title: The receptor for activated C-kinase-I (RACK-I) anchors activated PKC-beta on melanosomes.
abstract: Protein kinase C (PKC), a family of at least eleven isoforms, mediates numerous cell functions. In human melanocytes, alpha, beta, delta, epsilon and zeta isoforms of PKC are expressed, but uniquely PKC-beta 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-beta phosphorylates tyrosinase, we examined the expression of receptor for activated C-kinase-I (RACK-I), a receptor specific for activated PKC-beta, 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-beta, revealed abundant PKC-beta, whereas PKC-alpha was not detected when immunoblot analysis was performed using antibody against PKC-alpha. Activation of PKC in melanocytes increased the level of PKC-beta 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-beta in the presence of UV-activated 1, 1'-decamethylenebis-4-aminoquinaldinium di-iodide (DECA), known to disrupt the interaction between activated PKC-beta 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-beta on the melanosome membrane, allowing PKC-beta to phosphorylate tyrosinase.
authors.affiliation: Department of Dermatology, Boston University School of Medicine, 609 Albany Street, Boston, MA 02118, USA. hypark@bu.edu
authors.forename: Hee-Young
authors.initials: HY
authors.lastname: Park
authors.forename: Heng
authors.initials: H
authors.lastname: Wu
authors.forename: Christina E
authors.initials: CE
authors.lastname: Killoran
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Gilchrest
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D003868:Dequalinium / Q000031:analogs & derivatives* / Q000494:pharmacology; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D020460:Melanosomes / Q000201:enzymology / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D034622:RNA Interference; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: C500008:1,1'-decamethylenebis-4-aminoquinaldinium di-iodide; D017931:DNA Primers; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D003868:Dequalinium; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D013755:Tetradecanoylphorbol Acetate