title: The mechanism of insulin-stimulated 4E-BP protein binding to mammalian target of rapamycin (mTOR) complex 1 and its contribution to mTOR complex 1 signaling.
abstract: Insulin activation of mTOR complex 1 is accompanied by enhanced binding of substrates. We examined the mechanism and contribution of this enhancement to insulin activation of mTORC1 signaling in 293E and HeLa cells. In 293E, insulin increased the amount of mTORC1 retrieved by the transiently expressed nonphosphorylatable 4E-BP[5A] to an extent that varied inversely with the amount of PRAS40 bound to mTORC1. RNAi depletion of PRAS40 enhanced 4E-BP[5A] binding to ∼70% the extent of maximal insulin, and PRAS40 RNAi and insulin together did not increase 4E-BP[5A] binding beyond insulin alone, suggesting that removal of PRAS40 from mTORC1 is the predominant mechanism of an insulin-induced increase in substrate access. As regards the role of increased substrate access in mTORC1 signaling, RNAi depletion of PRAS40, although increasing 4E-BP[5A] binding, did not stimulate phosphorylation of endogenous mTORC1 substrates S6K1(Thr(389)) or 4E-BP (Thr(37)/Thr(46)), the latter already ∼70% of maximal in amino acid replete, serum-deprived 293E cells. In HeLa cells, insulin and PRAS40 RNAi also both enhanced the binding of 4E-BP[5A] to raptor but only insulin stimulated S6K1 and 4E-BP phosphorylation. Furthermore, Rheb overexpression in 293E activated mTORC1 signaling completely without causing PRAS40 release. In the presence of Rheb and insulin, PRAS40 release is abolished by Akt inhibition without diminishing mTORC1 signaling. In conclusion, dissociation of PRAS40 from mTORC1 and enhanced mTORC1 substrate binding results from Akt and mTORC1 activation and makes little or no contribution to mTORC1 signaling, which rather is determined by Rheb activation of mTOR catalytic activity, through mechanisms that remain to be fully elucidated.
authors.affiliation: Department of Molecular Biology and Diabetes Unit of the Medical Services, Massachusetts General Hospital and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114.
authors.forename: Joseph
authors.initials: J
authors.lastname: Rapley
authors.affiliation: Department of Molecular Biology and Diabetes Unit of the Medical Services, Massachusetts General Hospital and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114.
authors.forename: Noriko
authors.initials: N
authors.lastname: Oshiro
authors.affiliation: Department of Molecular Biology and Diabetes Unit of the Medical Services, Massachusetts General Hospital and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114.
authors.forename: Sara
authors.initials: S
authors.lastname: Ortiz-Vega
authors.affiliation: Department of Molecular Biology and Diabetes Unit of the Medical Services, Massachusetts General Hospital and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114. Electronic address: avruch@molbio.mgh.harvard.edu.
authors.forename: Joseph
authors.initials: J
authors.lastname: Avruch
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
chemical_list: C484523:AKT1S1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D007328:Insulin; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D011506:Proteins; D034741:RNA, Small Interfering; D005982:Glutathione Transferase; D000076222:Mechanistic Target of Rapamycin Complex 1; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases