Peroxynitrite induces covalent dimerization of epidermal growth factor receptors in A431 epidermoid carcinoma cells.
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.
van der Vliet
A
A
Center for Comparative Respiratory Biology and Medicine, Department of Internal Medicine, University of California, Davis, California 95616, USA.
Hristova
M
M
Cross
C E
CE
Eiserich
J P
JP
Goldkorn
T
T
D002234:Carbodiimides; D002294:Carcinoma, Squamous Cell; D002455:Cell Division / Q000187:drug effects; D003432:Cross-Linking Reagents; D019281:Dimerization; D066246:ErbB Receptors / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009566:Nitrates / Q000494:pharmacology*; D051966:Phospholipase C gamma; D010766:Phosphorylation; D019000:Phosphotyrosine; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000187:drug effects / Q000378:metabolism
D002234:Carbodiimides; D003432:Cross-Linking Reagents; D007527:Isoenzymes; D009566:Nitrates; C074139:1-ethyl-3-(3-dimethylaminoethyl)carbodiimide; D019000:Phosphotyrosine; C039642:peroxynitric acid; D066246:ErbB Receptors; D010738:Type C Phospholipases; D051966:Phospholipase C gamma