A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity.
cRaf-1 is a mitogen-activated protein kinase that is the main effector recruited by GTP-bound Ras in order to activate the MAP kinase pathway. Inactive Raf is found in the cytosol in a complex with Hsp90, Hsp50 (Cdc37) and the 14-3-3 proteins. GTP-bound Ras binds Raf and is necessary but not sufficient for the stable activation of Raf that occurs in response to serum, epidermal growth factor, platelet-derived growth factor or insulin. These agents cause a two- to threefold increase in overall phosphorylation of Raf on serine/threonine residues, and treatment of cRaf-1 with protein (serine/threonine) phosphatases can deactivate it, at least partially. The role of 14-3-3 proteins in the regulation of Raf's kinase activity is uncertain and is investigated here. Active Raf can be almost completely deactivated in vitro by displacement of 14-3-3 using synthetic phosphopeptides. Deactivation can be substantially reversed by addition of purified recombinant bacterial 14-3-3; however, Raf must have been previously activated in vivo to be reactivated by 14-3-3 in vitro. The ability of 14-3-3 to support Raf activity is dependent on phosphorylation of serine residues on Raf and on the integrity of the 14-3-3 dimer; mutant monomeric forms of 14-3-3, although able to bind Raf in vivo, do not enable Raf to be activated in vivo or restore Raf activity after displacement of 14-3-3 in vitro. The 14-3-3 protein is not required to induce dimerization of Raf. We propose that dimeric 14-3-3 is needed both to maintain Raf in an inactive state in the absence of GTP-bound Ras and to stabilize an active conformation of Raf produced during activation in vivo.
Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston 02114, USA.
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D048948:14-3-3 Proteins; D000818:Animals; D019556:COS Cells; D019281:Dimerization; D004789:Enzyme Activation; D009154:Mutation; D010748:Phosphopeptides / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D010748:Phosphopeptides; D011506:Proteins; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C