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

Abstract

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.

Specifying protein kinase C functions in melanoma

“…Consistent with this, PKC activators can stimulate melanocyte pigmentation and PKC inhibitors block pigment production in mouse skin (Park et al, 1993(Park et al, , 2004a. Selectivity for PKCb phosphorylation of tyrosinase on melanosomes is dependent on the anchoring protein RACK1 (Park et al, 2004b;Stebbins and Mochly-Rosen, 2001). The loss of PKCb expression and pigment production would be predicted to increase damage from UV radiation, the principle etiological agent for both melanoma and non-melanoma skin cancers.…”

“…In addition, PKCb expression is reduced or undetectable in 90% of melanoma cell lines and 50% of benign nevi and melanomas (Gilhooly et al, 2001;Oka et al, 1996Oka et al, , 2006Powell et al, 1993;Ryu et al, 2007;Voris et al, 2010;Yamanishi et al, 1991). The function of PKCb in melanin biosynthesis is linked to its ability to phosphorylate and activate tyrosinase, the rate-limiting enzyme in melanin production (Park et al, 1999(Park et al, , 2004b. Consistent with this, PKC activators can stimulate melanocyte pigmentation and PKC inhibitors block pigment production in mouse skin (Park et al, 1993(Park et al, , 2004a.…”

“…The subcellular locations of individual PKC enzymes are highly variable and dependent on the cell type, activation mechanism, scaffolding ⁄ adaptor proteins present, and even the stage of cell cycle, making it difficult to generalize about PKC enzyme subcellular localization (Perego et al, 2002;Schechtman and Mochly-Rosen, 2001;Wang et al, 1999Wang et al, , 2000. Studies in melanocytes have localized PKCb to melanosomes dependent on the scaffolding protein RACK1 and have found mitochondrial localization of PKCb-II (Park et al, 2004b;Voris et al, 2010). Nuclear PKCd is associated with apoptosis in melanoma, and PKC enzymes have also been localized to the cytoskeleton, consistent with their role in cell migration (Szalay et al, 2001;Zhang et al, 2005).…”

A Spatiotemporally Coordinated Cascade of Protein Kinase C Activation Controls Isoform-Selective Translocation

“…Indeed, PKCs are known to interact with many partners (39), some of which are adaptor proteins able to anchor specific isoforms to selective subcellular locations and thus to maintain each PKC isoform next to a subset of proteins and away from the other substrates (29,30,48). A recent example of this has been provided by Park et al who show that RACK1 (named RACK1 for receptor for activated C kinase I) anchors PKC␤ on melanosomes where it phosphorylates tyrosinase (36). In PKCε-induced heart failure, PKC␤2-RACK1 interactions are enhanced (37).…”

Diacylglycerol kinase regulates tyrosinase expression and function in human melanocytes

“…DGKs are also known to associate with receptor for activated Ckinase-1 (RACK-1) (Imai et al, 2009). Interestingly, many of those signaling pathways have been shown to regulate melanogenesis (Hemesath et al, 1998;Khaled et al, 2003;Park et al, 2004). DAG exerts its effects by modulating the activity of a number of enzymes, such as PKC, DGK, α-and β-chimerins, Ras guanyl releasing protein (RasGRP), and protein kinase D (Blumberg et al, 2008).…”