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.
Mukherjee
J J
JJ
Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA.
Chung
T
T
Ways
D K
DK
Kiss
Z
Z
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