ratio. This observation is consistent with previous studies in Drosophila, which show that co-overexpression of Tsc1 and Tsc2, but not either gene alone, results in growth inhibition. The weaker effect of Tsc2 alone on Rheb GTP loading is caused, at least in part, by the lower level of Tsc2 when expressed alone, as compared with Tsc1 co-expression. Mutual stabilization between Drosophila Tsc1 and Tsc2 has been documented previously (Zhang, 2003).

To demonstrate that the effect of Tsc1-Tsc2 overexpression on Rheb GTP loading was caused by the GAP activity of Tsc2, similar in vivo labelling experiments were performed with Tsc2 variants carrying point mutations in the GAP domain. The mutations Tsc2^K1693A and Tsc2^N1698K changed residues in the GAP domain that are conserved in Drosophila, human and a probable Schizosaccharomyces pombe Tsc2 homolog. In addition, a mutation analogous to Tsc2^K1693A has been shown to abolish Rap1-GAP activity, whereas Tsc2^N1698K mimics a disease-causing mutation in human TSC patients. The activity of Tsc2-N, a construct that contains just the amino-terminal half of Tsc2 and thus lacks the carboxy-terminal GAP domain, was also examined. Tsc2-N can associate with Tsc1 normally, but does not interact with Rheb in co-immunoprecipitation assays. Similar to Tsc2-N, neither Tsc2^K1693A nor Tsc2^N1698K affects the ability of Tsc2 to associate with Tsc1. Despite their ability to associate with Tsc1, these mutants all abolished the effect of Tsc1-Tsc2 overexpression on Rheb GTP loading. Complementary to the results from Tsc1-Tsc2 overexpression, RNAi of Tsc2 increases the ratio of GTP:GDP bound to Rheb. The smaller change in GTP:GDP ratio after Tsc2 RNAi, compared with Tsc1-Tsc2 overexpression, is not surprising given that Rheb is already at a relatively active state in wild-type cells. Taken together, these results provide strong evidence that Rheb is a physiological target of Tsc2 GAP activity (Zhang, 2003).

To test whether Rheb is a direct substrate of Tsc2 GAP in vitro, a fusion protein of glutathione S-transferase (GST) and the Tsc2 GAP domain against GTP-loaded Rheb protein was tested using a nitrocellulose filter assay. alpha-³²P-GTP- or gamma-³²P-GTP-loaded GST-Rheb was incubated with GST-Tsc2 and the remaining radioactive GTP bound on Rheb was measured at different time intervals. GST-Tsc2 results in a dramatic decrease of Rheb-associated radioactive counts when gamma-³²P-GTP, but not alpha-³²P-GTP, was used in the assay. Thus, Tsc2 functions as a Rheb GAP in vitro. This GAP activity is highly specific, and no activity was detected, using as a substrate Drosophila Ras1, the closest relative of Rheb among all GTPases. In addition, the K1693A or the N1698K point mutation abrogates the in vitro GAP activity of Tsc2 towards Rheb. These results provide further evidence that Tsc2 functions as a Rheb GAP (Zhang, 2003).

The data presented so far suggest a model in which the tuberous sclerosis tumor suppressor proteins negatively regulate Rheb through the Rheb GAP activity of Tsc2. To further substantiate this model, whether there are any genetic interactions between TSC1-TSC2 and Rheb was tested. Flies homozygous for a null allele of TSC1, TSC1²⁹, do not survive beyond the second-instar larval stage. Strikingly, the lethality of TSC1 null animals was partially rescued by removing one of the two copies of Rheb gene from the diploid genome: 61% of TSC1²⁹ homozygotes that were also heterozygous for a null allele of Rheb, Rheb^PDelta1, survived to third-instar larval stage, and 21% of the third-instar survivors continued development and arrested at the pupal stage. Such dose-sensitive interactions are reminiscent of those observed between TSC1-TSC2 and TOR, further supporting the model that Tsc1-Tsc2 negatively regulates Rheb during cell growth (Zhang, 2003).

Finally, how the Tsc-Rheb pathway interacts with the amino acid-TOR-S6K signalling network was investigated. Tsc and Rheb could either function as obligatory components between amino acids and TOR in a linear amino-acid sensing pathway, or in a parallel pathway that converges on TOR. The former (but not the latter) model predicts that the activity of Rheb is dependent on the presence of amino acids. The ratio of GTP:GDP bound to Rheb is not reduced after 5 h of amino-acid starvation. Thus, a model is favored in which TSC and Rheb function in a parallel pathway that converges on TOR. According to this model, loss of Tsc1-Tsc2 or ectopic activation of Rheb results in constitutive activation of TOR, which bypasses the requirement for amino acids and renders S6K activity resistant to amino-acid starvation. How Rheb signals to TOR will be an important question for future investigation (Zhang, 2003).

In summary, the small GTPase Rheb is a direct target of the tuberous sclerosis tumor suppressor proteins. Wild-type Tsc2, but not mutant Tsc2 carrying point mutations in the GAP domain, shows GAP activity towards Rheb both in vitro and in vivo. The importance of Tsc2's GAP activity is further supported by the high proportion of mis-sense mutations localized to the Tsc2 GAP domain among TSC patients. Thus, the Tsc2 tumor suppressor functions as a Rheb-GAP in an analogous way to the neurofibromin (NF1) tumor suppressor as a Ras-GAP. These studies suggest that Rheb represents a novel target for therapeutic intervention in the TSC disease. The identification of a small GTPase as the direct target of the TSC tumor suppressors further implicates the existence of activators of GTPases, such as guanine nucleotide-exchange factors (GEFs), as potential regulators of this disease pathway. Identification of the putative Rheb-GEF represents an important goal for the next phase of TSC research (Zhang, 2003).

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

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

TCTP is a highly conserved protein upregulated in various tumours. Despite studies on the biochemical and structural properties of TCTP, the physiological significance of these findings has not been determined. Thus, the function of TCTP in vivo was studied using Drosophila as a model organism (Hsu, 2007).

First dTCTP expression was knocked down in developing flies by targeted expression of double-stranded RNA (dsRNA) for RNA interference (RNAi). Expression of dTCTP RNAi depleted endogenous dTCTP to nearly undetectable levels by different GAL4 drivers. Tissue-specific expression of dTCTP RNAi reduced the size of the eye, wing, notum, or a specific region in the wing pouch, corresponding to the expression domains of various GAL4 lines. The size reduction was caused by a decrease in both cell size and cell number, a typical phenotype for mutations in the insulin or TSC pathways. Ubiquitous expression of UAS-dTCTP RNAi by actin-GAL4 (act>dTCTPi) caused lethality around the third instar larval stage. A portion of these larvae was able to survive to the pupal stage with reduced body size, consistent with the organ size reduction. The lethality and phenotypes caused by dTCTP RNAi were rescued by co-expression of a dTCTP complementary DNA, indicating that these defects were due to a reduction of dTCTP levels. It is therefore concluded that dTCTP RNAi suppresses organ growth by affecting both cell size and number (Hsu, 2007).

The phenotypes of dTCTP loss-of-function mutants were further investigated because RNAi may represent a hypomorphic situation. dTCTP^Ey09182 is a hypomorphic allele of dTCTP resulting from a P-element insertion in its 5' untranslated region. Rare homozygous dTCTP^Ey09182 flies that escaped from lethality showed smaller body size compared with their heterozygous siblings. To create null alleles, imprecise excisions were generated from dTCTP^Ey09182. One imprecise excision line, dTCTP^h59, showed a 1.1-kilobase deletion downstream of the insertion site that removes the entire dTCTP coding sequence. Western blot analysis showed no detectable dTCTP protein in dTCTP^h59 early first instar larvae. Both dTCTP^h59 homozygotes and dTCTP^h59 heterozygotes for a deficiency chromosome uncovering the dTCTP region (dTCTP^h59/Df(3R)M-Kx1) showed 100% lethality at the late first instar stage, indicating that this allele is a genetic null. Expression of dTCTP cDNA or a genomic DNA construct was able to rescue dTCTP^h59 mutants, indicating that the lethality is due to deletion of the dTCTP gene (Hsu, 2007).

To examine the phenotypes of dTCTP null mutant cells, dTCTP mutant clones were generated using mitotic recombination. dTCTP^h59 mutant clones showed growth disadvantage compared to their wild-type twin spots. The sizes of dTCTP^h59 clones were similar to those of the twin spots 24 h after heat shock. However, the sizes of the twin spots were much larger than dTCTP^h59 clones 48 h after heat shock, and most dTCTP^h59 clones were eliminated by 60 h after heat shock. Similarly, using the EGUF/Hid technique (Stowers, 1999) to remove most wild-type cells in dTCTP^h59 mosaic eyes resulted in either a no-eye or small-eye phenotype. Therefore, the null phenotypes were qualitatively comparable to the dTCTP RNAi phenotype, but more severe (Hsu, 2007).

The reduction in cell number caused by dTCTP RNAi and the behaviour of dTCTP mutant cells may result from a proliferation defect or abnormal cell death. These possibilities were tested using the MARCM (mosaic analysis with a repressible cell marker) technique. Similar to clones generated by traditional mitotic recombination, numerous small dTCTP^h59 green-fluorescent-protein-expressing (GFP⁺) clones were observed at 24 h after heat shock. By 72 h after heat shock, very few GFP⁺ clones remained on the discs. In contrast, Cyclin E (CycE) overexpression, via the MARCM technique, within dTCTP^h59 clones resulted in four times more dTCTP^h59 cells at 72 h after heat shock. Similarly, CycE overexpression also suppressed the dTCTP RNAi phenotypes. Next whether the dTCTP^h59 phenotypes can be attributed to abnormal cell death was tested. Expression of the P35 cell death inhibitor also significantly suppressed the dTCTP^h59 phenotypes, leading to the presence of four times more GFP⁺ cells at 72 h after heat shock. These data indicate that loss of dTCTP causes defects in cell proliferation and triggers cell death (Hsu, 2007).

