Reactive oxygen species- and dimerization-induced activation of apoptosis signal-regulating kinase 1 in tumor necrosis factor-α signal transduction

Reactive oxygen species (ROS) have been implicated in the induction of apoptosis by tumor necrosis factor-alpha (TNF alpha) and other cytotoxic insults, although the molecule(s) regulated by ROS in TNF alpha signaling have not been identified. Apoptosis signal regulating kinase 1 (ASK1) is a member of the mitogen activated protein kinase kinase kinase (MAPKKK) superfamily that has been shown to be activated during TNF alpha-induced apoptosis. ASK1 increases apoptosis when overexpressed, but the mechanism of ASK1 activation and the mechanisms of ASK1-induced apoptosis are unclear. We now report that hydrogen peroxide induces the activation of ASK1 in 293 cells. TNF alpha induced activation of ASK1 was inhibited by antioxidants. Hydrogen peroxide-induced apoptosis was markedly enhanced by the expression of ASK1. These results suggest that TNF alpha-induced activation of ASK1 is mediated by ROS. We also examined how ASK1 activity is regulated by ROS. We found that ASK1 formed dimers or higher order oligomers in 293 cells. TNF alpha or hydrogen peroxide treatment increased the dimeric form of ASK1, and pretreatment with N-acetylcysteine decreased it. Furthermore, synthetic dimerization of an ASK1-gyrase B fusion protein by coumermycin resulted in substantial activation of ASK1, suggesting that dimerization of ASK1 is sufficient for its activation. These results taken together suggest that TNF alpha causes ASK1 activation via ROS-mediated dimerization of ASK1.