Protein kinase C-stimulated formation of ethanolamine from phosphatidylethanolamine involves a protein phosphorylation mechanism: negative regulation by p21 Ras protein.
Mammalian cells express a phospholipase D (PLD)-like enzyme which forms ethanolamine from phosphatidylethanolamine (PtdEtn) by a protein kinase C-alpha (PKC-alpha)-activated, presently unknown, mechanism. Now we report that addition of a PKC-alpha-enriched purified PKC preparation or recombinant PKC-alpha to a plasma membrane-enriched membrane fraction, isolated from leukemic HL60 cells, greatly ( approximately 6.5-fold stimulation) enhanced PtdEtn hydrolysis if the PKC activator phorbol 12-myristate 13-acetate (PMA) and ATP were both present; this was accompanied by PKC-mediated phosphorylation of several membrane proteins. The combined effects of PKC-alpha, ATP, and PMA on [(14)C]PtdEtn hydrolysis were inhibited by GF 109203X (10 microM), an inhibitor of catalytic activity of PKC. In this membrane fraction, PMA alone also had a smaller ( approximately 3.5-fold) stimulatory effect on PtdEtn hydrolysis which was not affected by adding ATP or GF 109203X to the membranes. These results suggest that PMA can stimulate PtdEtn hydrolysis via a PKC-catalyzed phosphorylation mechanism as well as by a phosphorylation-independent process. Transformation of NIH 3T3 fibroblasts by H-ras reduced the effect of PMA on PtdEtn hydrolysis. Furthermore, in NIH 3T3 fibroblasts, scrape-loaded Y13-259 anti Ras antibody enhanced PMA-stimulated hydrolysis of PtdEtn. These results suggest that activation of the PtdEtn-hydrolyzing PLD enzyme by PKC-alpha is inhibited by p21 Ras.
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Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA.
Kiss
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D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000819:agonists / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000008:administration & dosage / Q000276:immunology; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D019856:Ethanolamine / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000819:agonists / Q000378:metabolism / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism* / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D015689:Oncogene Protein p21(ras) / Q000276:immunology; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism* / Q000494:pharmacology; D051571:Protein Kinase C-alpha; D016283:Proto-Oncogene Proteins p21(ras) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000819:agonists / Q000037:antagonists & inhibitors / Q000494:pharmacology
D000906:Antibodies; D007527:Isoenzymes; D008565:Membrane Proteins; D010714:Phosphatidylethanolamines; D010982:Platelet-Derived Growth Factor; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019856:Ethanolamine; D000255:Adenosine Triphosphate; C496628:PRKCA protein, human; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010739:Phospholipase D; C501009:HRAS protein, human; D015689:Oncogene Protein p21(ras); D016283:Proto-Oncogene Proteins p21(ras); D013755:Tetradecanoylphorbol Acetate