Specific activation of mTORC1 by Rheb G-protein in vitro involves enhanced recruitment of its substrate protein.
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.
Sato
Tatsuhiro
T
Department of Microbiology, Immunology & Molecular Genetics, Molecular Biology Institute, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095, USA.
Nakashima
Akio
A
Guo
Lea
L
Tamanoi
Fuyuhiko
F
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D000076222:Mechanistic Target of Rapamycin Complex 1; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D000076205:Ras Homolog Enriched in Brain Protein; D000076223:Regulatory-Associated Protein of mTOR; D058570:TOR Serine-Threonine Kinases; D022021:Tacrolimus Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C493245:CRTC2 protein, human; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; C471205:FKBP8 protein, human; D046912:Multiprotein Complexes; D009479:Neuropeptides; D010750:Phosphoproteins; D011506:Proteins; C490211:RHEB protein, human; D034741:RNA, Small Interfering; C463659:RPTOR protein, human; D000076205:Ras Homolog Enriched in Brain Protein; D000076223:Regulatory-Associated Protein of mTOR; D014157:Transcription Factors; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D019204:GTP-Binding Proteins; D020559:Monomeric GTP-Binding Proteins; D022021:Tacrolimus Binding Proteins