Sturani E, Zippel R, Toschi L, Morello L, Comoglio P M, Alberghina L
Dipartimento di Fisiologia e Biochimica Generali, Università, Milan, Italy.
Mol Cell Biol. 1988 Mar;8(3):1345-51. doi: 10.1128/mcb.8.3.1345-1351.1988.
We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986). In this report, the time course of receptor phosphorylation is investigated. In normal human fibroblasts, ligand-induced phosphorylation of PDGF and EGF receptors is followed by rapid dephosphorylation. However, in A431 cells the tyrosine-phosphorylated form of EGF receptor persists for many hours after EGF stimulation, allowing a detailed analysis of the conditions affecting receptor phosphorylation and dephosphorylation. In A431 cells, the number of receptor molecules phosphorylated on tyrosine was quantitated and found to be about 10% of total EGF receptors. The phosphorylated receptor molecules are localized on the cell surface, and they are rapidly dephosphorylated upon removal of EGF from binding sites by a short acid wash of intact cells and upon a mild treatment with trypsin. ATP depletion also results in rapid dephosphorylation, indicating that continuous phosphorylation-dephosphorylation reactions occur in the ligand-receptor complex at steady state. Phorbol 12-myristate 13-acetate added shortly before EGF reduces the rate and the final extent of receptor phosphorylation. Moreover, it also reduces the amount of phosphorylated receptors if it is added after EGF. Down-regulation of protein kinase C by chronic treatment with phorbol dibutyrate increases the receptor phosphorylation induced by EGF, suggesting a homologous feedback regulation of EGF receptor functions.
我们之前曾报道过，抗磷酸酪氨酸抗体可识别血小板衍生生长因子（PDGF）和表皮生长因子（EGF）受体的磷酸化形式（齐佩尔等人，《生物化学与生物物理学学报》881:54 - 61，1986年；以及斯图拉尼等人，《生物化学与生物物理学研究通讯》137:343 - 350，1986年）。在本报告中，对受体磷酸化的时间进程进行了研究。在正常人成纤维细胞中，配体诱导的PDGF和EGF受体磷酸化之后会迅速发生去磷酸化。然而，在A431细胞中，EGF受体的酪氨酸磷酸化形式在EGF刺激后会持续数小时，这使得能够对影响受体磷酸化和去磷酸化的条件进行详细分析。在A431细胞中，对酪氨酸磷酸化的受体分子数量进行了定量，发现约占总EGF受体的10%。磷酸化的受体分子定位于细胞表面，通过完整细胞的短时间酸洗将EGF从结合位点去除后，以及用胰蛋白酶进行温和处理后，它们会迅速去磷酸化。ATP耗竭也会导致迅速去磷酸化，这表明在稳态下配体 - 受体复合物中持续发生磷酸化 - 去磷酸化反应。在EGF之前不久添加佛波醇12 - 肉豆蔻酸酯13 - 乙酸盐会降低受体磷酸化的速率和最终程度。此外，如果在EGF之后添加，它还会减少磷酸化受体的数量。用佛波醇二丁酸酯长期处理使蛋白激酶C下调，会增加EGF诱导的受体磷酸化，这表明存在对EGF受体功能的同源反馈调节。
