The Proline-rich Akt Substrate of 40 kDa (PRAS40) Is a Physiological Substrate of Mammalian Target of Rapamycin Complex 1*

The proline-rich Akt substrate of 40 kilodaltons (PRAS40) was identified as a raptor-binding protein that is phosphorylated directly by mammalian target of rapamycin (mTOR) complex 1 (mTORC1) but not mTORC2 in vitro, predominantly at PRAS40 (Ser183). The binding of S6K1 and 4E-BP1 to raptor requires a TOR signaling (TOS) motif, which contains an essential Phe followed by four alternating acidic and small hydrophobic amino acids. PRAS40 binding to raptor was severely inhibited by mutation of PRAS40 (Phe129 to Ala). Immediately carboxyl-terminal to Phe129 are two small hydrophobic amino acid followed by two acidic residues. PRAS40 binding to raptor was also abolished by mutation of the major mTORC1 phosphorylation site, Ser183, to Asp. PRAS40 (Ser183) was phosphorylated in intact cells; this phosphorylation was inhibited by rapamycin, by 2-deoxyglucose, and by overexpression of the tuberous sclerosis complex heterodimer. PRAS40 (Ser183) phosphorylation was also inhibited reversibly by withdrawal of all or of only the branched chain amino acids; this inhibition was reversed by overexpression of the Rheb GTPase. Overexpressed PRAS40 suppressed the phosphorylation of S6K1 and 4E-BP1 at their rapamycin-sensitive phosphorylation sites, and reciprocally, overexpression of S6K1 or 4E-BP1 suppressed phosphorylation of PRAS40 (Ser183) and its binding to raptor. RNA interference-induced depletion of PRAS40 enhanced the amino acid-stimulated phosphorylation of both S6K1 and 4E-BP1. These results establish PRAS40 as a physiological mTORC1 substrate that contains a variant TOS motif. Moreover, they indicate that the ability of raptor to bind endogenous substrates is limiting for the activity of mTORC1 in vivo and is therefore a potential locus of regulation.

[1]  S. Carr,et al.  PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. , 2007, Molecular cell.

[2]  Timothy J. Griffin,et al.  Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40 , 2007, Nature Cell Biology.

[3]  J. Eckel,et al.  Insulin-Mediated Phosphorylation of the Proline-Rich Akt Substrate PRAS40 Is Impaired in Insulin Target Tissues of High-Fat Diet–Fed Rats , 2006, Diabetes.

[4]  Kwang-Wook Choi,et al.  Lobe and Serrate are required for cell survival during early eye development in Drosophila , 2006, Development.

[5]  Y. Ono,et al.  Phosphorylation and Up-regulation of Diacylglycerol Kinase γ via Its Interaction with Protein Kinase Cγ* , 2006, Journal of Biological Chemistry.

[6]  J. Lawrence,et al.  Activation of Mammalian Target of Rapamycin (mTOR) by Insulin Is Associated with Stimulation of 4EBP1 Binding to Dimeric mTOR Complex 1* , 2006, Journal of Biological Chemistry.

[7]  M. Hall,et al.  TOR Signaling in Growth and Metabolism , 2006, Cell.

[8]  A. Saito,et al.  Modulation of proline-rich akt substrate survival signaling pathways by oxidative stress in mouse brains after transient focal cerebral ischemia. , 2006, Stroke.

[9]  Gavin W. Porter,et al.  Expression of proline-rich Akt-substrate PRAS40 in cell survival pathway and carcinogenesis , 2005, Acta Pharmacologica Sinica.

[10]  Joseph Avruch,et al.  Rheb Binds and Regulates the mTOR Kinase , 2005, Current Biology.

[11]  N. Oshiro,et al.  Molecular Identification and Characterization of Xenopus Egg Uroplakin III, an Egg Raft-associated Transmembrane Protein That Is Tyrosine-phosphorylated upon Fertilization* , 2005, Journal of Biological Chemistry.

[12]  T. Hunter,et al.  Tuberous Sclerosis and Insulin Resistance: Unlikely Bedfellows Reveal A TORrid Affair , 2005, Cell cycle.

[13]  I. Gout,et al.  The TSC1-2 tumor suppressor controls insulin–PI3K signaling via regulation of IRS proteins , 2004, The Journal of cell biology.

[14]  C. MacKintosh,et al.  Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes. , 2004, The Biochemical journal.

[15]  J. Blenis,et al.  Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression , 2004, Oncogene.

[16]  P. Crino,et al.  Expression profiling in tuberous sclerosis complex (TSC) knockout mouse astrocytes to characterize human TSC brain pathology , 2004, Glia.

[17]  Atsushi Saito,et al.  Neuroprotective Role of a Proline-Rich Akt Substrate in Apoptotic Neuronal Cell Death after Stroke: Relationships with Nerve Growth Factor , 2004, The Journal of Neuroscience.

