Disruption of the 14-3-3 binding site within the B-Raf kinase domain uncouples catalytic activity from PC12 cell differentiation

A number of Raf-associated proteins have recently been identified, includin g members of the 14-3-3 family of phosphoserine-binding proteins. Although both positive and negative regulatory functions have been ascribed for 14-3 -3 interactions with Raf-1, the mechanisms by which 14-3-3 binding modulate s Raf activity have not been fully established. We report that mutational d isruption of 14-3-3 binding to the B-Raf catalytic domain inhibits B-Raf bi ological activity. Expression of the isolated B-Raf catalytic domain (B-Raf cat) induces PC12 cell differentiation in the absence of nerve growth facto r, By contrast, the B-Rafcat 14-3-3 binding mutant, B-Rafcat S728A, was sev erely compromised for the induction of PC12 cell differentiation. Interesti ngly, the B-Rafcat 14-3-3 binding mutant retained significant in vitro cata lytic activity. In Xenopus oocytes, the analogous full-length B-Raf 14-3-3 binding mutant blocked progesterone-stimulated maturation and the activatio n of endogenous mitogen-activated protein kinase kinase and mitogen-activat ed protein kinase. Similarly, the full-length B-Raf 14-3-3 binding mutant i nhibited nerve growth factor-stimulated PC12 cell differentiation. We concl ude that 14-3-3 interaction with the catalytic domain is not required for k inase activity per se but is essential to couple B-Raf catalytic activity t o downstream effector activation.