Title: Regulation of eIF-4E BP1 Phosphorylation by mTOR
Abstract: The proteins eIF-4E BP1 and p70 S6 kinase each undergo an insulin/mitogen-stimulated phosphorylation in situ that is partially inhibited by rapamycin. Previous work has established that the protein known as mTOR/RAFT-1/FRAP is the target through which the rapamycin·FKBP12 complex acts to dephosphorylate/deactivate the p70 S6 kinase; thus, some mTOR mutants that have lost the ability to bind to the rapamycin·FKBP12 complex in vitro can protect the p70 S6 kinase against rapamycin-induced dephosphorylation/deactivationin situ. We show herein that such mTOR mutants also protect eIF-4E BP1 against rapamycin-induced dephosphorylation, and for both p70 S6 kinase and eIF-4E BP1, such protection requires that the rapamycin-resistant mTOR variant retains an active catalytic domain. In contrast, mutants of p70 S6 kinase rendered intrinsically resistant to inhibition by rapamycin in situ are not able to protect coexpressed eIF-4E BP1 from rapamycin-induced dephosphorylation. We conclude that mTOR is an upstream regulator of eIF-4E BP1 as well as the p70 S6 kinase; moreover, these two mTOR targets are regulated in a parallel rather than sequential manner. The proteins eIF-4E BP1 and p70 S6 kinase each undergo an insulin/mitogen-stimulated phosphorylation in situ that is partially inhibited by rapamycin. Previous work has established that the protein known as mTOR/RAFT-1/FRAP is the target through which the rapamycin·FKBP12 complex acts to dephosphorylate/deactivate the p70 S6 kinase; thus, some mTOR mutants that have lost the ability to bind to the rapamycin·FKBP12 complex in vitro can protect the p70 S6 kinase against rapamycin-induced dephosphorylation/deactivationin situ. We show herein that such mTOR mutants also protect eIF-4E BP1 against rapamycin-induced dephosphorylation, and for both p70 S6 kinase and eIF-4E BP1, such protection requires that the rapamycin-resistant mTOR variant retains an active catalytic domain. In contrast, mutants of p70 S6 kinase rendered intrinsically resistant to inhibition by rapamycin in situ are not able to protect coexpressed eIF-4E BP1 from rapamycin-induced dephosphorylation. We conclude that mTOR is an upstream regulator of eIF-4E BP1 as well as the p70 S6 kinase; moreover, these two mTOR targets are regulated in a parallel rather than sequential manner. Rapamycin is an immunosurpressive macrolide whose major cellular receptor is the cytosolic 12-kDa FK506-binding protein (FKBP12) 1The abbreviations used are: FKBP12, 12-kDa FK506-binding protein; PI, phosphatidylinositol; PCR, polymerase chain reaction; bp, base pair(s); HA, hemagglutinin; GST, glutathioneS-transferase; CHO-IR, Chinese hamster ovary cells overexpressing human insulin receptors; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; DMEM, Dulbecco's modified Eagle's medium. 1The abbreviations used are: FKBP12, 12-kDa FK506-binding protein; PI, phosphatidylinositol; PCR, polymerase chain reaction; bp, base pair(s); HA, hemagglutinin; GST, glutathioneS-transferase; CHO-IR, Chinese hamster ovary cells overexpressing human insulin receptors; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; DMEM, Dulbecco's modified Eagle's medium. (1Kunz J. Hall M.N. Trends Biochem. Sci. 1993; 18: 334-338Abstract Full Text PDF PubMed Scopus (249) Google Scholar, 2Schreiber S.L. Science. 1991; 251: 283-287Crossref PubMed Scopus (1336) Google Scholar). Rapamycin binds to FKBP12 at a single site, identical to the site bound by the structurally related drug FK506, and both agents, acting in situ as a drug-protein complex, are immunosurpressive through inhibition of T-cell proliferation. Despite these similarities, the two drugs operate through distinct mechanisms. The FK506·FKBP12 complex blocks T-cell receptor signal transduction by directly inhibiting protein phosphatase 2B/calcineurin. The rapamycin·FKBP12 complex does not inhibit T-cell receptor signal transduction or calcineurin activity but rather inhibits interleukin-2-stimulated signal transduction, concomitant with a potent (>95%) and selective inhibition in situ of the p70 S6 kinase (3Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (585) Google Scholar, 4Kuo C.J. Chung J. Fiorentino D.F. Flanagan W.M. Blenis J. Crabtree G.R. Nature. 1992; 358: 70-73Crossref PubMed Scopus (561) Google Scholar), an enzyme critical for the G1 to S transition, at least in some cells (5Lane H.A. Fernandez A. Lamb N.J. Thomas G. Nature. 1993; 363: 170-172Crossref PubMed Scopus (318) Google Scholar, 6Reinhard G. Fernandez A. Lamb N.J.C. Thomas G. EMBO J. 1994; 1: 1557-1565Crossref Scopus (179) Google Scholar). Rapamycin, acting indirectly in situ, causes a partial dephosphorylation and deactivation of p70; the direct target of rapamycin·FKBP12 complexes in situ relevant to the rapamycin-inhibition of the p70 S6 kinase is the protein known variously as RAFT-1/FRAP/RAPT-1 or mTOR.The TOR proteins were first identified in Saccharomyces cerevisiae, where rapamycin (but not FK506) is growth-inhibitory. The TOR proteins are the product of one class of mutant genes that confer resistance to rapamycin-induced growth inhibition as a dominant phenotype (7Kunz J. Henriquez R. Schneider U. Deuter-Reinhard M. Movva N.R. Hall M.N. Cell. 1993; 73: 585-596Abstract Full Text PDF PubMed Scopus (721) Google Scholar, 8Cafferkey R. Young P.R. McLaughlin M.M. Bergsma D.J. Koltin Y. Sathe G.M. Faucette L. Eng W.K. Johnson R.K. Livi G.P. Mol. Cell. Biol. 1993; 13: 6012-6023Crossref PubMed Scopus (256) Google Scholar). The yeast (7Kunz J. Henriquez R. Schneider U. Deuter-Reinhard M. Movva N.R. Hall M.N. Cell. 1993; 73: 585-596Abstract Full Text PDF PubMed Scopus (721) Google Scholar, 8Cafferkey R. Young P.R. McLaughlin M.M. Bergsma D.J. Koltin Y. Sathe G.M. Faucette L. Eng W.K. Johnson R.K. Livi G.P. Mol. Cell. Biol. 1993; 13: 6012-6023Crossref PubMed Scopus (256) Google Scholar, 9Helliwell S.B. Wagner P. Kunz J. Deuter-Reinhard M. Henriquez R. Hall M.N. Mol. Cell. Biol. 1994; 5: 105-118Crossref Scopus (312) Google Scholar) and mammalian TOR proteins (specifically, FRAP (10Brown E.J. Albers M.W. Shin T.B. Ichikawa K. Keith C.T. Lane W.S. Schreiber S.L. Nature. 1994; 369: 756-758Crossref PubMed Scopus (1640) Google Scholar); RAFT-1 (11Sabatini D.M. Erdjument-Bromage H. Lui M. Tempst P. Snyder S.H. Cell. 1994; 78: 35-43Abstract Full Text PDF PubMed Scopus (1205) Google Scholar); RAPT-1 (12Chiu M.I. Katz H. Berlin V. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12574-12578Crossref PubMed Scopus (405) Google Scholar); and mTOR (13Sabers C.J. Martin M.M. Brunn G.J. Williams J.M. Dumont F.J. Wiederrecht G. Abraham R.T. J. Biol. Chem. 1995; 270: 815-822Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar)) are >250-kDa polypeptides that contain at their carboxyl terminus a protein and/or lipid kinase catalytic domain, most closely related to those of the DNA protein kinase and the ATM, MEC1, and Tel1 checkpoint gene products, and somewhat more distantly related to the PI-3 kinases (14Keith C.T. Schreiber S.L. Science. 1995; 270: 50-51Crossref PubMed Scopus (442) Google Scholar). The rapamycin/FKBP12 complex binds directly to TOR, and the TOR mutations that confer rapamycin resistance in situ result in a loss of rapamycin/FKBP12 binding (15Cardenas M.E. Heitman J. EMBO J. 1995; 14: 5892-5907Crossref PubMed Scopus (108) Google Scholar, 16Stan R. McLaughlin M.M. Cafferkey R. Johnson R.K. Rosenberg M. Livi G.P. J. Biol. Chem. 1994; 269: 32027-32030Abstract Full Text PDF PubMed Google Scholar, 17Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (616) Google Scholar). The role of TOR as the rapamycin target responsible for inhibition of p70 S6 kinase has been established by the work of Brown et al. (17Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (616) Google Scholar), who showed that coexpression of p70 with certain mutant TORs, which lack the ability to bind rapamycin/FKBP12 complexes, confers partial resistance to the rapamycin-induced inhibition of coexpressed p70; a further mutation that inactivates the TOR catalytic domain abrogates this rescue of p70.PHAS-1/eIF-4E BP1 is another rapamycin-sensitive protein; this 12-kDa polypeptide binds to the 7-methylguanosine cap-binding protein, eIF-4E, and prevents eIF-4E binding to p220/eIF-4G (18Haghighat A. Mader S. Pause A. Sonenberg N. EMBO J. 1995; 14: 5701-5709Crossref PubMed Scopus (525) Google Scholar). The assembly of eIF-4E with the RNA helicase eIF-4A and the RNA binding protein eIF-4B on the p220 eIF-4G polypeptide creates a functional eIF-4F complex. Mitogens stimulate the phosphorylation of eIF-4E BP1 resulting in its disassociation from eIF-4E; the latter can then interact with p220/eIF-4G (19Sonenberg N. Hershey W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 245-269Google Scholar). Mitogen-stimulated phosphorylation eIF-4E BP1 is potently inhibited by rapamycin (20Alarcon C.M. Cardenas M.E. Heitman J. Genes Dev. 1996; 10: 279-288Crossref PubMed Scopus (35) Google Scholar, 21Beretta L. Gingras A.-C. Suitkein Y.V. Hall M.N. Sonenberg N. EMBO J. 1996; 15: 658-664Crossref PubMed Scopus (601) Google Scholar, 22Azipiazu I. Saltiel A.R. DePaoli-Roach A.A. Lawrence Jr., J.C J. Biol. Chem. 1996; 271: 5033-5039Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 23Diggle T.A. Moule S.K. Avison M.B. Flynn A. Foulstone E.J. Proud C.G. Denton R.M. Biochem J. 1996; 315: 146-162Google Scholar); inasmuch as eIF-4F activity is limiting for the translation of certain mitogen-sensitive mRNAs (e.g. ornithine decarboxylase), it is likely that TOR controls the expression of these mRNA at least in part by regulating eIF-4E BP1 phosphorylation (19Sonenberg N. Hershey W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 245-269Google Scholar). The identity of the kinases operating upstream of eIF-4E BP1 in situ is not known. Although eIF-4E BP1 is phosphorylated by p42 mitogen-activated protein kinase in vitro, the MEK inhibitor PD098059 does not inhibit mitogen-stimulated eIF-4E BP1 phosphorylation. Recombinant eIF-4E BP1 is not a substrate for the p70 S6 kinase in vitro; however, the activity of p70 S6 kinase and the phosphorylation of eIF-4E BP1 respond in a parallel fashion to a variety of inhibitors (wortmannin, SQ20006, cAMP congeners) (20Alarcon C.M. Cardenas M.E. Heitman J. Genes Dev. 1996; 10: 279-288Crossref PubMed Scopus (35) Google Scholar, 21Beretta L. Gingras A.