The synergistic activation of Raf-1 kinase by phorbol myristate acetate and hydrogen peroxide in NIH3T3 cells.
We have previously demonstrated that a 33kDa C-terminal fragment of c-Raf-1 underwent a mobility shift in response to hydrogen peroxide (H(2)O(2)) and phorbol myristate acetate (PMA), respectively. In this study, we have demonstrated that H(2)O(2) induced the activation of N-terminal deletion mutant as well as full length Raf-1 kinase. The pharmacological PKC activator PMA also induced a weak increase in Raf-1 kinase activity through PKC-epsilon activation as determined by the transient expression of dominant negative mutants of PKC-epsilon-K436R. Interestingly, H(2)O(2) produced synergistic increase of PMA-stimulated Raf-1 kinase activation after simultaneous treatment of PMA and H(2)O(2). This synergistic activation of Raf-1 kinase was further enhanced by cypermethrin (an inhibitor of protein phosphatase 2B) and dephostatin (tyrosine kinase inhibitor) implying an inhibitory role for these phosphatases in the Raf-1 signaling pathway. Taken together, our data suggest that the synergistic activation of Raf-1 kinase in response to PMA and H(2)O(2) occurs via mechanisms that involve an interaction of Raf-1 kinase and PKC-epsilon, along with a transient phosphorylation of both Raf-1 kinase and PKC.
Lee
Michael
M
Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, P.O. Box 123, Yusong, 305-600, Daejeon, Republic of Korea. mikelee@kitox.re.kr
Petrovics
Gyorgy
G
Anderson
Wayne B
WB
D000222:Adaptation, Physiological / Q000187:drug effects; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004789:Enzyme Activation; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D051379:Mice; D041681:NIH 3T3 Cells; D019908:Proto-Oncogene Proteins c-raf / Q000187:drug effects* / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D006861:Hydrogen Peroxide; D019908:Proto-Oncogene Proteins c-raf; D013755:Tetradecanoylphorbol Acetate