Decker S J, Ellis C, Pawson T, Velu T
Rockefeller University, New York, New York 10021.
J Biol Chem. 1990 Apr 25;265(12):7009-15.
Addition of epidermal growth factor (EGF) to many cell types activates phospholipase C resulting in increased levels of diacylglycerol and intracellular Ca2+ which may lead to activation of protein kinase C. EGF treatment of cells can also lead to phosphorylation of the EGF receptor at threonine 654 (a protein kinase C phosphorylation site) which appears to attenuate some aspects of receptor signaling. Thus, a feedback loop involving the EGF receptor, phospholipase C, and protein kinase C may regulate EGF receptor function. In this report, the role of phosphorylation of threonine 654 of the EGF receptor in regulation of EGF-stimulated activation of phospholipase C was investigated. NIH-3T3 cells expressing the normal human EGF receptor or expressing EGF receptor in which an alanine residue had been substituted at residue 654 of the receptor were used. Addition of EGF to cells expressing wild-type receptor induced a rapid, but transient, increase in phosphorylation of threonine 654. EGF addition also caused the rapid accumulation of inositol phosphates in these cells. EGF-stimulated accumulation of inositol phosphates was significantly higher in cells expressing Ala-654 receptors compared to control cells. Treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulated phosphorylation of threonine 654 to a greater degree than EGF, completely inhibited EGF-dependent inositol phosphate accumulation in cells expressing wild-type receptor, but caused only a 20-30% inhibition in Ala-654 expressing cells. EGF stimulated phosphorylation of phospholipase C-gamma on serine and tyrosine residues in cells expressing wild-type of Ala-654 receptors. However, TPA treatment of cells inhibited EGF-induced tyrosine phosphorylation of phospholipase C-gamma only in cells expressing wild-type receptors. Similarly, TPA inhibited tyrosine-specific autophosphorylation of the EGF receptor and tyrosine phosphorylation of several other proteins in wild-type receptor cells, but not in Ala-654 cells. TPA treatment abolished high affinity binding of EGF to cells expressing wild-type receptors, while decreasing the number of high affinity binding sites 20-30% in Ala-654 cells. These data suggest that phosphorylation of threonine 654 can regulate early events in EGF receptor signal transduction such as phosphoinositide turnover, probably through a feedback mechanism involving protein kinase C. Subsequent dephosphorylation of threonine 654 could reactivate the EGF receptor for participation in later signaling events.
向多种细胞类型中添加表皮生长因子（EGF）会激活磷脂酶C，导致二酰基甘油和细胞内Ca2+水平升高，这可能会激活蛋白激酶C。用EGF处理细胞还会导致EGF受体在苏氨酸654（一个蛋白激酶C磷酸化位点）处发生磷酸化，这似乎会减弱受体信号传导的某些方面。因此，一个涉及EGF受体、磷脂酶C和蛋白激酶C的反馈回路可能会调节EGF受体的功能。在本报告中，研究了EGF受体苏氨酸654磷酸化在调节EGF刺激的磷脂酶C激活中的作用。使用了表达正常人EGF受体的NIH-3T3细胞或表达在受体654位残基处用丙氨酸残基替代的EGF受体的细胞。向表达野生型受体的细胞中添加EGF会诱导苏氨酸654磷酸化迅速但短暂地增加。添加EGF还会导致这些细胞中肌醇磷酸迅速积累。与对照细胞相比，在表达Ala-654受体的细胞中，EGF刺激的肌醇磷酸积累明显更高。用12-O-十四烷酰佛波醇13-乙酸酯（TPA）处理细胞，TPA刺激苏氨酸654磷酸化的程度比EGF更高，它完全抑制了表达野生型受体的细胞中EGF依赖性肌醇磷酸积累，但在表达Ala-654的细胞中仅导致20-30%的抑制。EGF刺激了表达野生型或Ala-654受体的细胞中磷脂酶C-γ丝氨酸和酪氨酸残基的磷酸化。然而，用TPA处理细胞仅在表达野生型受体的细胞中抑制了EGF诱导的磷脂酶C-γ酪氨酸磷酸化。同样，TPA抑制了野生型受体细胞中EGF受体的酪氨酸特异性自磷酸化以及其他几种蛋白质的酪氨酸磷酸化，但在Ala-654细胞中没有。TPA处理消除了EGF与表达野生型受体的细胞的高亲和力结合，同时使Ala-654细胞中的高亲和力结合位点数量减少20-30%。这些数据表明，苏氨酸654磷酸化可能通过涉及蛋白激酶C的反馈机制调节EGF受体信号转导中的早期事件，如磷酸肌醇周转。随后苏氨酸654的去磷酸化可能会重新激活EGF受体以参与后续的信号传导事件。
