CELL-PERMEABLE CERAMIDES PREVENT THE ACTIVATION OF PHOSPHOLIPASE-D BYADP-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 phosphati dylcholine by membrane-bound PLD depended upon phosphatidylinositol 4, 5-bisphosphate, guanosine 5'-3-O-(thio)triphosphate) (GTP gamma S), an d cytosolic factors including ADP-ribosylating factor (ARF) and RhoA. C-2(N-acetyl-), C-8-(N-octanoyl-), and long-chain ceramides, but not d ihydro-C-2-ceramide inhibited PLD activity. Apyrase or okadaic acid di d not modify the inhibition of PLD by ceramides, indicating that the e ffect in the cell-free system was unlikely to be dependent upon a cera mide-stimulated kinase or phosphoprotein phosphatases. C-2- and C-8-ce ramides prevented the GTP gamma S-induced translocation of ARF1 and Rh oA from the cytosol to the membrane fraction. In whole cells, C-2-cera mide, but not dihydro-C-2-ceramide, inhibited the stimulation of PLD b y N-formylmethionylleucylphenylalanine and decreased the amounts of AR F1, RhoA, CDC42, Rab4, and protein kinase C-alpha and -beta(1) that we re associated with the membrane fraction, but did not alter the distri bution of protein kinase C-epsilon and -zeta. It is concluded that one mechanism by which ceramides prevent the activation of PLD is inhibit ion of the translocation to membranes of G-proteins and protein kinase C isoforms that are required for PLD activity.