Peroxides of vanadate induce activation of phospholipase D in HL-60 cells. Role of tyrosine phosphorylation.
To determine the role of protein tyrosine phosphorylation in the activation of phospholipase D (PLD), electropermeabilized HL-60 cells labeled in [3H]alkyl-phosphatidylcholine were treated with vanadate derivatives. Micromolar concentrations of vanadyl hydroperoxide (V(4+)-OOH) induced accumulation of tyrosine-phosphorylated proteins. Concomitantly, V(4+)-OOH or a combination of vanadate and NADPH elicited a concentration- and time-dependent accumulation of phosphatidic acid (PtdOH). In the presence of ethanol a sustained formation of phosphatidylethanol was observed, indicating that a type D phospholipase was activated. A good correlation was found to exist between the accumulation of tyrosine-phosphorylated proteins and activation of PLD. The V(4+)-OOH concentration dependence of the two responses was nearly identical, and the time course of activation was similar, with tyrosine phosphorylation preceding PLD activation by approximately 1 min. The ability of V(4+)-OOH to induce both responses was found to be strictly dependent on the presence of ATP and/or Mg2+, suggesting that PLD activation involves phosphotransferase reactions. Accordingly, ST638, a tyrosine kinase inhibitor, reduced concomitantly tyrosine phosphorylation and PLD activation elicited by V(4+)-OOH. The mechanism of action of V(4+)-OOH was investigated. The diacylglycerol kinase inhibitors, dioctanoylethylene glycol and R59022 potentiated PLD stimulation by exogenous diacylglycerol but not by V(4+)-OOH. Moreover, stimulation by V(4+)-OOH and by phorbol esters was synergystic. Therefore, diacylglycerol-induced activation of protein kinase C is unlikely to mediate the effects of V(4+)-OOH. The response of PLD to V(4+)-OOH was larger than that to guanosine 5'-(gamma-thio)triphosphate. Moreover, the effects of GTP gamma S and V(4+)-OOH were additive. Hence, activation of G proteins cannot account for the stimulation of PLD by V(4+)-OOH. V(4+)-OOH also triggers a burst of O2 consumption by the NADPH oxidase. Inhibition of PtdOH accumulation by addition of ethanol or by ST638 abolished this respiratory burst. Together, the results establish a strong correlation between tyrosine phosphorylation, PLD activation, and stimulation of the NADPH oxidase in HL-60 cells, suggesting a causal relationship.
Bourgoin
S
S
Division of Cell Biology, Hospital for Sick Children, Toronto, Canada.
Grinstein
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D005026:Ethylene Glycols / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010101:Oxygen Consumption; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology*
D005026:Ethylene Glycols; C069276:peroxovanadate; D014638:Vanadates; D014443:Tyrosine; C056214:dioctanoyl ethylene glycol; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010739:Phospholipase D; D019204:GTP-Binding Proteins