Further identification of protein kinase C isozymes in mouse epidermis

Abstract

In the current study, the protein kinase C (PKC) isozymes present in mouse epidermis have been identified using immunological and chromatographic methods. Six PKC isozymes, PKCα, PKCβ, PKCγ, PKCδ, PKCε, and PKCζ, were identified in unfractionated epidermal preparations by protein immunoblotting. The subcellular distribution and presence of these isozymes was further verified by hydroxyapatite (HA) chromatography with the exception of PKEε, which could not be detected following HA chromatography. The five PKC isozymes recovered following HA chromatography were detected in both epidermal cytosol and particulate fractions, although PKCδ was found in a much higher proportion relative to the other PKC isozymes in the particulate fraction using histone H1 as the substrate. The biochemical properties of the epidermal PKC isozymes partially purified by HA chromatography agreed with those reported for other tissues and further supported their immunological identification in epidermal preparations. The activities of HA chromatography peaks corresponding to PKCα, PKCβ, and PKCγ were found to be dependent on both Ca²⁺ and phosphatidylserine (PtdSer), whereas, the activities of HA peaks corresponding to PKCδ and PKCζ were Ca²⁺-independent but PtdSer-dependent. The HA peak corresponding to PKCγ also displayed a characteristic biphasic modulation by arachidonic acid (activation at low, inactivation at high concentrations) and inactivation by preincubation with PtdSer. PKCζ activity was also characteristic, in that it was dependent on PtdSer and was not increased by the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. Some differences in substrate specificity were also observed between the epidermal PKC isozymes. The presence of multiple isozymes of PKC in mouse epidermis suggests that the different isozymes may play distinct roles in signal transduction and tumor promotion in this tissue.