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

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. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). The increase in activity produced by insulin with an increase in the phosphorylation of mTOR. the effect of insulin was by mTOR in vitro with protein indicating that the effect of insulin was to phosphorylation of mTOR (18Scott P.H. Brunn G.J. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). site for activation is the of mTOR in a of that the epitope for the mTAb1 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). phosphorylation of mTOR inhibited binding of mTAb1, and mTOR with binding (18Scott P.H. Brunn G.J. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). The effects of insulin on mTOR in 3T3-L1 adipocytes by the PI 3-kinase inhibitor, activating with in which a protein that is when binds A. A. P.H. Lawrence Jr., J.C. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), mimicked insulin by increasing both the phosphorylation and activity of mTOR (18Scott P.H. Brunn G.J. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). These findings that PI 3-kinase and are regulators of mTOR. or of mTOR function have effects on mRNA translation and cell (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 of mTOR activity is a for of of mTOR by regulators has not been cAMP has been shown to the phosphorylation of PHAS-I and p70S6K in certain cell Kong X. Lin T.-A. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, T.-A. Lawrence Jr., J.C. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar, P.H. J. Biochem. J. 1996; PubMed Scopus Google Scholar). In that these effects of cAMP from of mTOR. 3T3-L1 adipocytes in and in the (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). for in the to cells at for in and 10 and with insulin and other as To the the was and the cells and in of which 10 10 10 10 and at for and the for The protein was by using Biochem. PubMed Scopus Google Scholar). to PHAS-I C. S. Kong X. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google and mTOR 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 To generate to a having a to that of the in was to and the to as Kong X. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The affinity by using containing by the to using or the to protein for at with protein of a in and The and in with for at and PHAS-I with of 10 10 with and with A. mTOR with with and with B and 10 with and and with and To PHAS-I from immune the with of in of and 10 and at for the at for 10 the was for of of mTOR or to be to proteins in Nature. PubMed Scopus Google Scholar). protein kinase to be in of the mTOR and measured in the with the mTOR 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), to protein the the for activity and the containing the mTOR immune as To assess mTOR the in of mTOR that the 10 B.E. Pearson R.B. J. Biol. Chem. Full Text PDF PubMed Google Scholar), T.A.J. Haystead C. Lin T.-A. Lawrence Jr., J.C. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), 10 10 and the mTOR immune with to protein The and in a to that EMBO J. 1995; 14: PubMed Scopus Google Scholar), that was for protein. mTOR and for at and the by of PHAS-I that been from 3T3-L1 adipocytes with and by performance liquid chromatography as for phosphorylation site of 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). to Nature. PubMed Scopus Google Scholar), and proteins by of the mTOR 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 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 by was in and as Brunn G.J. Williams J.M. Wiederrecht G. Abraham R.T. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). insulin was from theophylline, and CPT-cAMP from Rapamycin was from was from and was from Both rapamycin and increasing cAMP 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, T.-A. Lawrence Jr., J.C. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). a in rapamycin and cAMP a of PHAS-I, to forskolin, an agent that increases intracellular affected the sites in PHAS-I as by was to and 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 as as the sites for by the in the (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 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, C. S. Kong X. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). for containing 3T3-L1 PHAS-I be to to those from rat able to phosphorylation sites in PHAS-I from 3T3-L1 adipocytes by using the for of the rat protein. To sites in PHAS-I, 3T3-L1 adipocytes in containing with the of the cells was and with The to and in the as 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 of rat C. S. Kong X. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google 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 PHAS-I proteins it to that the the phosphorylation sites (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 are to and and are in a in These two sites be with (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 and to containing and containing a with phosphorylated was from rat PHAS-I with (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 was in from the 3T3-L1 protein. does not to be phosphorylated to a in 3T3-L1 The effects of rapamycin, and forskolin on the phosphorylation of sites was assessed by the of the the in containing and Rapamycin and forskolin effects on the phosphorylation of PHAS-I in the of the two inhibited the effect of insulin on increasing the phosphorylation of the four sites. increasing cAMP with forskolin was with a in the pattern of PHAS-I phosphorylation that was to that produced by inhibiting mTOR with The phosphorylation of four sites also by with the effect of the PI 3-kinase to activation of mTOR (18Scott P.H. Brunn G.J. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). To increasing cAMP in cells inhibited mTOR, adipocytes with forskolin, and mTOR activity was measured in an immune complex assay with recombinant as (18Scott P.H. Brunn G.J. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar), insulin the PHAS-I kinase activity of mTOR by Forskolin and CPT-cAMP effect on mTOR activity when cells in the of but both inhibited the activation of mTOR by insulin. Like forskolin and prevented the activation of mTOR by also mTOR that the of effect from that of the other two for mechanisms of was in a control in which in the to mTOR activity. forskolin CPT-cAMP affected the PHAS-I kinase activity of mTOR from rat or 3T3-L1 adipocytes This was as the of these are to of cAMP and/or activation of which not in vitro kinase in the assay the PHAS-I kinase activity of mTOR, indicating that is of inhibiting mTOR by a that does not of mTOR with forskolin, and on mTOR activity. mTOR was from of 3T3-L1 adipocytes or rat 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 by using mTAb1 and protein in mTOR or with forskolin, or are as of the control is for 3T3-L1 mTOR. The with rat mTOR are from three of mTOR in response to insulin is with phosphorylation of a that the of mTAb1 to bind mTOR (18Scott P.H. Brunn G.J. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). able to increasing cAMP prevented phosphorylation of site by with Incubating cells with insulin mTAb1 binding which as an of the of mTOR and effect on mTAb1 binding in the of but three prevented the effect of insulin on mTAb1 binding The support the hypothesis that the the activation of mTOR by the phosphorylation of the protein. increasing cAMP affected the activity of an in the mTOR signaling pathway (18Scott P.H. Brunn G.J. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). Phosphorylation and activation of is with a in the of the when by of cells the of of the which was by The produced by insulin was with an increase of in activity assessed in an immune complex assay with as forskolin CPT-cAMP activity when added to adipocytes insulin or when added to cells to insulin with these neither agent affected the of In contrast, blocked the gel produced by insulin and the effect of the on activity. The with forskolin and CPT-cAMP that increasing intracellular cAMP leads to of the effect of insulin on activating mTOR. mTOR is known to have a in the control of both mRNA translation and cell an is that of mTOR is involved in the effects of cAMP on these in certain cell Although the control by mTOR be of the mechanisms involved have been and at least a of cAMP-dependent of mTOR to of protein and on with rapamycin, it is that inhibiting mTOR to of the PHAS-I and p70S6K C.G. Trends Biochem. Sci. 1996; 21: 181-185Abstract Full Text PDF PubMed Scopus (199) Google Scholar, Jr., J.C. Abraham R.T. Trends Biochem. Sci. 1997; 22: 345-349Abstract Full Text PDF PubMed Scopus (186) Google Scholar). phosphorylation of both proteins has been shown to in response to at least in certain cell Kong X. Lin T.-A. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, T.-A. Lawrence Jr., J.C. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar, P.H. J. Biochem. J. 1996; PubMed Scopus Google Scholar). of the sites in p70S6K in of the phosphorylation of ribosomal protein and of those having the polypyrimidine tract motif C.G. Trends Biochem. Sci. 1996; 21: 181-185Abstract Full Text PDF PubMed Scopus (199) Google Scholar). of PHAS-I in binding of PHAS-I to eIF4E and of the translation of mRNA (15Lawrence Jr., J.C. Abraham R.T. Trends Biochem. Sci. 1997; 22: 345-349Abstract Full Text PDF PubMed Scopus (186) Google Scholar). eIF4E in cells have a that eIF4E is involved in the control of cell A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. Sonenberg N. Nature. PubMed Scopus Google Scholar). not directly but it is to that of mTOR and the in eIF4E function from of PHAS-I to the effects of cAMP. which mTOR signaling cell cycle progression a of mTOR with rapamycin in of the increase in needed for progression J. S. J. J.M. Nature. 