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

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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.

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