Rheb homologs have been identified in the budding yeast Saccharomyces cerevisiae (ScRheb) as well as in Schizosaccharomyces pombe, Drosophila melanogaster, zebrafish, and Ciona intestinalis. These proteins define a new class of G-proteins based on (1) their overall sequence similarity, (2) high conservation of their effector domain sequence, (3) presence of a unique arginine in their G1 box, and (4) presence of a conserved CAAX farnesylation motif. Characterization of an S. cerevisiae strain deficient in ScRheb shows that it is hypersensitive to growth inhibitory effects of canavanine and thialysine, which are analogues of arginine and lysine, respectively. Accordingly, the uptake of arginine and lysine is increased in the ScRheb-deficient strain. This increased arginine uptake requires the arginine-specific permease Can1p. The function of ScRheb is dependent on having an intact effector domain since mutations in the effector domain of ScRheb are incapable of complementing canavanine hypersensitivity of scrheb disruptant cells. Furthermore, the conserved arginine in the G1 box plays a role in the activity of ScRheb, since a mutation of this arginine to glycine significantly reduces the ability of ScRheb to complement canavanine hypersensitivity of ScRheb-deficient yeast. Finally, a mutation in the C-terminal CAAX farnesylation motif results in a loss of ScRheb function. This result, in combination with the finding that ScRheb is farnesylated, suggests that farnesylation plays a key role in ScRheb function. These findings assign the regulation of arginine and lysine uptake as the first physiological function for this new farnesylated Ras superfamily G-protein (Urano, 2000).
A fission yeast homolog of mammalian Rheb, which has been designated Rhb1, was identified by genome sequencing. rhb1- cells arrest cell growth and division with a terminal phenotype similar to that of nitrogen-starved cells. In particular, cells depleted of Rhb1 arrest as small, round cells with 1N DNA content, arrest more quickly in low-nitrogen medium, and induce expression of fnx1 and mei2 mRNAs, two mRNAs that were normally induced by nitrogen starvation. Since mammalian Rheb binds and may regulate Raf-1, a Ras effector, functional overlap between Ras1 and Rhb1 was tested in fission yeast. This analysis shows that Ras1 overexpression does not suppress rhb1- mutant phenotypes, Rhb1 overexpression does not suppress ras1- mutant phenotypes, and ras1- rhb1- double mutants have phenotypes equal to the sum of the corresponding single-mutant phenotypes. Hence, there is no evidence for overlapping functions between Ras1 and Rhb1. On the basis of this study, it is hypothesized that Rhb1 negatively regulates entry into stationary phase when extracellular nitrogen levels are adequate for growth. If this hypothesis is correct, then Rhb1 and Ras1 regulate alternative responses to limiting nutrients (Mach, 2000).
A gene encoding a ras protein with homology to the rheb family was cloned from Aspergillus fumigatus. Although conserved ras domains are present, the predicted RhbA protein sequence deviates from the ras consensus in a manner that is characteristic of rheb proteins. The invariant Gly-Gly in the first GTP-binding domain of ras proteins is replaced by Arg-Ser in RhbA, and a conserved Asp in the effector region of ras proteins is replaced by Asn in RhbA. The rhbA mRNA is detected throughout the A. fumigatus asexual developmental cycle, and accumulates over 5-fold in response to nitrogen starvation. The rhbA gene is able to complement the canavanine hypersensitivity of Saccharomyces cerevisiae Deltarhb1 mutants, suggesting that the two proteins share overlapping function (Panepinto, 2002).
Protein farnesylation is important for a number of physiological processes, including proliferation and cell morphology. The Schizosaccharomyces pombe mutant, cpp1-, defective in farnesylation, exhibits distinct phenotypes, including morphological changes and sensitivity to the arginine analogue, canavanine. A novel phenotype of this mutant is reported -- enrichment of G0/G1 phase cells. This phenotype results mainly from the inability to farnesylate the Rheb G-protein, since normal cell cycle progression can be restored to the mutant by expressing a mutant form of SpRheb (SpRheb-CVIL) that can bypass farnesylation. In contrast, a farnesylation-defective mutant of SpRheb (SpRheb-SVIA) is incapable of restoring the normal cell cycle profile to the cpp1- mutant. Inhibition of SpRheb expression leads to the accumulation of cells at the G0/G1 phase of the cell cycle. This growth arrest phenotype of the sprheb- disruption can be complemented by the introduction of wild-type sprheb+. The complementation is dependent on farnesylation, since the farnesylation-defective SpRheb-SVIA mutant is incapable of complementing the sprheb-disruption. Other mutants of SpRheb, E40K and S20N, are also incapable of complementing the sprheb- disruption. Furthermore, efficient complementation can be obtained by the expression of human Rheb but not Saccharomyces cerevisiae Rheb. These findings suggest that protein farnesylation is important for cell cycle progression of S. pombe cells and that farnesylated SpRheb is critical in this process (Yang, 2001).
