title: Kinetic analysis of a mammalian phospholipase D: allosteric modulation by monomeric GTPases, protein kinase C, and polyphosphoinositides.
abstract: In mammalian cells, phospholipase D activity is tightly regulated by diverse cellular signals, including hormones, neurotransmitters, and growth factors. Multiple signaling pathways converge upon phospholipase D to modulate cellular actions, such as cell growth, shape, and secretion. We examined the kinetics of protein kinase C and G-protein regulation of mammalian phospholipase D1 (PLD1) in order to better understand interactions between PLD1 and its regulators. Activation by Arf-1, RhoA, Rac1, Cdc42, protein kinase Calpha, and phosphatidylinositol 4,5-bisphosphate displayed surface dilution kinetics, but these effectors modulated different kinetic parameters. PKCalpha activation of PLD1 involves N- and C-terminal PLD domains. Rho GTPases were binding activators, enhancing the catalytic efficiency of a purified PLD1 catalytic domain via effects on Km. Arf-1, a catalytic activator, stimulated PLD1 by enhancing the catalytic constant, kcat. A kinetic description of PLD1 activation by multiple modulators reveals a mechanism for apparent synergy between activators. Synergy was observed only when PLD1 was simultaneously stimulated by a binding activator and a catalytic activator. Surprisingly, synergistic activation was steeply dependent on phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine. Together, these findings suggest a role for PLD1 as a signaling node, in which integration of convergent signals occurs within discrete locales of the cellular membrane.
authors.affiliation: Department of Pharmacology, Institute for Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232-6600, USA.
authors.forename: Lee G
authors.initials: LG
authors.lastname: Henage
authors.forename: John H
authors.initials: JH
authors.lastname: Exton
authors.forename: H Alex
authors.initials: HA
authors.lastname: Brown
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000737:chemistry; D000495:Allosteric Site; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry; D005822:Genetic Vectors; D007700:Kinetics; D008956:Models, Chemical; D016366:Open Reading Frames; D010713:Phosphatidylcholines / Q000737:chemistry; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry*; D010739:Phospholipase D / Q000737:chemistry*; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D013395:Sucrose / Q000737:chemistry; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D010713:Phosphatidylcholines; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D011994:Recombinant Proteins; D013395:Sucrose; D000244:Adenosine Diphosphate; D011493:Protein Kinase C; D010739:Phospholipase D; C106801:phospholipase D1; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein