Overexpression of protein kinase C-epsilon 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-alpha-mediated nonphosphorylating and phosphorylating mechanisms. Here we have used NIH 3T3 fibroblasts overexpressing holo PKC-epsilon and its regulatory, catalytic, and zinc finger domain fragments to determine if this isozyme also regulates PLD activity. Overexpression of holo PKC-epsilon inhibited the stimulatory effects of PMA (5-100 nM) on both PtdCho and PtdEtn hydrolysis. Overexpression of PKC-epsilon also was found to inhibit platelet-derived growth factor-induced PLD activity. Expression of the catalytic unit of PKC-epsilon had no effect on PMA-induced PLD activity. In contrast, expression of both the regulatory domain fragment and the zinc finger domain of PKC-epsilon resulted in significant inhibition of PMA-stimulated PtdCho and PtdEtn hydrolysis. Interestingly, although PKC-alpha also mediates the stimulatory effect of PMA on the synthesis of PtdCho by a phosphorylation mechanism, overexpression of holo PKC-epsilon or its regulatory domain fragments did not affect PMA-induced PtdCho synthesis. These results indicate that the PKC-epsilon system can act as a negative regulator of PLD activity and that this inhibition is mediated by its regulatory domain.
Kiss
Z
Z
The Hormel Institute, University of Minnesota, Austin, Minnesota, 55912, USA. kissx001@maroon.tc.umn.edu
Petrovics
G
G
Olàh
Z
Z
Lehel
C
C
Anderson
W B
WB
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D020035:Holoenzymes / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000096:biosynthesis / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D020035:Holoenzymes; D007527:Isoenzymes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010982:Platelet-Derived Growth Factor; D011994:Recombinant Proteins; C497256:Prkce protein, mouse; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate