A soluble protein negatively regulates phospholipase D activity. Partial purification and characterization.
Phosphatidylcholine-specific phospholipase D (PLD) is an important signalling phospholipase in mammalian cells. Recently, PLD activity has been shown to be positively regulated by the GTP-binding protein ARF (ADP-ribosylating factor). In the present work, we document the presence of a factor negatively regulating PLD activity in bovine brain cytosol. The inhibitory factor is characterized as a large protein or a complex of proteins with a molecular mass higher than 300 kDa. Using permeabilized and pre-permeabilized HL-60 cells depleted of their cytosol, we demonstrate that the inhibitor acts on GTP[S]-stimulated PLD activity. This effect is immediate, persistent and dose dependent for GTP[S]-stimulated PLD. Different possibilities for a mechanism of action of the inhibitory factor on the regulation of GTP binding of ARF were investigated. This inhibitory factor is not the guanine-dissociating inhibitor (GDI) for the small G-binding proteins Rho (Rho-GDI), reported to be a PLD inhibitor, since specific antibodies against this protein did not recognize a protein in the peak containing the inhibitory factor for PLD activity. Furthermore, the inhibitory factor does not prevent the binding of GTP[S] to ARF in the presence of HL-60 membranes. This excludes its possible role as an inhibitor of an ARF/guanine exchange factor. The inhibitory factor not only inhibits a pathway of PLD through GTP[S] activation in particular of the small GTP-binding protein, ARF, but it also inhibits PLD activated via either protein kinase C (PKC) or tyrosine kinase activation. The inhibitory factor also decreases PLC activity and this effect seems to be secondary to the inhibition of PLD activity. We discuss a mechanism of action of the inhibitor on PLD and the importance of this enzyme activity for membrane traffic.
Geny
B
B
INSERM, unité 332, Institut Cochin de Génétique Moléculaire, Paris, France.
Paris
S
S
Dubois
T
T
Franco
M
M
Lukowski
S
S
Chardin
P
P
Russo Marie
F
F
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D006150:Guanine Nucleotides / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008563:Membrane Lipids / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D006150:Guanine Nucleotides; D008563:Membrane Lipids; D009419:Nerve Tissue Proteins; D010716:Phosphatidylinositols; C076715:pervanadate; D014638:Vanadates; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate