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Cyclic AMP Inhibits Extracellular Signal-regulated Kinase and Phosphatidylinositol 3-Kinase/Akt Pathways by Inhibiting Rap1*

  • Lai Wang
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  • Feng Liu
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  • Martin L. Adamo
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, Mail Code 7760, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, Texas 78229-3900. Tel.: 210-567-3742; Fax: 210-567-6595; E-mail: [email protected]
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  • Author Footnotes
    * This study was supported by Grant DK-47357 from NIDDK, National Institutes of Health, Grant AQ-1385 from the Robert A. Welch Foundation, and Grant 07 from the Children's Cancer Research Center of University of Texas Health Science Center at San Antonio (to M. L. A.) and by Grant DK-56166 (to F. L.) from NIDDK, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:October 05, 2001DOI:https://doi.org/10.1074/jbc.M105089200
      Cyclic AMP inhibited both ERK and Akt activities in rat C6 glioma cells. A constitutively active form of phosphatidylinositol 3-kinase (PI3K) prevented cAMP from inhibiting Akt, suggesting that the inactivation of Akt by cAMP is a consequence of PI3K inhibition. Neither protein kinase A nor Epac (Exchange protein directlyactivated by cAMP), two known direct effectors of cAMP, mediated the cAMP-induced inhibition of ERK and Akt phosphorylation. Cyclic AMP inhibited Rap1 activation in C6 cells. Moreover, inhibition of Rap1 by a Rap1 GTPase-activating protein-1 also resulted in a decrease in ERK and Akt phosphorylation, which was not further decreased by cAMP, suggesting that cAMP inhibits ERK and Akt by inhibiting Rap1. The role of Rap1 in ERK and Akt activity was further demonstrated by our observation that an active form of Epac, which activated Rap1 in the absence of cAMP, increased ERK and Akt phosphorylation. Inhibition of ERK and/or PI3K pathways mediated the inhibitory effects of cAMP on insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 gene expression. Moreover, cAMP, as well as ERK and PI3K inhibitors produced equivalent stimulation and inhibition, respectively, of p27Kip1 and cyclin D2 protein levels, potentially explaining the observation that cAMP prevented C6 cells from entering S phase.
      cAMP
      cyclic AMP
      8-CPT-cAMP
      8-(4-chloropenylthio)-cAMP
      Cdk
      cyclin-dependent kinase
      Epac
      exchange protein directly activated by cAMP
      ERK
      extracellular signal-regulated kinase
      FBS
      fetal bovine serum
      GAP
      GTPase activating protein
      GEF
      guanine nucleotide exchange factor
      GST-RBD
      glutathioneS-transferase Ras binding domain
      HA
      hemagglutinin
      IGF-I
      insulin-like growth factor I
      IGFBP
      insulin-like growth factor binding protein
      MAPK
      mitogen-activated protein kinase
      MEK
      MAPK/ERK kinase
      PAGE
      polyacrylamide gel electrophoresis
      PI3K
      phosphatidylinositol 3-kinase
      PKA
      protein kinase A
      Rap1GAP1
      Rap1 GTPase-activating protein 1
      Cyclic AMP stimulates the proliferation of various epithelial cells, hepatocytes, keratinocytes, pancreatic islet β cells, Schwann cells, and Swiss 3T3 cells (
      • Dumont J.E.
      • Jauniaux J.C.
      • Roger P.P.
      ). On the other hand, cAMP1 inhibits proliferation of normal fibroblasts, smooth muscle cells, lymphoid cells, neuronal cells, and glial cells (
      • Hollenberg M.D.
      • Cuatrecasas P.
      ,
      • Nilsson J.
      • Olsson A.G.
      ,
      • Blomhoff H.K.
      • Blomhoff R.
      • Stokke T.
      • deLange Davies C.
      • Brevik K.
      • Smeland E.B.
      • Funderud S.
      • Godal T.
      ,
      • Mark M.D.
      • Storm D.R.
      ,
      • Dugan L.L.
      • Kim J.S.
      • Zhang Y.
      • Bart R.D.
      • Sun Y.
      • Holtzman D.M.
      • Gutmann D.H.
      ). The growth-inhibitory effects of cAMP are proposed to be mediated at least in part through cAMP-dependent inactivation of MAPK, also known as ERK1/2 (
      • Dugan L.L.
      • Kim J.S.
      • Zhang Y.
      • Bart R.D.
      • Sun Y.
      • Holtzman D.M.
      • Gutmann D.H.
      ,
      • Cook S.J.
      • McCormick F.
      ,
      • Hordijk P.L.
      • Verlaan I.
      • Jalink K.
      • Van Corven E.J.
      • Moolenaar W.H.
      ,
      • Schmitt J.M.
      • Stork P.J.
      ). ERK1/2 are phosphorylated and activated by MEK-1 and -2, which are phosphorylated and activated by members of the Raf family of protein kinases (Raf-1, A-Raf, and B-Raf) (
      • Hagemann C.
      • Rapp U.R.
      ). The activities of Raf kinases are regulated by the Ras family of small GTP-binding proteins, including Ras and Rap1 (
      • Zwartkruis F.J.
      • Bos J.L.