Neuronal activity results in long term cellular changes that underlie normal brain development and synaptic plasticity. To examine the molecular basis of activity-dependent plasticity, differential cloning techniques were used to identify genes that are rapidly induced in brain neurons by synaptic activity. An inducible novel member of the Ras family of small GTP-binding proteins has been termed Rheb. rheb mRNA is rapidly and transiently induced in hippocampal granule cells by seizures and by NMDA-dependent synaptic activity in the long term potentiation paradigm. The predicted amino acid sequence of Rheb is most closely homologous to yeast Ras1 and human Rap2. The putative GTP binding regions are highly conserved. A bacterial fusion protein of Rheb binds GTP and exhibits intrinsic GTPase activity. Like Ha-Ras, the carboxylterminal sequence encodes a CAAX box that is predicted to signal post-translational farnesylation and to target Rheb to specific membranes. rheb mRNA is expressed at comparatively high levels in normal adult cortex as well as a number of peripheral tissues, including lung and intestine. In the developing brain, rheb mRNA is expressed at relatively high levels in embryonic day 19 cortical plate, and expression remains at stable levels throughout the remainder of prenatal and postnatal development. Its close homology with ras and its rapid inducibility by receptor-dependent synaptic activity suggest that rheb may play an important role in long term activity-dependent neuronal responses (Yamagata, 1994).
Dominant negative mutants of S. pombe Rheb (SpRheb) are reported. Screens of a randomly mutagenized SpRheb library yielded a mutant, SpRhebD60V, whose expression in S. pombe results in growth inhibition, G1 arrest, and induction of fnx1+, a gene whose expression is induced by the disruption of Rheb. Alteration of the Asp-60 residue to all possible amino acids by site-directed mutagenesis led to the identification of two particularly strong dominant negative mutants, D60I and D60K. Characterization of these dominant negative mutant proteins revealed that D60V and D60I exhibit preferential binding of GDP, while D60K lost the ability to bind both GTP and GDP. A possible use of the dominant negative mutants in the study of mammalian Rheb was explored by introducing dominant negative mutations into human Rheb. Transient expression of the wild type Rheb1 or Rheb2 causes activation of p70S6K, while expression of Rheb1D60K mutant results in inhibition of basal level activity of p70S6K. In addition, Rheb1D60K and Rheb1D60V mutants block nutrient- or serum-induced activation of p70S6K. This provides critical evidence that Rheb plays a role in the mTOR/S6K pathway in mammalian cells (Tabancay, 2003).
Tumor suppressor genes evolved as negative effectors of mitogen and nutrient signaling pathways, such that mutations in these genes can lead to pathological states of growth. Tuberous sclerosis (TSC) is a potentially devastating disease associated with mutations in two tumor suppressor genes, TSC1 and 2, that function as a complex to suppress signaling in the mTOR/S6K/4E-BP pathway. However, the inhibitory target of TSC1/2 and the mechanism by which it acts are unknown. Evidence is provided that TSC1/2 is a GAP for the small GTPase Rheb and that insulin-mediated Rheb activation is PI3K dependent. Moreover, Rheb overexpression induces S6K1 phosphorylation and inhibits PKB phosphorylation, as do loss-of-function mutations in TSC1/2, but contrary to earlier reports Rheb has no effect on MAPK phosphorylation. Finally, coexpression of a human TSC2 cDNA harboring a disease-associated point mutation in the GAP domain, failed to stimulate Rheb GTPase activity or block Rheb activation of S6K1 (Garami, 2003).
