van der Vliet A, Hristova M, Cross C E, Eiserich J P, Goldkorn T
Center for Comparative Respiratory Biology and Medicine, Department of Internal Medicine, University of California, Davis, California 95616, USA.
J Biol Chem. 1998 Nov 27;273(48):31860-6. doi: 10.1074/jbc.273.48.31860.
Irreversible tyrosine modifications by inflammatory oxidants such as peroxynitrite (ONOO-) can affect signal transduction pathways involving tyrosine phosphorylation. The epidermal growth factor receptor (EGFR), a member of the c-ErbB receptor tyrosine kinase family, is involved in regulation of epithelial cell growth and differentiation, and possible modulation of EGFR-dependent signaling by ONOO- was studied. Exposure of epidermoid carcinoma A431 cells to 0.1-1.0 mM ONOO- resulted in tyrosine nitration on EGFR and other proteins but did not significantly affect EGFR tyrosine autophosphorylation. A high molecular mass tyrosine-phosphorylated protein (approximately 340 kDa) was detected in A431 cell lysates after exposure to ONOO-, most likely representing a covalently dimerized form of EGFR, based on immunoprecipitation and/or immunoblotting with alpha-EGFR antibodies and co-migration with ligand-induced EGFR dimers cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Covalent EGFR dimerization by ONOO- probably involved intermolecular dityrosine cross-linking and was enhanced after receptor activation with epidermal growth factor. Furthermore, irreversibly cross-linked EGFR was more extensively tyrosine-phosphorylated compared with the monomeric form, indicating that ONOO- preferentially cross-links activated EGFR. Exposure of A431 cells to ONOO- markedly reduced the kinetics of tyrosine phosphorylation of a downstream EGFR substrate, phospholipase C-gamma1, which may be related to covalent alterations in EGFR. Alteration of EGFR signaling by covalent EGFR dimerization by inflammatory oxidants such as ONOO- may affect conditions of increased EGFR activation such as epithelial repair or tumorigenesis.
炎症性氧化剂如过氧亚硝酸盐（ONOO-）引起的不可逆酪氨酸修饰可影响涉及酪氨酸磷酸化的信号转导途径。表皮生长因子受体（EGFR）是c-ErbB受体酪氨酸激酶家族的成员，参与上皮细胞生长和分化的调控，并且对ONOO-可能调节EGFR依赖性信号传导进行了研究。将表皮样癌A431细胞暴露于0.1 - 1.0 mM的ONOO-会导致EGFR和其他蛋白质上的酪氨酸硝化，但对EGFR酪氨酸自身磷酸化没有显著影响。在暴露于ONOO-后的A431细胞裂解物中检测到一种高分子量的酪氨酸磷酸化蛋白（约340 kDa），基于用α-EGFR抗体进行免疫沉淀和/或免疫印迹以及与用1-乙基-3-（3-二甲基氨基丙基）碳二亚胺交联的配体诱导的EGFR二聚体共迁移，最有可能代表EGFR的共价二聚体形式。ONOO-引起的EGFR共价二聚化可能涉及分子间二酪氨酸交联，并且在用表皮生长因子激活受体后增强。此外，与单体形式相比，不可逆交联的EGFR酪氨酸磷酸化程度更高，表明ONOO-优先交联活化的EGFR。将A431细胞暴露于ONOO-会显著降低下游EGFR底物磷脂酶C-γ1的酪氨酸磷酸化动力学，这可能与EGFR的共价改变有关。炎症性氧化剂如ONOO-通过EGFR共价二聚化改变EGFR信号传导可能会影响EGFR活化增加的情况，如上皮修复或肿瘤发生。
