1. Diabetes Unit and Medical Services and the Department of Molecular Biology, Massachusetts General Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114, USA

Correspondence to: Joseph Avruch¹ Correspondence and requests for materials should be addressed to J. A. (e-mail: Email: avruch@helix.mgh.harvard.edu).

Abstract

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 pathway¹. Inactive Raf is found in the cytosol in a complex with Hsp90, Hsp50 (Cdc37)²,³ and the 14-3-3 proteins⁴. 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^{5, 6, 7, 8}. These agents cause a two- to threefold increase in overall phosphorylation of Raf on serine/threonine residues⁸,⁹, and treatment of cRaf-1 with protein (serine/threonine) phosphatases can deactivate it, at least partially¹⁰. The role of 14-3-3 proteins in the regulation of Raf's kinase activity is uncertain⁴,¹¹ 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.