Mechanism of Oxidative Stress-Induced ASK1-Catalyzed MKK6 Phosphorylation

Apoptosis signal-regulating kinase 1 (ASK1) is a serine/threonine kinase that responds to a plethora of stress-inducing signals. In turn, activation of ASK1 is associated with a number of human pathological conditions, including neurodegenerative disease, inflammation, and heart failure. In response to oxidative stress, ASK1 activates the cell death-associated p38 MAPK pathway by phosphorylating M K K6. Here, we investigated the regulation of oxidative stress-induced ASK1-catalyzed phosphorylation of M K K6. M K K6 phosphorylation levels increased immediately after H2O2 treatment in intact cells and decreased Following treatment for 30 min. When expressed in HEK293T cells, ASK1 was reproducibly purified within a high-molecular mass complex (similar to 1500 kDa) known as the ASK1 signalosome. Measurement of the in vitro kinetic parameters revealed that the catalytic efficiency (k(cat)/K-m) of ASK1 was 4000-fold greater in cells treated with H2O2 for 3 min than in untreated cells. Interestingly, although the K-m(ATP) values were found to be unchanged, the K-m(M (K) (K6)) was dramatically decreased (similar to 1000-fold). The increased affinity was specific for M K K6 and short-lived, as the K-m(M (K) (K6)) returned to basal levels 30 min after treatment. Consistently, endogenous MKK6 was found within the ASK1 signalosome in intact cells and in addition copurified with ASK1 following treatment for 3 min. In contrast, proteins modulating ASK I activity and degradation were found to interact with the ASK1 signalosome once M K K6 activation was completed. Taken together, these data suggest that oxidative stress rapidly increases ASK1 catalytic efficiency for M K K6 phosphorylation by increasing M K K6 binding affinity within the ASK1 signalosome prior to induction of inactivation and degradation of the complex.