Huckle W R, Hepler J R, Rhee S G, Harden T K, Earp H S
Lineberger Cancer Research Center, School of Medicine, University of North Carolina, Chapel Hill 27599.
Endocrinology. 1990 Oct;127(4):1697-705. doi: 10.1210/endo-127-4-1697.
Recent studies have shown that the receptor for epidermal growth factor (EGF) can associate with and tyrosine-phosphorylate the gamma-isozyme of phosphoinositide (PtdIns)-specific phospholipase C (PLC gamma), suggesting a possible mechanism for activation of PtdIns hydrolysis by EGF. In the present study, the coupling between PtdIns hydrolysis and PLC gamma tyrosine phosphorylation in WB liver epithelial cells was examined. Peak levels of [P-Tyr]PLC gamma, measured by anti-P-Tyr immunoblotting, occurred at 0.5-2 min of EGF treatment and coincided with the onset of [3H]inositol phosphate production. The termination of PtdIns hydrolysis after EGF stimulation was accompanied by return of [P-Tyr]PLC gamma to near-basal levels. Activation of protein kinase C (PKC) with a phorbol ester inhibited (IC50 = 3-10 nM) both EGF-dependent PtdIns hydrolysis and PLC gamma phosphorylation by more than 90%. Both EGF-stimulated responses were potentiated in cells depleted of PKC by prolonged phorbol ester treatment. At physiological ionic strength, monoclonal antibodies to PLC gamma specifically precipitated (in addition to PLC gamma) the EGF receptor and at least six other [P-Tyr]proteins from extracts of EGF-treated cells. PKC activation had differential effects on the tyrosine phosphorylation of these coprecipitating proteins, i.e. the relative abundance of certain [P-Tyr] proteins decreased, whereas that of another protein increased. In conclusion, EGF-stimulated tyrosine phosphorylation of PLC gamma is broadly correlated with stimulation of PtdIns hydrolysis, consistent with a role for tyrosine phosphorylation in PLC activation. The attendant diacylglycerol release and activation of PKC may terminate PLC gamma activation, in part by inhibiting PLC gamma phosphorylation by the EGF receptor. Our results suggest further that PKC may exert regulatory effects by altering the relationship of PLC gamma to its associated [P-Tyr]proteins.
最近的研究表明，表皮生长因子（EGF）受体可与磷脂酰肌醇（PtdIns）特异性磷脂酶C（PLCγ）的γ同工酶结合并使其酪氨酸磷酸化，提示EGF激活PtdIns水解的一种可能机制。在本研究中，检测了WB肝上皮细胞中PtdIns水解与PLCγ酪氨酸磷酸化之间的偶联关系。通过抗磷酸酪氨酸免疫印迹法测定，[磷酸酪氨酸]PLCγ的峰值水平在EGF处理0.5 - 2分钟时出现，与[3H]肌醇磷酸生成的起始时间一致。EGF刺激后PtdIns水解的终止伴随着[磷酸酪氨酸]PLCγ恢复到接近基础水平。用佛波酯激活蛋白激酶C（PKC）可抑制（IC50 = 3 - 10 nM）EGF依赖性PtdIns水解和PLCγ磷酸化达90%以上。通过延长佛波酯处理使PKC缺失的细胞中，两种EGF刺激的反应均增强。在生理离子强度下，针对PLCγ的单克隆抗体特异性沉淀（除PLCγ外）来自EGF处理细胞提取物中的EGF受体和至少六种其他[磷酸酪氨酸]蛋白。PKC激活对这些共沉淀蛋白的酪氨酸磷酸化有不同影响，即某些[磷酸酪氨酸]蛋白的相对丰度降低，而另一种蛋白的相对丰度增加。总之，EGF刺激的PLCγ酪氨酸磷酸化与PtdIns水解的刺激广泛相关，这与酪氨酸磷酸化在PLC激活中的作用一致。伴随的二酰基甘油释放和PKC激活可能部分通过抑制EGF受体对PLCγ的磷酸化来终止PLCγ的激活。我们的结果进一步表明，PKC可能通过改变PLCγ与其相关[磷酸酪氨酸]蛋白的关系发挥调节作用。
