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 Gilchrest, B. A. (1993) J. Biol. Chem. 268, 11742-11749), the rate-limiting enzyme in melanogenesis. We now examine its mechanism of activation in human melanocytes. In vivo phosphorylation experiments revealed that tyrosinase is phosphorylated through the PKC-dependent pathway and that introduction of PKC-beta into nonpigmented human melanoma cells lacking PKC-beta lead to the phosphorylation and activation of tyrosinase. Preincubation of intact melanosomes with purified active PKC-beta in vitro increased tyrosinase activity 3-fold. By immunoelectron microscopy, PKC-beta but not PKC-alpha was closely associated with tyrosinase on the outer surface of melanosomes. Western blot analysis confirmed the association of PKC-beta with melanosomes. Only the cytoplasmic (extra-melanosomal) domain of tyrosinase, which contains two serines but no threonines, was phosphorylated by the serine/threonine kinase PKC-beta. These two serines at positions 505 and 509 both are present in the C-terminal peptide generated by trypsin digestion of tyrosinase. Co-migration experiments comparing synthetic peptide standards of all three possible phosphorylated tryptic peptides, a diphosphopeptide and two monophosphopeptides, to tyrosinase-phosphorylated in intact melanocytes by PKC-beta and then subjected to trypsin digestion revealed that both serine residues are phosphorylated by PKC-beta. We conclude that PKC-beta activates tyrosinase directly by phosphorylating serine residues at positions 505 and 509 in the cytoplasmic domain of this melanosome-associated protein.
Park
H Y
HY
Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts 02118, USA. hypark@acs.bu.edu
Perez
J M
JM
Laursen
R
R
Hara
M
M
Gilchrest
B A
BA
D000818:Animals; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D020460:Melanosomes / Q000201:enzymology; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D011817:Rabbits; D012694:Serine / Q000378:metabolism*
D007527:Isoenzymes; D012694:Serine; D014442:Monophenol Monooxygenase; D011493:Protein Kinase C; D064546:Protein Kinase C beta