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Cell Type-specific Regulation of B-Raf Kinase by cAMP and 14-3-3 Proteins

2000; Elsevier BV; Volume: 275; Issue: 41 Linguagem: Inglês

10.1074/jbc.m003327200

ISSN

1083-351X

Autores

W. Qiu, Shunhui Zhuang, Friederike C. von Lintig, Gerry R. Boss, Renate B. Pilz,

Tópico(s)

Protein Kinase Regulation and GTPase Signaling

Resumo

Cyclic AMP can either activate or inhibit the mitogen-activated protein kinase (MAPK) pathway in different cell types; MAPK activation has been observed in B-Raf-expressing cells and has been attributed to Rap1 activation with subsequent B-Raf activation, whereas MAPK inhibition has been observed in cells lacking B-Raf and has been attributed to cAMP-dependent protein kinase (protein kinase A)-mediated phosphorylation and inhibition of Raf-1 kinase. We found that cAMP stimulated MAPK activity in CHO-K1 and PC12 cells but inhibited MAPK activity in C6 and NB2A cells. In all four cell types, cAMP activated Rap1, and the 95- and 68-kDa isoforms of B-Raf were expressed. cAMP activation or inhibition of MAPK correlated with activation or inhibition of endogenous and transfected B-Raf kinase. Although all cell types expressed similar amounts of 14-3-3 proteins, approximately 5-fold less 14-3-3 was associated with B-Raf in cells in which cAMP was inhibitory than in cells in which cAMP was stimulatory. We found that the cell type-specific inhibition of B-Raf could be completely prevented by overexpression of 14-3-3 isoforms, whereas expression of a dominant negative 14-3-3 mutant resulted in partial loss of B-Raf activity. Our data suggest that 14-3-3 bound to B-Raf protects the enzyme from protein kinase A-mediated inhibition; the amount of 14-3-3 associated with B-Raf may explain the tissue-specific effects of cAMP on B-Raf kinase activity. Cyclic AMP can either activate or inhibit the mitogen-activated protein kinase (MAPK) pathway in different cell types; MAPK activation has been observed in B-Raf-expressing cells and has been attributed to Rap1 activation with subsequent B-Raf activation, whereas MAPK inhibition has been observed in cells lacking B-Raf and has been attributed to cAMP-dependent protein kinase (protein kinase A)-mediated phosphorylation and inhibition of Raf-1 kinase. We found that cAMP stimulated MAPK activity in CHO-K1 and PC12 cells but inhibited MAPK activity in C6 and NB2A cells. In all four cell types, cAMP activated Rap1, and the 95- and 68-kDa isoforms of B-Raf were expressed. cAMP activation or inhibition of MAPK correlated with activation or inhibition of endogenous and transfected B-Raf kinase. Although all cell types expressed similar amounts of 14-3-3 proteins, approximately 5-fold less 14-3-3 was associated with B-Raf in cells in which cAMP was inhibitory than in cells in which cAMP was stimulatory. We found that the cell type-specific inhibition of B-Raf could be completely prevented by overexpression of 14-3-3 isoforms, whereas expression of a dominant negative 14-3-3 mutant resulted in partial loss of B-Raf activity. 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Cell Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar). These data have led to a model in which 14-3-3 binding to Raf-1 is necessary to keep the enzyme in an inactive but activation-competent conformation (56Hancock J.F. Roy S. McPherson R.A. Appoloni A. Yan J. Lane A. Clyde-Smith J. Mol. Cell Biol. 1998; 18: 3947-3955Crossref PubMed Scopus (116) Google Scholar, 59Tzivion G. Luo Z. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (388) Google Scholar, 60Li S. Janosch P. Tanji M. Rosenfeld G.C. Waymire J.C. Mischak H. Kolch W. Sedivy J.M. EMBO J. 1995; 14: 685-696Crossref PubMed Scopus (154) Google Scholar). Some investigators have reported that overexpression of 14-3-3 potentiates Raf-1 activation, whereas others have found no effect (53Thorson J.A., Yu, L.W.K. Hsu A.L. Shih N.