Overexpression of Protein Kinase C-ε and Its Regulatory Domains in Fibroblasts Inhibits Phorbol Ester-Induced Phospholipase D Activity

In fibroblasts, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) stimulates phospholipase D (PLD)-mediated hydrolysis of both phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) by PKC-α-mediated nonphosphorylating and phosphorylating mechanisms. Here we have used NIH 3T3 fibroblasts overexpressing holo PKC-ε and its regulatory, catalytic, and zinc finger domain fragments to determine if this isozyme also regulates PLD activity. Overexpression of holo PKC-ε inhibited the stimulatory effects of PMA (5–100 nM) on both PtdCho and PtdEtn hydrolysis. Overexpression of PKC-ε also was found to inhibit platelet-derived growth factor-induced PLD activity. Expression of the catalytic unit of PKC-ε had no effect on PMA-induced PLD activity. In contrast, expression of both the regulatory domain fragment and the zinc finger domain of PKC-ε resulted in significant inhibition of PMA-stimulated PtdCho and PtdEtn hydrolysis. Interestingly, although PKC-α also mediates the stimulatory effect of PMA on the synthesis of PtdCho by a phosphorylation mechanism, overexpression of holo PKC-ε or its regulatory domain fragments did not affect PMA-induced PtdCho synthesis. These results indicate that the PKC-ε system can act as a negative regulator of PLD activity and that this inhibition is mediated by its regulatory domain.