1994; PubMed Scopus Google Scholar, J. J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). cAMP has also been shown to increase J. J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar), and it is an hypothesis that the of both rapamycin and cAMP on the from of mTOR. hypothesis is that the mechanisms by which rapamycin and cAMP increase J. J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). Rapamycin to the at which is J. S. J. J.M. Nature. 1994; PubMed Scopus Google Scholar), cAMP has been to increase the of the J. J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). The that of mTOR contributes to the effects of cAMP on PHAS-I and p70S6K does not that cAMP effects on the phosphorylation of these proteins by other The that forskolin a rapamycin in the phosphorylation of and be with the of an mTOR-independent effect of cAMP on these two sites in The activation of p70S6K in 3T3-L1 adipocytes is by the kinase kinase inhibitor, that one or more of the kinase family be involved in the control of p70S6K P.H. Lawrence Jr., J.C. 1997; PubMed Scopus Google Scholar). cAMP has been shown to inhibit the kinase signaling pathway in cell Lawrence Jr., J.C. Trends 1996; Full Text PDF PubMed Scopus Google Scholar). the effect of cAMP on kinase activation is not in in to findings in 3T3-L1 adipocytes, neither forskolin cAMP the activation of p70S6K in 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, V. S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). In these the activation of p70S6K was inhibited by phosphodiesterase cAMP not These support the that methylxanthines by a to activation of on the that phosphodiesterase to cAMP in a of the effect of cAMP on J. J. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar), it was that the effects on kinase activation to the of 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, V. S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). This is to be the as in cell 3T3-L1 adipocytes, have shown that cAMP and that increase cAMP inhibit activation of p70S6K in the of methylxanthines Kong X. Lin T.-A. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, T.-A. Lawrence Jr., J.C. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, P.H. J. Biochem. J. 1996; PubMed Scopus Google Scholar). The interpretation is that the effects of methylxanthines and cAMP are mediated by at least two inhibit activation of mTOR by activation of the on the finding that inhibited mTOR vitro that methylxanthines directly inhibit mTOR. in cells mTOR, it for the effects of methylxanthines on the phosphorylation of p70S6K and PHAS-I in cells the response to cAMP. increasing cAMP by activating which phosphorylates an of mTOR The the response to cAMP is not in cell as the of of elements or the of mTOR activity was from have activation of in the effects of insulin on PHAS-I A. A. P.H. Lawrence Jr., J.C. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Sonenberg N. N. 1998; PubMed Scopus Google Scholar, P.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), and have recently in the activation of mTOR by insulin (18Scott P.H. Brunn G.J. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). the possibility that increasing cAMP inhibit activation of mTOR by the activation of This hypothesis be neither forskolin CPT-cAMP affected the activation of in response to insulin in which the the activation of mTOR by the The failure to activation also that of signaling from the insulin the of cAMP. mTOR contains consensus sites for phosphorylation by P.J. J. Biol. Chem. 266: Full Text PDF PubMed Google Scholar), and have to the hypothesis that mTOR is by not able to phosphorylate mTOR in vitro with catalytic subunit of or to mTOR kinase activity by mTOR immune with the catalytic subunit. H. and J. C. Jr., These findings not the possibility that phosphorylate mTOR the the finding that cAMP the effect of insulin on mTOR phosphorylation, as assessed by mTAb1 binding (18Scott P.H. Brunn G.J. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar), support for the interpretation that the effects of cAMP are not mediated by phosphorylation of mTOR by The finding that activation of by in cells in activation of mTOR and the of mTAb1 binding that activation of is for the activation of mTOR, at least in cell (18Scott P.H. Brunn G.J. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). (18Scott P.H. Brunn G.J. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar), the mTAb1 epitope contains an consensus site for phosphorylation by but have not been able to phosphorylation of site by is a for of mTOR in the signaling not directly phosphorylate mTOR. In to the of it be to the protein for the phosphorylation and activation of mTOR. a that is an and mTOR. in which activation of or in activation or of be with the to the of mTOR by insulin and cAMP.