The Proline-rich Akt Substrate of 40 kDa (PRAS40) Is a Physiological Substrate of Mammalian Target of Rapamycin Complex 1
2007; Elsevier BV; Volume: 282; Issue: 28 Linguagem: Inglês
10.1074/jbc.m702636200
ISSN1083-351X
AutoresNoriko Oshiro, Rinako Takahashi, Kenichi Yoshino, Keiko Tanimura, Akio Nakashima, Satoshi Eguchi, Takafumi Miyamoto, Kenta Hara, Kenji Takehana, Joseph Avruch, Ushio Kikkawa, Kazuyoshi Yonezawa,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe 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. 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. The mammalian target of rapamycin (mTOR) 3The abbreviations used are: mTOR, mammalian target of rapamycin; mTORC1 and 2, mTOR complex 1 and 2, respectively; TOS, TOR signaling; DMEM, Dulbecco's modified Eagle's medium; siRNA, small interfering RNA; ESI, electrospray ionization; MS, mass spectrometry; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. is the founding member of the PI-3 kinase-related family of protein (Ser/Thr) kinases (PIKKs) and controls many important aspects of the cellular response to nutrient sufficiency and growth factors (1Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4771) Google Scholar). The mTOR polypeptide is now known to function in two distinct, independently regulated hetero-oligomeric complexes, called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Both complexes contain mTOR and the polypeptide mLST8/GβL; mTORC1 in addition contains raptor (an ortholog of Saccharomyces cerevisiae KOG1), which binds directly to the known mTORC1 substrates S6K1 and 4E-BP1 and is indispensable for their phosphorylation by mTOR in vivo and in vitro. mTORC2 lacks raptor but contains the polypeptides rictor (an ortholog to ScAVO3) and mSin1 (an ortholog of ScAVO1). mTORC2 is one of the activating kinases for Akt, previously called PDK2, and also regulates the actin cytoskeleton through as yet unidentified effectors. Although rapamycin, in complex with FKBP12, binds directly to mTOR in a segment just amino-terminal to the catalytic domain, only mTORC1 binds the FKBP12-rapamycin complex, and thus only mTORC1 is directly susceptible to inhibition by rapamycin. Rapamycin is among the most selective kinase inhibitors known (2Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3962) Google Scholar), and its longtime availability has enabled a detailed accounting of the cellular responses to inhibition of mTORC1. Perhaps the best appreciated and most general action of rapamycin in cell culture is its ability to inhibit cell growth (i.e. to suppress the accumulation of cellular mass) (3Fingar D.C. Blenis J. Oncogene. 2004; 23: 3151-3171Crossref PubMed Scopus (1077) Google Scholar). This is accomplished by a selective inhibition of mRNA translation and by suppression of ribosomal biogenesis at both a transcriptional and translational level (1Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4771) Google Scholar, 3Fingar D.C. Blenis J. Oncogene. 2004; 23: 3151-3171Crossref PubMed Scopus (1077) Google Scholar). In addition, rapamycin activates autophagy and in some cells acts as a powerful inhibitor of proliferation (4Dumont F.J. Staruch M.J. Koprak S.L. 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Biol. 2002; 12: 632-639Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar) pointed out that the ability of these substrates to be regulated by mTORC1 in vivo depended on a short sequence (F(D/E)(F/I/L/M)(D/E)(L/I)) present in the noncatalytic amino-terminal flanking region of S6K1 and at the carboxyl terminus of 4E-BP1, which they named the TOR signaling (TOS) motif. Such a motif is also present in STAT3, another rapamycin-sensitive phosphoprotein (14Yokogami K. Wakisaka S. Avruch J. Reeves S.A. Curr. Biol. 2000; 10: 47-50Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Subsequent work established that an intact TOS motif is required for the binding of S6K1 and 4E-BP1 to raptor (15Beugnet A. Wang X. Proud C.G. J. Biol. Chem. 2003; 278: 40717-40722Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 16Choi K.M. McMahon L.P. Lawrence Jr., J.C. J. Biol. Chem. 2003; 278: 19667-19673Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 17Nojima H. Tokunaga C. Eguchi S. Oshiro N. Hidayat S. Yoshino K. Hara K. Tanaka N. Avruch J. Yonezawa K. J. Biol. Chem. 2003; 278: 15461-15464Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar, 18Schalm S.S. Fingar D.C. Sabatini D.M. Blenis J. Curr. Biol. 2003; 13: 797-806Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar), consistent with the view that raptor serves a necessary substrate-binding function in mTOR complex 1 (19Hara K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1470) Google Scholar). Although many potential TOS motifs are evident by BLAST analysis, we are unaware of validated mTORC1 substrates that have been identified thereby. Consequently, we sought novel candidate mTORC1 substrates by analyzing the cellular polypeptides that bound to recombinant raptor overexpressed in HEK293 cells. Herein, we describe the identification of PRAS40 (proline-rich Akt substrate of 40 kDa), previously identified as an Akt substrate and 14-3-3 binding partner (20Kovacina K.S. Park G.Y. Bae S.S. Guzzetta A.W. Schaefer E. Birnbaum M.J. Roth R.A. J. Biol. Chem. 2003; 278: 10189-10194Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar), as a raptor-binding protein and a physiological substrate of mTORC1. During the preparation of this report, two papers (21Vander Haar E.V. Lee S.I. Bandhakavi S. Griffin T.J. Kim D.H. Nat. Cell Biol. 2007; 9: 316-323Crossref PubMed Scopus (943) Google Scholar, 22Sancak Y. Thoreen C.C. Peterson T.R. Lindquist R.A. Kang S.A. Spooner E. Carr S.A. Sabatini D.M. Mol. Cell. 2007; 25: 903-915Abstract Full Text Full Text PDF PubMed Scopus (1012) Google Scholar) appeared describing the ability of PRAS40 to bind raptor. Moreover, based on the ability of PRAS40 to antagonize the mTORC1-catalyzed phosphorylation of S6K1 and 4E-BP1, it was proposed that a primary function of PRAS40 is to inhibit mTORC1 signaling to its physiological substrates, a function that is ameliorated by Akt-catalyzed PRAS40 phosphorylation; the latter was claimed to reduce, in a 14-3-3-dependent manner, PRAS40 binding to raptor. In contrast, we demonstrate that PRAS40 is itself a physiological substrate of mTORC1 and, like other mTORC1 substrates, competes for a pool of raptor substrate-binding sites whose abundance is apparently limiting for mTORC1-catalyzed substrate phosphorylation in vivo. PRAS40 binding to raptor is abolished by mTORC1-catalyzed PRAS40 phosphorylation but is unaffected by mutation of the PRAS40 Akt phosphorylation site, Thr246. We propose that PRAS40 functions primarily to regulate an as yet poorly defined cellular process, under the joint control of Akt and mTORC1. cDNAs and siRNAs—The expression vectors of FLAG-tagged raptor (pcDNA1-FLAG-raptor) (19Hara K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1470) Google Scholar), HA-tagged wild-type mTOR (pcDNA1-HA-mTOR), HA-tagged kinase-negative mTOR (pcDNA1-HA-mTOR NK), FLAG-tagged wild-type mTOR (pCMV5-FLAG-mTOR), FLAG-tagged rapamycin-resistant mTOR (pCMV5-FLAG-mTOR ST), FLAG-tagged rapamycin-resistant/kinase-negative mTOR (pCMV5-FLAG-mTOR ST/NK), FLAG-tagged 4E-BP1 (pCMV5-FLAG-4E-BP1) (23Hara K. Yonezawa K. Kozlowski M.T. Sugimoto T. Andrabi K. Weng Q.P. Kasuga M. Nishimoto I. Avruch J. J. Biol. Chem. 1997; 272: 26457-26463Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar), HA-tagged S6K1 (pMT2-HA-S6K1), HA-tagged kinase-negative S6K1 (pMT2-HA-S6K1 KM) (24Weng Q.P. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (211) Google Scholar), GST-fused S6K1 (pEBG2T-S6K1) (25Alessi D.R. Kozlowski M.T. Weng Q.P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar), and GST-fused 4E-BP1 (pGEX4T1–4E-BP1) (19Hara K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1470) Google Scholar) were constructed as described. His6-tagged human PRAS40 (pReceiver-MO1-His6-PRAS40) was purchased from GeneCopoeia. The coding region was amplified by PCR with the addition of EcoRI and XhoI sites at 5′ and 3′ ends, respectively, and the PCR product was cloned into pcDNA3-Myc and pcDNA3-FLAG. The insert was mutated using a QuikChange™ site-directed mutagenesis kit (Stratagene). The mutants having Ala at Phe129 (F129A), Ser183 (S183A), and Thr246 (T246A) or Asp at Ser183 (S183D) and Thr246 (T246D) were generated in pcDNA3-Myc. The wild-type PRAS40 and the S183A mutant were also cloned into pGEX4T1 to produce GST fusion proteins in Escherichia coli. The human Rheb sequence was amplified by PCR from the QUICK-Clone™ cDNA of human brain (Clontech) with the addition of EcoRI and XhoI sites at 5′ and 3′ ends, respectively, and the PCR product was cloned into pcDNA3-Myc. The expression vectors of FLAG-tagged TSC1 (pcDNA3-FLAG-TSC1) and FLAG-tagged TSC2 (pcDNA3-FLAG-TSC2) were kindly provided by Dr. David J. Kwiatkowski (Brigham and Women's Hospital, Boston). Antibodies—The anti-mTOR antibody was produced as described previously (26Nishiuma T. Hara K. Tsujishita Y. Kaneko K. Shii K. Yonezawa K. Biochem. Biophys. Res. Commun. 1998; 252: 440-444Crossref PubMed Scopus (24) Google Scholar). Rabbit polyclonal anti-peptide antibodies recognizing PRAS40, phosphorylated PRAS40 at Ser183, raptor, and rictor were produced against the following peptides by Immuno-Biological Laboratories: PRAS40 (P238), DLPRPRLNTSDFQKLKRKY (amino acids 238–256); phosphorylated PRAS40 (Ser(P)183), QYAKpSLPVS (amino acids 179–187) (in which pS represents phospho-Ser; raptor (R1), MESEMLQSPLLGLGEEDEA (amino acids 1–19); raptor (R984), IRKEREWRFLRNSRVRRQA (amino acids 984–1002); and rictor (R1291), GSSHTLPRRAQSLKA (amino acids 1291–1305). In brief, rabbits were immunized with each synthetic peptide coupled with thyroglobulin, and the immunoglobulin G fractions against these peptide sequences were obtained from the immune sera using a column of antigen-coupled Activated Thiol Sepharose™ 4B (GE Healthcare Bio-Sciences). The following antibodies were purchased from commercial sources. Anti-FLAG (M2) was from Sigma; anti-Myc (9E10) and anti-HA (12CA5) were from Roche Applied Science; normal rabbit immunoglobulin, normal mouse immunoglobulin, anti-S6K1 (H-9), and anti-4E-BP1 (C-19) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-phospho-AMPK (Thr(P)172) (40H9), anti-phospho-S6K1 (Thr(P)389) (1A5), anti-phospho-4E-BP1 (Ser(P)65), and anti-phospho-4E-BP1 (Thr(P)37/46) were from Cell Signaling Technology; anti-PRAS40 (73P21) and anti-phospho-PRAS40 (Thr(P)246) were from BioSource International; anti-GST was from Upstate Biotechnology; anti-β-actin was from Abcam. Cell Culture and Treatment—HEK293 and HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma) containing 10% fetal bovine serum at 37 °C in a 5% CO2 incubator. HEK293 cells were transfected with each expression vector by the lipofection method using Lipofectamine reagent (Invitrogen) according to the manufacturer's protocol. For the small interfering RNA (siRNA) studies, HEK293 cells or HeLa cells were transfected with PRAS40 siRNA duplexes with 3′ dTdT overhangs corresponding to human PRAS40 mRNA (5′-GGGCAUUAGUGAUAAUGGA-3′) or (5′-GCGACUUCCAGAAGCUGAA-3′) (Qiagen) using Nucleofector 3 (AMAXA Biosystems). Scramble siRNA duplexes (iGENE Therapeutics) were employed as a control. For starvation of nutrients, cells were deprived of serum for 15 h, and the medium was replaced with fresh DMEM without serum for 1 h. For starvation of amino acids, the cells were washed twice with DMEM lacking amino acids, further incubated in the same medium for 2 h, and then stimulated with DMEM containing amino acids for 30 min in the presence or absence of 200 nm rapamycin. Where indicated, the amino acid mixture lacking branched-chain amino acids was employed instead of amino acids. For starvation of glucose, the cells were washed twice with DMEM lacking glucose (Sigma) and stimulated with DMEM lacking glucose in the presence