Protein kinase C-stimulated formation of ethanolamine from phosphatidylethanolamine involves a protein phosphorylation mechanism: Negative regulationby p21 Ras protein

Mammalian cells express a phospholipase D (PLD)-like enzyme which forms eth anolamine from phosphatidylethanolamine (PtdEtn) by a protein kinase C-alph a (PHC-alpha)-activated, presently unknown, mechanism. Now we report that a ddition of a PKC-cw-enriched purified PKC preparation or recombinant PKC-al pha to a plasma membrane-enriched membrane fraction, isolated from leukemic HL60 cells, greatly (similar to 6.5-fold stimulation) enhanced PtdEtn hydr olysis if the PKC activator phorbol 12-myristate 13-acetate (PMA) and ATP w ere both present; this was accompanied by PKC-mediated phosphorylation of s everal membrane proteins. The combined effects of PKC-alpha, ATP, and PMA o n [C-14]PtdEtn hydrolysis were inhibited by GF 109203X (10 mu M), an inhibi tor of catalytic activity of PKC. In this membrane fraction, PMA alone also had a smaller (similar to 3.5-fold) stimulatory effect on PtdEtn hydrolysi s which was not affected by adding ATP or GF 109203X to the membranes. Thes e results suggest that PMA can stimulate PtdEtn hydrolysis via a PKC-cataly zed phosphorylation mechanism as well as by a phosphorylation-independent p rocess. Transformation of NIH 3T3 fibroblasts by H-ras reduced the effect o f PMA on PtdEtn hydrolysis. Furthermore, in NIH 3T3 fibroblasts, scrape-loa ded 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. (C) 2000 Academic Press.