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, PKCalpha and epsilon were activated upon ONOO(-) exposure. When cells were treated with ONOO(-) in a calcium-free buffer, no activation of PKCalpha was detected. Concomitantly, a reduction of ERK1/2 phosphorylation was observed suggesting that calcium was required for translocation of PKCalpha 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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Institute of Biomembranes, Department of Molecular Cell Biology, Universiteit Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands. s.bapat@bio.uu.nl
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D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009566:Nitrates / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D051381:Rats
D009566:Nitrates; C039642:peroxynitric acid; D015240:Phorbol 12,13-Dibutyrate; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D002118:Calcium