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-α (PKC-α)-activated, presently unknown, mechanism. Now we report that addition of a PKC-α-enriched purified PKC preparation or recombinant PKC-α to a plasma membrane-enriched membrane fraction, isolated from leukemic HL60 cells, greatly (∼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-α, ATP, and PMA on [14C]PtdEtn hydrolysis were inhibited by GF 109203X (10 μM), an inhibitor of catalytic activity of PKC. In this membrane fraction, PMA alone also had a smaller (∼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-α is inhibited by p21 Ras.