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Attenuation of Mammalian Target of Rapamycin Activity by Increased cAMP in 3T3-L1 Adipocytes

1998; Elsevier BV; Volume: 273; Issue: 51 Linguagem: Inglês

10.1074/jbc.273.51.34496

1083-351X

Pamela Scott, John C. Lawrence,

Phosphodiesterase function and regulation

Incubating 3T3-L1 adipocytes with forskolin, which increases intracellular cAMP by activating adenylate cyclase, mimicked rapamycin by attenuating the effect of insulin on stimulating the phosphorylation of four (S/T)P sites in PHAS-I, a downstream target of the mammalian target of rapamycin (mTOR) signaling pathway. To investigate the hypothesis that increasing cAMP inhibits mTOR, the protein kinase activity of mTOR was measured in an immune complex assay with recombinant PHAS-I as substrate. Both forskolin and 8-(4-chlorophenylthio)adenosine 3′-5′-monophosphate (CPT-cAMP) prevented the activation of mTOR by insulin in adipocytes, but neither agent affected mTOR activity when added directly to the immunopurified protein. In contrast, the cAMP phosphodiesterase inhibitor, theophylline, inhibited mTOR activity not only when added to intact adipocytes but also when added to immunopurified mTOR in vitro, demonstrating that certain methylxanthines are able to inhibit mTOR independently of increasing cAMP. Forskolin and CPT-cAMP blocked the effect of insulin on increasing mTOR phosphorylation, which was assessed using mTAb1, an antibody whose binding is inhibited by phosphorylation of mTOR. Although the mTAb1 epitope contains a consensus site for protein kinase B, neither agent inhibited the activation of protein kinase B produced by insulin. These findings support the interpretation that increasing cAMP attenuates the effects of insulin on PHAS-I, p70S6K, and other downstream targets of the mTOR signaling pathway by inhibiting the phosphorylation and activation of mTOR. Incubating 3T3-L1 adipocytes with forskolin, which increases intracellular cAMP by activating adenylate cyclase, mimicked rapamycin by attenuating the effect of insulin on stimulating the phosphorylation of four (S/T)P sites in PHAS-I, a downstream target of the mammalian target of rapamycin (mTOR) signaling pathway. To investigate the hypothesis that increasing cAMP inhibits mTOR, the protein kinase activity of mTOR was measured in an immune complex assay with recombinant PHAS-I as substrate. Both forskolin and 8-(4-chlorophenylthio)adenosine 3′-5′-monophosphate (CPT-cAMP) prevented the activation of mTOR by insulin in adipocytes, but neither agent affected mTOR activity when added directly to the immunopurified protein. In contrast, the cAMP phosphodiesterase inhibitor, theophylline, inhibited mTOR activity not only when added to intact adipocytes but also when added to immunopurified mTOR in vitro, demonstrating that certain methylxanthines are able to inhibit mTOR independently of increasing cAMP. Forskolin and CPT-cAMP blocked the effect of insulin on increasing mTOR phosphorylation, which was assessed using mTAb1, an antibody whose binding is inhibited by phosphorylation of mTOR. Although the mTAb1 epitope contains a consensus site for protein kinase B, neither agent inhibited the activation of protein kinase B produced by insulin. These findings support the interpretation that increasing cAMP attenuates the effects of insulin on PHAS-I, p70S6K, and other downstream targets of the mTOR signaling pathway by inhibiting the phosphorylation and activation of mTOR. mammalian target of rapamycin 8-(4-chlorophenylthio)adenosine 3′-5′-monophosphate eukaryotic initiation factor 4E eukaryotic initiation factor 4G FK506-binding protein ofM r = 12,000 high performance liquid chromatography mitogen-activated protein kinase polyacrylamide gel electrophoresis an eIF4E-binding protein also known as 4E-BP1 phosphatidylinositol 3-kinase cAMP-dependent protein kinase protein kinase B the M r ≈ 70,000 ribosomal S6 protein kinase mTOR kinase. Translation of certain classes of mRNA in mammalian cells is regulated by a signaling pathway containing mTOR,1 the mammalian target of rapamycin (1Abraham R.T. Wiederrecht G.J. Annu. Rev. Immunol. 1996; 14: 483-510Crossref PubMed Scopus (575) Google Scholar, 2Brown E.J. Schreiber S.L. Cell. 1996; 86: 517-520Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). mTOR is the counterpart of Tor1p and Tor2p, two proteins required for cell cycle progression in Saccharomyces cerevisiae. Like the yeast proteins, mTOR contains a high affinity binding site for rapamycin-FKBP12. The function of mTOR in cells is potently inhibited by rapamycin, which has proven to be a useful pharmacological tool for identifying downstream elements in the mTOR signaling pathway. Rapamycin attenuates the phosphorylation of p70S6K and PHAS-I that occurs in response to insulin (3Lin 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,4Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (590) Google Scholar), and overexpression of mTOR increases the phosphorylation of both proteins (5Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (619) Google Scholar, 6Brunn G.J. Hudson C.C. Sekulic A. Williams J.M. Hosoi H. Houghton P.J. Lawrence Jr., J.C. Abraham R.T. Science. 