Ceramide inhibits phospholipase D in a cell-free system.
Recent evidence in whole cells has implicated ceramide in the regulation of phospholipase D (PLD). In intact HL-60 cells, phorbol myristate acetate (PMA) activated PLD as measured by [3H]palmitate-labeled phosphatidylcholine conversion to phosphatidylethanol in the presence of 2% ethanol. C6-Ceramide completely inhibited PLD activation after 4 h of treatment and was maximally active at 10 microM. The activity was structurally specific in that the structural analogs 4,5-dihydro-C6-ceramide and dioctanoylglycerol were inactive. Although ceramide inhibited PMA-induced activation of PLD, it did not inhibit translocation of protein kinase C (PKC) to the membrane in response to PMA. In a cell-free system, we confirmed that PLD is activated by guanosine 5'-O-(3-thiotriphosphate (GTPgammaS); however, ceramide had no effect on this activity under a variety of conditions. Activation of PLD by GTPgammaS was synergistically enhanced by the addition of PKC activators. This upstream effect was inhibited rapidly and specifically by ceramide (30 microM). Recombinant ARF plus PKCalpha substituted for crude cytosol in the activation of PLD, and this activity was inhibited by C6-ceramide. Taken together, these data show that ceramide interferes with PKC-mediated activation of PLD.
Venable
M E
ME
Departments of Medicine and Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Bielawska
A
A
Obeid
L M
LM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001692:Biological Transport; D002474:Cell-Free System; D002518:Ceramides / Q000378:metabolism*; D004789:Enzyme Activation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D010739:Phospholipase D / Q000037:antagonists & inhibitors*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002518:Ceramides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate