Peroxynitrite targets the epidermal growth factor receptor, Raf-1, and MEK independently to activate MAPK

Activation of ERK-1 and -2 by H2O2 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-phos-pho-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 mu M ONOO-. Activation of ERK by ONOO- and H2O2 was blocked by the antioxidant N-acetyl-L-cysteine. Catalase blocked ERK activation by H2O2, but not by ONOO-, demonstrating that the effect of ONOO- was not due to the generation of H2O2, Both H2O2 and ONOO- induced phosphorylation of EGFR in Western blot experiments using an anti-phos-pho-EGFR antibody. However, the EGFR tyrosine kinase inhibitor AG1478 abolished ERK activation by H2O2, but not by ONOO-. Both H2O2 and ONOO- activated Raf-1, However, the Raf inhibitor forskolin blocked ERK activation by H2O2, but not by ONOO-. The MEK inhibitor PD98059 inhibited ERK activation by both H2O2 and ONOO-. Moreover, ONOO- or H2O2 caused a cytotoxic response of myofibroblasts that was prevented by preincubation with PD98059, In a cell-free kinase assay, ONOO- (but not H2O2) 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, H2O2-induced ERK activation is dependent on EGFR activation, which then leads to downstream Raf-1 and MEK-1 activation.