1997; 277: 99-101Crossref PubMed Scopus (813) Google Scholar). These and other findings have established that mTOR controls these two regulators of mRNA translation. p70S6K phosphorylates ribosomal protein S6 and increases translation of mRNAs having the polypyrimidine tract (TOP) motif (7Jeffries H.B.J. Reinhard C. Kozma S.C. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4441-4445Crossref PubMed Scopus (560) Google Scholar). The activation of p70S6K is a function of a complex pattern of phosphorylation mediated by three or more protein kinases that phosphorylate at least 10 sites (8Avruch J. Mol. Cell. Biochem. 1998; 182: 31-48Crossref PubMed Scopus (324) Google Scholar, 9Proud C.G. Trends Biochem. Sci. 1996; 21: 181-185Abstract Full Text PDF PubMed Scopus (199) Google Scholar). The rapamycin-sensitive sites, which by inference are those regulated by mTOR, include three in which the phosphorylated Ser/Thr is flanked by aromatic residues and one which fits a (Ser/Thr)-Pro motif (10Han J.-W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Nonphosphorylated PHAS-I binds tightly to eIF4E (11Lin T.-A. Kong X. Haystead T.A.J. Pause A. Belsham G.J. Sonenberg N. Lawrence Jr., J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (602) Google Scholar, 12Pause A. Belsham G.J. Gringas A.C. Donze O. Lin T.-A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1062) Google Scholar), the mRNA cap-binding protein, and prevents eIF4E from interacting with eIF4G (13Haghighat A. Mader S. Pause A. Sonenberg N. EMBO J. 1995; 14: 5701-5709Crossref PubMed Scopus (533) Google Scholar). Phosphorylation of PHAS-I leads to the dissociation of the PHAS-I·eIF4E complex (11Lin T.-A. Kong X. Haystead T.A.J. Pause A. Belsham G.J. Sonenberg N. Lawrence Jr., J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (602) Google Scholar,12Pause A. Belsham G.J. Gringas A.C. Donze O. Lin T.-A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1062) Google Scholar). This allows eIF4E to bind eIF4G to generate the complex that is needed for the efficient binding and/or scanning by the 40 S ribosomal subunit. In rat adipocytes, five (Ser/Thr)-Pro sites in PHAS-I are phosphorylated in response to insulin (14Fadden 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). Rapamycin attenuates, but does not abolish, the effects of insulin on the phosphorylation of these sites. The failure of rapamycin to inhibit fully the effect of insulin supports the view that an mTOR-independent pathway also contributes to the control of PHAS-I (15Lawrence Jr., J.C. Abraham R.T. Trends Biochem. Sci. 1997; 22: 345-349Abstract Full Text PDF PubMed Scopus (186) Google Scholar). Significant progress has been made in understanding mechanisms involved in mTOR signaling. mTOR contains an essential COOH-terminal catalytic domain that is homologous to the catalytic subunit of PI 3-kinase (1Abraham R.T. Wiederrecht G.J. Annu. Rev. Immunol. 1996; 14: 483-510Crossref PubMed Scopus (575) Google Scholar,2Brown E.J. Schreiber S.L. Cell. 1996; 86: 517-520Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Although the possibility that mTOR functions as a lipid kinase has not been eliminated, mTOR has been shown to phosphorylate PHAS-Iin vitro (6Brunn G.J. Hudson C.C. Sekulic A. Williams J.M. Hosoi H. Houghton P.J. Lawrence Jr., J.C. Abraham R.T. Science. 1997; 277: 99-101Crossref PubMed Scopus (813) Google Scholar, 16Brunn 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, 17Burnett 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 (946) Google Scholar), indicating that it is a member of the family of PI 3-kinase-related enzymes that signal as protein kinases. The finding that mTOR phosphorylates PHAS-I is also of practical importance as it has provided a means to assess mTOR activity. Incubating 3T3-L1 adipocytes with insulin was recently found to increase the PHAS-I kinase activity of mTOR (18Scott 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 (414) Google Scholar). The increase in activity produced by insulin correlated closely with an increase in the phosphorylation of mTOR. Furthermore, the effect of insulin was reversed by incubating mTOR in vitro with protein phosphatase 1, indicating that the effect of insulin was due to phosphorylation of mTOR (18Scott 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 (414) Google Scholar). A site potentially responsible for activation is near the COOH terminus of mTOR in a stretch of 20 amino acids that form the epitope for the antibody, mTAb1 (16Brunn 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). Insulin-stimulated phosphorylation of mTOR markedly inhibited binding of mTAb1, and incubating mTOR with phosphatase restored binding (18Scott 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 (414) Google Scholar). The effects of insulin on mTOR in 3T3-L1 adipocytes were abolished by the PI 3-kinase inhibitor, wortmannin. Moreover, selectively activating PKB with tamoxifen in MER-Akt cells, which express a PKB-estrogen receptor fusion protein that is rapidly activated when tamoxifen binds (19Kohn A.D. Barthel A. Kovacina K.S. Boge A. Wallach B. Summers S.A. Birnbaum M.J. Scott P.H. Lawrence Jr., J.C. Roth R.A. J. Biol. Chem. 1998; 273: 11937-11943Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), mimicked insulin by increasing both the phosphorylation and activity of mTOR (18Scott 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 (414) Google Scholar). These findings indicate that PI 3-kinase and PKB are upstream regulators of mTOR. Pharmacological or genetic disruption of mTOR function may have striking inhibitory effects on mRNA translation and cell proliferation (1Abraham R.T. Wiederrecht G.J. Annu. Rev. Immunol. 1996; 14: 483-510Crossref PubMed Scopus (575) Google Scholar, 2Brown E.J. Schreiber S.L. Cell. 1996; 86: 517-520Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Although inhibition of mTOR activity is clearly a potential mechanism for regulation of cellular function, inactivation of mTOR by physiological regulators has not been described. Increasing cAMP has been shown to decrease the phosphorylation of PHAS-I and p70S6K in certain cell types (20Graves 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, 21Lin T.-A. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 30199-30204Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 22Monfar M. Lemon K.P. Grammer T.C. Cheatham L. Chung J. Vlahos C.J. Blenis J. Mol. Cell. Biol. 1995; 15: 326-337Crossref PubMed Scopus (158) Google Scholar, 23Scott P.H. Belham C.M. Al-Hafidh J. Chilvers E.R. Peacock A.J. Gould G.W. Plevin R. Biochem. J. 1996; 318: 965-971Crossref PubMed Scopus (99) Google Scholar). In this report, we present evidence that these effects of cAMP result from inactivation of mTOR. 3T3-L1 adipocytes were cultured in 10-cm diameter dishes and used in experiments 8–10 days after removing the differentiation medium (3Lin 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). Except for the32P-labeling experiments (described in the legend to Fig.1), cells were incubated at 37 °C for 2.5 h in HEPES-buffered saline (145 mm NaCl, 5.4 mm KCl, 1.4 mm CaCl2, 1.4 mm MgCl2, 0.1 mm NaPi, 5 mm glucose, 0.5% bovine serum albumin, and 10 mm Na-HEPES, pH 7.4) and then incubated with insulin and other agents as indicated. To terminate the incubation, the medium was aspirated, and the cells were rinsed and homogenized in 1 ml of Homogenization Buffer, which contained 50 mm NaF, 10 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm sodium vanadate, 0.1 μm microcystin-LR, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm benzamidine, 1 mmphenylmethylsulfonyl fluoride, 10 mm KPi, and 50 mm β-glycerophosphate, pH 7.4. Homogenates were centrifuged at 10,000 × g for 20 min, and the supernatants were retained for analyses. The protein content was determined by using bicinchoninic acid (24Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18702) Google Scholar). Antibodies to PHAS-I (25Hu C. Pang S. Kong X. Velleca M. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3730-3734Crossref PubMed Scopus (126) Google Scholar) and mTOR (16Brunn 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) were described previously. To generate antibodies to PKB, a peptide (CVDSERRPHFPQFSYSASSTA) having a sequence identical to that of the last 21 amino acids in PKBα was coupled to keyhole limpet hemocyanin, and the peptide-hemocyanin conjugates were used to immunize rabbits as described previously (26Sevetson B.R. Kong X. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10305-10309Crossref PubMed Scopus (345) Google Scholar). The PKB antibodies were affinity purified by using columns containing resin prepared by coupling the peptide to Sulfolink beads (Pierce). Immunoprecipitations were conducted using either non-immune IgG or the appropriate antibodies coupled to protein A-agarose (Life Technologies, Inc.). Briefly, antibodies (5 μg) were incubated for 1 h at 23 °C with protein A-agarose beads (20 μl of a 1:1 suspension) in Tris-buffered saline (TBS) (150 mm NaCl and 50 mm Tris-HCl, pH 7.4). The beads were then washed 3 times (1 ml/wash) and suspended in TBS (20 μl). Extract samples (800 μl) were incubated with beads for 90 min at 4 °C and then washed (1 ml buffer/wash). For PHAS-I immunoprecipitations, beads were washed twice with 1 ml of Buffer A (1 mm EDTA, 1 mm EGTA, 10 mm MgCl2, 10 mm HEPES, pH 7.4), twice with Buffer A plus 0.5m NaCl, and then twice with Buffer A. For mTOR immunoprecipitations, beads were washed twice with Buffer A, twice with Buffer