Park H Y, Perez J M, Laursen R, Hara M, Gilchrest B A
Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
J Biol Chem. 1999 Jun 4;274(23):16470-8. doi: 10.1074/jbc.274.23.16470.
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.
我们之前已经表明，蛋白激酶C-β（PKC-β）是激活酪氨酸酶所必需的（Park, H. Y., Russakovsky, V., Ohno, S., and Gilchrest, B. A. (1993) J. Biol. Chem. 268, 11742 - 11749），酪氨酸酶是黑色素生成中的限速酶。我们现在研究其在人黑素细胞中的激活机制。体内磷酸化实验表明，酪氨酸酶通过PKC依赖途径被磷酸化，并且将PKC-β导入缺乏PKC-β的无色素人黑素瘤细胞会导致酪氨酸酶的磷酸化和激活。完整的黑素小体与纯化的活性PKC-β在体外预孵育可使酪氨酸酶活性增加3倍。通过免疫电子显微镜观察，PKC-β而非PKC-α与黑素小体外表面的酪氨酸酶紧密相关。蛋白质印迹分析证实了PKC-β与黑素小体的关联。只有酪氨酸酶的细胞质（黑素小体外）结构域被丝氨酸/苏氨酸激酶PKC-β磷酸化，该结构域含有两个丝氨酸但不含苏氨酸。这两个位于505和509位的丝氨酸都存在于酪氨酸酶经胰蛋白酶消化产生的C末端肽中。通过共迁移实验，将所有三种可能的磷酸化胰蛋白酶肽（一种双磷酸肽和两种单磷酸肽）的合成肽标准品与在完整黑素细胞中被PKC-β磷酸化然后经胰蛋白酶消化的酪氨酸酶进行比较，结果表明这两个丝氨酸残基都被PKC-β磷酸化。我们得出结论，PKC-β通过磷酸化这种与黑素小体相关蛋白细胞质结构域中505和509位的丝氨酸残基直接激活酪氨酸酶。