-C. Suitkein Y.V. Hall M.N. Sonenberg N. EMBO J. 1996; 15: 658-664Crossref PubMed Scopus (601) Google Scholar, 22Azipiazu I. Saltiel A.R. DePaoli-Roach A.A. Lawrence Jr., J.C J. Biol. Chem. 1996; 271: 5033-5039Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 23Diggle T.A. Moule S.K. Avison M.B. Flynn A. Foulstone E.J. Proud C.G. Denton R.M. Biochem J. 1996; 315: 146-162Google Scholar). We therefore inquired whether 1) TOR is the rapamycin-sensitive element that regulates eIF-4E BP1 phosphorylation, and if so, whether 2) TOR signals to eIF-4E BP1 through the p70 kinase. We find that, as with p70, a rapamycin-resistant mutant of mTOR (S2035T) substantially overcomes the ability of rapamycin to inhibit the phosphorylation of eIF-4E BP1; in contrast, rapamycin-resistant variants of p70 are unable to rescue eIF-4E BP1 phosphorylation from inhibition by rapamycin. We conclude that TOR regulates the phosphorylation of both eIF-4E BP1 and the p70 S6 kinase; however, p70 is not upstream of eIF-4E BP1 but is regulated in parallel.MATERIALS AND METHODSDulbecco's modified Eagle's minimal essential medium and fetal calf serum were purchased from Sigma. Protein G-agarose was from Life Technologies, Inc. Radioisotopes were obtained from NEN Life Science Products, and ECL reagents from Amersham Corp. The antibodies used were: 12CA5, a monoclonal antibody against influenza virus hemagglutinin (24Field J. Nikawa J.-I. Broek D. MacDonald B. Rogers L. Wilson I.A. Lerner R.A. Wigler M. Mol. Cell. Biol. 1988; 8: 2159-2165Crossref PubMed Scopus (728) Google Scholar); a monoclonal anti-FLAG antibody (M2) (Eastman Kodak Co.); and a polyclonal antiserum raised against a synthetic peptide corresponding to amino acids 337–352 of p70α1 S6 kinase (25Grove J.R. Banerjee P Balasubramanyam A. Coffer P.J. Price D.J. Avruch J. Woodgett J.R. Mol. Cell. Biol. 1991; 11: 5541-5550Crossref PubMed Scopus (147) Google Scholar).cDNA Cloning and ConstructionsA cDNA fragment encoding mammalian TOR was isolated by PCR, using first strand cDNA from mouse brain and degenerate oligonucleotide 5′-GAIGA(C/T)ITI(A/C)GICA(A/G)GA and 5′-ICC(A/G)AAA/GTCIAT(A/G)TG based on two short sequences that are well conserved in the catalytic domains of bovine p110 PI-3 kinase and yeast TOR-1 and TOR-2 ((E/D)D(L/I)RQD and HIDFG). Two major DNA products, 500 and 600 bp, were generated, purified, subcloned into pUC vectors, and subjected to DNA sequencing. The 500-bp product was found to be identical to the kinase domain of human p110β (26Hu P. Mondino A. Skolnik E.Y. Schlessinger J. Mol. Cell. Biol. 1993; 13: 7677-7688Crossref PubMed Scopus (235) Google Scholar), and the 600-bp product was 76% identical to the kinase domain of yeast TOR2. The 600-bp PCR product was used to screen a λ Zap rat brain cDNA library (a gift from Dr. Ivan Gout, Ludwig Institute for Cancer Research, London). A set of overlapping clones were obtained of which the two largest clones contained an open reading frame of 2549 amino acids (289 kDa), 43% identical to yeast TOR-2 and identical in DNA sequence to RAFT-1 (11Sabatini D.M. Erdjument-Bromage H. Lui M. Tempst P. Snyder S.H. Cell. 1994; 78: 35-43Abstract Full Text PDF PubMed Scopus (1205) Google Scholar). To facilitate recovery after transient expression in mammalian cells, the mTOR cDNA was modified by insertion of epitopes at its amino terminus; a 9-amino acid epitope from the influenza virus hemagglutinin (HA) (24Field J. Nikawa J.-I. Broek D. MacDonald B. Rogers L. Wilson I.A. Lerner R.A. Wigler M. Mol. Cell. Biol. 1988; 8: 2159-2165Crossref PubMed Scopus (728) Google Scholar) was introduced immediately after the initiator methionine using PCR, and the HA-tagged mTOR was ligated into the vector pCDNA1 (Invitrogen) to give pCDNAI-HA-mTOR. The mTOR cDNA was also ligated into vector pCMV-FLAG so as to introduce a FLAG epitope at the amino terminus (pCMV-FLAG-mTOR). Mutants of mTOR were constructed by insertion of aKpnI-XbaI fragment, encoding bp 5897–8434, into the M13 vector; site-directed mutagenesis was performed by the Kunkel (27Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4886) Google Scholar) method. Mutagenesis primers for each mutant were as follows: 5′-GTACAAGCGAGTGGCCTCTTC-3′ for Ser2035 → Thr, 5′-GTACAAGCGAGCGGCCTCTTC-3′ for Ser2035 → Ala, 5′-CCCCAAAGTACAAGCGACGGGCCTCTTCTAGGCC-3′ for Ser2035 → Arg, 5′-GGTCCAGCATAAGCTTGGATGGGTGC-3′ for Asn2343 → Lys. After performing mutagenesis, the KpnI-XbaI fragment of pCDNAI-HA-mTOR and pCMV-FLAG-mTOR was replaced by the mutant KpnI-XbaI fragment from M13. Each mutation was verified by DNA sequence analysis.A cDNA encoding PHAS-1/eIF-4E BP1 was isolated by PCR using a human B cell cDNA library as template and the oligonucleotides: 5′-CGCGAATCCATGTCCGGGGGCAGCAGC (forward) and 5′-TGCTCTAGATTAAATGTCCATCTC (reverse). The eIF-4E BP1 cDNA was verified by DNA sequence analysis and inserted into pCMV-FLAG.The cDNA encoding the wild-type α1 p70 S6 kinase and the Δ2–46/ΔCT104 mutant was described in Weng et al. (28Weng Q.-P. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (210) Google Scholar), tagged at the amino terminus with an HA or FLAG epitope, and introduced into the vector pMT2. A cDNA encoding FKBP12 (a gift from G. Crabtree) was ligated into the pGEX vector and expressed inEscherichia coli as a GST fusion protein.Cell Culture and cDNA ExpressionChinese hamster ovary cells overexpressing human insulin receptors (CHO-IR) (29Yonezawa K. Ueda H. Hara K. Nishida K. Ando A. Chavanieu A. Matsuba H. Shii K. Yokono K. Fukui Y. Calas B. Grigorescue F. Dhand R. Gout I. Otsu M. Waterfield M.D. Kasuga M. J. Biol. Chem. 1992; 267: 25958-25966Abstract Full Text PDF PubMed Google Scholar), HEK293, and COS7 cells were maintained and cultured as described previously (28Weng Q.-P. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (210) Google Scholar). Transient expression of the recombinant proteins in mammalian cells (COS7, HEK293, or CHO-IR cells) employed transfection of plasmids by the lipofection method using LipofectAMINE (Life Technologies, Inc.). When multiple plasmids were co-transfected and subjected to different treatments prior to harvest, the cells were split 24 h after transfection, replated on the number of plates appropriate to that experiment, and harvested 24–48 h later.Immunoprecipitation and AutophosphorylationCells were lysed in ice-cold buffer A (50 mm Tris/HCl (pH 8.0), 1% Nonidet P-40, 120 mm NaCl, 20 mm NaF, 1 mm benzamidine, 1 mm EDTA, 6 mmEGTA, 20 mm β-glycerophosphate, 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin), and the extracts were centrifuged at 10,000 × g for 20 min. Aliquots of the supernatants were subjected to immunoprecipitation with monoclonal antibodies. The immunoprecipitates were adsorbed to protein G-agarose beads, washed twice with buffer A containing 0.5m NaCl, and twice with the buffer consisting of 25 mm Hepes (pH 7.6) and 0.1% Nonidet P-40. The autophosphorylation reactions were initiated by addition of 25 mm Hepes (pH 7.6), 50 mm KCl, 10 mmMgCl2, 0.1% Nonidet P-40, 20% glycerol, 1 mmDTT, and 100 μm ATP (6 μCi of [γ-32P]ATP), incubated for 15 min at 30 °C, and terminated by adding SDS sample buffer. The phosphorylated proteins were analyzed by SDS-PAGE and subsequent autoradiography.Immunoblot and p70 S6 Kinase Assayp70 S6 kinase activity was determined in immunoprecipitates by using 40 S ribosomal subunits (25Grove J.R. Banerjee P Balasubramanyam A. Coffer P.J. Price D.J. Avruch J. Woodgett J.R. Mol. Cell. Biol. 1991; 11: 5541-5550Crossref PubMed Scopus (147) Google Scholar, 28Weng Q.