Partial characterization of epidermal protein kinase C in mice sensitive or resistant to phorbol ester

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Abstract
The present study has characterized several aspects of the mouse epidermal protein kinase C (PKC) system and compared phorbol ester‐sensitive and‐resistant mice. Protein immunoblots of partially purified epidermal PKC preparations from SENCAR and C57BL/6 mice indicated the presence of the γ‐, β‐, and α‐isozymes of PKC in both strains. Hydroxylapatite chromatography profiles of epidermal PKC isozymes from SENCAR and C57BL/6 mice revealed three major peaks of PKC activity eluting in fractions similar to those observed in chromatograms of brain tissue and corresponding to PKC‐γ, ‐β, and ‐α. Further analyses of hydroxylapatite chromatography fractions revealed that PKC‐γ and ‐β were present in approximately similar proportions and were much more abundant than PKC‐α. This distribution of epidermal PKC isozymes was similar in both strains. After a single topical application of 3.4 nmol 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) to SENCAR mouse epidermis, total PKC activity in the cytosol fraction decreased rapidly to about 50% of control within 15 min and was accompanied by an increase (∼150% of control) of PKC activity in the membrane fraction. At 4 h, PKC activities were significantly lower than the control levels and remained downregulated through 96 h with a maximal decrease (to ∼25–30% of the control) in both cytosol and membrane fractions at 48 h. PKC activity returned to control levels by 168 h. Ca++/phospholipid‐independent kinase activity was the same as control levels at 15 min, 1 h, and 4 h after TPA treatment but was elevated above control levels at 24 h, 48 h, and 96 h, and by 168 h returned essentially to control levels. No differences were found in the magnitude or kinetics of TPA‐induced translocation and downregulation of total PKC or appearance of Ca++/phospholipid‐independent kinase activity between SENCAR, DBA/2, and C57BL/6 mice. Scatchard analyses using a two binding site model revealed that the apparent Kd and Bmax values for binding of PDBu to epidermal cytosol and membrane fractions were similar between SSIn, SENCAR, DBA/2, and C57BL/6 mice. The present results demonstrate for the first time that mouse epidermis contains significant amounts of the three major PKC isozymes that are present in brain, especially PKC‐γ. In addition, topical application of a promoting dose of TPA did not lead to complete loss of PKC activity in either the membrane or cytosol fractions of mouse epidermis. In conclusion, no differences were observed between phorbol ester‐sensitive and ‐resistant mice in any aspect of epidermal PKC examined.