Specific Activation of mTORC1 by Rheb G-protein in Vitro Involves Enhanced Recruitment of Its Substrate Protein

Abstract

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. 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. Rheb defines a unique member of the Ras superfamily G-proteins (1Aspuria P.J. Tamanoi F. Cell Signal. 2004; 16: 1105-1112Crossref PubMed Scopus (167) Google Scholar). We have shown that Rheb proteins are conserved and are found from yeast to human (2Patel P.H. Thapar N. Guo L. Martinez M. Maris J. Gau C.L. Lengyel J.A. Tamanoi F. J. Cell Sci. 2003; 116: 3601-3610Crossref PubMed Scopus (132) Google Scholar). Although yeast and fruit fly have one Rheb, mouse and human have two Rheb proteins termed Rheb1 (or simply Rheb) and Rheb2 (RhebL1) (2Patel P.H. Thapar N. Guo L. Martinez M. Maris J. Gau C.L. Lengyel J.A. Tamanoi F. J. Cell Sci. 2003; 116: 3601-3610Crossref PubMed Scopus (132) Google Scholar). Structurally, these proteins contain G1-G5 boxes, short stretches of amino acids that define the function of the Ras superfamily G-proteins including guanine nucleotide binding (1Aspuria P.J. Tamanoi F. Cell Signal. 2004; 16: 1105-1112Crossref PubMed Scopus (167) Google Scholar, 3Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1418) Google Scholar, 4Wennerberg K. Rossman K.L. Der C.J. J. Cell Sci. 2005; 118: 843-846Crossref PubMed Scopus (1065) Google Scholar). Rheb proteins have a conserved arginine at residue 15 that corresponds to residue 12 of Ras (1Aspuria P.J. Tamanoi F. Cell Signal. 2004; 16: 1105-1112Crossref PubMed Scopus (167) Google Scholar). The effector domain required for the binding with downstream effectors encompasses the G2 box and its adjacent sequences (1Aspuria P.J. Tamanoi F. Cell Signal. 2004; 16: 1105-1112Crossref PubMed Scopus (167) Google Scholar, 5McCormick F. Wittinghofer A. Curr. Opin. Biotechnol. 1996; 7: 449-456Crossref PubMed Scopus (78) Google Scholar). Structural analysis by x-ray crystallography further shows that the effector domain is exposed to solvent, is located close to the phosphates of GTP especially at residues 35–38, and undergoes conformational change during GTP/GDP exchange (6Yu Y. Li S. Xu X. Li Y. Guan K. Arnold E. Ding J. J. Biol. Chem. 2005; 280: 17093-17100Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In addition, all Rheb proteins end with the CAAX (C is cysteine, A is an aliphatic amino acid, and X is the C-terminal amino acid) motif that signals farnesylation. In fact, we as well as others have shown that these proteins are farnesylated (7Clark G.J. Kinch M.S. Rogers-Graham K. Sebti S.M. Hamilton A.D. Der C.J. J. Biol. Chem. 1997; 272: 10608-10615Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 8Urano J. Tabancay A.P. Yang W. Tamanoi F. J. Biol. Chem. 2000; 275: 11198-11206Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 9Yang W. Tabancay Jr., A.P. Urano J. Tamanoi F. Mol. Microbiol. 2001; 41: 1339-1347Crossref PubMed Scopus (61) Google Scholar). Rheb plays critical roles in the TSC/Rheb/mTOR signaling, a signaling pathway that plays central roles in regulating protein synthesis and growth in response to nutrient, energy, and growth conditions (10Reiling J.H. Sabatini D.M. Oncogene. 2006; 25: 6373-6383Crossref PubMed Scopus (303) Google Scholar, 11Corradetti M.N. Guan K.L. Oncogene. 2006; 25: 6347-6360Crossref PubMed Scopus (337) Google Scholar, 12Avruch J. Hara K. Lin Y. Liu M. Long X. Ortiz-Vega S. Yonezawa K. Oncogene. 2006; 25: 6361-6372Crossref PubMed Scopus (269) Google Scholar, 13Huang J. Manning B.D. Biochem. J. 2008; 412: 179-190Crossref PubMed Scopus (964) Google Scholar, 14Tabancay Jr., A.P. Gau C.L. Machado I.M. Uhlmann E.J. Gutmann Guo L. Tamanoi F. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Rheb is by a that as a protein for Rheb A. M. M. E. Mol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Manning B.D. J. Curr. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Li Y. Xu Guan K.L. 2003; PubMed Scopus Google Scholar, Y. X. Cell Biol. 2003; PubMed Scopus Google Scholar, G.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). that the domain of defines the domain for the of Rheb A. Wittinghofer A. J. 2008; PubMed Scopus Google Scholar). in the to the of at of the as well as 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. The Scholar). of Rheb results in activation of in the of A. M. M. E. Mol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Manning B.D. J. Curr. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). mTORC1 and have D.M. 2006; PubMed Scopus Google Scholar, S. M.N. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). mTORC1 is involved in protein synthesis activation by and mTORC2 is involved in the of in response to that Rheb is involved in the activation of mTORC1 not mTORC2 K. E. Guan K.L. Sci. S. A. 2006; PubMed Scopus Google Scholar). Although Rheb is involved in the activation of the of activation not We as well as others have a that the of Rheb with the TOR J. Guo L. P.J. Tabancay Jr., A.P. J. Tamanoi F. Mol. Microbiol. 2005; PubMed Scopus Google Scholar, X. Lin Y. Ortiz-Vega S. Yonezawa K. J. Curr. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, G.J. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). Rheb activation of kinase activity using immunoprecipitated mTORC1 Y. E. Sabatini D.M. Mol. 25: Full Text Full Text PDF PubMed Scopus Google Scholar). Rheb shown to with X. Lin Y. Ortiz-Vega S. Yonezawa K. J. Curr. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J. 2005; PubMed Scopus Google and this of Rheb with the kinase domain of X. Lin Y. Ortiz-Vega S. Yonezawa K. J. Curr. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). this is a and is not dependent on the presence of GTP bound to Rheb X. Lin Y. Ortiz-Vega S. Yonezawa K. J. Curr. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, G.J. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). a that FKBP38 protein Biochem. 2004; PubMed Scopus Google the activation of mTORC1 by Rheb proposed X. Guo S. Y. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, X. Liu A. X. Liu Y. Y. Science. PubMed Scopus Google Scholar). In this FKBP38 and and this is by the binding of Rheb to this X. B.D. Liu A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). further Rheb activation of we have an in vitro that activation of mTORC1 by the addition of recombinant Rheb. We mTORC1 immunoprecipitated from cells using anti-raptor antibody and have shown that its kinase activity against 4E-BP1 is dramatically increased by the addition of recombinant Rheb. the activation of mTORC1 is specific to Rheb and is dependent on the presence of bound GTP as well as an effector FKBP38 is not in preparation and further that FKBP38 is not an for the activation of mTORC1 by Rheb. that Rheb the binding of a substrate 4E-BP1 with mTORC1 the kinase activity of mTOR. Cell and cells in with and at and the activity of cells in with and in and for of by using to the of by using to the antibody by against and from Cell antibody from antibody from Cell and antibody from Cell and and from 4E-BP1 and from and human substrate from and from and acids by as J. Y. M. Tamanoi F. Sci. S. A. PubMed Scopus Google Scholar). and Rheb by Rheb by of W. Tabancay Jr., A.P. Urano J. Tamanoi F. Mol. Microbiol. 