14-3-3 zeta negatively regulates Raf-1 activity by interactions with the Raf-1 cysteine-rich domain

Although Raf-l is a critical effector of nas signaling and transformation, the mechanism by which Ras promotes Raf-l activation is complex and remains pearly understood, We recently reported that Ras interaction with the Raf-1 cysteine-rich domain (Raf-CRD, residues 139-184) may be required for Raf-l activation, The Raf-CRD is located in the NH2-terminal negative regulatory domain of Raf-l and is highly homologous to cysteine-rich domains found in protein kinase C family members. Recent studies indicate that the structural integrity of the Raf-CRD is also critical for Raf-l interaction with 14-3-3 proteins. However, whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction may mediate Raf-1 function has not been determined. In the present study, we demonstrate that 14-3-3 zeta binds directly to the isolated Raf-CRD. Moreover, mutation of Raf-1 residues 143-145 impairs binding of 14-3-3, but not Ras, to the Raf-CRD. Introduction of mutations that impair 14-3-3 binding resulted in full-length Raf-1 mutants with enhanced transforming activity. Thus, 14-3-3 interaction with the Raf-CRD may serve in negative regulation of Raf-1 function by facilitating dissociation of 14-3-3 from the NH, terminus of Raf-P to promote subsequent events necessary for full activation of Raf-1.