Protein kinase Calpha 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 phosphatidylcholine (PtdCho) by phorbol 12-myristate 13-acetate (PMA) has been shown to be mediated by the alpha- and betaI-isoforms of protein kinase C (PKC). To determine the role of various PKC isozymes in the regulation of PLD-mediated phosphatidylethanolamine (PtdEtn) hydrolysis, MCF-7 human breast carcinoma cells overexpressing the alpha- and theta-isoforms, and R6 rat fibroblasts overexpressing the alpha-, betaI-, 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 on 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 betaI- and theta-isoforms as well, greatly enhanced both PMA-induced PLD-mediated formation of phosphatidylethanol (approximately 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 were significantly inhibited by 0.5-3 microM concentrations of Gö 6976, a selective inhibitor of the conventional PKC subfamily. Stable expression of PKC-alpha in R6 fibroblasts enhanced, at a shorter (10 min) incubation time, the effects of PMA on the hydrolysis of both PtdEtn and, to a lesser extent, PtdCho. In contrast, stable expression of PKC-betaI in R6 fibroblasts, which originally did not contain this enzyme, enhanced the effects of PMA only 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. Collectively, the results suggest that PKC-alpha has a major role in the mediation of phorbol ester action on PtdEtn hydrolysis, while PtdCho hydrolysis may be regulated by both the alpha and betaI isoforms.
Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA.
J J
JJ
Mukherjee
T
T
Chung
D K
DK
Ways
Z
Z
Kiss
D000818:Animals; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D010714:Phosphatidylethanolamines; D011493:Protein Kinase C; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate