PROTEIN-KINASE C-ALPHA IS A MAJOR MEDIATOR OF THE STIMULATORY EFFECT OF PHORBOL ESTER ON PHOSPHOLIPASE D-MEDIATED HYDROLYSIS OF PHOSPHATIDYLETHANOLAMINE

Stimulation of phospholipase D (PLD)-mediated hydrolysis of phosphatid ylcholine (PtdCho) by phorbol 12-myristate 13-acetate (PMA) has been s hown to be mediated by the alpha- and beta I-isoforms of protein kinas e C (PKC). To determine the role of various PKC isozymes in the regula tion of PLD-mediated phosphatidylethanolamine (PtdEtn) hydrolysis, MCF -7 human breast carcinoma cells overexpressing the alpha- and theta-is oforms, and R6 rat fibroblasts overexpressing the alpha-, beta I-, and epsilon-isoforms were used. In the vector control MCF-7 cells, which contain low levels of PKC-alpha, PMA (100 nM) had only small effects o n the hydrolysis of PtdEtn (1.1-1.35-fold) and PtdCho (1.15-1.6-fold). Stable expression of PKC-alpha in MCF-7 cells, which was accompanied by increased levels of the beta I- and theta-isoforms as well, greatly enhanced both PMA-induced PLD-mediated formation of phosphatidylethan ol (similar to 5-fold) and the hydrolysis of PtdEtn (2.5-2.9-fold) and PtdCho (5.5-7.2-fold). The effects of PMA on the hydrolysis of PtdEtn (and PtdCho) in MCF-7/PKC-alpha cells mere significantly inhibited by 0.5-3 mu M concentrations of Go 6976, a selective inhibitor of the co nventional PKC subfamily. Stable expression of PKC-alpha in R6 fibrobl asts enhanced, at a shorter (10 min) incubation time, the effects of P MA on the hydrolysis of both PtdEtn and, to a lesser extent, PtdCho. I n contrast, stable expression of PKC-beta I in R6 fibroblasts, which o riginally did not contain this enzyme, enhanced the effects of PMA onl y on PtdCho, but not PtdEtn, hydrolysis. Overexpression of either PKC- theta in MCF-7 cells or PKC-epsilon in R6 and NIH 3T3 fibroblasts had no detectable effects on PMA-induced hydrolysis of PtdEtn. Collectivel y, the results suggest that PKC-alpha has a major role in the mediatio n of phorbol ester action on PtdEtn hydrolysis, while PtdCho hydrolysi s may be regulated by both the alpha and beta I isoforms.