or absence of 5.5 mm 2-deoxyglucose for 30 min. Where indicated, DMEM containing glucose was employed as a control. Immunoprecipitation—This procedure was carried out essentially as described (19Hara K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1470) Google Scholar). All cells were lysed in ice-cold buffer A (20 mm Tris-HCl (pH 7.4), 120 mm NaCl, 1 mm EDTA, 5 mm EGTA, 50 mm β-glycerophosphate, 50 mm NaF, 0.3% CHAPS, 1 mm dithiothreitol, 4 μg/ml leupeptin, 4 μg/ml aprotinin). The immunoprecipitates were washed three times with buffer A and used for silver staining analysis or immunoblot. Where indicated, buffer A contained 1% Nonidet P-40 instead of 0.3% CHAPS. For the kinase assay, the immunoprecipitates were washed twice with buffer A and twice in buffer B (10 mm HEPES (pH 7.5), 50 mm β-glycerophosphate, 50 mm NaCl). Immunoblot—The lysates and immunoprecipitates were separated by SDS-PAGE, and the proteins were transferred onto a polyvinylidene difluoride membrane. The membranes were blocked at room temperature with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) and were then incubated with each primary antibody, diluted in the 2% skim milk in TBST, for 1 h at room temperature or overnight at 4 °C. The commercial antibodies were used at dilutions of 1:500–1:1000, except for the anti-β-actin antibody employed at a dilution of 1:5000. After incubation with the horseradish peroxidase-conjugated secondary antibody, the proteins were visualized by the enhanced chemiluminescence method. When the same sample was analyzed with different antibodies, the membrane was stripped and employed for the subsequent immunoblot analysis. When the endogenous 4E-BP1 proteins were analyzed, the cell lysates were heated at 100 °C for 5 min and centrifuged at 18,000 × g for 30 min, and the heat-stable proteins were subjected to immunoblot (27Shigemitsu K. Tsujishita Y. Hara K. Nanahoshi M. Avruch J. Yonezawa K. J. Biol. Chem. 1999; 274: 1058-1065Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Kinase Assay—The mTOR kinase assay was performed as previously described (19Hara K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1470) Google Scholar). GST and GST fusion proteins were prepared for substrates of the kinase assay, as previously described (17Nojima H. Tokunaga C. Eguchi S. Oshiro N. Hidayat S. Yoshino K. Hara K. Tanaka N. Avruch J. Yonezawa K. J. Biol. Chem. 2003; 278: 15461-15464Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar, 19Hara K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1470) Google Scholar). The purified MBP-PRAS40 was purchased from BioSource International. After the kinase reaction, the samples were separated by SDS-PAGE, transferred onto a polyvinylidene difluoride membrane, and analyzed by autoradiography using x-ray film or the Bioimaging Analyzer BAS2500 (Fujix). Then the membrane was immunoblotted with the appropriate antibody and visualized as described above. Mass Spectrometry—The analysis was carried out as described previously (28Yamaguchi Y. Shirai Y. Matsubara T. Sanse K. Kuriyama M. Oshiro N. Yoshino K. Yonezawa K. Ono Y. Saito N. J. Biol. Chem. 2006; 281: 31627-31637Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 29Sakakibara K. Sato K. Yoshino K. Oshiro N. Hirahara S. Mahbub Hasan A.K. Iwasaki T. Ueda Y. Iwao Y. Yonezawa K. Fukami Y. J. Biol. Chem. 2005; 280: 15029-15037Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Briefly, FLAG-raptor immunoprecipitates were separated by SDS-PAGE and visualized by silver staining. The band corresponding to p40 polypeptide was cut out and destained, and the proteins in gels were reduced and alkylated, followed by in-gel digestion with trypsin in 25 mm ammonium bicarbonate for 15 h at 37 °C. The resulting peptides were then subjected to the liquid chromatography electrospray ionization (ESI) mass spectrometry/mass spectrometry (MS/MS) by using a LCQ Advantage ion trap mass spectrometer (Thermo Finnigan). Protein identification according to product ion