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 ₂O₂) and phorbol myristate acetate (PMA), respectively. In this study, we have demonstrated that H₂O₂ 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-ε activation as determined by the transient expression of dominant negative mutants of PKC-ε-K436R. Interestingly, H₂O₂ produced synergistic increase of PMA-stimulated Raf-1 kinase activation after simultaneous treatment of PMA and H ₂O₂. 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 ₂O₂ occurs via mechanisms that involve an interaction of Raf-1 kinase and PKC-ε, along with a transient phosphorylation of both Raf-1 kinase and PKC.