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J. Biol. Chem., Vol. 267, Issue 17, 11908-11916, Jun, 1992
S Bourgoin and S Grinstein
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.
Peroxides of vanadate induce activation of phospholipase D in HL-60 cells. Role of tyrosine phosphorylation
Division of Cell Biology, Hospital for Sick Children, Toronto, Canada.
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