mass lists was performed by the product ion mass fingerprinting using MASCOT "MS/MS ion search." For identification of in vitro phosphorylation sites in PRAS40, GST-PRAS40 phosphorylated by mTOR was separated by SDS-PAGE. The GST-PRAS40 visualized by reverse staining was cut out from a gel. Destained proteins were digested with trypsin in a gel as described above and subjected to nano-ESI mass spectrometry by using a Q-Tof2 quadrupole time-of-flight mass spectrometer (Micromass), as described previously. Sequence Analysis—The amino acid sequence identity and similarity were analyzed by using the GENETYX program, version 8 (GENETYX). Recombinant Raptor Binds Endogenous PRAS40 in an Amino Acid-dependent Manner—Using one-dimensional SDS-PAGE and silver staining, we compared the polypeptides retrieved in anti-FLAG immunoprecipitates prepared from HEK293 cells transfected with FLAG-raptor or with an empty vector. The most abundant cellular polypeptide recovered in the FLAG-raptor immmunoprecipitate was a protein of ∼290 kDa (Fig. 1A), shown to be mTOR by immunoblot as well as by the peptide mass fingerprinting analysis (data not shown). Many of the other prominent polypeptide bands in the FLAG-raptor immunoprecipitate were identified by mass spectrometric analysis as raptor, presumably reflecting FLAG-raptor proteolysis. A polypeptide of ∼40 kDa, however, contained several peptide fragments distinct from raptor, which were consistent with the amino acid sequence of PRAS40 (NCBI accession number NP_115751), a proline-rich protein first identified as an Akt substrate and 14-3-3 binding partner (20Kovacina K.S. Park G.Y. Bae S.S. Guzzetta A.W. Schaefer E. Birnbaum M.J. Roth R.A. J. Biol. Chem. 2003; 278: 10189-10194Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). To verify the ability of recombinant raptor to bind endogenous PRAS40, the FLAG-raptor immunoprecipitate was immunoblotted with an anti-PRAS40 antibody (Fig. 1B, top). A small amount of PRAS40 was detected in the FLAG-raptor immunoprecipitate prepared from serum-deprived cells, but the additional removal of ambient amino acids resulted in a large increase in FLAG-raptor-associated PRAS40. A similar pattern was observed if an immunoprecipitate of endogenous PRAS40 was probed for endogenous raptor (Fig. 1B, bottom). Whereas no raptor was coprecipitated with normal rabbit IgG, endogenous raptor was present in the anti-PRAS40 immunoprecipitate; moreover, amino acid withdrawal substantially increased the amount of raptor recovered with PRAS40, whereas rapamycin resulted in a modest decrease in PRAS40-associated raptor. To determine whether the association of PRAS40 with raptor required mTOR or mLST8, we expressed recombinant His6-PRAS40 in HEK293 cells, retrieved the His6-PRAS40 on Ni2+-nitrilotriacetic acid beads, and washed the beads with buffer containing either 0.3% CHAPS or 1% Nonidet P-40; an immunoblot of the eluate from these beads was shown in Fig. 1C. Nonidet P-40, which causes the dissociation of an mTOR-mLST8 complex from raptor, did not diminish the recovery of raptor with His6-PRAS40. An amount of rapamycin sufficient to promote dissociation in vivo of raptor from mTORC1 modestly diminished the recovery of raptor with His6-PRAS40 (Fig. 1C). Thus, PRAS40 binds directly to raptor; this interaction occurs between the endogenous polypeptides, does not require mTOR, and is regulated by amino acid sufficiency. PRAS40 (Phe129) Is Important for the Association with Raptor—The binding of the mTORC1 substrates S6K1 and 4E-BP1 to raptor has been shown to require a specific five-amino acid sequence (F(D/E)(F/I/L/M)(D/E)(L/I)) called the TOS motif (13Schalm S.S. Blenis J. Curr. Biol. 2002; 12: 632-639Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 15Beugnet A. Wang X. Proud C.G. J. Biol. Chem. 2003; 278: 40717-40722Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 16Choi K.M. McMahon L.P. Lawrence Jr., J.C. J. Biol. Chem. 2003; 278: 19667-19673Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 