A plus 0.5 m NaCl, and twice with Buffer B (50 mm NaCl, 50 mm β-glycerol phosphate, 1 mm dithiothreitol, 0.2 μm microcystin-LR, and 10 mm HEPES, pH 7.4). For PKB immunoprecipitations, beads were washed twice with Buffer C (0.5 m NaCl, 10% glycerol, 0.1% bovine serum albumin, 0.5% Triton X-100, and 25 mm HEPES, pH 7.4) and twice with Buffer D (10 mm MgCl2, 1 mm dithiothreitol, and 50 mm Tris-Cl, pH 7.5). To elute PHAS-I from immune complexes, the beads were rinsed with 1 ml of H2O, suspended in 150 μl of 1% β-mercaptoethanol, 1 mm EDTA, and 10 mm Tris-HCl, pH 7.5, and incubated at 95 °C for 15 min. After centrifuging the samples at 10,000 × g for 10 min, the supernatant was retained for analyses of PHAS-I. When samples of mTOR or PKB were to be subjected to SDS-PAGE, proteins were eluted in SDS-sample buffer (27Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207479) Google Scholar). When protein kinase activities were to be assessed, beads were suspended in 50 μl of the appropriate reaction mixture. mTOR and PKB activities were measured in the same extract samples. Extracts (800 μl) were first incubated with the mTOR antibody, mTAb2 (16Brunn 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), coupled to protein A-agarose. After centrifuging the samples, the supernatants were retained for measuring PKB activity (described below), and the pellets containing the mTOR immune complexes were washed as described above. To assess mTOR activity, the beads were suspended in 50 μl of mTOR Reaction Mix that contained the following: 50 mm NaCl, 1 mm dithiothreitol, 10 μm PKA inhibitory peptide (28Cheng H.-C. Kemp B.E. Pearson R.B. Smith A.J. Misconi L. Van Patten S.M. Walsh D.A. J. Biol. Chem. 1986; 261: 989-992Abstract Full Text PDF PubMed Google Scholar), 0.2 μm microcystin-LR, 20 μg/ml [H6]PHAS-I (29Haystead T.A.J. Haystead C.M.M. Hu C. Lin T.-A. Lawrence Jr., J.C. J. Biol. Chem. 1994; 269: 23185-23191Abstract Full Text PDF PubMed Google Scholar), 0.2 mm[γ-32P]ATP (1 μCi/nmol), 10 mmMnCl2, 10 mm Na-HEPES, and 50 mmβ-glycerophosphate, pH 7.4. Supernatants remaining after removing the mTOR immune complexes were incubated with PKB antibodies coupled to protein A-agarose. The beads were then washed and suspended in a reaction mixture identical to that described previously (30Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (320) Google Scholar), except that histone 2B was substituted for myelin basic protein. mTOR and PKB samples were incubated for 30 min at 30 and 23 °C, respectively, before the reactions were terminated by adding SDS sample buffer. Samples of PHAS-I that had been immunoprecipitated from 32P-labeled 3T3-L1 adipocytes were digested with lysyl endopeptidase C (Wako Pure Chemicals) and chymotrypsin (Boehringer Mannheim) before phosphopeptides were resolved by reverse phase-high performance liquid chromatography (HPLC) as described previously for phosphorylation site analysis of rat adipocyte PHAS-I (14Fadden 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). Samples were subjected to SDS-PAGE (27Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207479) Google Scholar), and 32P-labeled proteins were detected by autoradiography of the dried gels. mTOR (16Brunn 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) and PHAS-I (11Lin T.-A. Kong X. Haystead T.A.J. Pause A. Belsham G.J. Sonenberg N. Lawrence Jr., J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (602) Google Scholar) were detected by immunoblotting. Glutathione S-transferase-FKBP12 was expressed in bacteria and purified 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 (714) Google Scholar). Recombinant human insulin was from Eli Lilly. Forskolin, theophylline, and CPT-cAMP were from Sigma. Rapamycin was from Calbiochem. [γ-32P]ATP was from NEN Life Science Products, and Na32Pi was from ICN Pharmaceuticals. Both rapamycin and increasing cAMP promote dephosphorylation of PHAS-I in 3T3-L1 adipocytes (3Lin 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, 21Lin T.-A. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 30199-30204Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). As a step in determining whether rapamycin and cAMP affect a common upstream regulator of PHAS-I, experiments were conducted to determine whether forskolin, an agent that increases intracellular cAMP, affected the same sites in PHAS-I as rapamycin. Peptide mapping followed by amino acid sequence analyses was previously used to identify Thr36, Thr45, Ser64, Thr69, and Ser82 as sites of phosphorylation in PHAS-I in rat adipocytes (14Fadden 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). These five residues, as well as the sites for cleavage by the proteases used in the previous mapping studies (14Fadden 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), are present in 3T3-L1 PHAS-I (3Lin 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, 25Hu C. Pang S. Kong X. Velleca M. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3730-3734Crossref PubMed Scopus (126) Google Scholar). Indeed, except for peptides containing Ser82, 3T3-L1 PHAS-I would be expected to yield fragments identical to those derived from rat PHAS-I. Thus, we were able to resolve phosphorylation sites in PHAS-I from 3T3-L1 adipocytes by using the strategies developed for analyses of the rat protein. To label sites in PHAS-I, 3T3-L1 adipocytes were incubated in medium containing 32Pi. After incubating with the indicated additions, extracts of the cells were prepared. [32P]PHAS-I was then immunoprecipitated and digested with lysyl endopeptidase. The resulting phosphopeptides were subjected to reverse phase-HPLC (Fig. 1 A). Three peaks, designated LE-P1, LE-P2, and LE-P3, eluted in the same position as peaks derived from rat PHAS-I (14Fadden 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). In view of the near identity of rat (25Hu C. Pang S. Kong X. Velleca M. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3730-3734Crossref PubMed Scopus (126) Google Scholar) and 3T3-L1 (3Lin 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) PHAS-I proteins (Fig.1 C), it seems reasonable to assume that the peaks represent the phosphorylation sites identified previously (14Fadden 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). Thus, LE-P1 and LE-P2 are presumed to contain Thr69 and Ser64, respectively. Thr36 and Thr45 are recovered in a fragment eluting in LE-P3. These two sites may be resolved after digesting this fragment with chymotrypsin (14Fadden 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), and the peaks designated CT-P1 and CT-P2 correspond to peptides containing Thr45 and Thr36, respectively (Fig.1 B). A relatively small third peak containing a peptide with phosphorylated Ser82 was previously detected after digesting LE-P3 from rat adipocyte PHAS-I with chymotrypsin (14Fadden 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). This peak was absent in samples derived from the 3T3-L1 protein. Thus, Ser82 does not seem to be phosphorylated to a significant level in 3T3-L1 adipocytes. The effects of insulin, rapamycin, and forskolin on the phosphorylation of sites was assessed by measuring the 32P contents of the appropriate peak fractions. Insulin increased the amount of32P recovered in peptides containing Thr36, Thr45, Ser64, and Thr69 (Fig.2). Rapamycin and forskolin had relatively small effects on the phosphorylation of PHAS-I in the absence of insulin; however, the two agents inhibited the effect of insulin on increasing the phosphorylation of the four sites. Thus, increasing cAMP with forskolin was associated with a change in the pattern of PHAS-I phosphorylation that was similar to that produced by inhibiting mTOR with rapamycin. The phosphorylation states of all four sites were also decreased by wortmannin, consistent with the effect of the PI 3-kinase inhibitor to block activation of mTOR (18Scott 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 (414) Google Scholar). To determine whether increasing cAMP in cells inhibited mTOR, adipocytes were incubated with forskolin, CPT-cAMP, and theophylline before mTOR activity was measured in an immune complex assay with recombinant [H6]PHAS-I as substrate (Fig.3). As previously reported (18Scott 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 (414) Google Scholar), insulin increased the [H6] PHAS-I kinase activity of mTOR by approximately 2.5-fold. Forskolin and CPT-cAMP were without effect on mTOR activity when cells were incubated in the absence of insulin, but both agents markedly inhibited the activation of mTOR by insulin. Like forskolin and CPT-cAMP, theophylline prevented the activation of mTOR by insulin; however, theophylline also decreased basal mTOR activity, suggesting that the mechanism of its inhibitory effect differed from that of the other two agents. Further evidence for different mechanisms of action was obtained in a control experiment in which agents were included in the reaction mixture used to measure mTOR activity. Neither forskolin nor CPT-cAMP affected the PHAS-I kinase activity of mTOR immunoprecipitated from either rat brain or 3T3-L1 adipocytes (Fig.4). This result was expected as the actions of these agents are presumed to require stimulation of cAMP production and/or activation of PKA, which would not occur in thein vitro kinase assay. Including theophylline in the assay markedly decreased the PHAS-I kinase activity of mTOR, indicating that theophylline is capable of inhibiting mTOR by a mechanism that does not involve increased cAMP.Figure 4Effects of incubating immunoprecipitated mTOR with forskolin, CPT-cAMP, and theophylline on mTOR activity. mTOR was immunoprecipitated from extracts of 3T3-L1 adipocytes or rat brain (16Brunn 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) by using mTAb1 and protein A-agarose beads. Immune complexes were incubated in mTOR Reaction Mix without additions or with 50 μm forskolin, 0.5 mm CPT-cAMP, or 5 mm theophylline. Results are expressed as percentages of the respective control activities. A representative experiment is presented for 3T3-L1 mTOR. The results with rat brain mTOR are mean values ± S.E. from three experiments.View Large Image Figure ViewerDownload (PPT) Activation of mTOR in response to insulin is associated with phosphorylation of a site(s) that decreases the ability of mTAb1 to bind mTOR (18Scott 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 (414) Google Scholar). Thus, we were able to determine whether increasing cAMP prevented phosphorylation of this site by performing immunoblotting experiments with mTAb1. Incubating cells with insulin markedly decreased mTAb1 binding without affecting mTAb2 binding, which serves as an index of the relative amount of mTOR present (Fig.5 A). Forskolin, CPT-cAMP, and theophylline were without effect on mTAb1 binding in the absence of insulin, but all three agents prevented the effect of insulin on decreasing mTAb1 binding (Fig. 5 B). The results support the hypothesis that the agents block the activation of mTOR by preventing the phosphorylation of the protein. We next determined whether increasing cAMP affected the activity of PKB (Fig. 6), an upstream element in the mTOR signaling pathway (18Scott 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 (414) Google Scholar). Phosphorylation and activation of PKB is associated with a decrease in the electrophoretic mobility of the enzyme when analyzed by SDS-PAGE. Insulin treatment of cells retarded the mobility of approximately half of the PKB, which was detected by immunoblotting (Fig. 6 A). The mobility shift produced by insulin was associated with an increase of approximately 5-fold in PKB activity (Fig. 6 B), assessed in an immune complex assay with histone 2B as substrate (Fig. 6 A). Neither forskolin nor CPT-cAMP decreased PKB activity when added to adipocytes incubated without insulin or when added to cells prior to insulin (Fig.6 B). Consistent with these observations, neither agent affected the electrophoretic mobility of PKB (Fig. 6 A). In contrast, theophylline blocked the gel shift produced by insulin and abolished the stimulatory effect of the hormone on PKB activity. The present results with forskolin and CPT-cAMP indicate that increasing intracellular cAMP leads to attenuation of the effect of insulin on activating mTOR. As mTOR is known to have a central role in the control of both mRNA translation and cell proliferation, an important implication is that inhibition of mTOR is involved in the potent inhibitory effects of cAMP on these processes in certain cell types. Although the control by mTOR may be complex, some of the mechanisms involved have been elucidated and provide at least a partial explanation of how cAMP-dependent inhibition of mTOR could lead to inhibition of protein synthesis and mitogenesis. Based on studies with rapamycin, it is clear that inhibiting mTOR would lead to dephosphorylation of the translational regulators, PHAS-I and p70S6K (9Proud C.G. Trends Biochem. Sci. 1996; 21: 181-185Abstract Full Text PDF PubMed Scopus (199) Google Scholar, 15Lawrence Jr., J.C. Abraham R.T. Trends Biochem. Sci. 1997; 22: 345-349Abstract Full Text PDF PubMed Scopus (186) Google Scholar). Decreased phosphorylation of both proteins has been shown to occur in response to increased cAMP, at least in certain cell types (20Graves 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, 21Lin T.-A. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 30199-30204Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 22Monfar M. Lemon K.P. Grammer T.C. Cheatham L. Chung J. Vlahos C.J. Blenis J. Mol. Cell. Biol. 1995; 15: 326-337Crossref PubMed Scopus (158) Google Scholar, 23Scott P.H. Belham C.M. Al-Hafidh J. Chilvers E.R. Peacock A.J. Gould G.W. Plevin R. Biochem. J. 1996; 318: 965-971Crossref PubMed Scopus (99) Google Scholar). Dephosphorylation of the appropriate sites in p70S6K results in inactivation of the kinase, less phosphorylation of ribosomal protein S6, and reduced synthesis of those messages having the polypyrimidine tract motif (9Proud C.G. Trends Biochem. Sci. 1996; 21: 181-185Abstract Full Text PDF PubMed Scopus (199) Google Scholar). Dephosphorylation of PHAS-I results in increased binding of PHAS-I to eIF4E and inhibition of the translation of capped mRNA (15Lawrence Jr., J.C. Abraham R.T. Trends Biochem. Sci. 1997; 22: 345-349Abstract Full Text PDF PubMed Scopus (186) Google Scholar). Overexpressing eIF4E in cells may have a potent mitogenic effect, suggesting that eIF4E is involved in the control of cell proliferation (32De Benedetti A. Rhoads R.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8212-8216Crossref PubMed Scopus (256) Google Scholar, 33Lazaris-Karatzas A. Montine K.S. Sonenberg N. Nature. 1990; 345: 544-547Crossref PubMed Scopus (809) Google Scholar). Our studies do not directly address this possibility, but it is interesting to speculate that cAMP-induced inhibition of mTOR and the decrease in eIF4E function resulting from dephosphorylation of PHAS-I might contribute to the antiproliferative effects of increased cAMP. Another mechanism through which compromised mTOR signaling could affect cell cycle progression involves p27Kip1, a heat-stable inhibitor of G1 cyclin-Cdk activities. Inhibiting mTOR with rapamycin results in stabilization of p27Kip1, thereby preventing the increase in cyclin-Cdk activities needed for G1 progression (34Nourse J. Firpo E. Flanagan W.M. Coats S. Polyak K. Lee M.-H. Massague J. Crabtree G.R. Roberts J.M. Nature. 1994; 372: 570-573Crossref PubMed Scopus (905) Google Scholar, 35Kato J. Matsuoka M. Polyak K. Massague J. Sherr C.J. Cell. 1994; 79: 487-496Abstract Full Text PDF PubMed Scopus (709) Google Scholar). Interestingly, cAMP has also been shown to increase p27Kip1 (35Kato J. Matsuoka M. Polyak K. Massague J. Sherr C.J. Cell. 1994; 79: 487-496Abstract Full Text PDF PubMed Scopus (709) Google Scholar), and it is an intriguing hypothesis that the actions of both rapamycin and cAMP on the inhibitor result from inhibition of mTOR. Arguing against this hypothesis is evidence that the mechanisms by which rapamycin and cAMP increase p27Kip1are different (35Kato J. Matsuoka M. Polyak K. Massague J. Sherr C.J. Cell. 1994; 79: 487-496Abstract Full Text PDF PubMed Scopus (709) Google Scholar). Rapamycin appears to decrease the rate at which p27Kip1 is degraded (34Nourse J. Firpo E. Flanagan W.M. Coats S. Polyak K. Lee M.-H. Massague J. Crabtree G.R. Roberts J.M. Nature. 1994; 372: 570-573Crossref PubMed Scopus (905) Google Scholar), whereas increased cAMP has been reported to increase the synthesis of the inhibitor (35Kato J. Matsuoka M. Polyak K. Massague J. Sherr C.J. Cell. 1994; 79: 487-496Abstract Full Text PDF PubMed Scopus (709) Google Scholar). The conclusion that inhibition of mTOR contributes to the effects of increased cAMP on PHAS-I and p70S6K does not imply that cAMP lacks effects on the phosphorylation of these proteins by other mechanisms. The observation that forskolin promoted a greater decrease than rapamycin in the phosphorylation of Thr45 and Thr36 would be consistent with the involvement of an mTOR-independent effect of cAMP on these two sites in PHAS-I. The activation of p70S6K in 3T3-L1 adipocytes is attenuated by the MAP kinase kinase inhibitor, PD 098059, suggesting that one or more members of the MAP kinase family may be involved in the control of p70S6K (36Scott P.H. Lawrence Jr., J.C. FEBS Lett. 1997; 409: 171-176Crossref PubMed Scopus (22) Google Scholar). Increasing cAMP has been shown to inhibit the MAP kinase signaling pathway in several cell types (37Graves L.M. Lawrence Jr., J.C. Trends Endocrinol. Metab. 1996; 7: 43-50Abstract Full Text PDF PubMed Scopus (44) Google Scholar). Interestingly, the inhibitory effect of cAMP on MAP kinase activation is not observed in all cells. Likewise, in contrast to our findings in 3T3-L1 adipocytes, neither forskolin nor cAMP derivatives decreased the activation of p70S6K in Swiss 3T3 cells (10Han J.-W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 38Frost V. Morley S.J. Mercep L. Meyer T. Fabbro D. Ferrari S. J. Biol. Chem. 1995; 270: 26698-26706Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 39Petritsch C. Woscholski R. Edelmann H.M.L. Ballou L.M. J. Biol. Chem. 1995; 270: 26619-26625Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In these cells, the activation of p70S6K was still markedly inhibited by methylxanthine phosphodiesterase inhibitors, even though cAMP levels were not significantly changed. These results support the conclusion that methylxanthines act by a cAMP-independent mechanism to block activation of p70S6K. Based on the fact that methylxanthine phosphodiesterase inhibitors were used to enhance cAMP production in a study of the effect of cAMP on p70S6K(22Monfar M. Lemon K.P. Grammer T.C. Cheatham L. Chung J. Vlahos C.J. Blenis J. Mol. Cell. Biol. 1995; 15: 326-337Crossref PubMed Scopus (158) Google Scholar), it was argued that the inhibitory effects on kinase activation were due to the methylxanthine instead of increased cAMP (10Han J.-W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 38Frost V. Morley S.J. Mercep L. Meyer T. Fabbro D. Ferrari S. J. Biol. Chem. 1995; 270: 26698-26706Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 39Petritsch C. Woscholski R. Edelmann H.M.L. Ballou L.M. J. Biol. Chem. 1995; 270: 26619-26625Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). This is unlikely to be the case, as experiments in several cell types, including 3T3-L1 adipocytes, have shown that cAMP derivatives and agents that increase cAMP markedly inhibit activation of p70S6K in the absence of methylxanthines (20Graves 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, 21Lin T.