Donato N J, Rosenblum M G, Steck P A
Department of Clinical Immunology, M. D. Anderson Cancer Center, Houston, Texas 77030.
Cell Growth Differ. 1992 May;3(5):259-68.
Previous studies of tumor necrosis factor (TNF) action on tumor cells revealed a possible role for tyrosine phosphorylation of epidermal growth factor (EGF) receptor in the growth-regulatory activities of this cytokine (N. J. Donato, G. E. Gallick, P. A. Steck, and M. G. Rosenblum, J. Biol. Chem., 264: 20474-20481, 1989). EGF receptor immunoprecipitated from [32P] phosphate-equilibrated A431 cells demonstrated that TNF treatment resulted in both a time- and concentration-dependent stimulation of EGF receptor phosphorylation, which was maximal (approximately 3-fold) after 10-20 min of TNF exposure (10 nM). Incubation of A431 cells with an equivalent concentration of EGF resulted in similar stimulation of EGF receptor phosphorylation, albeit at different phosphotyrosine levels. Antiphosphotyrosine immunoblot analysis confirmed these results but suggested that the extent and kinetics of TNF-induced tyrosine phosphorylation were distinct from those obtained in EGF-treated cells. Resolution of tryptic phosphopeptides from EGF receptor demonstrated that TNF-induced phosphorylation of EGF receptor was similar, but not identical, to profiles obtained from EGF-treated cells and distinct when compared to the actions of phorbol ester. Unlike EGF, TNF was unable to directly stimulate EGF receptor tyrosine kinase activity in membranes prepared from A431 cells. In addition, TNF treatment had no significant effect on either the high- or low-affinity ligand-binding sites on EGF receptor and did not alter the kinetics or extent of ligand-induced internalization of EGF receptors. However, EGF receptor biosynthesis was consistently increased upon prolonged treatment with TNF (4-12 h). Our results suggest that TNF regulates both phosphorylation and biosynthesis of EGF receptor in a manner distinct from that of both EGF and phorbol ester, and studies of the differential phosphorylation of EGF receptor may aid in understanding the molecular mode of TNF action.
先前关于肿瘤坏死因子（TNF）对肿瘤细胞作用的研究表明，表皮生长因子（EGF）受体的酪氨酸磷酸化在这种细胞因子的生长调节活性中可能发挥作用（N. J. 多纳托、G. E. 加利克、P. A. 斯特克和M. G. 罗森布卢姆，《生物化学杂志》，264: 20474 - 20481, 1989）。从经[32P]磷酸盐平衡的A431细胞中免疫沉淀的EGF受体表明，TNF处理导致EGF受体磷酸化呈现时间和浓度依赖性刺激，在TNF暴露（10 nM）10 - 20分钟后达到最大值（约3倍）。用等量浓度的EGF孵育A431细胞会导致类似的EGF受体磷酸化刺激，尽管磷酸酪氨酸水平不同。抗磷酸酪氨酸免疫印迹分析证实了这些结果，但表明TNF诱导的酪氨酸磷酸化的程度和动力学与EGF处理的细胞不同。对EGF受体的胰蛋白酶磷酸肽进行分析表明，TNF诱导的EGF受体磷酸化与EGF处理的细胞相似，但不完全相同，与佛波酯的作用相比则有所不同。与EGF不同，TNF无法直接刺激从A431细胞制备的膜中的EGF受体酪氨酸激酶活性。此外TNF处理对EGF受体上的高亲和力或低亲和力配体结合位点均无显著影响，也不会改变配体诱导的EGF受体内化的动力学或程度。然而，长时间用TNF处理（4 - 12小时）后，EGF受体的生物合成持续增加。我们的结果表明，TNF以一种不同于EGF和佛波酯的方式调节EGF受体的磷酸化和生物合成，对EGF受体磷酸化差异的研究可能有助于理解TNF作用的分子模式。
