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).
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, rhebPDelta1, 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, rhebPDelta2, 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 rhebPDelta1/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 rhebPDelta1/PDelta2 animals. With or without heat-shock, addition of these transgenes partially rescues the growth defect, enabling the rhebPDelta1/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 rhebPDelta1/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 (Ras1G12V) in Drosophila, Rheb is a far more potent promoter of growth, but affects none of the corresponding alterations of cell fate caused by Ras1G12V 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 RhebAV4 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 RhebAV4 chromosome results in a reversion to viability, confirming that the RhebAV4 lethal phenotype is due to disruption of Rheb activity resulting from the P-element insertion. To determine the lethal phase of RhebAV4 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 RhebAV4 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 RhebAV4 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).
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).
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 (SHIDN). 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 Ilp1, 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 SHIDN 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).
Bai, X., et al. (2007). Rheb activates mTOR by antagonizing its endogenous inhibitor, FKBP38. Science 318(5852): 977-80. PubMed citation: 17991864
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
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
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
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
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
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
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, 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 August 2011
Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.