Insulin and TSC signalling are two parallel but interacting pathways for growth control. Inactivation of positive regulatory components, such as Insulin receptor (InR), dRheb and Tor, leads to a decrease in organ size by affecting cell size and cell number. In contrast, overexpression of InR and dRheb, as well as inactivation of negative regulatory components such as Tsc1 and Pten, causes tissue overgrowth. Given the similar phenotypes between dTCTP mutants and mutants in the insulin/TSC pathways, genetic epistasis experiments were performed to test whether dTCTP has a role in these two pathways. Overexpression of InR by patched (ptc)-GAL4 caused weak but consistent expansion of the distance between L3 and L4 wing veins. In contrast, co-expression of InR and dTCTP RNAi reduced the L3-L4 distance, resembling the dTCTP RNAi phenotype. Therefore, dTCTP is epistatic to (acts downstream of) InR (Hsu, 2007).

Next, the relationship between Tsc1 and dTCTP was examined. Mutations in Tsc1 or Tsc2 cause similar phenotypes because they function as a complex. Mosaic eyes and heads consisting primarily of Tsc1 mutant cells were much larger than wild type. Expression of dTCTP RNAi in Tsc1 mutant cells suppressed this overgrowth both in the eye and head. Furthermore, when the eye was composed of Tsc1 and dTCTP double mutant cells, the flies displayed a small or no-eye phenotype indistinguishable from the dTCTP single mutant phenotype, suggesting that dTCTP acts downstream or in parallel to Tsc1 (Hsu, 2007).

The relationship between dTCTP and dRheb was tested. Ectopic expression of dRheb using ptc-GAL4 resulted in a 15% increase in the L3-L4 distance compared with the ptc>GFP control. However, co-expression of dTCTP RNAi and dRheb in the ptc expression region showed the dTCTP RNAi phenotype, suggesting that dTCTP is epistatic to dRheb (Hsu, 2007).

Finally, whether activation of dS6k, a downstream effector of the insulin/TSC pathway, is dependent on dTCTP was tested. The level of dS6k activation was measured using a phospho-specific antibody (dS6k p-Thr 398). Extracts from act>dTCTPi larvae showed a significantly lower amount of activated dS6k compared with the controls, indicating that dTCTP is required for dS6k activation. Consistent with this, the eyeless (ey)>dTCTPi phenotype was dominantly enhanced by heterozygosity for a null mutation of dS6k (dS6k^l-1). Removing a copy of dS6k caused an approximately 20% further reduction of eye size in ey>dTCTPi animals. Taken together, these data support a model wherein dTCTP functions either downstream or in parallel to Tsc and dRheb, but upstream of dS6k (Hsu, 2007).

Epistatic analysis suggests that dTCTP may be a new component in the TSC pathway. Because TCTP structurally resembles a small GTPase regulator, Mss4, it is proposed that dTCTP might directly associate with dRheb and positively regulate its activity. To test this, co-immunoprecipitation experiments were performed. Flag-tagged dTCTP immunoprecipitated together with Myc-tagged dRheb in 293T cell extracts, suggesting that dTCTP and dRheb form a complex in vivo. Furthermore, in vitro pull-down assays demonstrated direct binding of glutathione S-transferase (GST)-dTCTP to maltose binding protein (MBP)-dRheb. Notably, dTCTP showed preferential binding to nucleotide-free dRheb, a property shared among guanine nucleotide exchange factors (GEFs). To test whether dTCTP has GEF activity for dRheb, in vitro GDP release experiments were performed. MBP-dRheb alone showed weak intrinsic GDP dissociation. In contrast, addition of GST-dTCTP stimulated the GTP/GDP exchange on dRheb rapidly even when low amounts of dTCTP were used. The GEF-like activity is specific, as dTCTP did not accelerate the exchange reaction on dRas1, the closest GTPase to dRheb at sequence level. Moreover, a glutamic acid to valine mutation in the putative GTPase binding groove of dTCTP (dTCTP^E12V) abolished this GEF activity, even at a high concentration. Because dTCTP^E12V still retained binding activity to dRheb, this residue seems to have a critical function in catalytic reactions rather than binding between the two proteins. To determine whether E12 is critical for the function of dTCTP in vivo, genetic rescue experiments were performed. Whereas wild-type dTCTP was able to rescue fully the dTCTP RNAi phenotype, mutant dTCTP^E12V failed to rescue the RNAi defects even though the mutant protein was expressed at a high level. Therefore, the conserved E12 residue of dTCTP is essential for its normal function in development (Hsu, 2007).

To test whether this in vitro GEF activity has a physiological relevance, the in vivo level of GTP bound to dRheb was determined in Drosophila S2 cells. S2 cells treated with dTCTP dsRNA or a control EGFP dsRNA were transfected with Myc-tagged dRheb. dTCTP-dsRNA-treated cells consistently showed a lower percentage of GTP-bound dRheb, suggesting that dTCTP is required for dRheb activation in vivo. These GEF-like properties displayed by dTCTP towards dRheb are particularly intriguing, as no Rheb GEFs have been reported. Further kinetic and structural analysis will help to elucidate whether dTCTP is a bona fide GEF enzyme for dRheb (Hsu, 2007).

Human TCTP (hTCTP) and dTCTP are roughly 50% identical in their protein sequence. dTCTP RNAi and mutant phenotypes can be rescued by expression of hTCTP. Furthermore, hTCTP interacts most strongly with the nucleotide-free hRheb and stimulates the GDP/GTP exchange of hRheb in vitro. These data suggest that the function of TCTP in the TSC pathway is likely to be conserved throughout evolution (Hsu, 2007).

dTCTP controls cell growth and proliferation by positively regulating dRheb activity. The data suggest that dTCTP may function as a GEF or a related regulatory factor to activate dRheb. Given the strong epistatic effect of dTCTP to dRheb, it is also possible that dTCTP may have additional roles in the TSC pathway, acting downstream of dRheb but upstream of S6k (Hsu, 2007).

TCTP has been implicated in a wide range of cancers. Nevertheless, overgrowth phenotypes as a result of dTCTP overexpression were not observed, suggesting that dTCTP is not sufficient to induce growth. Notably, reduction of TCTP levels is sufficient for suppression of malignancy in tumour reversion models. This study provides a possible explanation for this phenomenon. It will be intriguing to learn whether lowering the insulin/TSC signalling output can be a general mechanism for tumour reversion (Hsu, 2007).

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).

DEVELOPMENTAL BIOLOGY

In situ hybridization to mRNA has revealed that Rheb is expressed ubiquitously throughout embryogenesis and in all tissues of L3 larvae, with highest expression in the testis (Saucedo, 2003).

In the developing embryo, Rheb expression correlates with DNA replication. Immediately after fertilization, as rapid, syncytial nuclear divisions take place, Rheb mRNA is present at a high, uniform level. This level decreases as rates of nuclear division slow, begins increasing again during stage 11 and becomes significantly higher in tissues undergoing endocycles (i.e. midgut) and mitoses (i.e. central nervous system). Later in embryogenesis (stages 13-16), as revealed by in situ hybridization and BrdU incorporation, the same regions that show strong Rheb expression are also carrying out DNA synthesis. Thus, embryonic mRNA expression patterns and levels are consistent with a role for Rheb in promoting S-phase (Patel, 2003).

EFFECTS OF MUTATION

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

A gain-of-function screen utilizing the GeneSearch (GS) P element was used to identify novel regulators of cell growth. Transcription from mobilized P elements was induced from bidirectional upstream activator sequence (UAS) enhancers using GMR-GAL4, which is expressed in post-mitotic cells of the developing eye. Of approximately 20,000 animals scored, 48 were found to have enlarged eyes. Insertion in a line that demonstrates one of the strongest overgrowth phenotypes (GSjE2) was mapped to cytological position 83B2, within the 5'-untranslated region (UTR) of CG1081. A full-length Rheb EST was cloned downstream of UAS sequences and transformed into naïve flies. Multiple independently derived transgenic animals recapitulated the eye overgrowth phenotype of GSjE2, confirming that induction of Rheb is sufficient to promote growth in the eye (Saucedo, 2003).

Imprecise excision of the GS element in the 5'-UTR of rheb yielded two lines that expressed no detectable rheb mRNA. One allele, rheb^PDelta1, removed all of the coding sequence for rheb and 13 bases of the 5'-UTR of the neighboring gene, CRMP (collapsin response mediator protein). The second allele, rheb^PDelta2, deleted sequences in the opposing direction, removing the promoter of rheb as well as coding sequence for two predicted genes located upstream. Trans-heterozygous animals containing these two opposing deletions survived embryogenesis, but spent an extended period in the first instar of larval development before dying approximately 6 days after hatching. Food was detected in the gut of these mutants, verifying that they ate. Thus, the inhibition of larval growth is probably caused by a cellular growth defect. Because rheb^{PDelta1/PDelta2} animals are homozygous for disruption of rheb only, loss of rheb is most probably responsible for lethality. To test this interpretation, UAS-Rheb and hs-GAL4 were introduced into rheb^{PDelta1/PDelta2} animals. With or without heat-shock, addition of these transgenes partially rescues the growth defect, enabling the rheb^{PDelta1/PDelta2} animals to reach the second larval stage before arresting. Given that Rheb is a dose-dependent regulator of cell growth, the failure to fully rescue the rheb^{PDelta1/PDelta2} animals using this artificial expression method is not unexpected (Saucedo, 2003).

To ascertain whether Rheb functions as a general growth promoter, the effect of Rheb overexpression was examined in multiple tissues. Expression of Rheb in the posterior compartment of the developing wing resulted in a 22% expansion of the posterior tissue of the adult wing with minimal disruption of patterning. In addition, wing hairs (trichomes) of the posterior wing were spaced further apart than controls. Because a single hair marks each wing cell, wing-hair density was enumerated as a means of gauging cell size. Overexpression of Rheb resulted in a 34% increase in adult wing cell area. To examine the effect of Rheb in larval tissues, cell clones overexpressing Rheb and marked with GFP were generated using the FLP/GAL4 method. Rheb overexpression increased cell size and nuclear DNA content in endoreduplicating tissues, including the gut, proventriculus and fat body. Fat body cells overexpressing Rheb encompassed approximately 2.5 times the area of control cells. Therefore, Rheb promotes growth in both mitotic and endoreduplicating cells of diverse tissues (Saucedo, 2003).

