Bourgoin S, Grinstein S
Division of Cell Biology, Hospital for Sick Children, Toronto, Canada.
J Biol Chem. 1992 Jun 15;267(17):11908-16.
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.
为确定蛋白质酪氨酸磷酸化在磷脂酶D（PLD）激活过程中的作用，用钒酸盐衍生物处理经电通透处理并用[3H]烷基磷脂酰胆碱标记的HL-60细胞。微摩尔浓度的氢过氧化钒酰（V(4+)-OOH）可诱导酪氨酸磷酸化蛋白的积累。与此同时，V(4+)-OOH或钒酸盐与NADPH的组合可引起磷脂酸（PtdOH）浓度和时间依赖性的积累。在乙醇存在的情况下，观察到磷脂酰乙醇的持续形成，表明D型磷脂酶被激活。发现酪氨酸磷酸化蛋白的积累与PLD的激活之间存在良好的相关性。两种反应的V(4+)-OOH浓度依赖性几乎相同，激活的时间进程相似，酪氨酸磷酸化比PLD激活提前约1分钟。发现V(4+)-OOH诱导两种反应的能力严格依赖于ATP和/或Mg2+的存在，这表明PLD激活涉及磷酸转移酶反应。因此，酪氨酸激酶抑制剂ST638可同时降低V(4+)-OOH引起的酪氨酸磷酸化和PLD激活。研究了V(4+)-OOH的作用机制。二酰基甘油激酶抑制剂二辛酰乙二醇和R59022可增强外源性二酰基甘油对PLD的刺激作用，但不能增强V(4+)-OOH对PLD的刺激作用。此外，V(4+)-OOH和佛波酯的刺激作用具有协同性。因此，二酰基甘油诱导的蛋白激酶C激活不太可能介导V(4+)-OOH的作用。PLD对V(4+)-OOH的反应大于对鸟苷5'-(γ-硫代)三磷酸的反应。此外，GTPγS和V(4+)-OOH的作用是相加的。因此，G蛋白的激活不能解释V(4+)-OOH对PLD 的刺激作用。V(4+)-OOH还可触发NADPH氧化酶的氧气消耗爆发。通过添加乙醇或ST638抑制PtdOH积累可消除这种呼吸爆发。总之，这些结果表明HL-60细胞中酪氨酸磷酸化、PLD激活和NADPH氧化酶刺激之间存在密切相关性，提示存在因果关系。