-P. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (210) Google Scholar) or a synthetic S6 peptide KRRRLSSLRASTSKSESSQK as substrates. The immobilized immunoprecipitates were washed twice with buffer A containing 0.5 m NaCl, twice with 20 mm MOPS (pH 7.2), 10 mm β-glycerophosphate, 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride. The S6 peptide kinase reaction was initiated by adding the reaction mixture I (25 mm MOPS (pH 7.2), 12 mm MgCl2, 2 mm EGTA, 0.5 mm DTT, 10 mmβ-glycerophosphate, 0.5 μm protein kinase inhibitor, 100 μm ATP(2 μCi of [γ-32P]ATP), and 0.1 mm S6 peptide). The 40 S S6 protein kinase assay used reaction mixture II (50 mm MOPS (pH 7.2), 12 mmMgCl2, 2 mm EGTA, 1 mm DTT, 10 mm β-glycerophosphate, 0.5 μm protein kinase inhibitor, 0.5 A 260 units of 40 S ribosomal subunits, and 60 μm ATP (5 μCi of [γ-32P]ATP). The reaction was continued for 20 min at 30 °C. The S6 peptide kinase assay was terminated by addition of 20 mm EDTA and 1.5 mm adenosine. The reaction mixtures were spotted on P81 phosphocellulose paper, followed by washes in 0.5% phosphoric acid; 32P-labeled peptides were measured by Cerenkov counting. The 40 S kinase assay was terminated by adding SDS sample buffer followed by SDS-PAGE and autoradiography. Immunoblot was performed using the ECL method as described by the manufacturer (Amersham Corp.).In Vitro Binding of mTOR to GST-FKBP12Prokaryotic recombinant GST or GST-FKBP12 (15 μg) were incubated with 15 μl of glutathione-Sepharose beads (60 min at 4 °C), washed three times with 50 mm Hepes (pH 7.6), 0.5 m NaCl, and 0.1% Triton X-100, once with PBS. Immobilized GST fusion proteins were incubated in 1 ml of PBS with or without 500 ng/ml rapamycin; after 1 h at 4 °C, the PBS was discarded, and the cell lysate expressing mTOR was added, with or without 200 ng/ml rapamycin, as indicated in Fig. 2 A. In Fig. 2 B, all GST proteins and lysates contained rapamycin at the concentrations indicated above. COS7 cells or HEK293 cells expressing FLAG- or HA-tagged wild-type or mutant mTOR were extracted, 72 h after transfection, with buffer B (50 mm Tris (pH 7.4), 150 mm NaCl, 1 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, 1% Triton X-100, 300 μl/10-cm plate) and centrifuged at 10,000 × g for 20 min. The supernatants were diluted by adding 700 μl of buffer B without detergent; aliquots from each extract were subjected to immunoprecipitation and immunoblot with anti-FLAG or anti-HA antibody to verify the expression of mTOR variants. The remaining supernatant was incubated with the immobilized GST proteins at 4 °C for 3 h. The beads were washed three times with buffer B containing 0.3% Triton X-100 and 0.5 m NaCl, once with 50 mmHepes (pH 7.6), 150 mm NaCl, 0.1% Triton X-100. In Fig.2 A, the bound proteins were eluted directly into SDS sample buffer, separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with monoclonal anti-FLAG antibody. The GST proteins were identified by Coomassie Blue stain. Alternatively, GST fusion proteins were eluted twice by the incubation in buffer that contained 100 mm Tris (pH 8.0), 150 mm NaCl, 1 mmDTT, and 20 mm glutathione. One-quarter volume of the eluate was subjected to SDS-PAGE on a 14% acrylamide gel, followed by Coomassie Blue staining to verify that the eluted GST proteins were almost equal. Three-quarters volume of the eluates were separated by SDS-PAGE on 6% polyacrylamide gels, transferred to PVDF membranes, and immunoblotted with anti-HA antibody to detect retained HA-TOR polypeptides.RESULTSThe mTOR cDNA was tagged at its amino terminus with an HA or FLAG epitope and expressed transiently after transfection into CHO-IR and COS cells. Anti-epitope immunoblot of anti-HA immunoprecipitates revealed a recombinant polypeptide of the expected size (Fig.1, upper panel). Incubation of the immunoprecipitated HA-mTOR with Mg2+ and [γ-32P]ATP was accompanied by 32P incorporation into the 290-kDa polypeptide (Fig. 1, lower panel, WT). The CHO-IR cells express approximately 106 recombinant human insulin receptors per cell; pretreatment of the cells with insulin (10−7m) for 15 min prior to harvest did not alter the extent ofin vitro phosphorylation of HA-mTOR (Fig. 1, lower panel). Evidence that the 32P incorporation into mTORin vitro reflected mTOR autophosphorylation was obtained by mutation of Asn2343 in the mTOR catalytic domain to Lys. This mutation corresponds in location to one previously reported to cause inactivation of the yeast VPS34 PI kinase (30Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (803) Google Scholar). The mTOR mutant N2343K, which is expressed at levels comparable to wild-type mTOR (Fig.1, upper panel, compare WT to NK), exhibits a complete loss of 32P incorporation from [γ-32P]ATP into the recombinant polypeptide in vitro, whereas mutation of Ser2035 → Arg, which corresponds to the mutation which confers rapamycin resistance to yeast TOR2 (7Kunz J. Henriquez R. Schneider U. Deuter-Reinhard M. Movva N.R. Hall M.N. Cell. 1993; 73: 585-596Abstract Full Text PDF PubMed Scopus (721) Google Scholar, 8Cafferkey R. Young P.R. McLaughlin M.M. Bergsma D.J. Koltin Y. Sathe G.M. Faucette L. Eng W.K. Johnson R.K. Livi G.P. Mol. Cell. Biol. 1993; 13: 6012-6023Crossref PubMed Scopus (256) Google Scholar), does not significantly affect mTOR autophosphorylationin vitro (Fig. 1, lower panel, SR). These results demonstrate that the recombinant wild-type mTOR is expressed and is catalytically active. In agreement with Sabatiniet al. (31Sabatini D.M. Pierchala B.A. Barrow R.K. Schell M.J. Snyder S.H. J. Biol. Chem. 1995; 270: 20875-20878Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), we observe that the recombinant wild-type mTOR, despite an ability to catalyze autophosphorylation, did not catalyze phosphatidylinositol phosphorylation in vitro. In contrast, an antiserum raised to an amino-terminal 100-amino acid fragment of mTOR, which precipitates endogenous mTOR, coprecipitated PI kinase activity (data not shown).Figure 1Expression and in vitroautophosphorylation of mTOR variants. CHO-IR cells were grown in 100-mm dishes to 80% confluence and transfected with 12 μg of pCDNA vector encoding HA-tagged mTOR wild type (WT), Asn2343 → Lys (NK), Ser2035 → Arg (SR), or pCDNA1 vector alone; 24 h later, each plate was split into three 60-mm dishes. At 60 h after transfection, media were replaced with Ham's F-12 medium without fetal calf serum. 72 h after transfection, cells were treated with carrier or insulin (10−7m) for 15 min as indicated. Cell extracts were prepared, and immunoprecipitation was carried out with the anti-HA antibody, 12CA5. One set of immunoprecipitates was subjected to anti-HA immunoblot to verify mTOR expression (upper panel). The immunoprecipitates from control and insulin treated cells were subjected to in vitroautophosphorylation as described under “Materials and Methods”; the32P-labeled immunoprecipitates were subjected to autoradiography after SDS-PAGE (lower panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The ability of recombinant TOR to associate in vitrowith