To determine if Rheb-induced growth is accompanied by accelerated cell cycle progression, cell clones overexpressing Rheb were examined in developing wing discs. Cell cycle profiles were obtained using flow cytometry on live cells from dissociated wing discs. Forward scatter (FSC) analysis was used to measure cell volume and confirmed the effect of Rheb on cell size, demonstrating a 65% increase in mean FSC in the line with the strongest transgene. DNA profiles have demonstrated that overexpression of Rheb profoundly alters cell cycle phasing, reducing the fraction of cells in G1 from 43% to 16%. Next, cell division times were calculated by counting the number of cells per clone and monitoring the time between clone induction and fixation of the wing disc. The doubling time of control cells and cells overexpressing Rheb was calculated to be 13.9 h and 13.7 h, respectively. Thus, although Rheb strongly promotes G1-S progression, a corresponding extension of S and G2 phase results in overall preservation of a normal cell division rate (Saucedo, 2003).

Because the growth and cell cycle phenotypes after Rheb overexpression are reminiscent of those caused by hyperactivation of insulin/phosphatidylinositol-3-OH kinase [PI(3)K] signalling, the potential role of Rheb in this network was tested. Using a pleckstrin homology (PH) domain-green fluorescent protein (GFP) fusion protein as a reporter of PI(3)K activity, it was found that Rheb dies not stimulate PI(3)K function, indicating that if Rheb has a role in insulin/PI(3)K signalling, it functions further downstream. The lipid phosphatase PTEN (phosphatase and tensin homolog deleted in chromosome 10) directly antagonizes the kinase function of PI(3)K and suppresses growth when overexpressed. Co-overexpression of Rheb bypasses PTEN-mediated growth inhibition in the adult eye, providing further evidence that Rheb functions downstream of PI(3)K activity. Whether PI(3)K signalling occurs in the absence of Rheb was tested. Animals overexpressing PI(3)K are sensitive to starvation, most probably because of inappropriate anabolic metabolism. Removal of one or both copies of rheb suppresses this hypersensitivity, suggesting that Rheb is required for PI(3)K signalling (Saucedo, 2003).

Tsc1-Tsc2 is a phosphorylation target of protein kinase B (PKB) and interferes with insulin/PI(3)K signalling. Overexpression of Tsc1-Tsc2 markedly reduced the size of the adult eye and this growth suppression is partially reversed by co-expression of Rheb. The Tsc1-Tsc2 complex probably antagonizes growth by suppressing TOR, a protein kinase implicated in the mediation of protein synthesis in response to nutrients. Tsc1-Tsc2 and TOR physically associate, overexpressed Tsc1-Tsc2 inhibits TOR signalling, and genetic epistasis tests place TOR downstream of Tsc1-Tsc2. In addition, TOR is necessary for insulin/PI(3)K-directed growth (Saucedo, 2003 and references therein).

The ability of Rheb to induce cell growth was tested in the absence of tor. Clones of cells lacking tor in the presence or absence of overexpressed Rheb were created in developing wing discs using FRT-mediated recombination and examined by flow cytometry. Loss of tor causes a marked reduction in cell size and an increase in the population of cells in G1. This phenotype persists when Rheb is overexpressed, demonstrating that tor is epistatic to (functions downstream of) overexpressed Rheb (Saucedo, 2003).

The role of S6 kinase (S6K), a protein involved in translation and an effector of TOR-mediated growth, was tested. In s6k null animals, Rheb is still able to produce enlarged eyes when expressed using GMR-GAL4. The puckering of eye tissue in s6k animals overexpressing Rheb is most probably caused by the substantial reduction in body and head capsule size of s6k animals. Consistently, it has been reported that overexpression of PI(3)K promotes growth in animals lacking S6K. Higher magnification has revealed that overexpression of Rheb is partially attenuated by loss of s6k. In conclusion, these genetic interaction tests strongly implicate Rheb as a downstream component of the insulin/PI(3)K signalling pathway that requires TOR (Saucedo, 2003).

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

In situ hybridization to mRNA has revealed that Rheb is expressed ubiquitously throughout embryogenesis and in all tissues of L3 larvae, with highest expression in the testis. Because transcription of Rheb is induced after nitrogen starvation in Aspergillus fumigatus, the nutritional responsiveness of rheb expression was examined. Microarray analyses reveal that rheb transcripts are upregulated in larvae that were starved on a protein-free diet. Induction of rheb is rapid (2.2-fold at 4 h) and persistent (2.4-fold at 48 h). After refeeding, levels of rheb decreased twofold. Of the six other components of the insulin/PI(3)K signalling pathway examined, only the insulin receptor substrate Chico demonstrated a similar pattern of nutritional responsiveness (Saucedo, 2003).

Rheb can promote cell growth, even in starved animals. Fat body and gut cells overexpressing Rheb were examined in second-instar larvae starved of protein for 72 h. Before starvation, cells expressing Rheb were approximately the same size as control cells in the same tissue. After three days of starvation, no growth of control cells was apparent, but cells overexpressing Rheb demonstrated marked growth. Thus, Rheb is capable of bypassing the dietary requirement for cell growth. This phenotype has also been demonstrated after overexpression of insulin receptor and PI(3)K. In contrast, constitutive expression of S6K in starved animals failed to promote cell growth, indicating that S6K alone cannot recapitulate the phenotype observed with Rheb or factors upstream of Rheb (Saucedo, 2003)..

This investigation of Drosophila Rheb has revealed a new function for this small GTP-binding protein in the regulation of cell growth. In comparison with similar studies of activated Ras (Ras1^G12V) in Drosophila, Rheb is a far more potent promoter of growth, but affects none of the corresponding alterations of cell fate caused by Ras1^G12V expression in the wing and eye. Because Raf-1 is an effector of Ras signalling in directing cell fate in Drosophila, these results suggest that Rheb does not affect Raf-1 function in vivo, in contradiction to in vitro binding studies (Saucedo, 2003).

RNAi studies in cultured cells demonstrate that Rheb is epistatic to Tsc1-Tsc2. Interestingly, Tsc2 contains a GTPase-activating domain (GAP). Zhang (2003) has provided compelling evidence that Tsc2 functions as a Rheb-GAP. Inactivation of Tsc1 or Tsc2 results in tumorigenesis in humans, and mutations in the GAP domain of Tsc2 have been identified in tuberous sclerosis patients. If Rheb is a physiological target of Tsc2, a greater proportion of Rheb should be GTP-bound and active in these patients. Tsc1-Tsc2 has been reported to be located at the cell membrane and this localization is disrupted by PKB signalling. Rheb has been shown to be farnesylated and also localizes to plasma membranes. Farnesylation of Rheb is critical for its activity, since Rheb constructs lacking the CAAX domain do not complement yeast deficient for rheb. One possibility is that membrane-associated Tsc1-Tsc2 impedes Rheb function. After activation of PKB, however, dissociation of Tsc1-Tsc2 may activate Rheb (Saucedo, 2003).

Thus Rheb-mediated cell growth requires TOR. How Rheb positively signals to TOR remains an important question. Two possible models can be invisioned: (1) Rheb directly regulates TOR function; (2) Rheb stimulates TOR indirectly by elevating nutrient import. Rheb has been implicated in regulating amino-acid import in Saccharomyces cerevisiae, but in a manner opposite that which would be expected of a growth promoter. Rheb mutants have increased uptake of arginine and lysine, suggesting that Rheb restricts amino-acid import. Another plausible interpretation is that the increase in amino-acid uptake is an indirect effect of losing Rheb. If Rheb normally stimulates nutrient import in S. cerevisiae, strains mutant for rheb may respond by up-regulating alternative pathways (Saucedo, 2003).

rheb mRNA levels are induced after protein starvation and subsequently reduced after refeeding. In addition, overexpressed Rheb can function as a growth promoter, even in animals starved for protein. On the basis of these results, the model is favored whereby Rheb directly promotes nutrient import. In Schizosaccharomyces pombe, Rheb has been shown to be required for cells to grow normally under limited amounts of nitrogen. Together, these data suggest that induction of Rheb in response to nitrogen or protein starvation may be a mechanism to mobilize limited resources and thereby maintain homeostasis under non-optimal conditions (Saucedo, 2003 and references therein).

Overexpression of Rheb leads to enlargement of the larval hindgut; the generality of this effect was tested by driving Rheb expression in other tissues. In the larval salivary glands, Rheb overexpression also results in significant enlargement. When Rheb is overexpressed in the developing eye imaginal disc, using the GMR-GAL4 driver, dramatically enlarged eyes are seen in the adult. Similarly, overexpression of Rheb in clones of cells in the eye and antennal discs, using the 'flip-out' method and eyFLP, results in an enlarged eye, antenna and head. When compared with control eyes, the ommatidia of these eyes are much larger than normal, are not organized into the normal hexagonal array and are frequently flanked by extra bristles. The larger overall eye and ommatidial size, and the extra bristles further support the notion that Rheb overexpression promotes growth. The fact that bristles in the Rheb-overexpressing eyes are larger (thicker) than normal, together with the fact that each bristle is derived from a single cell, suggests that Rheb might promote growth at the level of the individual cell (Patel, 2003).

Overexpression of Rheb also promotes growth in the wing, an organ consisting of two opposed, flat, single-layered epithelial sheets. Overexpression of Rheb in the dorsal epithelial compartment causes the wing to curve downwards, presumably owing to an increase in area of the dorsal compartment. When Rheb is overexpressed in the posterior compartment of the wing, the posterior area is increased by 36%, further supporting a role for Rheb in growth. To distinguish between effects on cell number versus cell size, wing hairs (each of which is produced by a single cell) were counted in normal and Rheb-overexpressing compartments. Because the cell density in the Rheb-overexpressing compartment is only 67% of that in the wild-type compartment, it is concluded that Rheb-overexpressing cells are significantly larger (i.e., each cell occupies more area) than wild-type cells. Wing hair density, combined with compartment area measurements, shows that total cell number in the posterior compartment is the same whether or not Rheb is being overexpressed. Thus, in the wing, Rheb overexpression results in an increase in cell size but not cell number. This is similar to what has been described for overexpression of activated Ras1 and Myc; in Drosophila, these genes are believed to affect cell cycle progression via their primary function as regulators of cell growth (Patel, 2003).

