Cell-permeable ceramides prevent the activation of phospholipase D by ADP-ribosylation factor and RhoA

The mechanism of inhibition of phospholipase D (PLD) by ceramides was determined using granulocytes differentiated from human promyelocytic leukemic (HL-60) cells. In a cell-free system, hydrolysis of phosphatidylcholine by membrane-bound PLD depended upon phosphatidylinositol 4,5-bisphosphate, guanosine 5'-3-O-(thio)triphosphate) (GTPgammaS), and cytosolic factors including ADP-ribosylating factor (ARF) and RhoA. C2-(N-acetyl-), C8- (N-octanoyl-), and long-chain ceramides, but not dihydro-C2-ceramide, inhibited PLD activity. Apyrase or okadaic acid did not modify the inhibition of PLD by ceramides, indicating that the effect in the cell-free system was unlikely to be dependent upon a ceramide-stimulated kinase or phosphoprotein phosphatases. C2- and C8-ceramides prevented the GTPgammaS-induced translocation of ARF1 and RhoA from the cytosol to the membrane fraction. In whole cells, C2-ceramide, but not dihydro-C2-ceramide, inhibited the stimulation of PLD by N-formylmethionylleucylphenylalanine and decreased the amounts of ARF1, RhoA, CDC42, Rab4, and protein kinase C-alpha and -beta1 that were associated with the membrane fraction, but did not alter the distribution of protein kinase C-epsilon and -zeta. It is concluded that one mechanism by which ceramides prevent the activation of PLD is inhibition of the translocation to membranes of G-proteins and protein kinase C isoforms that are required for PLD activity.