Peroxynitrite targets the epidermal growth factor receptor, Raf-1, and MEK independently to activate MAPK.
Activation of ERK-1 and -2 by H(2)O(2) in a variety of cell types requires epidermal growth factor receptor (EGFR) phosphorylation. In this study, we investigated the activation of ERK by ONOO(-) in cultured rat lung myofibroblasts. Western blot analysis using anti-phospho-ERK antibodies along with an ERK kinase assay using the phosphorylated heat- and acid-stable protein (PHAS-1) substrate demonstrated that ERK activation peaked within 15 min after ONOO(-) treatment and was maximally activated with 100 micrometer ONOO(-). Activation of ERK by ONOO(-) and H(2)O(2) was blocked by the antioxidant N-acetyl-l-cysteine. Catalase blocked ERK activation by H(2)O(2), but not by ONOO(-), demonstrating that the effect of ONOO(-) was not due to the generation of H(2)O(2). Both H(2)O(2) and ONOO(-) induced phosphorylation of EGFR in Western blot experiments using an anti-phospho-EGFR antibody. However, the EGFR tyrosine kinase inhibitor AG1478 abolished ERK activation by H(2)O(2), but not by ONOO(-). Both H(2)O(2) and ONOO(-) activated Raf-1. However, the Raf inhibitor forskolin blocked ERK activation by H(2)O(2), but not by ONOO(-). The MEK inhibitor PD98059 inhibited ERK activation by both H(2)O(2) and ONOO(-). Moreover, ONOO(-) or H(2)O(2) caused a cytotoxic response of myofibroblasts that was prevented by preincubation with PD98059. In a cell-free kinase assay, ONOO(-) (but not H(2)O(2)) induced autophosphorylation and nitration of a glutathione S-transferase-MEK-1 fusion protein. Collectively, these data indicate that ONOO(-) activates EGFR and Raf-1, but these signaling intermediates are not required for ONOO(-)-induced ERK activation. However, MEK-1 activation is required for ONOO(-)-induced ERK activation in myofibroblasts. In contrast, H(2)O(2)-induced ERK activation is dependent on EGFR activation, which then leads to downstream Raf-1 and MEK-1 activation.
Zhang
P
P
Laboratory of Pulmonary Pathobiology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Wang
Y Z
YZ
Kagan
E
E
Bonner
J C
JC
D000818:Animals; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000819:agonists / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D009566:Nitrates / Q000378:metabolism / Q000494:pharmacology*; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects*
D009566:Nitrates; D016877:Oxidants; C039642:peroxynitric acid; D066246:ErbB Receptors; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf