DIFFERENTIAL DOWN-REGULATION OF PROTEIN-KINASE-C SUBSPECIES IN NORMALHUMAN MELANOCYTES - POSSIBLE INVOLVEMENT OF THE ZETA-SUBSPECIES IN GROWTH-REGULATION

Normal human melanocytes are often grown in vitro in the continuous pr esence of 12-O-tetradecanoylphorbol-13-acetate (TPA) for growth in vit ro. The expression of protein kinase C (PKC) subspecies, which are the major cellular receptors for phorbol esters, was examined in melanocy tes after long-term treatment with TPA to investigate the role of PKC subspecies in TPA-dependent cell growth. The PKC enzyme activity detec ted in quiescent melanocytes was almost completely depleted in cells a fter incubation with 85 nM TPA for 48 h. Immunoblot analysis indicated that, among the PKC subspecies alpha, beta, delta, epsilon, and zeta expressed in quiescent cells, alpha-, beta-, delta-, and epsilon-PKC w ere significantly down-regulated, whereas zeta-PKC remained at detecta ble levels in TPA-treated cells. TPA did not significantly affect the expression or subcellular distribution of zeta-PKC in melanocytes. Imm unoprecipitation assay revealed that the enzyme activity of zeta-PKC w as increased in both the cytosol and particulate cell fractions, but t he increase was much greater in the latter. The activation of zeta-PKC lasted for 24 to 48 h after the addition of TPA; thereafter, zeta-PKC activity returned to basal levels. DNA synthesis was shown to change concomitantly with the activation of zeta-PKC in TPA-treated cells. Th ese results indicate that TPA induces not only the down-regulation of alpha-, beta-, delta-, and epsilon-PKC, but also long-term activation of zeta-PKC in melanocytes, and that activation of zeta-PKC parallels the growth of normal human melanocytes.