To characterize the effect of Rheb at the cellular level, Drosophila S2 cells were co-transfected with vectors expressing Rheb and GFP (a marker for transfection). Examination of the cell cycle profile of transfected (GFP plus Rheb-overexpressing) cells by FACS reveals that increased levels of Rheb result in an increase in the proportion of cells in S-phase. In addition, analysis of cell size by forward scatter analysis reveals that S2 cells overexpressing Rheb are slightly larger than control (GFP alone) cells (Patel, 2003).

The overexpression studies described above show that Rheb can promote two processes: increase in cell size and cell cycle progression. The correlation between Rheb expression and DNA synthesis in the embryo, and the effect of Rheb overexpression in cultured cells both suggest that Rheb promotes progression into S-phase. The increased size of cultured cells, eye bristles and wing cells overexpressing Rheb suggests that Rheb promotes increase in mass of individual cells (cell growth). These two distinct effects of Rheb raise the question of whether Rheb affects the cell cycle and cell growth independently or via a pathway that coordinates both of these processes (Patel, 2003).

Although the results of overexpression studies described above suggest a role for Rheb in regulation of cell growth and cell cycle progression, removal of gene activity is necessary unequivocally to establish required function. Of great utility is the fact that the Rheb^AV4 allele, in addition to allowing ectopic expression when combined with a GAL4 driver, is also homozygous lethal. Precise excision of the P-element in the Rheb^AV4 chromosome results in a reversion to viability, confirming that the Rheb^AV4 lethal phenotype is due to disruption of Rheb activity resulting from the P-element insertion. To determine the lethal phase of Rheb^AV4 homozygotes, eggs were collected for 2 hours from heterozygous parents and the number of surviving homozygous mutant larvae relative to heterozygous sibs was determined as a function of time. Embryos homozygous for Rheb^AV4 hatch into first instar larvae that grow very slowly, move lethargically, and die by 72 hours without molting into second instar larvae. Rheb is thus required for growth of the whole organism (Patel, 2003).

To assess the requirement for Rheb in individual cells, clones of cells lacking Rheb were made in the eye. For this purpose, FRT(82B) was recombined onto the Rheb^AV4 chromosome; combination of this chromosome with eyFLP should, in principle, allow generation of w;dRheb^-/- clones in the background of a w⁺;dRheb^+/- eye. In this background, however, Rheb^-/- clones were not detected, similar to what has been reported for clones lacking Ras1 function. In both cases, the absence of detectable w clones is probably due to cell competition, a process in which faster-growing cells out-compete slowly growing cells, which are then eliminated by apoptosis. To give Rheb^-/- clones a growth advantage, they were generated in a Minute (M^+/-) background (Minute genes regulate protein synthesis; thus M^+/+ cells grow more rapidly than surrounding M^+/- cells). Heads and eyes containing multiple Rheb^-/- M^+/+ clones are dramatically smaller than those containing Rheb^+/+clones. Sections of these eyes show that the Rheb^-/- ommatidia are smaller overall because they are composed of dramatically smaller cells (Patel, 2003).

Two important inferences can be drawn from analysis of these Rheb loss-of-function clones: (1) because eyes bearing multiple Rheb^-/- clones are smaller than wild-type, Rheb must play a required role in tissue growth; (2) the smaller size of individual Rheb^-/- M^+/+ cells indicates that Rheb is required in a cell autonomous manner for cell growth (increase in mass) (Patel, 2003).

To investigate whether these inferred roles of Rheb can be demonstrated at the cellular level, dsRNA was used to inhibit Rheb function in Drosophila S2 cells. Addition of dsRNA corresponding to the entire coding sequence of Rheb to the culture medium almost completely inhibits expression of FLAG-tagged dRheb. Characterization of cell cycle profiles by FACS showed a dramatic increase in the proportion of cells in G1-phase by day four to five in Rheb dsRNA-treated cells; this effect persisted to at least day 8. In addition to having an effect on the cell cycle, inhibition of Rheb also has a significant effect on cell growth. Forward scatter analysis reveals a dramatic reduction in cell size after the addition of Rheb dsRNA. Both the diminution in cell size and the accumulation of cells in G1-phase after Rheb inhibition follow roughly the same time course (i.e., both are maximal by day five) (Patel, 2003).

From the loss-of-function studies in the whole organism and in cultured cells described above, it is concluded that Rheb affects both cell growth (mass increase) and cell cycle progression promoting transition of cells from G1 to S phase (Patel, 2003).

Control of cell growth and cell cycle progression can, in principle, be regulated by parallel independent pathways or through a signal that coordinates both. In both yeast and Drosophila, mutations in cell-cycle-specific genes (such as cyclin E) result in cell cycle arrest with an associated increase in cell size owing to continued cell growth, although overexpression of these genes results in smaller cells. Because loss of Rheb function in both cultured cells and in the whole organism results in reduced cell size, it is likely that Rheb coordinates cell cycle and cell growth. Therefore the possibility was considered that Rheb might impinge on the insulin and TOR signaling pathways, which are major contributors to the regulation of cell growth in both Drosophila and mammalian cells. Because Rheb larvae exhibit a growth arrest similar to dTOR mutants and larvae starved for amino acids, rapamycin treatment was used to investigate whether Rheb interacts, either in the whole organism or in cultured cells, with dTOR. Under normal growth conditions, flies with either one or two copies of Rheb eclose (emerge from the pupal case) at the same time; in the presence of rapamycin, however, larvae with only one wild-type copy of Rheb grow more slowly, eclosing two days later than their wild-type sibs. Reduced Rheb thus sensitizes the organism to the growth-inhibiting effect of rapamycin. Possible involvement of dTOR in Rheb function was examined in S2 cells, in which, as expected, treatment with rapamycin causes cells to decrease in size. Significantly, the cell-growth-promoting effect of overexpressing Rheb is blocked by rapamycin. This latter result indicates that the effect of Rheb overexpression depends on the functional activity of dTOR; in other words, dTOR is epistatic to (downstream of) Rheb (Patel, 2003).

Mechanisms of TSC-mediated control of synapse assembly and axon guidance

Tuberous sclerosis complex is a dominant genetic disorder produced by mutations in either of two tumor suppressor genes, TSC1 and TSC2; it is characterized by hamartomatous tumors, and is associated with severe neurological and behavioral disturbances. Mutations in TSC1 or TSC2 deregulate a conserved growth control pathway that includes Ras homolog enriched in brain (Rheb) and Target of Rapamycin (TOR). To understand the function of this pathway in neural development, this study examined the contributions of multiple components of this pathway in both neuromuscular junction assembly and photoreceptor axon guidance in Drosophila. Expression of Rheb in the motoneuron, but not the muscle of the larval neuromuscular junction produced synaptic overgrowth and enhanced synaptic function, while reductions in Rheb function compromised synapse development. Synapse growth produced by Rheb is insensitive to rapamycin, an inhibitor of Tor complex 1, and requires wishful thinking, a bone morphogenetic protein receptor critical for functional synapse expansion. In the visual system, loss of Tsc1 in the developing retina disrupted axon guidance independently of cellular growth. Inhibiting Tor complex 1 with rapamycin or eliminating the Tor complex 1 effector, S6 kinase (S6k), did not rescue axon guidance abnormalities of Tsc1 mosaics, while reductions in Tor function suppressed those phenotypes. These findings show that Tsc-mediated control of axon guidance and synapse assembly occurs via growth-independent signaling mechanisms, and suggest that Tor complex 2, a regulator of actin organization, is critical in these aspects of neuronal development (Knox, 2007).

The Tsc-Rheb-Tor pathway is critical for integrating a variety of signals that govern cellular and organismal growth. Inappropriate activation of the pathway also leads to severe neurological and behavioral abnormalities, including mental retardation, autism, and epilepsy. While TSC mutations produce hamartomatous growths in the brain, recent evidence has suggested that these benign tumors may not be solely responsible for the nervous system dysfunction that is a hallmark of tuberous sclerosis complex. Loss of TSC2 in hippocampal neurons produces changes in neuronal morphology and synaptic transmission. Heterozygosity for TSC2 in the rat compromises several measures of hippocampal long term potentiation. Loss of Pten, an important upstream regulator of Tsc-Rheb-Tor signaling, in a limited set of neurons also affects neuronal morphology and socialization behavior. These findings collectively provide evidence that Tsc-Rheb-Tor signaling is critical for the morphological and functional development of the nervous system. It is not clear, however, if the entire Tsc-Rheb-Tor signaling network is critical for nervous system development, or if neural function is strictly a consequence of altered growth regulation. It is also not known if loss of signaling is as detrimental to neuronal development as inappropriately elevated signaling, such as occurs with loss of TSC function. This study has taken advantage of the genetic and molecular tools available in Drosophila to address these questions. The findings demonstrate that appropriate levels of Tsc-Rheb-Tor signaling are critical for both NMJ development and for axon guidance in the visual system. In both these contexts, effects are independent of growth, implicating TORC2 (which includes Rictor in addition to Tor and mLST8; in both yeast and mammalian cells TORC2 influences the actin cytoskeleton) rather than TORC1 (which includes Raptor and mLST8, and regulates translation via phosphorylation of S6 kinase and 4E-binding protein) as the complex mediating Tsc-Rheb-Tor signaling influences in the nervous system (Knox, 2007).

Given the importance of Tsc-Rheb-Tor signaling in regulating cellular and tissue growth, it was important to determine if disruption of this pathway affects neural development via its effects on growth or through signaling components independent of those that govern cellular size and growth. To address this issue both pharmacological and genetic methods were used to block the increased growth produced by pathway activation. The immunosuppressant rapamycin is a TORC1-specific inhibitor that prevents activation of S6k and blocks growth mediated by loss of Tsc1. Rapamycin treatment retarded growth in larvae with pan-neuronal expression of Rheb, but failed to reduce the synapse expansion characteristic of these animals. Similarly, while rapamycin effectively reduced the retinal overgrowth of Tsc1 mosaic animals, it failed to suppress the photoreceptor axon guidance defects seen in the visual system. Loss of S6k function also failed to ameliorate axon guidance defects in Tsc1 mosaic animals. This contrasts with effects of Tor partial loss-of-function mutations, which effectively rescued axon guidance defects of Tsc1 mutants. Collectively, these findings demonstrate that the role of Tsc-Rheb-Tor signaling in synapse assembly and axon guidance is largely independent of TORC1, S6k, and their effects on growth. Indeed, while animals bearing null alleles of S6k have some axon pathfinding defects, the effects are relatively modest compared to Tsc1 mosaics, indicating that S6k does not provide the critical outputs affecting axon guidance (Knox, 2007).

