Yeo E J, Exton J H
Howard Hughes Medical Institute, Nashville, Tennessee.
J Biol Chem. 1995 Feb 24;270(8):3980-8. doi: 10.1074/jbc.270.8.3980.
The proposal that epidermal growth factor (EGF) activates phospholipase D (PLD) by a mechanism(s) not involving phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis was examined in Swiss 3T3 fibroblasts. EGF, basic fibroblast growth factor (bFGF), bombesin, and platelet-derived growth factor (PDGF) activated PLD as measured by transphosphatidylation of butanol to phosphatidylbutanol. The increase in inositol phosphates induced by bFGF, EGF, or bombesin was significantly enhanced by Ro-31-8220, an inhibitor of protein kinase C (PKC), suggesting that PtdIns(4,5)P2-hydrolyzing phospholipase is coupled to the receptors for these agonists but that the response is down-regulated by PKC. Activation of PLD by EGF was inhibited dose dependently by the PKC inhibitors bis-indolylmaleimide and Ro-31-8220, which also inhibited the effects of bFGF, bombesin, and PDGF. Down-regulation of PKC by prolonged treatment with 4 beta-phorbol 12-myristate 13-acetate also abolished EGF- and PDGF-stimulated phosphatidylbutanol formation. EGF and bombesin induced biphasic translocations of PKC delta and epsilon to the membrane that were detectable at 15 s. In the presence of Ro-31-8220, translocation of PKC alpha became evident, and membrane association of the delta- and epsilon-isozymes was enhanced and/or sustained in response to the two agonists. The inhibitor also enhanced EGF-stimulated [3H]diacylglycerol formation in cells preincubated with [3H]arachidonic acid, which labeled predominantly phosphatidylinositol, but inhibited [3H]diacylglycerol production in cells preincubated with [3H]myristic acid, which labeled mainly phosphatidylcholine. These data support the conclusion that EGF can stimulate diacylglycerol formation from PtdIns(4,5)P2 and that PKC performs the dual role of down-regulating this response as well as mediating phosphatidylcholine hydrolysis. In summary, all of the results of the study indicate that PLD activation by EGF is downstream of PtdIns(4,5)P2-hydrolyzing phospholipase and is dependent upon subsequent PKC activation.
在瑞士3T3成纤维细胞中研究了表皮生长因子（EGF）通过不涉及磷脂酰肌醇4,5-二磷酸（PtdIns(4,5)P2）水解的机制激活磷脂酶D（PLD）的提议。通过丁醇向磷脂酰丁醇的转磷脂酰基作用测定，EGF、碱性成纤维细胞生长因子（bFGF）、蛙皮素和血小板衍生生长因子（PDGF）均可激活PLD。蛋白激酶C（PKC）抑制剂Ro-31-8220显著增强了bFGF、EGF或蛙皮素诱导的肌醇磷酸增加，这表明水解PtdIns(4,5)P2的磷脂酶与这些激动剂的受体偶联，但该反应受到PKC的负调控。PKC抑制剂双吲哚基马来酰亚胺和Ro-31-8220剂量依赖性地抑制了EGF对PLD的激活，它们也抑制了bFGF、蛙皮素和PDGF的作用。用4β-佛波醇12-肉豆蔻酸酯13-乙酸酯长期处理导致PKC下调，这也消除了EGF和PDGF刺激的磷脂酰丁醇形成。EGF和蛙皮素诱导PKCδ和ε向膜的双相转位，在15秒时可检测到。在存在Ro-31-8220的情况下，PKCα的转位变得明显，并且δ和ε同工酶的膜结合因这两种激动剂而增强和/或持续。该抑制剂还增强了用[3H]花生四烯酸预孵育的细胞中EGF刺激的[3H]二酰基甘油形成，[3H]花生四烯酸主要标记磷脂酰肌醇，但抑制了用[3H]肉豆蔻酸预孵育的细胞中[3H]二酰基甘油的产生，[3H]肉豆蔻酸主要标记磷脂酰胆碱。这些数据支持以下结论：EGF可刺激从PtdIns(4,5)P2形成二酰基甘油，并且PKC在下调该反应以及介导磷脂酰胆碱水解方面发挥双重作用。总之，该研究的所有结果表明，EGF激活PLD是在水解PtdIns(4,5)P2的磷脂酶下游，并且依赖于随后的PKC激活。
