Kinetic analysis of a mammalian phospholipase D: allosteric modulation by monomeric GTPases, protein kinase C, and polyphosphoinositides.

Henage LG, Exton JH, Brown HA
J Biol Chem. 2006 281 (6): 3408-17

PMID: 16339153 · PMCID: PMC3800466 · DOI:10.1074/jbc.M508800200

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.

MeSH Terms (30)

Adenosine Diphosphate ADP-Ribosylation Factor 1 Allosteric Site Animals Catalysis cdc42 GTP-Binding Protein Cell Membrane Cercopithecus aethiops COS Cells Dose-Response Relationship, Drug Enzyme Activation Escherichia coli Genetic Vectors GTP-Binding Proteins Kinetics Models, Chemical Open Reading Frames Phosphatidylcholines Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositol Phosphates Phospholipase D Protein Binding Protein Kinase C Protein Structure, Tertiary rac1 GTP-Binding Protein Rats Recombinant Proteins rhoA GTP-Binding Protein Signal Transduction Sucrose

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