-A. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 30199-30204Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 23Scott P.H. Belham C.M. Al-Hafidh J. Chilvers E.R. Peacock A.J. Gould G.W. Plevin R. Biochem. J. 1996; 318: 965-971Crossref PubMed Scopus (99) Google Scholar). The most straightforward interpretation is that the inhibitory effects of methylxanthines and increased cAMP are mediated by at least two different mechanisms. Methylxanthines could inhibit activation of mTOR by blocking activation of the upstream effector, PKB (Fig. 6). However, based on the finding that theophylline markedly inhibited mTOR activityin vitro (Fig. 4), we propose that methylxanthines directly inhibit mTOR. Because this direct mechanism would presumably operate in all cells expressing mTOR, it could account for the inhibitory effects of methylxanthines on the phosphorylation of p70S6K and PHAS-I in cells lacking the response to cAMP. As discussed below, increasing cAMP presumably acts by activating PKA, which phosphorylates an upstream regulator of mTOR phosphorylation. The indirect mechanism could explain why the response to cAMP is not observed in all cell types, since factors such as the level of expression of regulatory elements or the presence of opposing pathways could determine whether mTOR activity was decreased. Studies from several laboratories have implicated activation of PKB in the effects of insulin on PHAS-I (19Kohn A.D. Barthel A. Kovacina K.S. Boge A. Wallach B. Summers S.A. Birnbaum M.J. Scott P.H. Lawrence Jr., J.C. Roth R.A. J. Biol. Chem. 1998; 273: 11937-11943Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 40Gingras A.-C. Kennedy S.G. O'Leary M.A. Sonenberg N. Hay N. Genes Dev. 1998; 12: 502-513Crossref PubMed Scopus (730) Google Scholar, 41Ueki K. Yamamoto-Honda R. Kaburagi Y. Yamauchi T. Tobe K. Burgering B.M.T. Coffer P.J. Komuro I. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1998; 273: 5315-5322Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), and we have recently obtained evidence implicating PKB in the activation of mTOR by insulin (18Scott 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 (414) Google Scholar). Therefore, we considered the possibility that increasing cAMP might inhibit activation of mTOR by preventing the activation of PKB. This hypothesis can be eliminated since neither forskolin nor CPT-cAMP affected the activation of PKB in response to insulin (Fig. 6) under conditions in which the agents markedly attenuated the activation of mTOR by the hormone (Fig. 3). The failure to affect PKB activation also indicates that inhibition of upstream signaling from the insulin receptor cannot explain the actions of cAMP. mTOR contains consensus sites for phosphorylation by PKA (42Kennelly P.J. Krebs E.G. J. Biol. Chem. 1991; 266: 15555-15558Abstract Full Text PDF PubMed Google Scholar), and we have attempted to test the hypothesis that mTOR is inactivated by PKA. However, we were not able to phosphorylate mTOR in vitro with purified catalytic subunit of PKA or to affect mTOR kinase activity by incubating mTOR immune complexes with the catalytic subunit. 2P. H. Scott and J. C. Lawrence, Jr., unpublished observations. These negative findings do not eliminate the possibility that PKA can phosphorylate mTOR under the appropriate conditions. However, the finding that increased cAMP opposes the effect of insulin on mTOR phosphorylation, as assessed by mTAb1 binding (18Scott 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 (414) Google Scholar), provides further support for the interpretation that the effects of cAMP are not mediated by direct phosphorylation of mTOR by PKA. The recent finding that activation of PKB by tamoxifen in MER-Akt cells resulted in activation of mTOR and the loss of mTAb1 binding indicates that activation of PKB is sufficient for the activation of mTOR, at least in some cell types (18Scott 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 (414) Google Scholar). As discussed previously (18Scott 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 (414) Google Scholar), the mTAb1 epitope contains an almost ideal consensus site for phosphorylation by PKB, but we have not been able to demonstrate direct phosphorylation of this site by PKB. Thus, although there is a strong argument for placing PKB upstream of mTOR in the signaling pathway, PKB may not directly phosphorylate mTOR. In order to understand the action of cAMP, it will be necessary to identify the protein kinase(s) responsible for the phosphorylation and activation of mTOR. As a working hypothesis, we suggest that there is an intervening kinase, designated TORK, between PKB and mTOR. A model in which activation of PKB or PKA resulted in activation or inhibition of TORK would be consistent with the available results pertaining to the regulation of mTOR by insulin and cAMP.