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Volume 272, Number 2, Issue of January 10, 1997 pp. 1069-1075
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Cell-permeable Ceramides Prevent the Activation of Phospholipase D by ADP-ribosylation Factor and RhoA

(Received for publication, May 24, 1996, and in revised form, October 30, 1996)

From the Department of Biochemistry (Signal Transduction Laboratories) and the Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

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) (GTPS), 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 GTPS-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- and -₁ that were associated with the membrane fraction, but did not alter the distribution of protein kinase C- and -. 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.

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