GST-FKBP12 was determined. COS cell extracts containing recombinant FLAG-mTOR were incubated in vitro with purified prokaryotic recombinant GST or GST-FKBP12 immobilized on GSH-Sepharose. Little or no binding of FLAG-mTOR to GST-FKBP12 is detected, unless rapamycin (200 ng/ml) is present (Fig.2 A). Thus recombinant mTOR binds to FKBP12 in vitro in a rapamycin-dependent way. Previous work has defined the FKBP12 binding domain in yeast and mammalian TOR and shown that most mutations at Ser2035 (Ser1972/1975 in yeast TOR 1/2) can block the binding of mTOR to FKBP12/rapamycin (15Cardenas M.E. Heitman J. EMBO J. 1995; 14: 5892-5907Crossref PubMed Scopus (108) Google Scholar, 16Stan R. McLaughlin M.M. Cafferkey R. Johnson R.K. Rosenberg M. Livi G.P. J. Biol. Chem. 1994; 269: 32027-32030Abstract Full Text PDF PubMed Google Scholar, 17Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (616) Google Scholar, 32Chen J. Zheng X-f. Brown E.J. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4947-4951Crossref PubMed Scopus (443) Google Scholar). We observe, consistent with previous reports (17Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (616) Google Scholar, 32Chen J. Zheng X-f. Brown E.J. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4947-4951Crossref PubMed Scopus (443) Google Scholar), that substitution of mTOR Ser2035 by Arg or Thr (Fig. 2 B) results in the complete loss of binding of COS recombinant mTOR to GST-FKBP12·rapamycin in vitro, whereas conversion of mTOR Ser2035 → Ala does not cause detectable impairment of mTOR binding to GST-FKBP12·rapamycin. The catalytically inactive HA-mTOR N2343K mutant also binds to the GST-FKBP12·rapamycin complex to an extent comparable to wild-type HA-mTOR.Inasmuch as the biochemical function of mTOR relevant to its biologic role(s) is not known, we attempted to establish the functionality of recombinant mTOR in situ by its ability to confer resistance toward the rapamycin inhibition of p70 kinase in situ. The activity of recombinant, wild-type HAp70 kinase toward an S6 peptide substrate (and 40 S subunits, see Fig. 5), assayed in vitroafter transient expression in COS cells, is inhibited >95% by pretreatment of cells with rapamycin prior to extraction (>20 nm, 15 min). Half-maximal p70 inhibition is observed at approximately 2 nm (3Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (585) Google Scholar, 28Weng Q.-P. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (210) Google Scholar). Coexpression of p70 with wild-type FLAG-mTOR does not alter p70 activity in vitro as compared with FLAG vector control, 2In five experiments, the activity of p70 coexpressed with wild-type FLAG-mTOR was 93.4 ± 13% that of p70 coexpressed with FLAG vector. nor does the coexpressed wild-type mTOR alter the 95% inhibition induced by a supramaximal concentration of rapamycin (Fig.3). The mTOR mutant S2035A, which is unimpaired in its binding to GST-FKBP12·rapamycin, like wild-type mTOR, is also unable to protect p70 against rapamycin inhibition. In contrast FLAG-mTOR S2035T, despite comparable polypeptide expression to wild-type and S2035A mTOR, does provide substantial protection against rapamycin inhibition of p70 kinase activity. The ability of mTOR S2035T to protect p70 from rapamycin inhibition requires a functional TOR catalytic domain; mutation of mTOR Asn2343 → Lys, which abolishes mTOR autophosphorylation in vitro (Fig. 1), also abolishes the ability of mTOR S2035T to rescue p70 from rapamycin inhibition (Fig. 3). Surprisingly, the mTOR S2035R mutant, which is capable of autophosphorylation i