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).

GENE STRUCTURE

cDNA clone length - 1021

Bases in 5' UTR - 98

Exons - 5

Bases in 3' UTR - 374

PROTEIN STRUCTURE

Amino Acids - 182

Structural Domains

Drosophila Rheb gene is predicted to encode a 182 amino acid protein that contains G-boxes characteristic of the Ras superfamily of G-proteins. Yeast and fly each have a single Rheb gene; the most similar human gene is hRheb1, which maps to chromosome 7. An additional, more divergent human Rheb gene, designated hRheb2, maps to chromosome 12. The overall sequence identity between Drosophila and S. pombe Rheb is 51%, and that between Drosophila and human Rheb1 is 63%. Consistent with this high sequence conservation, dRheb, hRheb1 and hRheb2 can replace the function of S. pombe Rheb (Patel, 2003).

Sequence alignments indicate that CG1081 is the Drosophila homolog of rheb, a member of the Ras superfamily of GTP-binding proteins. Similar to its mammalian and yeast homologs, Drosophila Rheb encodes a carboxy-terminal CAAX farnesylation motif and contains highly conserved arginine and serine residues (amino acids 15 and 16, respectively); mutations in the homologous positions of Ras (amino acids 12 and 13, respectively) are oncogenic and confer GTPase insensitivity and constitutive activity (Saucedo, 2003).

date revised: 10 September 2003

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