17Nojima H. Tokunaga C. Eguchi S. Oshiro N. Hidayat S. Yoshino K. Hara K. Tanaka N. Avruch J. Yonezawa K. J. Biol. Chem. 2003; 278: 15461-15464Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar, 18Schalm S.S. Fingar D.C. Sabatini D.M. Blenis J. Curr. Biol. 2003; 13: 797-806Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Human PRAS40 has six Phe residues, none of which are followed by a TOS motif consensus corresponding to those found in S6K1, 4E-BP1, or STAT3. The sequence in PRAS40 most similar to the TOS motif begins with Phe129, 129FVMDE133; this motif is conserved down through Xenopus tropicalis, slightly altered in Danio rerio (222FSMDE226) and lacking entirely in the putative Drosophila melanogaster ortholog, Lobe (supplemental Fig. 1). Nevertheless, we generated a PRAS40 mutant replacing Phe129 by Ala (F129A) and found that this mutant was greatly deficient in its ability to bind raptor as compared with wild-type PRAS40 (Fig. 2). Moreover, as seen in Figs. 1C and 2, the ability of PRAS40, when overexpressed, to bind endogenous raptor was not noticeably enhanced by amino acid withdrawal, whereas rapamycin, at concentrations sufficient to promote dissociation of the raptor-mTOR complex, did decrease the amount of raptor recovered with recombinant PRAS40. mTORC1 Phosphorylates PRAS40 in Vitro at Ser183—The ability of PRAS40 to associate with raptor through a TOS-like motif raised the possibility that PRAS40, like S6K1 and 4E-BP1, could serve as a substrate for the mTORC1 kinase. As seen in Fig. 3A, an immunoprecipitate of endogenous mTOR phosphorylated PRAS40 (as a prokaryotic recombinant GST or MBP fusion protein) in vitro to an extent comparable with GST-S6K1 or GST-4E-BP1 (the former purified from serum-deprived, rapamycin-treated HEK293 cells). This phosphorylation of PRAS40 was catalyzed by mTOR itself, inas- much as only the active recombinant mTOR but not the kinase-negative mTOR (N2343K) mutant (23Hara K. Yonezawa K. Kozlowski M.T. Sugimoto T. Andrabi K. Weng Q.P. Kasuga M. Nishimoto I. Avruch J. J. Biol. Chem. 1997; 272: 26457-26463Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar) catalyzed GST-PRAS40 phosphorylation in vitro (Fig. 3B). Tryptic digestion of PRAS40 followed by nano-ESI mass spectrometry was employed to identify the site(s) phosphorylated by mTOR in vitro. The experimental mass value (1395.75 Da) of the protonated peptide corresponding to the ion at m/z 698.38, which was observed only in the mass spectrum of GST-PRAS40 phosphorylated by mTOR (supplemental Fig. 2), was nearly identical to the calculated mass value (1395.71 Da) of a singly phosphorylated form of the PRAS40 tryptic peptide, 183SLPVSVPVWGFK194. Subjecting the m/z 698.38 ion to MS/MS established that Ser183, which is highly conserved (supplemental Fig. 1), is the phosphorylated residue (data not shown). In support of this identification, a PRAS40 (S183A) mutant was phosphorylated by mTOR in vitro to a greatly reduced extent as compared with wild-type PRAS40 (Fig. 4A), indicating that Ser183 is a major although not the sole site of mTOR-catalyzed PRAS40 phosphorylation in vitro.FIGURE 4Identification of an in vitro mTORC1-catalyzed phosphorylation site in PRAS40. A, mutation of PRAS40 Ser183 to Ala reduces PRAS40 phosphorylation by mTOR in vitro. mTOR immunoprecipitated from HEK293 cells was used to phosphorylate GST, GST-PRAS40, and GST-PRAS40 (S183A). The immunoprecipitate by the NMG was employed as a control. The top panel shows autoradiography, and the lower three panels show immunoblot by using the anti-mTOR, anti-GST, and phospho-specific anti-PRAS40 (Ser(P)183) antibodies, respectively. B, mTORC1-specific phosphorylation of PRAS40 in vitro. mTORC1 and mTORC2 were immunoprecipitated from HEK293 cells by the anti-raptor and anti-rictor antibodies, respectively, and the immunoprecipitates were used to phosphorylate GST-PRAS40 in vitro. The immunoprecipitate by the N
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