Protein kinase C-beta activates tyrosinase by phosphorylating serine residues in its cytoplasmic domain

We have previously shown that protein kinase C-beta (PKC-beta) is required for activation of tyrosinase (Park, H. Y,, Russakovsky, V., Ohno, S., and G ilchrest, B. A. (1993) J. Biol. Chem. 268, 11742-11749), the rate-limiting enzyme in melanogenesis, We now examine its mechanism of activation in huma n melanocytes. lit vivo phosphorylation experiments revealed that tyrosinas e is phosphorylated through the PKC-dependent pathway and that introduction of PKC-beta into nonpigmented human melanoma cells lacking PKC-beta lead t o the phosphorylation and activation of tyrosinase. Preincubation of intact melanosomes with purified active PKC-beta in vitro increased tyrosinase ac tivity S-fold. By immunoelectron microscopy, PKC-beta but not PRC-alpha was closely associated with tyrosinase on the outer surface of melanosomes. We stern blot analysis confirmed the association of PKC-beta with melanosomes. Only the cytoplasmic (extra-melanosomal) domain of tyrosinase, which conta ins two serines but no threonines, was phosphorylated by the serine/threoni ne kinase PKC-beta. These two serines at positions 505 and 509 both are pre sent in the C-terminal peptide generated by trypsin digestion of tyrosinase . Go-migration experiments comparing synthetic peptide standards of all thr ee possible phosphorylated tryptic peptides, a diphosphopeptide and two mon ophosphopeptides, to tyrosinase-phosphorylated in intact melanocytes by PKC -beta and then subjected to trypsin digestion revealed that both serine res idues are phosphorylated by PKC-beta. We conclude that PKC-beta activates t yrosinase directly by phosphorylating serine residues at positions 505 and 509 in the cytoplasmic domain of this melanosome-associated protein.