Protein kinase Cα activates RAF-1 by direct phosphorylation

THE kinase Raf-1 can be activated by treatment of cells with mitogens and by the protein kinase C (PKC) activator 12-0-tetradecanoyl-phorbol-13-acetate (TPA) (reviewed in refs 1,2). Activated Raf-1 triggers a protein kinase cascade by direct phosphorylation of MAP kinase kinase^3-5, resulting in phosphorylation of ternary complex factor⁶ and Jim^7,8 by MAP kinase. Here we investigate the molecular mechanism and biological consequences of PKCα-mediated Raf-1 activation in NIH3T³ fibroblasts. PKCα directly phosphorylates and activates Raf-1 both in vitro and in vivo. PKCα induces Raf-1 phosphorylation at several sites, including a serine residue at position 499. Mutation of serine at this position or at residue 259 does not abrogate Raf-1 stimulation by a combination of Ras plus the src tyrosine kinase Lck, but severely impedes Raf-1 activation by PKCα. Consistent with such a direct interaction is the observation that Raf-1 and PKCα cooperate in the transformation of NIH3T3 cells. The Ser499 phosphorylation site is necessary for this synergism.