title: Synergistic activation of phospholipase D by protein kinase C- and G-protein-mediated pathways in streptolysin O-permeabilized HL60 cells.
abstract: Stimulation of phospholipase D (PLD) by cell surface receptors has been observed in many cell types. We have investigated the mechanism of activation of this enzyme in undifferentiated HL60 cells. GTP analogues and Ca2+ (buffered in the nanomolar to micromolar range) were introduced into HL60 cells in the presence of the permeabilizing agent, streptolysin O. We report that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) is a potent activator of phospholipase D when Ca2+ is available at micromolar levels. Phorbol 12-myristate 13-acetate or Ca2+ alone can also stimulate PLD, but to a limited extent. The activation of PLD by GTP[S] can be partially dissociated from GTP[S]-stimulated phosphoinositide-specific phospholipase C, suggesting that a G-protein may be directly involved in regulating PLD. However, maximal activation of PLD only occurs under conditions that are permissive to phospholipase C stimulation. We conclude that PLD activation is under dual control, i.e. protein kinase C- as well as G-protein-mediated regulation. Synergistic activation occurs when both pathways are simultaneously stimulated. We conclude that full activation of PLD requires protein kinase C, increased Ca2+ and a GTP-binding protein. Evidence for cytosolic components that may also be involved in obtaining full activation of PLD is also presented.
authors.affiliation: Department of Physiology, University College London, London, U.K.
authors.forename: B
authors.initials: B
authors.lastname: Geny
authors.forename: S
authors.initials: S
authors.lastname: Cockcroft
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D001426:Bacterial Proteins; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D004357:Drug Synergism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020404:Glycerophospholipids; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D009355:Neomycin / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019311:Staurosporine; D013301:Streptolysins / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D000470:Alkaloids; D001426:Bacterial Proteins; D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D013301:Streptolysins; C051521:phosphatidylethanol; C025964:streptolysin O; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010738:Type C Phospholipases; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D019311:Staurosporine; D009355:Neomycin; D013755:Tetradecanoylphorbol Acetate