-Y. Graves P.R. Tanner J.W. Allen P.M. Piwnica-Worms H. Shaw A.S. Mol. Cell Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar, 54Michaud N.R. Fabian J.R. Mathes K.D. Morrison D.K. Mol. Cell Biol. 1995; 15: 3390-3397Crossref PubMed Scopus (190) Google Scholar, 56Hancock J.F. Roy S. McPherson R.A. Appoloni A. Yan J. Lane A. Clyde-Smith J. Mol. Cell Biol. 1998; 18: 3947-3955Crossref PubMed Scopus (116) Google Scholar, 60Li S. Janosch P. Tanji M. Rosenfeld G.C. Waymire J.C. Mischak H. Kolch W. Sedivy J.M. EMBO J. 1995; 14: 685-696Crossref PubMed Scopus (154) Google Scholar). All three 14-3-3 binding sites of Raf-1 are highly conserved in B-Raf, and intracellularly, B-Raf appears to exists in a high molecular weight complex with 14-3-3 proteins, HSP90, and MEK-1 and -2 (8Moodie S.A. Paris M.J. Kolch W. Wolfman A. Mol. Cell Biol. 1994; 14: 7153-7162Crossref PubMed Scopus (69) Google Scholar, 49Sithanandam G. Kolch W. Duh F.-M. Rapp U.R. Oncogene. 1990; 5: 1775-1780PubMed Google Scholar, 50Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1188) Google Scholar, 61Yamamori B. Kuroda S. Shimizu K. Fukui K. Ohtsuka T. Takai Y. J. Biol. Chem. 1995; 270: 11723-11726Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 62Jaiswal R.K. Weissinger E. Kolch W. Landreth G.E. J. Biol. Chem. 1996; 271: 23626-23629Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 63Papin C. Eychene A. Brunet A. Pages G. Pouyssegur J. Calothy G. Barnier J.V. Oncogene. 1995; 10: 1647-1651PubMed Google Scholar). Ser728 of B-Raf is a 14-3-3 binding site that appears to be necessary for the biological activity of B-Raf; a Ser728 to Ala substitution in the isolated B-Raf catalytic domain dramatically reduces the ability of the enzyme to induce Xenopus oocyte maturation or PC12 cell differentiation, although the mutant enzyme retains significant catalytic activity in vitro (64MacNicol M.C. Muslin A.J. MacNicol A.M. J. Biol. Chem. 2000; 275: 3803-3809Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). B-Raf purified fromXenopus oocytes is synergistically activated by a combination of 14-3-3 and Ras·GTP (14Shimizu K. Kuroda S. Yamamori B. Matsuda S. Kaibuchi K. Yamauchi T. Isobe T. Irie K. Matsumoto K. Takai Y. J. Biol. Chem. 1994; 269: 22917-22920Abstract Full Text PDF PubMed Google Scholar). We hypothesized that the variable results concerning cAMP regulation of B-Raf could be secondary to cell type-specific factors and, therefore, studied the effect of cAMP on B-Raf and MAPK activity in CHO-K1, PC12, C6 glioma, and NB2A neuroblastoma cells. In all four cell types, the 95-kDa isoform of B-Raf was expressed and membrane-permeable cAMP analogs increased the activation state of Rap1. However, the cAMP analogs stimulated B-Raf and MAPK activity in CHO-K1 and PC12 cells while inhibiting B-Raf and MAPK activity in C6 and NB2A cells. We found that inhibition of B-Raf by cAMP correlated with significantly lower amounts of enzyme-associated 14-3-3, and the cAMP-mediated inhibition was completely prevented by overexpression of 14-3-3; expression of a dominant negative 14-3-3 resulted in partial loss of B-Raf kinase activity. Our data suggest a model in which 14-3-3 protects B-Raf from protein kinase A inhibition and may explain the varying response of B-Raf to cAMP in different cell types. Rabbit polyclonal antibodies specific for Erk-1/2 (C16, SC93), for B-Raf kinase (C19, SC166), for Rap1 (121, SC65), and for 14-3-3 (K19, SC629) were from Santa Cruz Biotechnology, as was recombinant MEK-1. An anti-active MAPK antibody (V803A) was from Promega, an actin-specific antibody (A2066) was from Sigma, and a Raf-1 phospho-Ser621-specific antibody was from A. S. Shaw (53Thorson J.A., Yu, L.W.K. Hsu A.L. Shih N.-Y. Graves P.R. Tanner J.W. Allen P.M. Piwnica-Worms H. Shaw A.S. Mol. Cell Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar). The glutathione S-transferase (GST)-tagged peptide encoding the Rap-binding domain of Ral-GDS (RBD peptide) was purified from bacteria transformed with the expression plasmid pGEX-RGF97 (provided by J. L. Bos; Ref. 65Franke B. Akkerman J.-W. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar). The membrane-permeable cAMP analogs 8-(4-chlorophenylthio)cAMP (8-pCPT-cAMP) and 8-bromo-cAMP were from Biolog, and forskolin was from Calbiochem. LipofectAMINE PlusTM and LipofectAMINE 2000TM were from Life Technologies, Inc. An expression vector encoding the 95-kDa isoform of B-Raf but lacking exons 8b and 10 was from P. J. S. Stork (pcDNA3-Braf; Ref. 33Vossler M.R. Yao H. York R.D. Pan M.-G. Rim C.S. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar). Either full-length B-Raf or the catalytic domain of B-Raf (amino acids 385–765) was fused in frame to GST in the vector pCMV-GST usingBamHI (66Tsai R.Y.L. Reed R.R. BioTechniques. 1997; 23: 794-800Crossref PubMed Scopus (29) Google Scholar). Wild type 14-3-3β,14-3-3τ, and the dominant negative mutant 14-3-3(R56, 60A) were from A. S. Shaw (53Thorson J.A., Yu, L.W.K. Hsu A.L. Shih N.-Y. Graves P.R. Tanner J.W. Allen P.M. Piwnica-Worms H. Shaw A.S. Mol. Cell Biol. 1998; 18: 5229-5238Crossref PubMed Scopus (184) Google Scholar). The reporter plasmid pGAL4-Luc was from M. Karin, and pElk-Gal4 was from G. L. Johnson (67Johnson N.L. Gardner A.M. Diener K.M. Lange-Carter C.A. Gleavy J. Jarpe M.B. Minden A. Karin M. Zon L.I. Johnson G.L. J. Biol. Chem. 1996; 271: 3229-3237Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). CHO-K1 and PC12 cells were from the American Type Culture Collection, and C6 and NB2A cells were provided by M. H. Ellisman and E. Koo, respectively. CHO-K1 and NB2A cells were grown in F-12K medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS); C6 and PC12 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% or 5% FBS plus 5% horse serum, respectively. Cells were transfected using 9 μl of LipofectAMINE PlusTM and 1.2 μg of DNA (for C6, CHO-K1, and PC12 cells) or 9 μl of LipofectAMINE 2000TM and 1.2 μg of DNA (for NB2A cells) in 1 ml of OptiMEMTM medium according to the manufacturer's protocol. After 5 h of transfection, cells were placed for 1 h in serum-containing medium and then some of the cells were transferred to medium containing 0.1% FBS and 0.1% bovine serum albumin (referred to as "low serum-containing medium"). Cells were cultured for 48 h in either full (10%) serum-containing medium or in low serum-containing medium prior to adding 250 μm 8-CPT-cAMP for the indicated time. Cells were lysed in SDS-polyacrylamide electrophoresis (PAGE) sample buffer, and Western blots were generated as described previously (68Suhasini M. Li H. Lohmann S.M. Boss G.R. Pilz R.B. Mol. Cell Biol. 1998; 18: 6983-6994Crossref PubMed Scopus (100) Google Scholar) using an anti-active MAPK antibody that specifically recognizes the dually phosphorylated, active form of Erk-1 and -2 (69Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.-H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (841) Google Scholar). Equal loading of protein was verified by reprobing the blot with an Erk-1 and -2-specific antibody. Western blots were developed using horseradish peroxidase-coupled secondary antibodies and enhanced chemiluminescence. In some experiments, MAPK activity was measured in Erk-1/2 immunoprecipitates using myelin basic protein as substrate (68Suhasini M. Li H. Lohmann S.M. Boss G.R. Pilz R.B. Mol. Cell Biol. 1998; 18: 6983-6994Crossref PubMed Scopus (100) Google Scholar). Rap1·GTP was captured using RBD peptide (65Franke B. Akkerman J.-W. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar) and GTP eluted from the isolated Rap was measured as described previously (70Sharma P.M. Egawa K. Huang Y. Martin J.L. Huvar I. Boss G.R. Olefsky J.M. J. Biol. Chem. 1998; 273: 18528-18537Abstract Full Text Full Text PDF PubMed Scopus (108) Google Schol

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