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).
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date revised: 1 August 2008
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