      ). Three different pathways have been suggested to be involved in the inhibition of ERK by cAMP: 1) cAMP-activated PKA phosphorylates Rap1, which induces Rap1 GTP-binding by an unknown mechanism, resulting in competition with Ras for the binding of Raf-1 and thereby blocking Ras-induced Raf-1 activation (
      • Schmitt J.M.
      • Stork P.J.
      ,
      • Vossler M.R.
      • Yao H.
      • York R.D.
      • Pan M.G.
      • Rim C.S.
      • Stork P.J.
      ); 2) phosphorylation of Raf-1 directly by PKA at two serine residues inhibits both Ras binding and Raf-1 activity (
      • Wu J.
      • Dent P.
      • Jelinek T.
      • Wolfman A.
      • Weber M.J.
      • Sturgill T.W.
      ,
      • Mischak H.
      • Seitz T.
      • Janosch P.
      • Eulitz M.
      • Steen H.
      • Schellerer M.
      • Philipp A.
      • Kolch W.
      ,
      • Häfner S.
      • Adler H.S.
      • Mischak H.
      • Janosch P.
      • Heidecker G.
      • Wolfman A.
      • Pippig S.
      • Lohse M.
      • Ueffing M.
      • Kolch W.
      ); 3) cAMP inhibits ERK by inhibiting B-Raf using an unknown mechanism (
      • Qiu W.
      • Zhuang S.
      • von Lintig F.C.
      • Boss G.R.
      • Pilz R.B.
      ), although in other studies, cAMP is reported to activate B-Raf through PKA/Rap1, which leads to ERK activation (
      • Vossler M.R.
      • Yao H.
      • York R.D.
      • Pan M.G.
      • Rim C.S.
      • Stork P.J.
      ,
      • Seidel M.G.
      • Klinger M.
      • Freissmuth M.
      • Holler C.
      ).
      Cyclic AMP and trophic hormones in which the second messenger is cAMP activate PI3K, Akt, and/or p70 S6 kinase in thyroid cells and ovarian granulosa cells, which may mediate the cAMP-induced growth of these cells (
      • Cass L.A.
      • Meinkoth J.L.
      ,
      • Cass L.A.
      • Summers S.A.
      • Prendergast G.V.
      • Backer J.M.
      • Birnbaum M.J.
      • Meinkoth J.L.
      ,
      • Gonzalez-Robayna I.J.
      • Falender A.E.
      • Ochsner S.
      • Firestone G.L.
      • Richards J.S.
      ). Cyclic AMP activates Akt through a PI3K-dependent mechanism (
      • Gonzalez-Robayna I.J.
      • Falender A.E.
      • Ochsner S.
      • Firestone G.L.
      • Richards J.S.
      ,
      • Tsygankova O.M.
      • Saavedra A.
      • Rebhun J.F.
      • Quilliam L.A.
      • Meinkoth J.L.
      ). Moreover, activation of Rap1 is suggested to mediate cAMP-induced Akt activation (
      • Tsygankova O.M.
      • Saavedra A.
      • Rebhun J.F.
      • Quilliam L.A.
      • Meinkoth J.L.
      ). Inhibition of PI3K/Akt pathways by cAMP has been reported in Swiss 3T3, HEK293, COS, and Rat2 cells, although the mechanism(s) by which this occurs is largely uncharacterized (
      • Kim S.
      • Jee K.
      • Kim D.
      • Koh H.
      • Chung J.
      ).
      Rat C6 glioma cells are one of the well established glioma cell lines used for a variety of studies related to glioma cell biology (
      • Barth R.F.
      ). Our previous study showed that cAMP inhibits both C6 cell growth and expression of the autocrine growth factor IGF-I (
      • Wang L.
      • Adamo M.L.
      ). The addition of exogenous IGF-I peptide to cAMP-treated C6 cells only partially prevents the decrease in cell growth caused by cAMP treatment, suggesting that cAMP inhibits C6 cell growth by another mechanism(s) in addition to down-regulation of IGF-I gene expression. It has been reported that cAMP inhibits ERK activity in both primary astrocytes and C6 cells (
      • Dugan L.L.
      • Kim J.S.
      • Zhang Y.
      • Bart R.D.
      • Sun Y.
      • Holtzman D.M.
      • Gutmann D.H.
      ,
      • Qiu W.
      • Zhuang S.
      • von Lintig F.C.
      • Boss G.R.
      • Pilz R.B.
      ,
      • Kurino M.
      • Fukunaga K.
      • Ushio Y.
      • Miyamoto E.
      ). This inactivation of ERK has been proposed to mediate the inhibitory effect of cAMP on astrocyte or C6 cell growth, although as described above, the mechanism(s) by which cAMP inhibits ERK remains to be clarified. In this study, we showed that cAMP inhibited ERK and PI3K/Akt pathways by inhibiting Rap1 activity in C6 cells. Neither PKA nor Epac (Exchangeprotein directly activated by cAMP) was involved in the cAMP-dependent inactivation of ERK and Akt. Moreover, the inhibition of these two pathways contributes to cAMP effects on cellular gene expression and growth.

      Acknowledgments

      We thank Xiuye Ma and Michael Wick (University of Texas Health Science Center at San Antonio (UTHSCSA)) for their excellent technical support. We thank Charles A. Thomas III (Dept. of Medicine, UTHSCSA) for his technical support with the fluorescence-activated cell sorting analyses. We thank Dr. J. Silvio Gutkind for the ERK2 expression vector, Drs. Anke Klippel and Lewis T. Williams for the p110* plasmid, Dr. Stanley McKnight for the PKA expression vectors, Dr. Johannes L. Bos for the active Epac and RalGDS GST-RBD constructs, and Dr. Philip J. Stork for the Rap1 and Rap1GAP1 expression vectors.

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