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First published online July 13, 2004
doi: 10.1242/10.1242/jcs.01219

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

Department of Dermatology, Boston University School of Medicine, 609 Albany Street, Boston, MA 02118, USA

^* Author for correspondence (e-mail: hypark{at}bu.edu)

Accepted 11 March 2004

Protein kinase C (PKC), a family of at least eleven isoforms, mediates numerous cell functions. In human melanocytes, , ß, , and isoforms of PKC are expressed, but uniquely PKC-ß 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-ß phosphorylates tyrosinase, we examined the expression of receptor for activated C-kinase-I (RACK-I), a receptor specific for activated PKC-ß, 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-ß, revealed abundant PKC-ß, whereas PKC- was not detected when immunoblot analysis was performed using antibody against PKC-. Activation of PKC in melanocytes increased the level of PKC-ß 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-ß in the presence of UV-activated 1, 1'-decamethylenebis-4-aminoquinaldinium di-iodide (DECA), known to disrupt the interaction between activated PKC-ß 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-ß on the melanosome membrane, allowing PKC-ß to phosphorylate tyrosinase.

Key words: RACK-I, PKC-ß, Melanocyte, Tyrosinase, Melanosomes, Pigmentation

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