Disulfide Bond-mediated multimerization of Ask1 and its reduction by thioredoxin-1 regulate H(2)O(2)-induced c-Jun NH(2)-terminal kinase activation and apoptosis

Apoptosis signal-regulated kinase-1 (Ask1) lies upstream of a major redox-sensitive pathway leading to the activation of Jun NH(2)-terminal kinase (JNK) and the induction of apoptosis. We found that cell exposure to H(2)O(2) caused the rapid oxidation of Ask1, leading to its multimerization through the formation of interchain disulfide bonds. Oxidized Ask1 was fully reduced within minutes after induction by H(2)O(2). During this reduction, the thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) became covalently associated with Ask1. Overexpression of Trx1 accelerated the reduction of Ask1, and a redox-inactive mutant of Trx1 (C35S) remained trapped with Ask1, blocking its reduction. Preventing the oxidation of Ask1 by either overexpressing Trx1 or using an Ask1 mutant in which the sensitive cysteines were mutated (Ask1-DeltaCys) impaired the activation of JNK and the induction of apoptosis while having little effect on Ask1 activation. These results indicate that Ask1 oxidation is required at a step subsequent to activation for signaling downstream of Ask1 after H(2)O(2) treatment.