科研之友
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摘要
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 H(2)O(2) 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 H(2)O(2) 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.
- 出版日期2010-5-18
