E129,A131 , which causes a morphological abnormality, was expressed. These results provide a direct molecular basis for the action of Rho3 on exocytosis and the actin cytoskeleton.

Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully reproduced in vitro by using mTORC1 immunoprecipitated by the use of anti-raptor antibody from mammalian cells starved for nutrients. The low in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is dramatically increased by the addition of recombinant Rheb. On the other hand, the addition of Rheb does not activate mTORC2 immunoprecipitated from mammalian cells by the use of anti-rictor antibody. The activation of mTORC1 is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42 did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition, the activation is dependent on the presence of bound GTP. We also find that the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a recently proposed mediator of Rheb action, appears not to be involved in the Rheb-dependent activation of mTORC1 in vitro, because the preparation of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of Rheb results in a significant increase of binding of the substrate protein 4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated by Rheb. Rheb does not induce autophosphorylation of mTOR. These results suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to regulate mTORC1 activation.

No abstract

Precise body and organ sizes in the adult animal are ensured by a range of signaling pathways. In a screen to identify genes affecting hindgut morphogenesis in Drosophila, we identified a P-element insertion in dRheb, a novel, highly conserved member of the Ras superfamily of G-proteins. Overexpression of dRheb in the developing fly (using the GAL4:UAS system) causes dramatic overgrowth of multiple tissues: in the wing,this is due to an increase in cell size; in cultured cells, dRheboverexpression results in accumulation of cells in S phase and an increase in cell size. Using a loss-of-function mutation we show that dRheb is required in the whole organism for viability (growth) and for the growth of individual cells. Inhibition of dRheb activity in cultured cells results in their arrest in G1 and a reduction in size. These data demonstrate that dRheb is required for both cell growth (increase in mass) and cell cycle progression; one explanation for this dual role would be that dRheb promotes cell cycle progression by affecting cell growth. Consistent with this interpretation, we find that flies with reduced dRheb activity are hypersensitive to rapamycin, an inhibitor of the growth regulator TOR. In cultured cells, the effect of overexpressing dRheb was blocked by the addition of rapamycin. These results imply that dRheb is involved in TOR signaling.

SummaryRheb GTPase is a key player in the control of growth, cell cycle and nutrient uptake that is conserved from yeast to humans. To further our understanding of the Rheb pathway, we sought to identify hyperactivating mutations in the Schizosaccharomyces pombe Rheb, Rhb1. Hyperactive forms of Rhb1 were found to result from single amino acid changes at valine-17, serine-21, lysine-120 or asparagine-153. Expression of these mutants confers resistance to canavanine and thialysine, phenotypes which are similar to phenotypes exhibited by cells lacking the Tsc1/Tsc2 complex that negatively regulates Rhb1. The thialysine-resistant phenotype of the hyperactive Rhb1 mutants is suppressed by a second mutation in the effector domain. Purified mutant proteins exhibit dramatically decreased binding of GDP, while their GTP binding is not drastically affected. In addition, some of the mutant proteins show significantly decreased GTPase activities. Thus the hyperactivating mutations are expected to result in an increase in the GTP-bound/ GDP-bound ratio of Rhb1. By using the hyperactive mutant, Rhb1 K120R , we have been able to demonstrate that Rhb1 interacts with Tor2, one of the two S. pombe TOR (Target of Rapamycin) proteins. These fission yeast results provide the first evidence for a GTPdependent association of Rheb with Tor.