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

Abstract

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, PKCα and ϵ were activated upon ONOO − exposure. When cells were treated with ONOO − in a calcium-free buffer, no activation of PKCα was detected. Concomitantly, a reduction of ERK1/2 phosphorylation was observed suggesting that calcium was required for translocation of PKCα and ERK phosphorylation by ONOO −. Indeed, ONOO − exposure resulted in increased cytosolic calcium, which depended on the presence of extracellular calcium. Finally, data using Gö6976, an inhibitor of calcium-dependent PKC activation, implied that ONOO −-mediated ERK1/2 phosphorylation depends on activation of a calcium-dependent PKC.