A dimeric 14-3-3 protein is an essential cofactor for Raf kinase activity

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 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.

Mechanistic principles of RAF kinase signaling

“…Ser621 is conserved in all RAF proteins and is also a docking site for 14-3-3 proteins [113,114]. Binding of 14-3-3 to phospho-Ser621 appears to have multiple roles in RAF regulation.…”

Phosphoserine/Threonine Binding Domains

“…For example, 14-3-3 helps to maintain the mitogenstimulated kinase Raf in an inactive but activatable conformation in resting cells. Upon cellular stimulation with growth factors, 14-3-3 is partly displaced from Raf to allow Raf activation, but 14-3-3 binding is not completely eliminated, because its continued presence is necessary for full catalytic activity [23][24][25][26][27].…”

“…A phosphopeptide binding site present within each monomer suggests that a dimeric 14-3-3 molecule might engage two distinct motifs within a single molecule to make use of an avidity effect. This type of bidentate interaction has been directly observed in 14-3-3 binding to serotonin N-acetyl transferase [48] and may occur in other 14-3-3 substrates that contain two or more phosphorylated 14-3-3 binding sites, such as Raf and BAD [5,18,[26][27][28]. A synthetic phosphopeptide that contains tandem 14-3-3 consensus motifs binds to 14-3-3 over 30 times more tightly than the same peptide containing only one phospho-motif [6].…”

Establishment and Characterization of a Murine Mast Cell Line Derived from NC/Nga Mice

“…Immunoprecipitation and immunoblotting were perfomed as previously described [9]following the manufacturer’s instruction using the anti Raf-1 (Ser259) Ab, and phospho-p44/42 MAPK (Erk1/2) Ab (New England Biolabs, Mass., USA) [10, 11]. …”