title: Cell-permeable ceramides prevent the activation of phospholipase D by ADP-ribosylation factor and RhoA.
abstract: 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.
authors.affiliation: Department of Biochemistry (Signal Transduction Laboratories), University of Alberta, Edmonton, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Abousalham
authors.forename: C
authors.initials: C
authors.lastname: Liossis
authors.forename: L
authors.initials: L
authors.lastname: O'Brien
authors.forename: D N
authors.initials: DN
authors.lastname: Brindley
mesh_terms: D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D002454:Cell Differentiation; D002518:Ceramides / Q000378:metabolism*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D020742:rhoA GTP-Binding Protein
chemical_list: D002518:Ceramides; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D020742:rhoA GTP-Binding Protein