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• W1668086213 abstract "The insulin-signaling pathway leading to the activation of Akt/protein kinase B has been well characterized except for a single step, the phosphorylation of Akt at Ser-473. Double-stranded DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) gene product, integrin-linked kinase (ILK), protein kinase Cα (PKCα), and mammalian target of rapamycin (mTOR), when complexed to rapamycin-insensitive companion of mTOR (RICTOR), have all been identified as playing a critical role in Akt Ser-473 phosphorylation. However, the apparently disparate results reported in these studies are difficult to evaluate, given that different stimuli and cell types were examined and that all of the candidate proteins have never been systematically studied in a single system. Additionally, none of these studies were performed in a classical insulin-responsive cell type or tissue such as muscle or fat. We therefore examined each of these candidates in 3T3-L1 adipocytes. In vitro kinase assays, using different subcellular fractions of 3T3-L1 adipocytes, revealed that phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 phosphorylation correlated well with the amount of DNA-PK, mTOR, and RICTOR but did not correlate with levels of ATM, ILK, and PKCα. PKCα was completely absent from compartments with Ser-473 phosphorylation activity. Although purified DNA-PK could phosphorylate a peptide derived from Akt that contains amino acid Ser-473, it could not phosphorylate full-length Akt2. Vesicles immunoprecipitated from low density microsomes using antibodies directed against mTOR or RICTOR had phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 activity that was sensitive to wortmannin but not staurosporine. In contrast, immunopurified low density microsome vesicles containing ILK could not phosphorylate Akt on Ser-473 in vitro. Small interference RNA knockdown of RICTOR, but not DNA-PK, ATM, or ILK, suppressed insulin-activated Ser-473 phosphorylation and, to a lesser extent, Thr-308 phosphorylation in 3T3-L1 adipocytes. Based on our cell-free kinase and small interference RNA results, we conclude that mTOR complexed to RICTOR is the Ser-473 kinase in 3T3-L1 adipocytes. The insulin-signaling pathway leading to the activation of Akt/protein kinase B has been well characterized except for a single step, the phosphorylation of Akt at Ser-473. Double-stranded DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) gene product, integrin-linked kinase (ILK), protein kinase Cα (PKCα), and mammalian target of rapamycin (mTOR), when complexed to rapamycin-insensitive companion of mTOR (RICTOR), have all been identified as playing a critical role in Akt Ser-473 phosphorylation. However, the apparently disparate results reported in these studies are difficult to evaluate, given that different stimuli and cell types were examined and that all of the candidate proteins have never been systematically studied in a single system. Additionally, none of these studies were performed in a classical insulin-responsive cell type or tissue such as muscle or fat. We therefore examined each of these candidates in 3T3-L1 adipocytes. In vitro kinase assays, using different subcellular fractions of 3T3-L1 adipocytes, revealed that phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 phosphorylation correlated well with the amount of DNA-PK, mTOR, and RICTOR but did not correlate with levels of ATM, ILK, and PKCα. PKCα was completely absent from compartments with Ser-473 phosphorylation activity. Although purified DNA-PK could phosphorylate a peptide derived from Akt that contains amino acid Ser-473, it could not phosphorylate full-length Akt2. Vesicles immunoprecipitated from low density microsomes using antibodies directed against mTOR or RICTOR had phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 activity that was sensitive to wortmannin but not staurosporine. In contrast, immunopurified low density microsome vesicles containing ILK could not phosphorylate Akt on Ser-473 in vitro. Small interference RNA knockdown of RICTOR, but not DNA-PK, ATM, or ILK, suppressed insulin-activated Ser-473 phosphorylation and, to a lesser extent, Thr-308 phosphorylation in 3T3-L1 adipocytes. Based on our cell-free kinase and small interference RNA results, we conclude that mTOR complexed to RICTOR is the Ser-473 kinase in 3T3-L1 adipocytes. Insulin increases glucose transport in muscle and fat by stimulating the translocation of the glucose transporter isoform Glut 4 from intracellular storage pools to the cell surface (1Cushman S.W. Wardzala L.J. J. Biol. Chem. 1980; 255: 4758-4762Abstract Full Text PDF PubMed Google Scholar, 2Suzuki K. Kono T. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2542-2545Crossref PubMed Scopus (768) Google Scholar), a process that requires the activation of the serine/threonine kinase Akt (protein kinase B) (3Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar, 4Hill M.M. Clark S.F. Tucker D.F. Birnbaum M.J. James D.E. Macaulay S.L. Mol. Cell. Biol. 1999; 19: 7771-7781Crossref PubMed Google Scholar, 5Bae S.S. Cho H. Mu J. Birnbaum M.J. J. Biol. Chem. 2003; 278: 49530-49536Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 6Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7569-7574Crossref PubMed Scopus (303) Google Scholar, 7Zeigerer A. McBrayer M.K. McGraw T.E. Mol. Biol. Cell. 2004; 15: 4406-4415Crossref PubMed Scopus (188) Google Scholar, 8Welsh G.I. Hers I. Berwick D.C. Wherlock M. Birkin R. Leney S. Tavare J.M. Biochem. Soc. Trans. 2005; 33: 346-349Crossref PubMed Scopus (106) Google Scholar). Although the molecular events that occur after Akt activation are poorly understood, the early insulin signaling pathway has been well characterized except for one step, the phosphorylation of Akt at Ser-473. The signaling cascade is initiated when insulin binds to specific receptors at the plasma membrane (PM), 2The abbreviations used are: PMplasma membraneATMataxia telangiectasia mutated gene productCYTcytosolDNA-PKdouble-stranded DNA-dependent protein kinaseExtextractHDMhigh density microsome(s)HiPhigh speed pelletHMD-PKhydrophobic motif domain protein kinaseILKintegrin-linked kinaseLDMlow density microsomesmTORmammalian target of rapamycinNUCnuclearPIP3phosphatidylinositol 3,4,5-trisphosphatePKCprotein kinase CRAPTORregulatory associated protein of mTORRICTORrapamycin-insensitive companion of mTORsiRNAsmall interference RNAIRSinsulin receptor substratePIphosphoinositideHMhydrophobic motifPHpleckstrin homologyCHAPS3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid.2The abbreviations used are: PMplasma membraneATMataxia telangiectasia mutated gene productCYTcytosolDNA-PKdouble-stranded DNA-dependent protein kinaseExtextractHDMhigh density microsome(s)HiPhigh speed pelletHMD-PKhydrophobic motif domain protein kinaseILKintegrin-linked kinaseLDMlow density microsomesmTORmammalian target of rapamycinNUCnuclearPIP3phosphatidylinositol 3,4,5-trisphosphatePKCprotein kinase CRAPTORregulatory associated protein of mTORRICTORrapamycin-insensitive companion of mTORsiRNAsmall interference RNAIRSinsulin receptor substratePIphosphoinositideHMhydrophobic motifPHpleckstrin homologyCHAPS3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid. triggering the autophosphorylation of several critical intracellular tyrosine residues and thereby activating an intrinsic tyrosine kinase that can phosphorylate cellular substrates, most notably the insulin receptor substrate (IRS) proteins (9Saltiel A.R. Kahn C.R. Nature. 2001; 414: 799-806Crossref PubMed Scopus (3842) Google Scholar, 10White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 11White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Crossref PubMed Scopus (623) Google Scholar). Tyrosine-phosphorylated IRS proteins can recruit and activate phosphoinositide 3-kinase (PI 3-kinase), which generates phosphatidylinositol 3,4,5-trisphosphate (PIP3), using inositol-containing phospholipids in the PM as substrates (12Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (835) Google Scholar). The activity of Akt is markedly stimulated in a PI 3-kinase-dependent manner (13Lawlor M.A. Alessi D.R. J. Cell Sci. 2001; 114: 2903-2910Crossref PubMed Google Scholar). This phenomenon predominantly relies on the phosphorylation of Akt on two of its amino acid residues: 1) threonine 308 in the activation loop of the kinase catalytic domain and 2) serine 473 in the “hydrophobic motif” (HM) carboxyl-terminal domain (14Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1386) Google Scholar). The phosphorylations of both of these regulatory sites occur after the recruitment of Akt to the PM through the binding of its pleckstrin homology (PH) domain to PIP3 (15Andjekkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J.C. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar) and are completely ablated in vivo by the PI 3-kinase inhibitor wortmannin (16Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar). The protein kinase responsible for phosphorylating Akt on Thr-308 is phosphoinositide-dependent kinase 1 (17Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 18Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (910) Google Scholar, 19Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar). The identity of the kinase responsible for phosphorylating Akt on Ser-473 is controversial.Akt is part of the protein kinase AGC (cAMP-dependent, cGMP-dependent, and protein kinase C) subfamily whose members are activated in a manner similar to Akt (20Frodin M. Antal T.L. Dummler B.A. Jensen C.J. Deak M. Gammeltoft S. Biondi R.M. EMBO J. 2002; 21: 5396-5407Crossref PubMed Scopus (218) Google Scholar). All members possess a phosphorylation site in the activation loop equivalent to Thr-308, whereas only some require phosphorylation at the HM site. Some AGC members have a negatively charged acidic residue instead of a Ser/Thr in the C-terminal hydrophobic domain (21Parekh D.B. Ziegler W. Parker P.J. EMBO J. 2000; 19: 496-503Crossref PubMed Scopus (508) Google Scholar). Phosphorylation of the HM site or substitution with an acidic residue provides a docking site to recruit phosphoinositide-dependent kinase 1 and stimulate the phosphorylation of Thr-308 (20Frodin M. Antal T.L. Dummler B.A. Jensen C.J. Deak M. Gammeltoft S. Biondi R.M. EMBO J. 2002; 21: 5396-5407Crossref PubMed Scopus (218) Google Scholar). X-ray crystallographic studies have suggested that in the inactive state the activation loop of Akt adopts a disordered structure that prevents the binding of ATP and protein substrates (22Huang X. Begley M. Morgenstern K.A. Gu Y. Rose P. Zhao H. Zhu X. Structure (Camb.). 2003; 11: 21-30Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Phosphorylation of Ser-473 results in a disorder to order transition, allowing interaction between the HM domain and the N-terminal lobe leading to the activation of the kinase (23Yang J. Cron P. Good V.M. Thompson V. Hemmings B.A. Barford D. Nat. Struct. Biol. 2002; 9: 940-944Crossref PubMed Scopus (426) Google Scholar).At least 10 kinases have been proposed to function as the hydrophobic motif domain protein kinase (HMD-PK) that phosphorylates Akt on Ser-473, including integrin-linked kinase (ILK), protein kinase Cα (PKCα), double-stranded DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) gene product, and the mammalian target of rapamycin (mTOR) (24Dong L.Q. Liu F. Am. J. Physiol. Endocrinol. Metab. 2005; 289: E187-E196Crossref PubMed Scopus (111) Google Scholar). It has also been suggested that Akt can undergo autophosphorylation at Ser-473 (25Toker A. Newton A.C. J. Biol. Chem. 2000; 275: 8271-8274Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar); however, this is most likely not the mechanism by which insulin stimulates Ser-473 phosphorylation (15Andjekkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J.C. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar, 16Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar, 26Wick M.J. Dong L.Q. Riojas R.A. Ramos F. Liu F. J. Biol. Chem. 2000; 275: 40400-40406Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 27Hresko R.C. Murata H. Mueckler M. J. Biol. Chem. 2003; 278: 21615-21622Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Of all of the proposed candidates, evidence supporting DNA-PK and mTOR is the most compelling. Based on the observation that membrane localization is sufficient to activate Akt (15Andjekkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J.C. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar), Hemmings and co-workers used a biochemical approach to purify a constitutively active membrane kinase from HEK 293 cells capable of phosphorylating Akt on Ser-473 (28Hill M.M. Feng J. Hemmings B.A. Curr. Biol. 2002; 12: 1251-1255Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The kinase, DNA-PK, was found to associate and co-localize with Akt at the PM as well as phosphorylate and activate Akt 10-fold in vitro (29Feng J. Park J. Cron P. Hess D. Hemmings B.A. J. Biol. Chem. 2004; 279: 41189-41196Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). These results were surprising, since DNA-PK has been described as a nuclear protein involved in DNA replication, gene transcription, and DNA repair (30Smith G.C. Jackson S.P. Genes Dev. 1999; 13: 916-934Crossref PubMed Scopus (765) Google Scholar). Nevertheless, knockdown studies with small interference RNA (siRNA) and experiments using DNA-PK-deficient glioblastoma cells revealed that insulin-stimulated Ser-473 phosphorylation was greatly impaired in both cases (29Feng J. Park J. Cron P. Hess D. Hemmings B.A. J. Biol. Chem. 2004; 279: 41189-41196Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). Like DNA-PK, mTOR is a member of the family of PI 3-kinase-related kinases whose activities are strongly inhibited by wortmannin and LY294002 (31Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5156) Google Scholar, 32Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1018) Google Scholar). mTOR can regulate cell growth and proliferation through its activation by growth factors and nutrients (33Fingar D.C. Blenis J. Oncogene. 2004; 23: 3151-3171Crossref PubMed Scopus (1044) Google Scholar). It is also the best characterized HM kinase, capable of phosphorylating the hydrophobic motif of p70S6 kinase when mTOR is complexed with GβL (34Kim D.-H. Sarbassov D.D. Ali S.M. Latek R.R. Guntur K.V. P. Erdjument-Bromage H. Tempst P. Sabatini D.M. Mol. Cell. 2003; 11: 895-904Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar) and regulatory associated protein of mTOR (RAPTOR) (35Hara K. Maruki Y. Long X. Yoshino K.-i. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1430) Google Scholar). Phosphorylation at the HM site of p70S6K (32Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1018) Google Scholar) but not Akt (36Tremblay F. Gagnon A. Veilleux A. Sorisky A. Marette A. Endocrinology. 2005; 146: 1328-1337Crossref PubMed Scopus (144) Google Scholar, 37Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar) is inhibited by rapamycin. It is for this reason that mTOR was initially ruled out as being the HMD-PK for Akt. Recently, however, Sabatini and co-workers (31Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5156) Google Scholar, 38Sarbassov 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 (2122) Google Scholar) have shown that mTOR can associate with GβL and another protein called rapamycin-insensitive companion of mTOR (RICTOR), forming a rapamycin-insensitive complex capable of phosphorylating Akt at Ser-473. RNA interference experiments in Drosophila and human cell lines as well as several in vitro studies provide intriguing evidence that the mTOR·RICTOR complex can phosphorylate the HM site of Akt (31Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5156) Google Scholar).It is still not exactly clear which if any of the proposed candidate kinases are the HMD-PK for Akt in an insulin-responsive tissue such as muscle and fat. In fact, it has been suggested that phosphorylation of Ser-473 may be due to multiple kinases that are cell type- or signaling pathway-specific (24Dong L.Q. Liu F. Am. J. Physiol. Endocrinol. Metab. 2005; 289: E187-E196Crossref PubMed Scopus (111) Google Scholar). Several years ago, we characterized the phosphorylation of Akt at Ser-473 using a cell-free insulin-signaling assay that we developed using subcellular fractions from 3T3-L1 adipocytes (27Hresko R.C. Murata H. Mueckler M. J. Biol. Chem. 2003; 278: 21615-21622Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 39Murata H. Hresko R.C. Mueckler M. J. Biol. Chem. 2003; 278: 21607-21614Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). We found that insulin-stimulated Akt phosphorylation at Ser-473 was PI 3-kinase-dependent and due to a kinase localized in both low density microsomes (LDM) and a subpopulation of the PM fraction enriched in proteins associated with the actin cytoskeleton (Ext-HiP) (27Hresko R.C. Murata H. Mueckler M. J. Biol. Chem. 2003; 278: 21615-21622Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Utilizing the cell-free system as well as in vivo siRNA knockdown studies, we addressed whether ILK, PKCα, DNA-PK, ATM, or mTOR was involved in the insulin-stimulated phosphorylation of Akt at Ser-473 in 3T3-L1 adipocytes. Our results indicate that the mTOR·RICTOR complex is the HMD-PK for Akt in fat cells.EXPERIMENTAL PROCEDURESCell Culture of 3T3-L1 Adipocytes—3T3-L1 preadipocytes obtained from the American Type Culture Collection were grown to confluence and 48 h later subjected to differentiation as described previously (40Tordjman K.M. Leingang K.A. James D.E. Mueckler M.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7761-7765Crossref PubMed Scopus (115) Google Scholar). 3T3-L1 adipocytes were used 10-14 days after initiating differentiation except where otherwise noted.In Vitro Assay—Mature 3T3-L1 adipocytes were serum-starved overnight and then fractionated in the basal state as described previously, except that 1 mm dithiothreitol was included in HES (50 mm Hepes, pH 7.4, 255 mm sucrose, 1 mm EDTA, and protease inhibitors) and IC (20 mm Hepes, pH 7.4, 140 mm potassium glutamate, 5 mm NaCl, 1 mm EGTA, and protease inhibitors) buffers (39Murata H. Hresko R.C. Mueckler M. J. Biol. Chem. 2003; 278: 21607-21614Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 41Piper R.C. Hess L.J. James D.E. Am. J. Physiol. 1991; 260: C570-C580Crossref PubMed Google Scholar). Protease inhibitors (Sigma) were as described (39Murata H. Hresko R.C. Mueckler M. J. Biol. Chem. 2003; 278: 21607-21614Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Ext-HiP and salt-washed plasma membranes were isolated in the manner described previously (27Hresko R.C. Murata H. Mueckler M. J. Biol. Chem. 2003; 278: 21615-21622Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In vitro reactions were prepared by mixing various combinations of cytosol (CYT), PM, nuclear (NUC) enriched fraction, LDM, and Ext-HiP with 16 nm Akt2 (Upstate Biotechnology, Inc., Lake Placid, NY) and 10 μm PIP3 (Calbiochem), prepared in a sonicated mixture of 100 μm phosphatidylcholine (Avanti Polar Lipids) and 100 μm phosphatidylserine (Avanti Polar Lipids). Reactions (typically 100 μl, final volume) were initiated with the addition of an ATP-regenerating system (final reaction concentrations, 1 mm ATP, 8 mm creatine phosphate, 30 units/ml creatine phosphokinase, and 5 mm MgCl2) and then incubated with rotation at 37 °C for the indicated period of time. Assays were quenched by the addition of an equal volume of buffer A (50 mm Hepes, pH 7.4, 150 mm NaCl, 1 mm sodium vanadate, 100 mm NaF, 10 mm sodium pyrophosphate, and protease inhibitors) containing 2% SDS.Immunoblot Analysis—Protein samples were subjected to SDS-PAGE and transferred to nitrocellulose. Akt phosphospecific and PKCα (2056) antibodies were obtained from Cell Signaling Technology. DNAPKcs (H-163) and ATM (5C2) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Two mTOR antibodies were used for immunoblot analysis, a polyclonal antibody mTOR (mTab2) from Upstate Biotechnology and a monoclonal antibody RAFT1 (611132) from BD Biosciences. RAPTOR antibodies were either from Cell Signaling Technology (4978) or from Abcam (antibody 5454). RICTOR antibodies were a kind gift from Dr. David M. Sabatini (Whitehead Institute) and were also obtained from Bethyl Laboratories. Immunoblots visualized by ECL detection (Amersham Biosciences) were quantified using NIH Image 1.62f. 6 and 12 μg of control proteins were run on each gel to establish the linearity of the autoradiogram.Immunoprecipitation of LDM—500 μg of LDM in 500 μl of IC buffer (with 1 mm dithiothreitol) were precleared with 40 μl of protein A-agarose for 30 min. After centrifugation for 10 min at 4 °C in a microcentrifuge, the supernatant was rotated overnight with 4 μg of primary antibody. Samples were then centrifuged for 5 min at 4 °C in a microcentrifuge to remove nonspecific aggregates that formed overnight. 30 μl of protein A-agarose were added to the supernatants for 2 h at 4°C. Pellets were washed six times with IC buffer, resuspended in IC buffer, and then assayed in vitro for HMD-PK activity. ILK (3862) polyclonal antibodies (Cell Signaling Technology), mTOR (mTab1) polyclonal antibodies (Upstate Biotechnology), and RICTOR antibodies (D. M. Sabatini) were used in the immunoprecipitation studies.HMD-PK Peptide Assay—HMD-PK activity was assayed using a modified version of Hill et al. (28Hill M.M. Feng J. Hemmings B.A. Curr. Biol. 2002; 12: 1251-1255Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). 50-μl reactions were incubated for 20 min at 30 °C in a buffer containing IC buffer, 1 mm dithiothreitol, 10 mm MgCl2, 1 μm protein kinase A inhibitor peptide, 100 μm ATP, 1 μCi of [γ-32P]ATP, 0.5 mg/ml substrate (RRPHFPQFSYSASSTA), or control (RRPHFPQFAYSASSTA) peptide. Peptides were phosphorylated using 25 units DNA-PK (Promega), 0.75 mg/ml LDM, or 0.4 mg/ml Ext-HiP. 10 μg/ml salmon sperm DNA was added in some cases. Reactions were quenched by adding 5 μl of 100% (w/v) trichloroacetic acid and then centrifuged for 10 min in a microcentrifuge at room temperature. 35 μl of the supernatant were spotted on P81 paper (Whatman), washed extensively in 0.1% phosphoric acid, and then analyzed by scintillation counting. Specific counts were calculated from the difference between the Ser-473 peptide and the control peptide.siRNA Duplexes—Complementary sense and antisense strands of RNA oligonucleotides for DNA-PK, ATM, and ILK were synthesized, annealed, and purified (PAGE) by Ambion. Negative control 1 siRNA (Ambion) and the following mouse sequences of strands of siRNA were used: DNA-PK sense, AGGGCCAAGCUAUCAUUCUtt; DNA-PK antisense, AGAAUGAUAGCUUGGCCCUtt; ATM sense, CAUACUACUCAAAGACAUUtt; ATM antisense, AAUGUCUUUGAGUAGUAUGtt; ILK sense, UGUUAAGUUUUCUUUCCAGUGtt; ILK antisense, CACUGGAAAGAAAACUUAACAtt. siGENOME Smartpool siRNA for RICTOR (M-064598-00; mouse 4921505C17RIK) and siCONTROL nontargeting siRNA 1 were from Dharmacon RNA Technologies.siRNA Electroporation of 3T3-L1 Adipocytes—3T3-L1 adipocytes were electroporated according to the procedure of Jiang et al. (6Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7569-7574Crossref PubMed Scopus (303) Google Scholar). Five days after differentiation, 3T3-L1 adipocytes were detached from cell culture plates with 1 mg/ml collagenase and 0.05% trypsin, 0.02% EDTA. Cells were washed three times with ice-cold PBS and resuspended in PBS prior to electroporation. Approximately 5 million cells (half of the cells on a 150-mm dish) were resuspended in 0.5 ml of PBS, mixed with 20 nmol of siRNA duplexes, and electroporated at the setting of 0.18 kV and 950 microfarads using the Bio-Rad Gene Pulser Xcell electroporation system. Cells were immediately incubated with fresh media for 10 min before reseeding. Experiments were conducted 72 h after electroporation.RESULTSSubcellular Localizations of DNA-PK and mTOR·RICTOR Correlate with HMD-PK Activity—ILK, PKCα, DNA-PK, ATM, and mTOR have all been proposed to play a major role in Akt Ser-473 phosphorylation based on experiments carried out on a variety of different cell types and stimuli (24Dong L.Q. Liu F. Am. J. Physiol. Endocrinol. Metab. 2005; 289: E187-E196Crossref PubMed Scopus (111) Google Scholar). We have decided to directly compare the functions of these five proteins in terms of insulin-stimulated Akt Ser-473 phosphorylation in a single cell type, 3T3-L1 adipocytes. Previously, we have shown that LDM and Ext-HiP, purified from 3T3-L1 adipocytes, are greatly enriched in insulin-stimulated HMD-PK activity (27Hresko R.C. Murata H. Mueckler M. J. Biol. Chem. 2003; 278: 21615-21622Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). To determine whether any of these proteins are enriched in LDM and Ext-HiP, we carried out immunoblot analyses on subcellular fractions prepared from serum-starved 3T3-L1 adipocytes (Fig. 1). DNA-PK was found in the NUC fraction as expected due to its well documented nuclear functions (30Smith G.C. Jackson S.P. Genes Dev. 1999; 13: 916-934Crossref PubMed Scopus (765) Google Scholar). It was, however, more greatly enriched in LDM and Ext-HiP but absent (or below detection) in high density microsomes (HDM), mitochondrial, and CYT fractions. HDM is enriched in endoplasmic reticulum, and LDM contains Golgi markers (42Simpson A.A. Yver D.R. Hissin P.J. Wardzala L.J. Karnieli E. Salans L.B. Cuchman S.W. Biochim. Biophys. Acta. 1983; 763: 393-407Crossref PubMed Scopus (330) Google Scholar). ATM, another member of the PI 3-kinase-related kinase family, had the same distribution as DNA-PK but was more abundant in NUC than LDM and Ext-HiP as compared with DNA-PK. ILK had a ubiquitous distribution but was slightly more enriched in NUC, LDM, and Ext-HiP fractions. PKCα was found almost exclusively in CYT in the basal state. PKCs are cytosolic proteins that translocate to membranes upon hormone stimulation (43Liu W.S. Heckman C.A. Cell. Signal. 1998; 10: 529-542Crossref PubMed Scopus (440) Google Scholar). mTOR was greatly enriched in Ext-HiP but was also found in NUC, HDM, and LDM fractions. mTOR has been previously reported to be associated with the endoplasmic reticulum (44Drenan R.M. Liu X. Bertram P.G. Zheng X.F.S. J. Biol. Chem. 2004; 279: 772-778Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and Golgi (44Drenan R.M. Liu X. Bertram P.G. Zheng X.F.S. J. Biol. Chem. 2004; 279: 772-778Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and in the nucleus (45Kim J.E. Chen J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14340-14345Crossref PubMed Scopus (186) Google Scholar). RICTOR, the binding protein of mTOR that is necessary for Ser-473 phosphorylation (31Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5156) Google Scholar), was very abundant in LDM and less abundant in Ext-HiP but was also present in NUC and HDM fractions.Next, we wanted to directly correlate HMD-PK activity with the amount of each candidate protein using the cell-free assay. We have previously shown that PIP3-dependent Akt Ser-473 phosphorylation in our in vitro assay was entirely dependent on exogenous ATP and the time of reaction incubation (27Hresko R.C. Murata H. Mueckler M. J. Biol. Chem. 2003; 278: 21615-21622Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 39Murata H. Hresko R.C. Mueckler M. J. Biol. Chem. 2003; 278: 21607-21614Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In vitro reactions were performed by combining CYT, NUC, PM, and LDM fractions with purified recombinant Akt2 and ATP in the absence and presence of PIP3. The amount of the particular fraction and the time of incubation were such tha" @default.
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• W1668086213 date "2005-12-01" @default.
• W1668086213 modified "2023-10-13" @default.
• W1668086213 title "mTOR·RICTOR Is the Ser473 Kinase for Akt/Protein Kinase B in 3T3-L1 Adipocytes" @default.
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