2001; 41: 1339-1347Crossref PubMed Scopus (61) Google the of in the of FKBP38 and as a from such as Rheb1 and Rheb2 and the proteins for with The cells in the by The and to the with A with The proteins using with and in at guanine nucleotide binding on Rheb the proteins in A with at for GTP binding on other Ras of A. the proteins with and at nucleotide binding in binding of of protein and binding at for The binding to a and with using a with a and in of mTORC1 and in vitro kinase as in A. Tamanoi F. 2008; PubMed Scopus Google the cells with and with from Science. The from the at for at immunoprecipitated using and protein protein the FKBP38 bound to mTORC1, the cells with the and the of to The with the in vitro kinase the further with The with of recombinant 4E-BP1 for mTORC1 for mTORC2 in kinase and for at the of proteins such as Rheb on the proteins the activation of by of in the kinase of The kinase by the addition of and at for The proteins by and a Rheb and Rheb2 by with in proteins by 4E-BP1 cells with the as The from the at for at with anti-raptor antibody and protein at for The with the and with The with recombinant 4E-BP1 in the and at for The with the and at for in 4E-BP1 bound to mTORC1 by mTORC1, the proteins in at for by using The proteins by to the Rheb mTORC1 mTORC2 in the in of the that Rheb (1Aspuria P.J. Tamanoi F. Cell Signal. 2004; 16: 1105-1112Crossref PubMed Scopus (167) Google Scholar, 9Yang W. Tabancay Jr., A.P. Urano J. Tamanoi F. Mol. Microbiol. 2001; 41: 1339-1347Crossref PubMed Scopus (61) Google Scholar, J.H. Sabatini D.M. Oncogene. 2006; 25: 6373-6383Crossref PubMed Scopus (303) Google Scholar, 11Corradetti M.N. Guan K.L. Oncogene. 2006; 25: 6347-6360Crossref PubMed Scopus (337) Google Scholar, 12Avruch J. Hara K. Lin Y. Liu M. Long X. Ortiz-Vega S. Yonezawa K. Oncogene. 2006; 25: 6361-6372Crossref PubMed Scopus (269) Google Scholar, 13Huang J. Manning B.D. Biochem. J. 2008; 412: 179-190Crossref PubMed Scopus (964) Google Scholar, 14Tabancay Jr., A.P. Gau C.L. Machado I.M. Uhlmann E.J. Gutmann Guo L. Tamanoi F. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. M. E. Mol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Manning B.D. J. Curr. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Li Y. Xu Guan K.L. 2003; PubMed Scopus Google Scholar, Y. X. Cell Biol. 2003; PubMed Scopus Google Scholar). the by Rheb not We have in vitro in a of to the of Rheb on shows the for this is cells starved for to of are in the mTORC1 that raptor as an protein and mTORC2 that can be by using against the specific mTORC1 and mTORC2 and the kinase by using 4E-BP1 and as a These kinase activity as by the of substrate the addition of recombinant Rheb with GTP to mTORC1 dramatically the of 4E-BP1 and On the other hand, mTORC2 activity as by the of not increased by the addition of In the presence of Rheb, the of 4E-BP1 by mTORC1 of and increased in the of Rheb, the at a low These that Rheb mTORC1 activity in of mTORC1 to to the Ras superfamily the Ras are as shown in We and are from Rheb by in and the mTORC1 activation is specific to Rheb These G-proteins with residues by in E. and bound with and we to activate mTORC1. can be in Rheb the G-protein of activating mTORC1. The of this that the of and raptor for We have also Cdc42 and in a did not activate mTORC1 two We GTP of Rheb for mTORC1 activation. Rheb bound with GTP dramatically activated mTORC1, Rheb bound with Rheb did not activate mTORC1 to the of bound GTP on Rheb for mTORC1 activation. cells two Rheb proteins Rheb and Rheb2 (RhebL1) (2Patel P.H. Thapar N. Guo L. Martinez M. Maris J. Gau C.L. Lengyel J.A. Tamanoi F. J. Cell Sci. 2003; 116: 3601-3610Crossref PubMed Scopus (132) Google Scholar). Although have in amino and the the two proteins is Although the of these proteins is the in C-terminal of to to activate mTORC1. Both Rheb1 and Rheb2 and for to activate mTORC1. can be in we mTORC1 activation by Rheb1 and Rheb2 and that the of activation the The at the of Rheb for the Rheb of effector domain the is a critical for G-proteins to activate downstream effectors F. Wittinghofer A. Curr. Opin. Biotechnol. 