Tyrosine phosphorylation and Ras activation is required for hydrogen peroxide-mediated Raf-1 kinase activation

Reactive oxygen species (ROS), such as hydrogen peroxide (H(2)O(2)), have been shown to play a significant role in regulating transmembrane signaling pathways to modulate cell proliferation and differentiation. Here we report findings that indicate that treatment of Sf9 cells expressing Raf-1 with H(2)O(2) results in significant and sustained activation of Raf-1 kinase. The activation of Raf-1 in response to H(2)O(2) treatment of Raf-expressing Sf9 cells was found to involve tyrosine phosphorylation, detected by immunoblotting with anti-phosphotyrosine antibody. The addition of tyrosine-specific phosphatase (PTP1B) to Raf-1 immunoprecipitated from Sf9 cells infected with Raf-1 after H(2)O(2) stimulation partially decreased the kinase activity of Raf-1. In a mammalian cell system, we also identified that the overexpression of a kinase-negative Raf-1 fragment (which acts as a dominant-negative inhibitor of Ras-Raf interaction) resulted in the inhibition of the H(2)O(2)-induced activation of Raf-1. Moreover, the blocking of the Ras function by the farnesyltransferase inhibitor, alpha-hydroxyfarnesylphosphonic acid, led to a 40% or greater reduction in Raf-1 kinase activity, suggesting that Ras is involved in the signaling pathway mediating the H(2)O(2) activation of Raf-1. Taken together, these results suggest that tyrosine phosphorylation and Ras activation are essential components of the mechanism by which H(2)O(2) activates Raf-1 kinase activity.