[18]  K. Inoki,et al.  TSC2: filling the GAP in the mTOR signaling pathway. , 2004, Trends in biochemical sciences.

[19]  K. Inoki,et al.  TSC2 Mediates Cellular Energy Response to Control Cell Growth and Survival , 2003, Cell.

[20]  C. Proud,et al.  Target of Rapamycin (TOR)-signaling and RAIP Motifs Play Distinct Roles in the Mammalian TOR-dependent Phosphorylation of Initiation Factor 4E-binding Protein 1* , 2003, Journal of Biological Chemistry.

[21]  J. Lawrence,et al.  Two Motifs in the Translational Repressor PHAS-I Required for Efficient Phosphorylation by Mammalian Target of Rapamycin and for Recognition by Raptor* , 2003, Journal of Biological Chemistry.

[22]  J. Blenis,et al.  TOS Motif-Mediated Raptor Binding Regulates 4E-BP1 Multisite Phosphorylation and Function , 2003, Current Biology.

[23]  J. Avruch,et al.  The Mammalian Target of Rapamycin (mTOR) Partner, Raptor, Binds the mTOR Substrates p70 S6 Kinase and 4E-BP1 through Their TOR Signaling (TOS) Motif* , 2003, The Journal of Biological Chemistry.

[24]  M. Birnbaum,et al.  Identification of a Proline-rich Akt Substrate as a 14-3-3 Binding Partner* , 2003, The Journal of Biological Chemistry.

[25]  C. MacKintosh,et al.  Regulation of the 14-3-3-binding protein p39 by growth factors and nutrients in rat PC12 pheochromocytoma cells. , 2002, The Biochemical journal.

[26]  Kwang-Wook Choi,et al.  Lobe mediates Notch signaling to control domain-specific growth in the Drosophila eye disc. , 2002, Development.

[27]  J. Avruch,et al.  Raptor, a Binding Partner of Target of Rapamycin (TOR), Mediates TOR Action , 2002, Cell.

[28]  J. Blenis,et al.  Identification of a Conserved Motif Required for mTOR Signaling , 2002, Current Biology.

[29]  J. Avruch,et al.  14-3-3 Proteins: Active Cofactors in Cellular Regulation by Serine/Threonine Phosphorylation* , 2002, The Journal of Biological Chemistry.

[30]  T. Haruta,et al.  Mammalian Target of Rapamycin Pathway Regulates Insulin Signaling via Subcellular Redistribution of Insulin Receptor Substrate 1 and Integrates Nutritional Signals and Metabolic Signals of Insulin , 2001, Molecular and Cellular Biology.

[31]  P. Cohen,et al.  Specificity and mechanism of action of some commonly used protein kinase inhibitors , 2000 .

[32]  J. Avruch,et al.  Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin target mTOR , 2000, Current Biology.

[33]  J. Avruch,et al.  Regulation of Translational Effectors by Amino Acid and Mammalian Target of Rapamycin Signaling Pathways , 1999, The Journal of Biological Chemistry.

[34]  K. Kaneko,et al.  Characterization of the phosphoproteins and protein kinase activity in mTOR immunoprecipitates. , 1998, Biochemical and biophysical research communications.

[35]  J. Avruch,et al.  Amino Acid Sufficiency and mTOR Regulate p70 S6 Kinase and eIF-4E BP1 through a Common Effector Mechanism* , 1998, The Journal of Biological Chemistry.

[36]  S. Snyder,et al.  RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Dario R. Alessi,et al.  3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro , 1998, Current Biology.

[38]  M. Kasuga,et al.  Regulation of eIF-4E BP1 Phosphorylation by mTOR* , 1997, The Journal of Biological Chemistry.

[39]  A. Gingras,et al.  The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70s6k , 1997, Molecular and cellular biology.

[40]  Christine C. Hudson,et al.  Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin. , 1997, Science.

[41]  J. Badimón,et al.  Rapamycin inhibits vascular smooth muscle cell migration. , 1996, The Journal of clinical investigation.

[42]  A. Gingras,et al.  4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[43]  A. Gingras,et al.  Rapamycin blocks the phosphorylation of 4E‐BP1 and inhibits cap‐dependent initiation of translation. , 1996, The EMBO journal.

[44]  J. Avruch,et al.  Multiple independent inputs are required for activation of the p70 S6 kinase , 1995, Molecular and cellular biology.

[45]  S. Marx,et al.  Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells. , 1995, Circulation research.

[46]  J. Avruch,et al.  Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. , 1992, Science.

[47]  G. Crabtree,et al.  Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases , 1992, Cell.

[48]  N. Sigal,et al.  Distinct mechanisms of suppression of murine T cell activation by the related macrolides FK-506 and rapamycin. , 1990, Journal of immunology.