1996; 7: 449-456Crossref PubMed Scopus (78) Google Scholar). this domain the effector activation such as the binding of guanine In the of Rheb, the and the effector domain are to residues and further the Rheb domain for mTORC1 activation in the in vitro we recombinant Rheb proteins that have at the the and the effector is well conserved and These proteins and for to GTP and to activate mTORC1 in shown in at residue did not change guanine nucleotide binding a at residue the of nucleotide bound to Rheb. to activate in vitro a in these The still bound GTP and of activating mTORC1. These especially with and that the in the effector domain activation GTP binding FKBP38 a in the of mTORC1 by results that Rheb can activate mTORC1 in vitro the that activation of mTORC1 by Rheb is to a In X. Guo S. Y. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, X. Liu A. X. Liu Y. Y. Science. PubMed Scopus Google is proposed that Rheb protein FKBP38, and of by FKBP38, in the activation of mTORC1. is with because mTORC1 preparation appears not to contain In we FKBP38 can be in the mTORC1 preparation we The of FKBP38 in preparation the of to On the other hand, FKBP38 in the FKBP38 not by of the mTORC1 and raptor further the that a an of proteins is in the mTORC1 and this be to Rheb activation of mTORC1, we against FKBP38 to the of mTORC1 from these cells and for its to Rheb in the in vitro shows that the of cells with the of Although a increase in the of protein not and in the of 4E-BP1 We mTORC1, and the kinase activity in shown in the of the of low using mTORC1 from cells and and the addition of Rheb increased the of 4E-BP1 in the of FKBP38 on the of Rheb to activate mTORC1. of FKBP38 in cells also the that FKBP38 is not a of mTOR. shows the results of we the FKBP38 in cells and on mTORC1 can be by In the of amino that mTORC1 is by the of mTORC1 by the addition of amino acids increased the of of FKBP38 did not have a significant on the of further the of FKBP38 on Rheb-induced activation of mTORC1 by Rheb amino The of increased in amino cells the of the of of Rheb and FKBP38 of mTORC1 by Rheb of of 4E-BP1 by mTORC1 of the substrate protein to mTORC1 by the by kinase The of mTORC1 with its substrate proteins such as 4E-BP1 and kinase is by raptor J. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, K. N. K. Biochem. 2004; PubMed Scopus Google Scholar, Biochem. 2004; PubMed Scopus Google Scholar, K. Y. Long X. K. N. S. J. Yonezawa K. Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. Sabatini D.M. Full Text Full Text PDF PubMed Scopus Google Scholar). of raptor results in the of the mTORC1 the is to mTORC1 activation the of binding S.M. Sabatini D.M. Full Text Full Text PDF PubMed Scopus Google Scholar, N. K. S. Hara K. S. J. Yonezawa K. 2004; PubMed Scopus Google Scholar). In addition, that of 4E-BP1 is with the binding of 4E-BP1 to raptor L. C.J. Jr., J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). we in Rheb binding of mTORC1 with the binding of mTORC1 with the mTORC1 preparation for the in vitro kinase with 4E-BP1 in the presence of Rheb. the of 4E-BP1 bound to mTORC1 by in the of 4E-BP1 bound to mTORC1 4E-BP1 to mTORC1. binding increased by the addition of Rheb. On the other hand, in the presence of the of 4E-BP1 binding to mTORC1 by Rheb not We that the of 4E-BP1 in the with 4E-BP1 and is in with the that the of 4E-BP1 binding to raptor is by of 4E-BP1 S. S. N. K. Yonezawa K. 2006; PubMed Scopus Google Scholar). further the of Rheb on mTORC1 binding to we and effector domain that GTP activation shown in and did not increase 4E-BP1 binding to mTORC1, Rheb increased the of 4E-BP1 with mTORC1. an in the of mTORC1 by Rheb activation of mTORC1 is dependent on the raptor bound to the this immunoprecipitated using the with in raptor not is from mTORC1 K. Y. Long X. K. N. S. J. Yonezawa K. Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. Sabatini D.M. Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. Sabatini D.M. Mol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). On the other hand, immunoprecipitated is by using the as a because the and raptor is not by K. Y. Long X. K. N. S. J. Yonezawa K. Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. Sabatini D.M. Full Text Full Text PDF PubMed Scopus Google Scholar). in raptor in the by using the with not from that with The in vitro kinase that raptor the that with bound to raptor and The addition of Rheb increased the of 4E-BP1 in the Rheb did not increase 4E-BP1 in the raptor and The substrate of as a of a motif for the binding to raptor and competes with 4E-BP1 for raptor binding to of the signaling pathway Y. E. Sabatini D.M. Mol. 25: Full Text Full Text PDF PubMed Scopus Google Scholar, K. A. M.N. PubMed Scopus Google Scholar, B.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, N. K. K. A. S. Hara K. K. J. Yonezawa K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. Jr., J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. S. Cell Biol. PubMed Scopus Google Scholar). is to the activation of mTORC1 by Rheb Rheb the binding of 4E-BP1 to mTORC1 by the of the We recombinant competes with 4E-BP1 for mTORC1 binding in can be in the binding of to mTORC1 increased in a 4E-BP1 binding to mTORC1 we that the Rheb mTORC1 activation be by we Rheb-induced activation of mTORC1 in the in vitro by the addition of Rheb of have shown that TOR proteins kinase activity that is by the addition of G.J. J. Jr., J. 1996; PubMed Scopus Google Scholar, E.J. J. PubMed Scopus Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar). is in the shown in In the shown in mTORC1 preparation for with and the proteins by and the of by A a in the presence of We the addition of Rheb can induce this autophosphorylation in the presence of The results that Rheb is of shows that Rheb is of 4E-BP1 in the presence of with of 4E-BP1 can Rheb. We also found that can activate not mTORC1 also mTORC2 of by mTORC2 is in the presence of These results the that can activate kinase activity of Rheb does not activate the kinase activity of mTOR. Rheb and to on the of Although two of 4E-BP1 mTORC1 activated by Rheb in the presence of one mTORC1 activated by 4E-BP1 is at such as and by G.J. Jr., J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, G.J. Jr., J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, Yang Jr., Mol. Biol. 2000; PubMed Scopus Google we that this the 4E-BP1 is further the of we the at as well as at and the in vitro mTORC1 kinase with Rheb, Rheb and In the presence of Rheb, of 4E-BP1 at at as at at and increased On the other hand, in the presence of the of at at and increased with the of 4E-BP1 at at as a at the from of Rheb and increased the of 4E-BP1 at and These results suggest that Rheb and can mTORC1 activation that the of activation the results using an in vitro the that Rheb mTORC1 to increased of its substrate Rheb mTORC1 on for the of Rheb G-protein because other of the Ras G-proteins such as KRas, RalA/B, and did not activate mTORC1. G-proteins such as Cdc42 and also did not activate mTORC1. The activation of mTORC1 with Rheb1 and is in with that Rheb1 and Rheb2 can activate the signaling in Jr., A.P. Gau C.L. Machado I.M. Uhlmann E.J. Gutmann Guo L. Tamanoi F. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google and is also with the that these Rheb proteins have effector domain (2Patel P.H. Thapar N. Guo L. Martinez M. Maris J. Gau C.L. Lengyel J.A. Tamanoi F. J. Cell Sci. 2003; 116: 3601-3610Crossref PubMed Scopus (132) Google Scholar). Rheb1 and Rheb2 Rheb1 is Rheb2 N. M. S. S. M. M. K. S. M. A. S. K. M. A. 1996; PubMed Scopus Google Scholar, K. Y. K. J. Biochem. 2005; PubMed Scopus Google Scholar). appears that these two Rheb proteins function that The in vitro activation of mTORC1 by Rheb is dependent on the presence of an effector at residue to a in to activate mTORC1 in vitro, still to GTP. These results are in with in that Rheb proteins in and the effector domain J. 2005; PubMed Scopus Google Scholar, X. Lin Y. Ortiz-Vega S. S. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the effector domain for the function of Rheb also with the yeast protein J. Guo L. P.J. Tabancay Jr., A.P. J. Tamanoi F. Mol. Microbiol. 2005; PubMed Scopus Google Scholar). In addition, analysis of Rheb that a conformational change during GTP/GDP at the especially at residues (6Yu Y. Li S. Xu X. Li Y. Guan K. Arnold E. Ding J. J. Biol. Chem. 2005; 280: 17093-17100Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). the phosphates of is to be in binding of because of the of bound to Rheb as in of the of and are during GTP/GDP on GTP binding the of mTORC1 that these residues roles in the with mTORC1. and also to activate mTORC1 GTP binding also to the of the effector results not the that Rheb mTORC1 by with FKBP38 X. Liu A. X. Liu Y. Y. Science. PubMed Scopus Google Scholar). to this Rheb with FKBP38 and of by FKBP38 with mTOR. we have shown that the mTORC1 preparation that is devoid of FKBP38 can be activated by Rheb. In fact, Rheb mTORC1 from cells with to FKBP38 In we FKBP38 from the using antibody. mTORC1 from the cells activated by Rheb. and F. These results suggest that Rheb does not FKBP38 to activate mTORC1. In of 4E-BP1 and not FKBP38 cells starved for amino A increase in of FKBP38 did not activation by the of Rheb by amino of FKBP38 on signaling recently X. B.D. Liu A. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). On the other hand, recombinant FKBP38 4E-BP1 in the in vitro mTORC1 kinase and F. these results the that FKBP38 is not a to the mTORC1 not that FKBP38 a that Rheb a significant on the binding of 4E-BP1 to mTORC1. the binding of 4E-BP1 to mTORC1 in the of kinase we that the Rheb effector domain and not this The of raptor in the Rheb-induced activation of mTORC1 is from the of activation by Rheb preparation that is devoid of raptor These results with the raptor plays in the binding of 4E-BP1 to mTORC1 its with the We also that PRAS40, a motif-containing protein that competes with 4E-BP1 for binding to mTORC1, inhibits Rheb-induced activation of mTORC1. is with the of 4E-BP1 binding to mTORC1 by Although Rheb are by we not Rheb with We can binding of Rheb to not to and F. is that Rheb and the of raptor with the substrate protein to the of is with the L. C.J. Jr., J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google that activation of mTORC1 of the binding of 4E-BP1 with mTORC1. Rheb of to the activation of mTORC1 We have also shown that Rheb does not induce autophosphorylation of that Rheb does not activate the activity of mTOR. Although autophosphorylation can be in the presence of the addition of Rheb did not in the of mTOR. In addition, we found that can activate mTORC1 in the of Rheb and is of activation of yeast Ras effector proteins N. A. Tamanoi F. J. M. 41: Full Text PDF PubMed Scopus Google Scholar, S. S. J. K. M. Full Text PDF PubMed Scopus Google the for Ras to activate yeast can be is with We also in the that 4E-BP1 is and Rheb. Rheb the of 4E-BP1 at the of 4E-BP1 at and Rheb the of of 4E-BP1 to mTORC1. are to Rheb mTOR. is that the activation of by the use of recombinant Rheb protein in E. does not in E. is not required for the of Rheb to activate mTORC1. further this we have farnesylated Rheb protein by using cells in in vitro The results that the of the farnesylated Rheb protein that with the Rheb and F. is not required for the of Rheb to activate mTORC1. On the other hand, we as well as others have shown that is for Rheb function in J. Tabancay A.P. Yang W. Tamanoi F. J. Biol. Chem. 2000; 275: 11198-11206Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 9Yang W. Tabancay Jr., A.P. Urano J. Tamanoi F. Mol. Microbiol. 2001; 41: 1339-1347Crossref PubMed Scopus (61) Google Scholar, Manning B.D. J. Curr. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, G.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). These results suggest that is for the of Rheb not for activation of mTORC1. We for FKBP38 and antibody. with