The receptor for activated C-kinase-I (RACK-I) anchors activated PKC-β on melanosomes

Protein kinase C (PKC), a family of at least eleven isoforms, mediates numerous cell functions. In human melanocytes, α, β, δ, ϵ and ζ isoforms of PKC are expressed, but uniquely PKC-β activates tyrosinase, the key and the rate-limiting enzyme in melanogenesis, by phosphorylating specific serine residues on its cytoplasmic domain. To investigate the mechanism by which only PKC-β phosphorylates tyrosinase, we examined the expression of receptor for activated C-kinase-I (RACK-I), a receptor specific for activated PKC-β, on the surface of melanosomes, the specialized organelle in which melanogenesis occurs. Immunoblot analysis of purified melanosomes revealed that RACK-I is readily detectable. Immunoprecipitation of RACK-I from purified melanosomes, followed by immunoblot analysis using antibody against PKC-β, revealed abundant PKC-β, whereas PKC-α was not detected when immunoblot analysis was performed using antibody against PKC-α. Activation of PKC in melanocytes increased the level of PKC-β co-immunoprecipitated with RACK-I, while the level of melanosome-associated RACK-I decreased when melanocytes were treated chronically with the 12-0-tetradecanoyl-phorbol 13-Acetate (TPA), a condition known to deplete PKC and reduce tyrosinase activity. Immunoprecipitation with RACK-I antibody co-precipitated fewer PKC-β in the presence of UV-activated 1, 1′-decamethylenebis-4-aminoquinaldinium di-iodide (DECA), known to disrupt the interaction between activated PKC-β and RACK-I. Treatment of intact melanocytes with DECA also decreased tyrosinase activity. Moreover, suppression of RACK-I expression by transfecting melanocytes with siRNA against RACK-I reduced the basal tyrosinase activity and blocked TPA-induced increases in tyrosinase activity. Taken together, these results demonstrate that RACK-I anchors activated PKC-β on the melanosome membrane, allowing PKC-β to phosphorylate tyrosinase.