Peroxynitrite activates mitogen-activated protein kinase (MAPK) via a MEK-independent pathway: a role for protein kinase C

In this study we show that phosphorylation of extracellular signal-regulated kinase (ERK1/2; also known as p44/42MAPK) following peroxynitrite (ONOO-) exposure occurs via a MAPK kinase (MEK)-independent but PKC-dependent pathway in rat-1 fibroblasts, ONOO--mediated ERK1/2 phosphorylation was not blocked by MEK inhibitors PD98059 and U0126, Furthermore, no increase in MEK phosphorylation was detected upon ONOO- treatment. Staurosporine was used to investigate whether protein kinase C (PKC) is involved. This was confirmed by down-regulation of PKC by phorbol-12,13-dibutyrate, which resulted in significant reduction of ERK1/2 phosphorylation by ONOO-, implying that activation of ERK by ONOO depends on activation of PKC, Indeed, PKC alpha and epsilon were activated upon ONOO- exposure, When cells were treated with ONOO- in a calcium-free buffer, no activation of PKC alpha was detected, Concomitantly, a reduction of ERK/2 phosphorylation was observed suggesting that calcium was required for translocation of PKC alpha and ERK phosphorylation by ONOO-. Indeed, ONOO- exposure resulted in increased cytosolic calcium, which depended on the presence of extracellular calcium. Finally, data using Go6976, an inhibitor of calcium-dependent PKC activation, implied that ONOO--mediated ERK1/2 phosphorylation depends on activation of a calcium-dependent PKC. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.