The findings parallel recent work in the mouse, where neuronal hypertrophy produced by loss of Pten in granule neurons of the cerebellum and dentate gyrus was not rescued by loss of S6k1. It is also of note that some but not all Tsc1/2-mediated changes in dendritic morphology of hippocampal neurons in organotypic cultures were suppressed by rapamycin treatment. The current findings suggest that inhibition of growth regulatory components in tuberous sclerosis patients, such as achieved with rapamycin and related agents, may not affect all processes that are deranged in the nervous system (Knox, 2007).

Recent studies of Pi3 kinase, Akt and InR in Drosophila have shown that activation of signaling upstream of Tsc1/2 also produces increases in synapse size, both at the NMJ as well as central synapses. Expression of these components in adult neurons demonstrated that Pi3 kinase-mediated synaptogenesis is age-independent, and therefore not a developmentally restricted phenomenon. In agreement with studies reported in this paper, the expanded NMJs produced by activation of Pi3 kinase were functional, with increased stimulus-induced EJPs. Overexpression of the Drosophila ortholog of the epidermal growth factor receptor (EgfR) in central neurons increased neuronal cell size, without an increase in synapse number. These results are consistent with those reported in this study it was possible to to directly suppress growth mediated by Tsc-Rheb-Tor pathway activation without altering effects on synapse formation or axon guidance (Knox, 2007).

Recent studies have also demonstrated a link between Tsc1/Tsc2 and highwire, a gene known to effect synapse size and functionality in Drosophila. The highwire ortholog Pam was shown to bind Tsc2 in pull-down assays, and it has been suggested that Pam may function as an E3 ubiquitin ligase to regulate the intracellular levels of the Tsc1/Tsc2 complex. This concept of Highwire as a negative regulator of Tsc levels is consistent with the current findings, since highwire mutants have been shown to possess enlarged NMJs similar to what is seen for Rheb overexpression. Despite this, the enlarged synapses of highwire mutants display compromised synaptic function which is contrary to what was found when overexpressing Rheb, so Highwire is likely to have multiple functions at the synapse besides simply the regulation of Tsc (Knox, 2007).

Tor has a number of molecular outputs that influence many cellular processes; notable among these are cellular growth and cellular morphology. TORC1, which contains Raptor and is sensitive to the anti-proliferative agent rapamycin, is a major contributor to the regulation of cellular growth, in large measure due to its effects on protein synthesis. TORC2, which includes Rictor, is implicated in the control of cell morphology mediated by regulation of the actin cytoskeleton. Both pharmacological and genetic studies presented here argue in favor of Tor complex 2 providing an essential regulatory component of both synapse growth and axon guidance in Drosophila. The current results support recent work showing that changes in dendritic morphology of hippocampal neurons produced by loss of Tsc1 required regulation of the actin-depolymerizing factor Cofilin, implicating TORC2-mediated processes. There is a considerable body of work demonstrating that control of the actin cytoskeleton is critical for NMJ growth and function and TORC2 may provide an important component of that control. Regulation of actin is also essential for axon guidance in the visual system, and disruption of Tor-mediated control of actin may be the underlying molecular deficit in Tsc1 mosaics (Knox, 2007).

A number of studies have suggested that TOR activation produced by loss of TSC1/2 affects neuronal morphology and synaptic function. The current findings support these observations; elevated Rheb activity produces synaptic enlargement and enhanced physiological function at the Drosophila NMJ. However, it was not evident from earlier studies whether loss of signaling through Rheb and Tor is also important for neural development. Evidence is provided that this is the case. Partial loss-of-function mutations in Rheb compromise NMJ growth and function, as well as photoreceptor axon targeting in the visual system. Overexpression of Tsc1 and Tsc2 in the motoneuron also limited synaptic growth, supporting the conclusion that depressed levels of Rheb activity compromise synapse development (Knox, 2007).

The capacity of Tsc-Rheb-Tor signaling to affect neuronal morphology and synapse function begs the question of whether these effects are dependent on signaling systems known to be critical for synapse development. At the Drosophila NMJ, BMP signaling is critical for normal growth and function. Mutations in wit, a gene encoding a type II BMP receptor, produce a small and poorly functioning NMJ. These deficits can be rescued by motoneuron expression of wit⁺, demonstrating that BMP signaling in the motoneuron is critical for synaptic expansion during larval growth. To determine if Rheb-mediated synaptic growth requires BMP signaling, elav-Gal4 and UAS-Rheb transgenes were placed into a wit mutant background. While overexpression of Rheb and the accompanying activation of the Tor pathway partially rescued the defect in synapse growth produced by loss of wit function, it was unable to restore a normal EJP response or rescue quantal content. These findings establish that Tsc-Rheb-Tor mediated effects on synapse morphology are partially dependent on BMP signaling, and are fully dependent on BMP activity for a physiologically competent synapse. The findings also establish that the functional deficits in wit mutants are not simply the result of reduced synapse size, since restoration of synapse size by expression of UAS-Rheb does not restore physiological function. Intersection of BMP, and Akt/PTEN/TOR signaling has been noted for other systems, and the results indicate the relationship between these pathways is important for synapse growth and plasticity as well (Knox, 2007).

Previous analysis of gigas/Tsc2 mutants demonstrated that loss of this gene in mechanoreceptors affects axon targeting, producing projections to novel areas in the CNS in addition to innervation of normal targets (Canal, 1998). Genetic mosaics were used to evaluate the function of Tsc-Rheb-Tor signaling in photoreceptor axon guidance. Animals homozygous for Tsc1 in the retina showed grossly aberrant photoreceptor projections to both the lamina and medulla. R7 and R8 projections to the medulla in 40h pupae failed to terminate correctly and projected beyond normal targets to inappropriate regions within the brain. Somatic mosaics bearing retinal neurons mutant for Pten also showed photoreceptor axon guidance defects, but to a notably lesser degree. Since both Tsc1 and Pten alleles used for this analysis were nulls and show comparable effects on cellular growth and differentiation, it follows that Pten is not as critical for axon guidance as Tsc1. The distinctions between axon guidance phenotypes of Pten and Tsc1 null mutants indicate that altered timing of differentiation is not critical for axon guidance and that control of this pathway at the level of Pten or Tsc1 is not functionally equivalent. The findings that rapamycin arrests retinal overgrowth produced by loss of Tsc1 but not Pten in the retina supports earlier work demonstrating that retinal overgrowth mediated by loss of Tsc1, but not Pten, can be suppressed by reductions in S6k activity. Those results were interpreted as demonstrating that Pten is largely a regulator of Akt activity, whereas Tsc1/2 serves as a tumor suppressor and inhibitor affecting principally S6k. The current results support these relationships and emphasize that in the nervous system regulation of Tsc1/2 targets other than S6k are critical (Knox, 2007).

Two different genetic methods were used for activating the Tsc-Rheb-Tor pathway in the visual system; generating retinal mosaics with a loss of function allele of Tsc1, and pan-neuronal expression of Rheb using elav-Gal4 and UAS-Rheb. The comparison of these methods revealed that overexpression of Rheb produced milder axon guidance phenotypes in the visual system than complete loss of Tsc1 function. Of interest is that the degree of activation achieved with elav-Gal4>UAS-Rheb, a level that did not produce lethality, did result in discernable axon targeting defects in the visual system. This suggests that axon guidance controlled by Tsc-Rheb-Tor is sensitive to incremental changes in signaling. The range of neurological and behavioral phenotypes associated with loss of one copy of TSC1 or TSC2 is consistent with this model, where other environmental or genetic factors may affect signaling levels, producing a range of deficits. The findings indicate that Drosophila can serve as a useful model for identifying how graded changes in signaling can produce a spectrum of defects in neural development (Knox, 2007).

Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila

Many stem, progenitor and cancer cells undergo periods of mitotic quiescence from which they can be reactivated. The signals triggering entry into and exit from this reversible dormant state are not well understood. In the developing Drosophila central nervous system, multipotent self-renewing progenitors called neuroblasts undergo quiescence in a stereotypical spatiotemporal pattern. Entry into quiescence is regulated by Hox proteins and an internal neuroblast timer. Exit from quiescence (reactivation) is subject to a nutritional checkpoint requiring dietary amino acids. Organ co-cultures also implicate an unidentified signal from an adipose/hepatic-like tissue called the fat body. This study provides in vivo evidence that Slimfast amino-acid sensing and Target of rapamycin (TOR) signalling activate a fat-body-derived signal (FDS) required for neuroblast reactivation. Downstream of this signal, Insulin-like receptor signalling and the Phosphatidylinositol 3-kinase (PI3K)/TOR network are required in neuroblasts for exit from quiescence. Nutritionally regulated glial cells provide the source of Insulin-like peptides (ILPs) relevant for timely neuroblast reactivation but not for overall larval growth. Conversely, ILPs secreted into the haemolymph by median neurosecretory cells systemically control organismal size but do not reactivate neuroblasts. Drosophila thus contains two segregated ILP pools, one regulating proliferation within the central nervous system and the other controlling tissue growth systemically. These findings support a model in which amino acids trigger the cell cycle re-entry of neural progenitors via a fat-body-glia-neuroblasts relay. This mechanism indicates that dietary nutrients and remote organs, as well as local niches, are key regulators of transitions in stem-cell behaviour (Sousa-Nunes, 2011).

In fed larvae, Drosophila neuroblasts exit quiescence from the late first instar (L1) stage onwards. This reactivation involves cell enlargement and entry into S phase, monitored in this study using the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). Reactivated neuroblast lineages (neuroblasts and their progeny) reproducibly incorporated EdU in a characteristic spatiotemporal sequence: central brain --> thoracic --> abdominal neuromeres. Mushroom-body neuroblasts and one ventrolateral neuroblast, however, are known not to undergo quiescence and to continue dividing for several days in the absence of dietary amino acids. This indicates that dietary amino acids are more than mere 'fuel', providing a specific signal that reactivates neuroblasts. However, explanted central nervous systems (CNSs) incubated with amino acids do not undergo neuroblast reactivation unless co-cultured with fat bodies from larvae raised on a diet containing amino acids. Therefore the in vivo requirement for a fat-body-derived signal (FDS) in neuroblast reactivation was tested by blocking vesicular trafficking and thus signalling from this organ using a dominant-negative Shibire dynamin (SHI^DN). This strongly reduced neuroblast EdU incorporation, indicating that exit from quiescence in vivo requires an FDS. One candidate tested was Ilp6, known to be expressed by the fat body, but neither fat-body-specific overexpression nor RNA interference of this gene significantly affected neuroblast reactivation. Fat-body cells are known to sense amino acids via the cationic amino-acid transporter Slimfast (SLIF), which activates the TOR signalling pathway, in turn leading to the production of a systemic growth signal. Fat-body-specific overexpression of the TOR activator Ras homologue was shown to be enriched in brain (RHEB), or of an activated form of the p110 PI3K catalytic subunit, or of the p60 adaptor subunit, had no significant effect on neuroblast reactivation in fed animals or in larvae raised on a nutrient-restricted diet lacking amino acids. In contrast, global inactivation of Tor, fat-body-specific Slif knockdown or fat-body-specific expression of the TOR inhibitors Tuberous sclerosis complex 1 and 2 (Tsc1/2) all strongly reduced neuroblasts from exiting quiescence. Together, these results show that a SLIF/TOR-dependent FDS is required for neuroblasts to exit quiescence and that this may be equivalent to the FDS known to regulate larval growth (Sousa-Nunes, 2011).

Next, the signalling pathways essential within neuroblasts for their reactivation were investigated. Nutrient-dependent growth is regulated in many species by the interconnected TOR and PI3K pathways. In fed larvae, it was found that neuroblast inactivation of TOR signalling (by overexpression of TSC1/2), or PI3K signalling (by overexpression of p60, the Phosphatase and tensin homologue PTEN, the Forkhead box subgroup O transcription factor FOXO or dominant-negative p110), all inhibited reactivation. Conversely, stimulation of neuroblast TOR signalling (by overexpression of RHEB) or PI3K signalling [by overexpression of activated p110 or Phosphoinositide-dependent kinase 1 (PDK1)] triggered precocious exit from quiescence. RHEB overexpression had a particularly early effect, preventing some neuroblasts from undergoing quiescence even in newly hatched larvae. Hence, TOR/PI3K signalling in neuroblasts is required to trigger their timely exit from quiescence. Importantly, neuroblast overexpression of RHEB or activated p110 in nutrient-restricted larvae, which lack FDS activity, was sufficient to bypass the block to neuroblast reactivation. Notably, both genetic manipulations were even sufficient to reactivate neuroblasts in explanted CNSs, cultured without fat body or any other tissue. Together with the previous results this indicates that neuroblast TOR/PI3K signalling lies downstream of the amino-acid-dependent FDS during exit from quiescence (Sousa-Nunes, 2011).

To identify the mechanism bridging the FDS with neuroblast TOR/PI3K signalling, the role of the Insulin-like receptor (InR) in neuroblasts was tested. Importantly, a dominant-negative InR inhibited neuroblast reactivation, whereas an activated form stimulated premature exit from quiescence. Furthermore, InR activation was sufficient to bypass the nutrient restriction block to neuroblast reactivation. This indicates that at least one of the potential InR ligands, the seven ILPs, may be the neuroblast reactivating signal(s). By testing various combinations of targeted Ilp null alleles and genomic Ilp deficiencies, it was found that neuroblast reactivation was moderately delayed in larvae deficient for both Ilp2 and Ilp3 (Df(3L)Ilp2-3) or lacking Ilp6 activity. Stronger delays, as severe as those observed in InR³¹ mutants, were observed in larvae simultaneously lacking the activities of Ilp2, 3 and 5 [Df(3L)Ilp2-3, Ilp5] or Ilp1-5 [Df(3L)Ilp1-5]. Despite the developmental delay in Df(3L)Ilp1-5 homozygotes, neuroblast reactivation eventually begins in the normal spatial pattern -- albeit heterochronically -- in larvae with L3 morphology. Together, the genetic analysis shows that Ilp2, 3, 5 and 6 regulate the timing but not the spatial pattern of neuroblast exit from quiescence. However, as removal of some ILPs can induce compensatory regulation of others, the relative importance of each cannot be assessed from loss-of-function studies alone (Sousa-Nunes, 2011).

Brain median neurosecretory cells (mNSCs) are an important source of ILPs, secreted into the haemolymph in an FDS-dependent manner to regulate larval growth. They express xIlp1, 2, 3 and 5, although not all during the same development stages. However, this study found that none of the seven ILPs could reactivate neuroblasts during nutrient restriction when overexpressed in mNSCs. Similarly, increasing mNSC secretion using the NaChBac sodium channel or altering mNSC size using PI3K inhibitors/activators, which in turn alters body growth, did not significantly affect neuroblast reactivation under fed conditions. Surprisingly, therefore, mNSCs are not the relevant ILP source for neuroblast reactivation. Nonetheless, Ilp3 and Ilp6 messenger RNAs were detected in the CNS cortex, at the early L2 stage, in a domain distinct from the Ilp2⁺ mNSCs. Two different Ilp3-lacZ transgenes indicate that Ilp3 is expressed in some glia (Repo⁺ cells) and neurons (Elav⁺ cells). An Ilp6-GAL4 insertion indicates that Ilp6 is also expressed in glia, including the cortex glia surrounding neuroblasts and the glia of the blood-brain barrier (BBB) (Sousa-Nunes, 2011).

Next the ability of each of the seven ILPs to reactivate neuroblasts when overexpressed in glia or in neurons was assessed. Pan-glial or pan-neuronal overexpression of ILP4, 5 or 6 led to precocious reactivation under fed conditions. Each of these manipulations also bypassed the nutrient restriction block to neuroblast reactivation, as did overexpression of ILP2 in glia or in neurons, or ILP3 in neurons. In all of these ILP overexpressions, and even when ILP6 was expressed in the posterior Ultrabithorax domain, the temporal rather than the spatial pattern of reactivation was affected. Importantly, experiments blocking cell signalling with SHI^DN indicate that glia rather than neurons are critical for neuroblast reactivation. Interestingly, glial-specific overexpression of ILP3-6 did not significantly alter larval mass. Thus, in contrast to mNSC-derived ILPs, glial-derived ILPs promote CNS growth without affecting body growth (Sousa-Nunes, 2011).

Focusing on ILP6, CNS explant cultures were used to demonstrate directly that glial overexpression was sufficient to substitute for the FDS during neuroblast exit from quiescence. In vivo, ILP6 was sufficient to induce reactivation during nutrient restriction when overexpressed via its own promoter or specifically in cortex glia but not in the subperineurial BBB glia, nor in many other CNS cells that were tested. Hence, cortex glia possess the appropriate processing machinery and/or location to deliver reactivating ILP6 to neuroblasts. Ilp6 mRNA is known to be upregulated rather than downregulated in the larval fat body during starvation and, accordingly, Ilp6-GAL4 activity is increased in this tissue after nutrient restriction. Conversely, it was found that Ilp6-GAL4 is strongly downregulated in CNS glia during nutrient restriction. Thus, dietary nutrients stimulate glia to express Ilp6 at the transcriptional level. Consistent with this, an important transducer of nutrient signals, the TOR/PI3K network, is necessary and sufficient in glia (but not in neurons) for neuroblast reactivation. Together, the genetic and expression analyses indicate that nutritionally regulated glia relay the FDS to quiescent neuroblasts via ILPs (Sousa-Nunes, 2011).

This study used an integrative physiology approach to identify the relay mechanism regulating a nutritional checkpoint in neural progenitors. A central feature of the fat-body --> glia --> neuroblasts relay model is that glial insulin signalling bridges the amino-acid/TOR-dependent fat-body-derived signal (FDS) with InR/PI3K/TOR signalling in neuroblasts. The importance of glial ILP signalling during neuroblast reactivation is also underscored by an independent study, published while this work was under revision (Chell, 2010). As TOR signalling is also required in neuroblasts and glia, direct amino-acid sensing by these cell types may also impinge upon the linear tissue relay. This would then constitute a feed-forward persistence detector, ensuring that neuroblasts exit quiescence only if high amino-acid levels are sustained rather than transient. This study also showed that the CNS 'compartment' in which glial ILPs promote growth is functionally isolated, perhaps by the BBB, from the systemic compartment where mNSC ILPs regulate the growth of other tissues. The existence of two functionally separate ILP pools may explain why bovine insulin cannot reactivate neuroblasts in CNS organ culture, despite being able to activate Drosophila InR in vitro. Given that insulin/PI3K/TOR signalling components are highly conserved between insects and vertebrates, it will be important to address whether mammalian adipose or hepatic tissues signal to glia and whether or not this involves an insulin/IGF relay to CNS progenitors. In this regard, it is intriguing that brain-specific overexpression of IGF1 can stimulate cell-cycle re-entry of mammalian cortical neural progenitors, indicating utilization of at least part of the mechanism identified by this study in Drosophila (Sousa-Nunes, 2011).

The small GTPase Rheb affects central brain neuronal morphology and memory formation in Drosophila

Mutations in either of two tumor suppressor genes, TSC1 or TSC2, cause tuberous sclerosis complex (TSC), a syndrome resulting in benign hamartomatous tumors and neurological disorders. Cellular growth defects and neuronal disorganization associated with TSC are believed to be due to upregulated TOR signaling. This study overexpressed Rheb, an upstream regulator of TOR, in two different subsets of D. melanogaster central brain neurons in order to upregulate the Tsc-Rheb-TOR pathway. Overexpression of Rheb in either the mushroom bodies or the insulin producing cells resulted in enlarged axon projections and cell bodies, which continued to increase in size with prolonged Rheb expression as the animals aged. Additionally, Rheb overexpression in the mushroom bodies resulted in deficiencies in 3 hr but not immediate appetitive memory. Thus, Rheb overexpression in the central brain neurons of flies causes not only morphological phenotypes, but behavioral and aging phenotypes that may mirror symptoms of TSC (Brown, 2012).

