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Activation of Mammalian Target of Rapamycin (mTOR) by Insulin Is Associated with Stimulation of 4EBP1 Binding to Dimeric mTOR Complex 1

2006; Elsevier BV; Volume: 281; Issue: 34 Linguagem: Inglês

10.1074/jbc.m603566200

ISSN

1083-351X

Autores

Lifu Wang, Christopher J. Rhodes, John C. Lawrence,

Tópico(s)

Polyamine Metabolism and Applications

Resumo

Insulin stimulates protein synthesis by promoting phosphorylation of the eIF4E-binding protein, 4EBP1. This effect is rapamycin-sensitive and mediated by mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a signaling complex containing mTOR, raptor, and mLST8. Here we demonstrate that insulin produces a stable increase in the kinase activity of mTORC1 in 3T3-L1 adipocytes. The response was associated with a marked increase in 4EBP1 binding to raptor in mTORC1, and it was abolished by disrupting the TOR signaling motif in 4EBP1. The stimulatory effects of insulin on both 4EBP1 kinase activity and binding occurred rapidly and at physiological concentrations of insulin, and both effects required an intact mTORC1. Results of experiments involving size exclusion chromatography and coimmunoprecipitation of epitope-tagged subunits provide evidence that the major insulin-responsive form is dimeric mTORC1, a structure containing two heterotrimers of mTOR, raptor, and mLST8. Insulin stimulates protein synthesis by promoting phosphorylation of the eIF4E-binding protein, 4EBP1. This effect is rapamycin-sensitive and mediated by mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a signaling complex containing mTOR, raptor, and mLST8. Here we demonstrate that insulin produces a stable increase in the kinase activity of mTORC1 in 3T3-L1 adipocytes. The response was associated with a marked increase in 4EBP1 binding to raptor in mTORC1, and it was abolished by disrupting the TOR signaling motif in 4EBP1. The stimulatory effects of insulin on both 4EBP1 kinase activity and binding occurred rapidly and at physiological concentrations of insulin, and both effects required an intact mTORC1. Results of experiments involving size exclusion chromatography and coimmunoprecipitation of epitope-tagged subunits provide evidence that the major insulin-responsive form is dimeric mTORC1, a structure containing two heterotrimers of mTOR, raptor, and mLST8. As the major anabolic hormone in mammals, insulin stimulates protein synthesis in a wide variety of cell types. This response is mediated in part by mTOR, 2The abbreviations used are: mTOR, mammalian target of rapamycin; C-Rap Ab, antibody to the COOH-terminal region of raptor; eIF3, eIF4E, and eIF4G, eukaryotic initiation factors 3, 4E, and 4G, respectively; FKBP12, FK506-binding protein of Mr = 12,000; GST, glutathione S-transferase; mTAb2, mTOR antibody 2; mTORC1 and mTORC2, mTOR complex 1 and 2; TOS, TOR signaling; HA, hemagglutinin; ERK, extracellular signal-regulated kinase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. a phosphatidylinositol 3-kinase-related protein kinase that controls the phosphorylation of multiple factors involved in the control of cell growth and proliferation (1Martin D.E. Hall M.N. Curr. Opin. Cell Biol. 2005; 17: 158-166Crossref PubMed Scopus (452) Google Scholar, 2Harris T.E. Lawrence Jr., J.C. Sci. STKE. 2003; 2003: re15PubMed Google Scholar). mTOR functions in two signaling complexes, mTORC1 and mTORC2 (1Martin D.E. Hall M.N. Curr. Opin. Cell Biol. 2005; 17: 158-166Crossref PubMed Scopus (452) Google Scholar, 3Sarbassov D.D. Ali S.M. Sabatini D.M. Curr. Opin. Cell Biol. 2005; 17: 596-603Crossref PubMed Scopus (1346) Google Scholar). Both complexes contain mTOR and mLST8 (also known as GβL), a protein homologous to β subunits of heterotrimeric G proteins (4Kim D.H. Sarbassov D.D. Ali S.M. Latek R.R. Guntur K.V. Erdjument-Bromage H. Tempst P. Sabatini D.M. Mol. Cell. 2003; 11: 895-904Abstract Full Text Full Text PDF PubMed Scopus (803) Google Scholar, 5Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1515) Google Scholar). One defining feature of the complexes is the third subunit, either raptor in mTORC1 (5Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1515) Google Scholar, 6Hara K. Maruki Y. Long X.M. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1495) Google Scholar, 7Kim D.H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2438) Google Scholar) or rictor (also known as mAVO3) in mTORC2 (8Jacinto E. Loewith R. Schmidt A. Lin S. Ruegg M.A. Hall A. Hall M.N. Nat. Cell Biol. 2004; 6: 1122-1128Crossref PubMed Scopus (1734) Google Scholar, 9Sarbassov D.D. Ali S.M. Kim D.H. Guertin D.A. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Curr. Biol. 2004; 14: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (2232) Google Scholar). 4EBP1 (also known as PHAS-I) is an important target of mTOR signaling. 4EBP1 binds eIF4E, the mRNA cap-binding protein, and it represses cap-dependent translation by competitively blocking the binding of eIF4G to eIF4E (2Harris T.E. Lawrence Jr., J.C. Sci. STKE. 2003; 2003: re15PubMed Google Scholar, 10Gingras A.-C. Raught B. Sonenberg N. Annu. Rev. Biochem. 1999; 68: 913-963Crossref PubMed Scopus (1784) Google Scholar). Activating mTOR with insulin stimulates the phosphorylation of 4EBP1 in four sites (11Fadden P. Haystead T.A.J. Lawrence Jr., J.C. J. Biol. Chem. 1997; 272: 10240-10247Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 12Mothe-Satney I. Brunn G.J. McMahon L.P. Capaldo C.T. Abraham R.T. Lawrence Jr., J.C. J. Biol. Chem. 2000; 275: 33836-33843Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), including Thr-36 and Thr-45, the two sites preferred by mTOR in vitro (13Burnett P.E. Barrow R.K. Cohen N.A. Snyder S.H. Sabatini D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1432-1437Crossref PubMed Scopus (961) Google Scholar, 14McMahon L.P. Choi K.M. Lin T.A. Abraham R.T. Lawrence J.C. Mol. Cell. Biol. 2002; 22: 7428-7438Crossref PubMed Scopus (85) Google Scholar), causing 4EBP1 to dissociate from eIF4E. This allows eIF4E to engage eIF4G, a scaffolding protein that binds eIF3 and eIF4A (10Gingras A.-C. Raught B. Sonenberg N. Annu. Rev. Biochem. 1999; 68: 913-963Crossref PubMed Scopus (1784) Google Scholar, 15Keiper B.D. Gan W. Rhoads R.E. Int. J. Biochem. Cell Biol. 1999; 31: 37-41Crossref PubMed Scopus (58) Google Scholar). eIF3 is a complex initiation factor that binds the small ribosomal subunit and several key initiation factors, and eIF4A is a helicase that unwinds mRNA to facilitate binding and/or scanning by the 40 S ribosomal subunit (10Gingras A.-C. Raught B. Sonenberg N. Annu. Rev. Biochem. 1999; 68: 913-963Crossref PubMed Scopus (1784) Google Scholar, 15Keiper B.D. Gan W. Rhoads R.E. Int. J. Biochem. Cell Biol. 1999; 31: 37-41Crossref PubMed Scopus (58) Google Scholar). Thus, the phosphorylation of 4EBP1 leads to the recruitment of the small ribosomal subunit and important initiation factors to the 5′-end of the message to begin the processes of scanning and selection of the start codon. The finding that the effects of insulin and insulin-like growth factor 1 on 4EBP1 were attenuated by rapamycin provided the first evidence that mTOR controlled 4EBP1 (16Graves L.M. Bornfeldt K.E. Argast G.M. Krebs E.G. Kong X. Lin T.-A. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7222-7226Crossref PubMed Scopus (210) Google Scholar, 17Lin T.-A. Kong X. Saltiel A.R. Blackshear P.J. Lawrence Jr., J.C. J. Biol. Chem. 1995; 270: 18531-18538Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Because rapamycin inhibits mTORC1 but not mTORC2 (8Jacinto E. Loewith R. Schmidt A. Lin S. Ruegg M.A. Hall A. Hall M.N. Nat. Cell Biol. 2004; 6: 1122-1128Crossref PubMed Scopus (1734) Google Scholar, 9Sarbassov D.D. Ali S.M. Kim D.H. Guertin D.A. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Curr. Biol. 2004; 14: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (2232) Google Scholar), the sensitivity to rapamycin also implicates mTORC1. The functions of the mTORC1 subunits are not fully understood. mLST8, which consists almost entirely of seven WD40 repeats, binds near the catalytic domain of mTOR and is required for the full activity of the mTOR kinase (4Kim D.H. Sarbassov D.D. Ali S.M. Latek R.R. Guntur K.V. Erdjument-Bromage H. Tempst P. Sabatini D.M. Mol. Cell. 2003; 11: 895-904Abstract Full Text Full Text PDF PubMed Scopus (803) Google Scholar). Raptor possesses a unique NH2-terminal region followed by three HEAT motifs and seven WD40 repeats that are believed to mediate protein-protein interactions (7Kim D.H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2438) Google Scholar). Raptor binds the mTOR substrates, 4EBP1 and S6K1, and it has been suggested that raptor might function to present substrates to mTOR for phosphorylation (6Hara K. Maruki Y. Long X.M. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1495) Google Scholar). 4EBP1 can be readily phosphorylated in vitro by mTORC1 (6Hara K. Maruki Y. Long X.M. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1495) Google Scholar) but not by mTORC2, which lacks raptor (9Sarbassov D.D. Ali S.M. Kim D.H. Guertin D.A. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Curr. Biol. 2004; 14: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (2232) Google Scholar). The substrate interactions with raptor are mediated by TOR signaling (TOS) motifs (18Beugnet A. Wang X. Proud C.G. J. Biol. Chem. 2003; 278: 40717-40722Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 19Schalm S.S. Fingar D.C. Sabatini D.M. Blenis J. Curr. Biol. 2003; 13: 797-806Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 20Nojima 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 (536) Google Scholar, 21Schalm S.S. Blenis J. Curr. Biol. 2002; 12: 632-639Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 22Choi K.M. McMahon L.P. Lawrence Jr., J.C. J. Biol. Chem. 2003; 278: 19667-19673Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In 4EBP1 this motif is formed by the COOH-terminal five amino acids (FEMDI) (21Schalm S.S. Blenis J. Curr. Biol. 2002; 12: 632-639Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). Disrupting the TOS motif by a Phe → Ala point mutation markedly decreases phosphorylation of the protein in response to activation by mTOR signaling in cells (21Schalm S.S. Blenis J. Curr. Biol. 2002; 12: 632-639Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar) and by mTORC1 in vitro (20Nojima 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 (536) Google Scholar, 22Choi K.M. McMahon L.P. Lawrence Jr., J.C. J. Biol. Chem. 2003; 278: 19667-19673Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Incubating cells with insulin (23Scott P.H. Brunn G.J. Kohn A.D. Roth R.A. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7772-7777Crossref PubMed Scopus (417) Google Scholar), serum (13Burnett P.E. Barrow R.K. Cohen N.A. Snyder S.H. Sabatini D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1432-1437Crossref PubMed Scopus (961) Google Scholar), or certain growth factors (24Sekulie A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. Cancer Res. 2000; 60: 3504-3513PubMed Google Scholar, 25Yokogami K. Wakisaka S. Avruch J. Reeves S.A. Curr. Biol. 2000; 10: 47-50Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar) has been reported to increase the protein kinase activity of mTOR. However, such changes in mTOR activity have not been detected in other studies, and the conclusion that insulin produces a stable increase in the kinase activity of mTOR is controversial. Previous studies of insulin action on mTOR activity in vitro have not discriminated between the two mTOR signaling complexes, and in some cases the conditions used to extract mTOR would have disrupted mTORC1. Because mTORC1 mediates the effects of insulin on the phosphorylation of 4EBP1 in cells, we conducted experiments to measure mTORC1 activity and the interaction of mTORC1 with 4EBP1. Adipocyte Culture and Extract Preparation—3T3-L1 fibroblasts were grown in Dulbecco's modified Eagle's medium containing 10% newborn calf serum. Fibroblasts were converted to adipocytes by using differentiation medium as described previously (17Lin T.-A. Kong X. Saltiel A.R. Blackshear P.J. Lawrence Jr., J.C. J. Biol. Chem. 1995; 270: 18531-18538Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum for 10–12 days after adding the differentiation medium. For experiments, the culture medium was replaced with a solution containing 145 mm NaCl, 5.4 mm KCl, 1.4 mm CaCl2, 1.4 mm MgSO4, 25 mm NaHCO3, 5 mm glucose, 5 mg/ml bovine serum albumin, 0.2 mm sodium phosphate, and 10 mm HEPES, pH 7.4. The cells were incubated at 37 °C without or with a maximally effective concentration of insulin (0.6 μm) and/or other additions. To terminate the incubation the adipocytes were rinsed once with chilled phosphate-buffered saline (145 mm NaCl, 5.4 mm KCl, and 10 mm sodium phosphate, pH 7.4) and then homogenized (0.8 ml of buffer/10-cm-diameter dish) in a glass tissue grinder with a Teflon pestle driven at 1,000 rpm. Homogenization Buffer was composed of Buffer A supplemented with 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml pepstatin, and 0.5 μm microcystin. Buffer A contained 50 mm NaF, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.1% Tween 20 (unless otherwise indicated), 10 mm sodium phosphate, and 50 mm β-glycerophosphate, pH 7.4. Homogenates were centrifuged at 12,000 × g for 10 min, and the supernatants were retained for analyses. Antibodies—Antibodies to the COOH-terminal region of 4EBP1 (26Hu C. Pang S. Kong X. Velleca M. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3730-3734Crossref PubMed Scopus (128) Google Scholar) and the phosphospecific antibodies to the Thr-36 and Thr-45 sites (12Mothe-Satney I. Brunn G.J. McMahon L.P. Capaldo C.T. Abraham R.T. Lawrence Jr., J.C. J. Biol. Chem. 2000; 275: 33836-33843Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) have been described previously (12Mothe-Satney I. Brunn G.J. McMahon L.P. Capaldo C.T. Abraham R.T. Lawrence Jr., J.C. J. Biol. Chem. 2000; 275: 33836-33843Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 26Hu C. Pang S. Kong X. Velleca M. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3730-3734Crossref PubMed Scopus (128) Google Scholar). The 4EBP1 antibodies bind wild type 4EBP1 and F113A equally well (22Choi K.M. McMahon L.P. Lawrence Jr., J.C. J. Biol. Chem. 2003; 278: 19667-19673Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and the phosphospecific antibodies bind 4EBP1 phosphorylated in either Thr-36 or Thr-45 (12Mothe-Satney I. Brunn G.J. McMahon L.P. Capaldo C.T. Abraham R.T. Lawrence Jr., J.C. J. Biol. Chem. 2000; 275: 33836-33843Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) as the amino acid sequences surrounding these sites are almost identical. The mTOR antibodies, mTAb1 and mTAb2, were described previously (27Brunn G.J. Fadden P. Haystead T.A.J. Lawrence Jr., J.C. J. Biol. Chem. 1997; 272: 32547-32550Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Antibodies (designated N-Rap Ab) to the region in raptor (amino acids 36–53) originally targeted by Kim et al. (7Kim D.H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2438) Google Scholar) were generated as described previously (22Choi K.M. McMahon L.P. Lawrence Jr., J.C. J. Biol. Chem. 2003; 278: 19667-19673Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). mTAb2 and N-Rap Ab were used to detect mTOR and raptor, respectively, by immunoblotting. mLST8 antibodies were described previously (28McMahon L.P. Yue W. Santen R.J. Lawrence Jr., J.C. Mol. Endocrinol. 2005; 19: 175-183Crossref PubMed Scopus (50) Google Scholar) as were phosphospecific antibodies to the Ser-2448 site in mTOR (29Reynolds T.H. Bodine S.C. Lawrence Jr., J.C. J. Biol. Chem. 2002; 277: 17657-17662Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). In pilot experiments we attempted to immunoprecipitate mTORC1 by using N-Rap Ab. Although raptor was readily detected in immunoprecipitates obtained with this antibody, neither mTOR nor mLST8 were found (results not presented). Thus, N-Rap Ab either promoted dissociation of mTOR and raptor or was unable to bind raptor associated with mTOR. To generate raptor antibodies that could be used to immunoprecipitate mTORC1, a peptide having an NH2-terminal Cys followed by 12 amino acids (YISVYSVEKRVR) corresponding to the COOH-terminal region of raptor was coupled to keyhole limpet hemocyanin, and the conjugate was used to immunize rabbits. The resulting raptor antibodies (C-Rap Ab) were purified using a column containing an affinity resin prepared by coupling the peptide to Sulfolink beads (Pierce). Rictor antibodies were generated in a similar manner except that a peptide (CRHSPDTAEGQLKEDRE) based on amino acids 263–278 in mouse rictor was used. Monoclonal antibody 12CA5, which recognizes the HA epitope tag, was purified from hybridoma culture medium. Phosphospecific antibodies to the Ser-473 site in Akt2, the Thr-389 site in S6K1, and the activating sites in the ERK1 and ERK2 isoforms of mitogen-activated protein kinase were from Cell Signaling Technology Inc. Purification of Recombinant Proteins—His-tagged forms of wild type 4EBP1 and a 4EBP1 protein having Ala at position 113 (F113A) were expressed in bacteria and purified as described previously (22Choi K.M. McMahon L.P. Lawrence Jr., J.C. J. Biol. Chem. 2003; 278: 19667-19673Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). To assess purity and to confirm protein concentrations, samples were subjected to SDS-PAGE and then stained with Coomassie Blue (22Choi K.M. McMahon L.P. Lawrence Jr., J.C. J. Biol. Chem. 2003; 278: 19667-19673Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Complexes of eIF4E bound to His-tagged forms of either 4EBP1 or 4EBP2 were purified as described previously (30Lin T.A. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 30199-30204Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Glutathione S-transferase (GST)-FKBP12 was prepared as described previously (31Sabers 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 (735) Google Scholar). Immunoprecipitation of mTORC1—Two strategies were used to recover mTORC1. In the first, the raptor subunit of the endogenous complex was targeted by using C-Rap Ab. In the other, an HA antibody was used to capture mTORC1 containing an epitope-tagged raptor that had been overexpressed in the adipocytes. Several antibodies to mTOR, raptor, and mLST8 proved unsuitable either because they activated mTOR or because they failed to immunoprecipitate mTORC1 (results not shown). Adipocyte extract samples (800 μl) were incubated with C-Rap Ab (2 μg) bound to protein A-agarose beads (15 μl) or with 12CA5 (2 μg) bound to protein G-agarose beads (15 μl) at 4 °C for 12 h with constant mixing. As a control for specificity, rabbit or mouse nonimmune IgG was substituted for the C-Rap Ab or 12CA5, respectively. The beads were then washed once with 1 ml of Buffer A, once with 1 ml of Buffer A plus 0.5 mm NaCl, and then twice with 1 ml of Buffer A. Expression of HA-raptor in 3T3-L1 Adipocytes by Adenoviral Mediated Gene Transfer—Virus for expressing HA-raptor was prepared using the system developed by He et al. (32He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3276) Google Scholar). Briefly cDNA encoding HA-tagged raptor was excised with KpnI and NotI from the pBluescript construct described previously (22Choi K.M. McMahon L.P. Lawrence Jr., J.C. J. Biol. Chem. 2003; 278: 19667-19673Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and inserted between the KpnI and NotI sites in the shuttle vector, pAdTrack. The resulting plasmid was cotransformed with the adenoviral backbone plasmid, pAdEasy-1, into BJ5183 bacteria. Recombined plasmid was selected and transfected into human embryonic kidney 293 cells to generate virus, which was amplified and then purified by CsCl gradient centrifugation to create a high titer viral stock. 3T3-L1 adipocytes were infected essentially as described by Kasuga and co-workers (33Kotani K. Ogawa W. Matsumoto M. Kitamura T. Sakaue H. Hino Y. Miyake K. Sano W. Akimoto K. Ohno S. Kasuga M. Mol. Cell. Biol. 1998; 18: 6971-6982Crossref PubMed Google Scholar). The efficiency of infection judged by expression of green fluorescent protein, which is also encoded by the HA-raptor virus, was ∼50%. Virus encoding β-galactosidase was used as a control. Expression Constructs for mTOR, Raptor, and mLST8—Most of the expression constructs used have been described previously (14McMahon L.P. Choi K.M. Lin T.A. Abraham R.T. Lawrence J.C. Mol. Cell. Biol. 2002; 22: 7428-7438Crossref PubMed Scopus (85) Google Scholar, 22Choi K.M. McMahon L.P. Lawrence Jr., J.C. J. Biol. Chem. 2003; 278: 19667-19673Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 28McMahon L.P. Yue W. Santen R.J. Lawrence Jr., J.C. Mol. Endocrinol. 2005; 19: 175-183Crossref PubMed Scopus (50) Google Scholar). To generate a construct for expressing Myc-raptor, cDNA encoding raptor was excised from an HA-raptor-pcDNA3 vector (22Choi K.M. McMahon L.P. Lawrence Jr., J.C. J. Biol. Chem. 2003; 278: 19667-19673Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) by using EcoRI and inserted into the EcoRI site in pCMVTag3B (Stratagene). The construct for expressing untagged mLST8 was prepared by excising mLST8 cDNA from the HA-mLST8 vector (28McMahon L.P. Yue W. Santen R.J. Lawrence Jr., J.C. Mol. Endocrinol. 2005; 19: 175-183Crossref PubMed Scopus (50) Google Scholar) and then inserting the mLST8 fragment into pcDNA3. To generate vector for expressing FLAG-mTOR, a fragment corresponding to bp 1–632 of the mTOR coding region was amplified by PCR with AU1-mTOR pcDNA3 template and the following primers: 5′-GGCGGATCCCACCATGCTTGGGACAGGCCCTG-3′ and 5′-GGCAGTCGACTCTAGAGCCACAGCTCCTTCACGGATG-3′. The fragment was digested with BamHI and SalI, and the product was inserted between the BamHI and SalI sites in pCMVTag2A (Stratagene) to generate NT-pCMVTag2A. To complete construction of the FLAG-mTOR expression construct (mTOR-pCMVTag2A), EcoRI and XbaI were used to excise a fragment from AU1-mTOR pcDNA3 that was inserted between the EcoRI (which cuts in the mTOR coding region in NT-pCMVTag2A) and XbaI sites in the NT-pCMVTag2A construct. The region of the FLAG-mTOR cDNA generated by PCR was sequenced and found to be free of errors. Immune Complex Assay of mTORC1 Activity—Immune complex beads were rinsed with 1 ml of Buffer B (50 mm NaCl, 0.1 mm EGTA, 1 mm dithiothreitol, 0.5 μm microcystin LR, 10 mm HEPES, and 50 mm β-glycerophosphate, pH 7.4) and suspended in 60 μl of Buffer B. After removing a sample for immunoblotting mTOR and raptor, the kinase reactions were initiated by adding to 20 μl of the suspension 5 μl of Buffer A supplemented with 0.5 mm [γ-32P]ATP (PerkinElmer Life Sciences, 1,000 mCi/mmol), 50 mm MnCl2, and 1 μg of the His-tagged form of either wild type 4EBP1 or F113A. Unless otherwise stated, reactions were terminated after 30 min at 30 °C by adding SDS sample buffer. The relative amounts of 32P incorporated into the 4EBP1 proteins were determined by phosphorimaging following SDS-PAGE. Measurements under these conditions reflect the initial rate of phosphorylation as less than 5% of the available substrates were phosphorylated, and the reactions proceed linearly for 60 min (see later, Fig. 2b). 4EBP1 Binding to C-Rap Ab Immune Complexes—Immune complexes from 400 μl of extract (7.5 μl of beads) were suspended in Buffer A (500 μl) containing 50 ng of the His-tagged form of either 4EBP1 or F113A. After incubating at 21 °C for 1 h with constant mixing, the beads were washed four times with 1 ml of Buffer A, twice with Buffer A plus 0.5 m NaCl, and then once with a solution containing 1 mm EDTA, 1 mm EDTA, and 50 mm Tris-HCl, pH 7.4. The relative amounts of the 4EBP1 proteins retained by the beads were determined by immunoblotting. Binding of Raptor to 4EBP1 Affinity Resins—His-tagged 4EBP1 or F113A proteins (1 mg) were coupled to CNBr-activated Sepharose (86 mg) in 0.5 m NaCl and 0.1 m NaHCO3 (pH 8.3). After 12 h at 4 °C, the resins were washed exhaustively as directed by the supplier (Amersham Biosciences). The beads were then suspended in 0.5 ml of buffer (0.5 m NaCl and 50 mm Tris-HCl, pH 7.4) and stored at 4 °C prior to use. For binding, an aliquot (15 μl) of the beads was added to 800 μl of extract. After incubating at 4 °C for 12 h with constant mixing, the beads were washed as described above for 4EBP1 binding to C-Rap Ab immune complexes. The relative amounts of raptor and mTOR retained by the beads were determined by immunoblotting. Electrophoretic Analyses—SDS-PAGE and immunoblotting were conducted as described previously (12Mothe-Satney I. Brunn G.J. McMahon L.P. Capaldo C.T. Abraham R.T. Lawrence Jr., J.C. J. Biol. Chem. 2000; 275: 33836-33843Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Binding of primary antibodies was monitored by using the appropriate alkaline phosphatase-conjugated secondary antibodies, which were detected by using CDP-Star reagent (PerkinElmer Life Sciences). Relative signal intensities of bands in immunoblots were determined by scanning laser densitometry of x-ray films or by using a Fujifilm LAS 3000 LCD camera system. Gel Filtration—Adipocytes were incubated without or with insulin, rinsed three times with Buffer A (minus detergents and dithiothreitol) that had been chilled on ice, and scraped from the dishes (six 10-cm-diameter dishes per treatment). To enhance resolution by the column, the volume of extract was minimized by homogenizing cells in 500 μl of Homogenization Buffer. The homogenates were centrifuged at 12,000 × g for 10 min. The supernatants were retained and passed through a 0.45-μm filter. Extract samples (350 μl) were applied to a Superose 6 HR 10/30 column (Amersham Biosciences) that had been equilibrated in Buffer A. The flow rate was maintained at 0.2 ml/min, and 1-ml fractions were collected. Other Materials—Recombinant human insulin (Novolin R) was from Novo Nordisk. Rapamycin, LY294002, and U0126 were from Calbiochem-Novabiochem. Epidermal growth factor was from Upstate Biologicals. Fibroblast growth factor-1 was provided by Dr. David Ornitz (Washington University). Farnesylthiosalicylic acid was provided by Dr. Wayne Bardin (Thyreos, New York, NY). Tween 20 was from Fischer. CHAPS was from Roche Applied Science. Caffeine, insulin-like growth factor 1, Nonidet P-40, Triton X-100, and wortmannin were from Sigma. Stable Activation of mTORC1—mTORC1 was immunoprecipitated from extracts of 3T3-L1 adipocytes by using an antibody (C-Rap Ab) to the COOH-terminal region of raptor (Fig. 1A). Raptor, mTOR, and mLST8 were detected in the immune complexes, confirming that intact mTORC1 was recovered (Fig. 1B). To measure mTORC1 activity, immune complexes were incubated with [γ-32P]ATP and 4EBP1. Treating adipocytes with insulin increased 4EBP1 phosphorylation by mTORC1 (Fig. 1B). No activity was detected in complexes isolated with nonimmune IgG. The 3-fold increase in mTORC1 activity produced by insulin (Fig. 1B) is comparable to the increase in phosphorylation of endogenous 4EBP1 when 32P-labeled 3T3-L1 adipocytes are incubated with the hormone (17Lin T.-A. Kong X. Saltiel A.R. Blackshear P.J. Lawrence Jr., J.C. J. Biol. Chem. 1995; 270: 18531-18538Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). mTORC1 isolated with C-Rap Ab was not able to phosphorylate F113A (Fig. 1C), a 4EBP1 protein having a point mutation that disrupts the TOS motif (22Choi K.M. McMahon L.P. Lawrence Jr., J.C. J. Biol. Chem. 2003; 278: 19667-19673Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In the present experiments, 3T3-L1 adipocytes were incubated in buffer lacking amino acids to isolate the effects due to insulin from those mediated by amino acids, which also activate mTOR signaling (34Kimball S.R. Jefferson L.S. J. Nutr. 2006; 136: 227S-231SCrossref PubMed Google Scholar). Activating mTOR signaling with insulin has been shown previously to increase 4EBP1 phosphorylation in 3T3-L1 adipocytes incubated in buffer without added amino acids (11Fadden P. Haystead T.A.J. Lawrence Jr., J.C. J. Biol. Chem. 1997; 272: 10240-10247Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 17Lin T.-A. Kong X. Saltiel A.R. Blackshear P.J. Lawrence Jr., J.C. J. Biol. Chem. 1995; 270: 18531-18538Abstrac

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