The tuberous sclerosis complex 2 (TSC2) tumor suppressor gene product is a negative regulator of protein synthesis upstream of the mTOR and ribosomal S6 kinases. Because of the homology of TSC2 with GTPase-activating proteins for Rap1, whether a Ras/Rap-related GTPase might be involved in this process was examined. TSC2 was found to bind to Rheb-GTP in vitro and to reduce Rheb GTP levels in vivo. Over-expression of Rheb but not Rap1 promotes the activation of S6 kinase in a rapamycin-dependent manner, suggesting that Rheb acts upstream of mTOR. The ability of Rheb to induce S6 phosphorylation is also inhibited by a farnesyl transferase inhibitor, suggesting that Rheb may be responsible for the Ras-independent anti-neoplastic properties of this drug (Castro, 2003).
Tuberous sclerosis complex is a genetic disease caused by mutation in either TSC1 or TSC2. The TSC1 and TSC2 gene products form a functional complex and inhibit phosphorylation of S6K and 4EBP1. These functions of TSC1/TSC2 are likely mediated by mTOR. TSC2 is a GTPase-activating protein (GAP) toward Rheb, a Ras family GTPase. Rheb stimulates phosphorylation of S6K and 4EBP1. This function of Rheb is blocked by rapamycin and dominant-negative mTOR. Rheb stimulates the phosphorylation of mTOR and plays an essential role in regulation of S6K and 4EBP1 in response to nutrients and cellular energy status. These data demonstrate that Rheb acts downstream of TSC1/TSC2 and upstream of mTOR to regulate cell growth (Inoki, 2003).
Tuberous Sclerosis Complex is a genetic disorder that occurs through the loss of heterozygosity of either TSC1 or TSC2, which encode Hamartin or Tuberin, respectively. Tuberin and Hamartin form a tumor suppressor heterodimer that inhibits the mammalian target of rapamycin (mTOR) nutrient signaling input, but how this occurs is unclear. The small G protein Rheb (Ras homolog enriched in brain) is shown to be a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, membrane localization of Rheb is shown to be important for its biological activity because a farnesylation-defective mutant of Rheb stimulates S6K1 activation less efficiently. Thus, Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells (Tee, 2003).
Rheb (Ras homolog enriched in brain) is a member of the Ras family of proteins, and is in the immediate Ras/Rap/Ral subfamily. In three different mammalian cell lines, it was found that Rheb was highly activated, to levels much higher than for Ras or Rap 1, and that Rheb's activation state was unaffected by changes in growth conditions. Rheb's high activation is not secondary to unique glycine to arginine, or glycine to serine substitutions at positions 14 and 15, corresponding to Ras residues 12 and 13, since Rheb R14G and R14G, S15G mutants have similarly high activation levels as wild type Rheb. These data are consistent with earlier work which showed that purified Rheb has similar GTPase activity as Ras, and suggest a relative intracellular deficiency of Rheb GTPase activating proteins (GAPs) compared to Rheb activators. Further evidence for relatively low intracellular GAP activity is that increased Rheb expression leads to a marked increase in Rheb activation. Rheb, like Ras and Rap1, binds B-Raf kinase, but in contrast to Ras and Rap 1, Rheb inhibits B-Raf kinase activity and prevents B-Raf-dependent activation of the transcription factor Elk-1. Thus, Rheb appears to be a unique member of the Ras/Rap/Ral subfamily, and in mammalian systems may serve to regulate B-Raf kinase activity (Im, 2002).
The mammalian target of rapamycin, mTOR, is a central regulator of cell growth. Its activity is regulated by Rheb, a Ras-like small guanosine triphosphatase (GTPase), in response to growth factor stimulation and nutrient availability. Rheb regulates mTOR through FKBP38, a member of the FK506-binding protein (FKBP) family that is structurally related to FKBP12. FKBP38 binds to mTOR and inhibits its activity in a manner similar to that of the FKBP12-rapamycin complex. Rheb interacts directly with FKBP38 and prevents its association with mTOR in a guanosine 5'-triphosphate (GTP)-dependent manner. These findings suggest that FKBP38 is an endogenous inhibitor of mTOR, whose inhibitory activity is antagonized by Rheb in response to growth factor stimulation and nutrient availability (Bai, 2007).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.