Differential expression of protein kinase C isoforms in glial and neuronal cells. Translocation and down-regulation of PKC isoforms in C 6 glioma and ng 108-15 hybrid cells: Effects of extracellular Ca 2+-depletion

Abstract

Protein kinase C (PKC), the major receptor for tumor-promoting phorbol esters, consists of a family of at least 12 distinct lipid-regulated enzymes. We examined the expression and regulation of PKC isoforms in C 6-glioma and NG 108-15 hybrid cells. Western blot analysis indicated that both cell lines express four PKC isoforms, PKCα, PKCδ, PKCε and PKCζ. The expression of PKCα and PKCδ in C 6-glioma cells was more abundant than NG 108-15 cells, however, PKCε in NG 108-15 was more abundant than C 6-glioma cells in which PKCε was almost undetectable. Treatment of both cells with TPA for 10 min resulted in the translocation of PKCα, PKCδ and PKCε to the membrane fraction. When the intact cells were treated with Ca 2+-free, EGTA containing physiological saline solution, the membrane bound conventional PKCα (cPKCα) was greatly reduced and cytosolic cPKCα was only slightly increased. However, neither membrane bound nor cytosolic new PKCδ (nPKCδ), nPKCε and atypical PKCζ (aPKCζ) was affected by extracellular Ca 2+ depletion. In this condition, the translocation of cPKCα, nPKCδ and nPKCε induced by TPA still occurred, however, that of cPKCα was reduced more than in the normal condition. After long-term treatment (17 h) with TPA, cPKCα, nPKCδ and nPKCε were down-regulated both in the cytosol and membrane. The phenomena of cPKCα were confirmed by measuring the PKC activity with histone as the substrate. From in vitro endogenous phosphorylation studies, a 31 kDa substrate protein phosphorylation in C 6 glioma cell membrane and 31 and 26 kDa proteins in NG 108-15 cell membrane were increased in the translocation but disappeared in the down-regulation of PKC.