PROTEIN KINASE-C-ALPHA 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-O-tetradecanoyl-phorbol-13-acetate (TPA) (reviewed in refs 1,2). Activated Raf-1 triggers a protein kinase cascade by direct phosphorylation of MAP kinase kinase3-5, resulting in phosphorylation of ternary complex factor6 and Jun7,8 by MAP kinase. Here we investigate the molecular mechanism and biological consequences of PKCalpha-mediated Raf-1 activation in NIH3T3 fibroblasts. PKCalpha directly phosphorylates and activates Raf-1 both in vitro and in vivo. PKCalpha 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 PKCalpha. Consistent with such a direct interaction is the observation that Raf-1 and PKCalpha cooperate in the transformation of NIH3T3 cells. The Ser499 phosphorylation site is necessary for this synergism.