Insulin/IGF-Regulated Size Scaling of Neuroendocrine Cells Expressing the bHLH Transcription Factor Dimmed in Drosophila

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

EVOLUTIONARY HOMOLOGS

Yeast Rheb is responsive to nutritional status

Rheb homologs have been identified in the budding yeast Saccharomyces cerevisiae (ScRheb) as well as in Schizosaccharomyces pombe, Drosophila melanogaster, zebrafish, and Ciona intestinalis. These proteins define a new class of G-proteins based on (1) their overall sequence similarity, (2) high conservation of their effector domain sequence, (3) presence of a unique arginine in their G1 box, and (4) presence of a conserved CAAX farnesylation motif. Characterization of an S. cerevisiae strain deficient in ScRheb shows that it is hypersensitive to growth inhibitory effects of canavanine and thialysine, which are analogues of arginine and lysine, respectively. Accordingly, the uptake of arginine and lysine is increased in the ScRheb-deficient strain. This increased arginine uptake requires the arginine-specific permease Can1p. The function of ScRheb is dependent on having an intact effector domain since mutations in the effector domain of ScRheb are incapable of complementing canavanine hypersensitivity of scrheb disruptant cells. Furthermore, the conserved arginine in the G1 box plays a role in the activity of ScRheb, since a mutation of this arginine to glycine significantly reduces the ability of ScRheb to complement canavanine hypersensitivity of ScRheb-deficient yeast. Finally, a mutation in the C-terminal CAAX farnesylation motif results in a loss of ScRheb function. This result, in combination with the finding that ScRheb is farnesylated, suggests that farnesylation plays a key role in ScRheb function. These findings assign the regulation of arginine and lysine uptake as the first physiological function for this new farnesylated Ras superfamily G-protein (Urano, 2000).

A fission yeast homolog of mammalian Rheb, which has been designated Rhb1, was identified by genome sequencing. rhb1^- cells arrest cell growth and division with a terminal phenotype similar to that of nitrogen-starved cells. In particular, cells depleted of Rhb1 arrest as small, round cells with 1N DNA content, arrest more quickly in low-nitrogen medium, and induce expression of fnx1 and mei2 mRNAs, two mRNAs that were normally induced by nitrogen starvation. Since mammalian Rheb binds and may regulate Raf-1, a Ras effector, functional overlap between Ras1 and Rhb1 was tested in fission yeast. This analysis shows that Ras1 overexpression does not suppress rhb1^- mutant phenotypes, Rhb1 overexpression does not suppress ras1^- mutant phenotypes, and ras1^- rhb1^- double mutants have phenotypes equal to the sum of the corresponding single-mutant phenotypes. Hence, there is no evidence for overlapping functions between Ras1 and Rhb1. On the basis of this study, it is hypothesized that Rhb1 negatively regulates entry into stationary phase when extracellular nitrogen levels are adequate for growth. If this hypothesis is correct, then Rhb1 and Ras1 regulate alternative responses to limiting nutrients (Mach, 2000).

A gene encoding a ras protein with homology to the rheb family was cloned from Aspergillus fumigatus. Although conserved ras domains are present, the predicted RhbA protein sequence deviates from the ras consensus in a manner that is characteristic of rheb proteins. The invariant Gly-Gly in the first GTP-binding domain of ras proteins is replaced by Arg-Ser in RhbA, and a conserved Asp in the effector region of ras proteins is replaced by Asn in RhbA. The rhbA mRNA is detected throughout the A. fumigatus asexual developmental cycle, and accumulates over 5-fold in response to nitrogen starvation. The rhbA gene is able to complement the canavanine hypersensitivity of Saccharomyces cerevisiae Deltarhb1 mutants, suggesting that the two proteins share overlapping function (Panepinto, 2002).

Farnesylation of Rheb is important for cell cycle progression of S. pombe cells

Protein farnesylation is important for a number of physiological processes, including proliferation and cell morphology. The Schizosaccharomyces pombe mutant, cpp1-, defective in farnesylation, exhibits distinct phenotypes, including morphological changes and sensitivity to the arginine analogue, canavanine. A novel phenotype of this mutant is reported -- enrichment of G0/G1 phase cells. This phenotype results mainly from the inability to farnesylate the Rheb G-protein, since normal cell cycle progression can be restored to the mutant by expressing a mutant form of SpRheb (SpRheb-CVIL) that can bypass farnesylation. In contrast, a farnesylation-defective mutant of SpRheb (SpRheb-SVIA) is incapable of restoring the normal cell cycle profile to the cpp1- mutant. Inhibition of SpRheb expression leads to the accumulation of cells at the G0/G1 phase of the cell cycle. This growth arrest phenotype of the sprheb- disruption can be complemented by the introduction of wild-type sprheb+. The complementation is dependent on farnesylation, since the farnesylation-defective SpRheb-SVIA mutant is incapable of complementing the sprheb-disruption. Other mutants of SpRheb, E40K and S20N, are also incapable of complementing the sprheb- disruption. Furthermore, efficient complementation can be obtained by the expression of human Rheb but not Saccharomyces cerevisiae Rheb. These findings suggest that protein farnesylation is important for cell cycle progression of S. pombe cells and that farnesylated SpRheb is critical in this process (Yang, 2001).

TOR signaling couples oxygen sensing to lifespan in C. elegans

Metazoans adapt to a low-oxygen environment (hypoxia) through activation of stress-response pathways. This study reports that transient hypoxia exposure extends lifespan in C. elegans through mitochondrial reactive oxygen species (ROS)-dependent regulation of the nutrient-sensing kinase target of rapamycin (TOR; see Drosophila Tor) and its upstream activator, RHEB-1 (see Drosophila Rheb). The increase in lifespan during hypoxia requires the intestinal GATA-type transcription factor ELT-2 (see Drosophila GATAe) downstream of TOR signaling. Using RNA sequencing (RNA-seq), this study describes an ELT-2-dependent hypoxia response that includes an intestinal glutathione S-transferase, GSTO-1, and it was uncovered that GSTO-1 is required for lifespan under hypoxia. These results indicate mitochondrial ROS-dependent TOR signaling integrates metabolic adaptations in order to confer survival under hypoxia (Schieber, 2014).

Identification and characterization of mammalian Rheb

Neuronal activity results in long term cellular changes that underlie normal brain development and synaptic plasticity. To examine the molecular basis of activity-dependent plasticity, differential cloning techniques were used to identify genes that are rapidly induced in brain neurons by synaptic activity. An inducible novel member of the Ras family of small GTP-binding proteins has been termed Rheb. rheb mRNA is rapidly and transiently induced in hippocampal granule cells by seizures and by NMDA-dependent synaptic activity in the long term potentiation paradigm. The predicted amino acid sequence of Rheb is most closely homologous to yeast Ras1 and human Rap2. The putative GTP binding regions are highly conserved. A bacterial fusion protein of Rheb binds GTP and exhibits intrinsic GTPase activity. Like Ha-Ras, the carboxylterminal sequence encodes a CAAX box that is predicted to signal post-translational farnesylation and to target Rheb to specific membranes. rheb mRNA is expressed at comparatively high levels in normal adult cortex as well as a number of peripheral tissues, including lung and intestine. In the developing brain, rheb mRNA is expressed at relatively high levels in embryonic day 19 cortical plate, and expression remains at stable levels throughout the remainder of prenatal and postnatal development. Its close homology with ras and its rapid inducibility by receptor-dependent synaptic activity suggest that rheb may play an important role in long term activity-dependent neuronal responses (Yamagata, 1994).

Dominant negative mutants of Rheb implicate Rheb in the activation of p70S6K

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

Rheb interacts with tuberous sclerosis complex 2 (TSC2) and functions in the TOR pathway

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

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

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

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

Rheb interacts with B-Raf

Rheb (Ras homolog enriched in brain) is a member of the Ras family of proteins, and is in the immediate Ras/Rap/Ral subfamily. In three different mammalian cell lines, it was found that Rheb was highly activated, to levels much higher than for Ras or Rap 1, and that Rheb's activation state was unaffected by changes in growth conditions. Rheb's high activation is not secondary to unique glycine to arginine, or glycine to serine substitutions at positions 14 and 15, corresponding to Ras residues 12 and 13, since Rheb R14G and R14G, S15G mutants have similarly high activation levels as wild type Rheb. These data are consistent with earlier work which showed that purified Rheb has similar GTPase activity as Ras, and suggest a relative intracellular deficiency of Rheb GTPase activating proteins (GAPs) compared to Rheb activators. Further evidence for relatively low intracellular GAP activity is that increased Rheb expression leads to a marked increase in Rheb activation. Rheb, like Ras and Rap1, binds B-Raf kinase, but in contrast to Ras and Rap 1, Rheb inhibits B-Raf kinase activity and prevents B-Raf-dependent activation of the transcription factor Elk-1. Thus, Rheb appears to be a unique member of the Ras/Rap/Ral subfamily, and in mammalian systems may serve to regulate B-Raf kinase activity (Im, 2002).

Rheb activates mTOR by antagonizing its endogenous inhibitor, FKBP38

The mammalian target of rapamycin, mTOR, is a central regulator of cell growth. Its activity is regulated by Rheb, a Ras-like small guanosine triphosphatase (GTPase), in response to growth factor stimulation and nutrient availability. Rheb regulates mTOR through FKBP38, a member of the FK506-binding protein (FKBP) family that is structurally related to FKBP12. FKBP38 binds to mTOR and inhibits its activity in a manner similar to that of the FKBP12-rapamycin complex. Rheb interacts directly with FKBP38 and prevents its association with mTOR in a guanosine 5'-triphosphate (GTP)-dependent manner. These findings suggest that FKBP38 is an endogenous inhibitor of mTOR, whose inhibitory activity is antagonized by Rheb in response to growth factor stimulation and nutrient availability (Bai, 2007).

V-ATPase/mTOR Signaling Regulates Megalin-Mediated Apical Endocytosis

mTOR kinase is a master growth regulator that can be stimulated by multiple signals, including amino acids and the lysosomal small GTPase Rheb. Recent studies have proposed an important role for the V-ATPase in the sensing of amino acids in the lysosomal lumen. Using the Drosophila wing as a model epithelium, this study showa that the V-ATPase is required for Rheb-dependent epithelial growth. A positive feedback loop for the control of apical protein uptake was uncovered that depends on V-ATPase/mTOR signaling. This feedback loop includes Rheb-dependent transcriptional regulation of the multiligand receptor Megalin, which itself is required for Rheb-induced endocytosis. In addition, evidence is provided that long-term mTOR inhibition with rapamycin in mice causes reduction of Megalin levels and proteinuria in the proximal tubular epithelium of the kidney. Thus, these findings unravel a homeostatic mechanism that allows epithelial cells to promote protein uptake under normal conditions and to prevent uptake in lysosomal stress conditions (Gleixner, 2014).

Aberrant Rheb-mediated mTORC1 activation and Pten haploinsufficiency are cooperative oncogenic events

The mammalian target of rapamycin (mTOR) represents a critical signaling crossroad where pathways commonly disrupted in cancer converge. Rheb GTPase, the upstream activator of the mTOR complex 1 (mTORC1) is amplified in human prostate cancers. Rheb overexpression promotes hyperplasia and a low-grade neoplastic phenotype in the mouse prostate while eliciting a concomitant senescence response and a negative feedback loop limiting Akt activation. Importantly, Pten haploinsufficiency cooperates with Rheb overexpression to markedly promote prostate tumorigenesis. It is concluded that Rheb acts as a proto-oncogene in the appropriate genetic milieu and signaling context (Nardella, 2008).

Tumorigenic activity and therapeutic inhibition of Rheb GTPase

The AKT-mTOR pathway harbors several known and putative oncogenes and tumor suppressors. In a phenotypic screen for lymphomagenesis, candidate genes acting upstream of and downstream from mTOR were tested in vivo. Rheb, a proximal activator of mTORC1, can produce rapid development of aggressive and drug-resistant lymphomas. Rheb causes mTORC1-dependent effects on apoptosis, senescence, and treatment responses that resemble those of Akt. Moreover, Rheb activity toward mTORC1 requires farnesylation and is readily blocked by a pharmacological inhibitor of farnesyltransferase (FTI). In Pten-deficient tumor cells, inhibition of Rheb by FTI is responsible for the drug's anti-tumor effects, such that a farnesylation-independent mutant of Rheb renders these tumors resistant to FTI therapy. Notably, RHEB is highly expressed in some human lymphomas, resulting in mTORC1 activation and increased sensitivity to rapamycin and FTI. Downstream from mTOR, translation initiation factors were examined that have been implicated in transformation in vitro. Of these, only eIF4E was able to enhance lymphomagenesis in vivo. In summary, the Rheb GTPase is an oncogenic activity upstream of mTORC1 and eIF4E and a direct therapeutic target of farnesyltransferase inhibitors in cancer (Mavrakis, 2008).

REFERENCES

Search PubMed for articles about Drosophila Rheb

Bai, X., et al. (2007). Rheb activates mTOR by antagonizing its endogenous inhibitor, FKBP38. Science 318(5852): 977-80. PubMed citation: 17991864

Brown, H. L., Kaun, K. R. and Edgar, B. A. (2012). The small GTPase Rheb affects central brain neuronal morphology and memory formation in Drosophila. PLoS One 7(9): e44888. PubMed Citation: 23028662

Castro, A. F., Rebhun, J. F., Clark, G. J. and Quilliam, L. A. (2003). Rheb binds tuberous sclerosis complex 2 (TSC2) and promotes S6 kinase activation in a rapamycin- and farnesylation-dependent manner. J Biol Chem. 278(35): 32493-6. 12842888

Chell, J. M. and Brand, A. H. (2010). Nutrition-responsive glia control exit of neural stem cells from quiescence. Cell 143: 1161-1173. PubMed Citation: 21183078

Frias, M. A., Mukhopadhyay, S., Lehman, E., Walasek, A., Utter, M., Menon, D. and Foster, D. A. (2019). Phosphatidic acid drives mTORC lysosomal translocation in the absence of amino acids. J Biol Chem 295(1):263-274. PubMed ID: 31767684

Garami, A., et al. (2003). Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol. Cell 11(6): 1457-66. 12820960

Gleixner, E. M., Canaud, G., Hermle, T., Guida, M. C., Kretz, O., Helmstadter, M., Huber, T. B., Eimer, S., Terzi, F. and Simons, M. (2014). V-ATPase/mTOR Signaling Regulates Megalin-Mediated Apical Endocytosis. Cell Rep 8(1):10-9. PubMed ID: 24953654

Haruta, T. et al. (2000). A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1. Mol. Endocrinol. 14: 783-794. 10847581

Hsu, Y.-C., Chern, J. J., Cai, Y., Liu, M. and Choi, K.-W. (2007). Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase. Nature 445: 785-788. PubMed Citation: 17301792

Im, E., et al. (2002). Rheb is in a high activation state and inhibits B-Raf kinase in mammalian cells. Oncogene 21(41): 6356-65. 12214276

Inoki, K., Li, Y., Xu, T. and Guan, K. L. (2003). Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 17(15): 1829-34. 12869586

Knox, S., et al. (2007). Mechanisms of TSC-mediated control of synapse assembly and axon guidance. PLoS One. 2(4): e375. PubMed Citation: 17440611

Luo, J., Liu, Y. and Nassel, D. R. (2013). Insulin/IGF-Regulated Size Scaling of Neuroendocrine Cells Expressing the bHLH Transcription Factor Dimmed in Drosophila. PLoS Genet 9: e1004052. PubMed ID: 24385933

Mach, K. E., Furge, K. A. and Albright, C. F. (2000). Loss of Rhb1, a Rheb-related GTPase in fission yeast, causes growth arrest with a terminal phenotype similar to that caused by nitrogen starvation. Genetics 155: 611-622. 10835385

Mavrakis, K. J., et al. (2008). Tumorigenic activity and therapeutic inhibition of Rheb GTPase. Genes Dev. 22(16): 2178-88. PubMed Citation: 18708578

Montagne, J. et al. (1999). Drosophila S6 kinase: a regulator of cell size. Science 285: 2126-2129. 10497130

Nardella, C., et al. (2008). Aberrant Rheb-mediated mTORC1 activation and Pten haploinsufficiency are cooperative oncogenic events. Genes Dev. 2008 22(16): 2172-7. PubMed Citation: 18708577

Nie, Y., Li, Q., Amcheslavsky, A., Duhart, J. C., Veraksa, A., Stocker, H., Raftery, L. A. and Ip, Y. T. (2015). Bunched and Madm function downstream of Tuberous Sclerosis Complex to regulate the growth of intestinal stem cells in Drosophila. Stem Cell Rev 11: 813-825. PubMed ID: 26323255

Oldham, S., et al. (2000). Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev. 14: 2689-2694. 11069885

Panepinto, J. C., et al. (2002). Expression of the Aspergillus fumigatus rheb homolog, rhbA, is induced by nitrogen starvation. Fungal Genet. Biol. 36: 207-214. 12135576

Patel, P. H., et al. (2003). Drosophila Rheb GTPase is required for cell cycle progression and cell growth. J. Cell Sci. 116(Pt 17): 3601-10. 12893813

Radimerski, T. et al. (2002). dS6K regulated cell growth is dPKB/dPI3K independent, but requires dPDK1. Nature Cell Biol. 4: 251-255. 11862217

Saucedo, L. J., et al. (2003). Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nat. Cell Biol. 5(6):566-71. 12766776

Schieber, M. and Chandel, N. S. (2014). TOR signaling couples oxygen sensing to lifespan in C. elegans. Cell Rep 9: 9-15. PubMed ID: 25284791

Sousa-Nunes, R., Yee, L. L. and Gould, A. P. (2011). Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila. Nature 471(7339): 508-12. PubMed Citation: 21346761

Stocker, H., et al. (2003). Rheb is an essential regulator of S6K in controlling cell growth in Drosophila. Nat. Cell Biol. 5(6):559-65. 12766775

Tabancay, A. P., et al. (2003). Identification of dominant negative mutants of Rheb GTPase and their use to implicate the involvement of human Rheb in the activation of p70S6K. J. Biol. Chem. 2003. 12869548

Tee, A. R., et al. (2003). Tuberous sclerosis complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb. Curr. Biol. 13(15): 1259-68. 12906785

Tsokanos, F. F., Albert, M. A., Demetriades, C., Spirohn, K., Boutros, M. and Teleman, A. A. (2016). eIF4A inactivates TORC1 in response to amino acid starvation. EMBO J 35(10):1058-76. PubMed ID: 26988032

Urano, J., et al. (2000). The Saccharomyces cerevisiae Rheb G-protein is involved in regulating canavanine resistance and arginine uptake. J. Biol. Chem. 275(15): 198-206. 10753927

Yamagata, K. et al. (1994). rheb, a growth factor- and synaptic activity-regulated gene, encodes a novel Ras-related protein. J. Biol. Chem. 269: 16333-16339. 8206940

Yang, G., Humphrey, S. J., Murashige, D. S., Francis, D., Wang, Q. P., Cooke, K. C., Neely, G. and James, D. E. (2018). RagC phosphorylation autoregulates mTOR complex 1. EMBO J. PubMed ID: 30552228

Yang, W., Tabancay, A. P., Jr., Urano, J. and Tamanoi, F. (2001). Failure to farnesylate Rheb protein contributes to the enrichment of G0/G1 phase cells in the Schizosaccharomyces pombe farnesyltransferase mutant. Mol. Microbiol. 41: 1339-1347. 11580838

Zhang, H., et al. (2000). Regulation of cellular growth by the Drosophila target of rapamycin dTOR. Genes Dev. 14: 2712-2724. 11069888

Zhang, Y., et al. (2003). Rheb is a direct target of the tuberous sclerosis tumor suppressor proteins. Nat. Cell Biol. 5(6): 578-81. 12771962

date revised: 5 May 2022

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