The steady-state level and translocation of the protein kinase C (PKC) isozymes during the early stages of phorbol 12-myristate 13-acetate (PMA)-induced differentiation, was followed in AGF cells by Western blot analysis of various cell fractions, immunofluorescence staining and by confocal microscopy. By Western blot analysis, uninduced AGF cells express four PKC isoforms, PKC-alpha, PKC-gamma, PKC-epsilon and PKC-zeta with no expression of PKC-beta or PKC-delta. PKC-alpha was exclusively localized to the cytosol, whereas PKC-epsilon was localized predominantly in the cytosol. PKC-gamma and PKC-zeta were found in the cytosolic, as well as in the nuclear and the membrane fractions. Following stimulation with PMA from 15 min to 24 h, cytosolic PKC-alpha did not translocate to the membrane or nuclear fractions. PKC-gamma expression in the membrane and nuclear fractions was decreased following 1 h of PMA stimulation. The expression of PKC-zeta in the membrane and nuclear fractions was transiently increased (2-3 fold) between 3-6 h after PMA stimulation. The expression of PKC epsilon and delta was also affected by PMA treatment. While PKC epsilon, in the membrane fraction, was down-regulated by PMA treatment (3 h), the expression of PKC delta was induced by PMA. Confocal microscopy of the translocation of PKC isoforms during PMA-induced differentiation, confirmed the results obtained by Western blot analysis. Our results indicate that both Ca2+-dependent (PKC-alpha and PKC-gamma) and Ca2+-independent (PKC-epsilon, delta and PKC-zeta) isozymes are expressed in AGF cells and their pattern of expression differ in response to short and prolonged stimulation with phorbol ester. The demonstrated heterogeneity of PKC isozymes in AGF cells, suggests that each PKC isoform may provide a unique contribution to signal transduction pathways and growth control.

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