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Mechanism of oxidative stress-induced ASK1-catalyzed MKK6 phosphorylation.
Biochemistry. 2010 May 18; 49(19):4094-102.B

Abstract

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 MKK6. Here, we investigated the regulation of oxidative stress-induced ASK1-catalyzed phosphorylation of MKK6. MKK6 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 (approximately 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(MKK6)) was dramatically decreased (approximately 1000-fold). The increased affinity was specific for MKK6 and short-lived, as the K(m(MKK6)) 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 ASK1 activity and degradation were found to interact with the ASK1 signalosome once MKK6 activation was completed. Taken together, these data suggest that oxidative stress rapidly increases ASK1 catalytic efficiency for MKK6 phosphorylation by increasing MKK6 binding affinity within the ASK1 signalosome prior to induction of inactivation and degradation of the complex.

Authors+Show Affiliations

Department of Molecular Therapeutics and Translational Research Institute, Scripps Florida, Jupiter, Florida 33458, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

20364819

Citation

Sturchler, Emmanuel, et al. "Mechanism of Oxidative Stress-induced ASK1-catalyzed MKK6 Phosphorylation." Biochemistry, vol. 49, no. 19, 2010, pp. 4094-102.
Sturchler E, Feurstein D, McDonald P, et al. Mechanism of oxidative stress-induced ASK1-catalyzed MKK6 phosphorylation. Biochemistry. 2010;49(19):4094-102.
Sturchler, E., Feurstein, D., McDonald, P., & Duckett, D. (2010). Mechanism of oxidative stress-induced ASK1-catalyzed MKK6 phosphorylation. Biochemistry, 49(19), 4094-102. https://doi.org/10.1021/bi100010j
Sturchler E, et al. Mechanism of Oxidative Stress-induced ASK1-catalyzed MKK6 Phosphorylation. Biochemistry. 2010 May 18;49(19):4094-102. PubMed PMID: 20364819.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Mechanism of oxidative stress-induced ASK1-catalyzed MKK6 phosphorylation. AU - Sturchler,Emmanuel, AU - Feurstein,Daniel, AU - McDonald,Patricia, AU - Duckett,Derek, PY - 2010/4/7/entrez PY - 2010/4/7/pubmed PY - 2010/6/17/medline SP - 4094 EP - 102 JF - Biochemistry JO - Biochemistry VL - 49 IS - 19 N2 - 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 MKK6. Here, we investigated the regulation of oxidative stress-induced ASK1-catalyzed phosphorylation of MKK6. MKK6 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 (approximately 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(MKK6)) was dramatically decreased (approximately 1000-fold). The increased affinity was specific for MKK6 and short-lived, as the K(m(MKK6)) 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 ASK1 activity and degradation were found to interact with the ASK1 signalosome once MKK6 activation was completed. Taken together, these data suggest that oxidative stress rapidly increases ASK1 catalytic efficiency for MKK6 phosphorylation by increasing MKK6 binding affinity within the ASK1 signalosome prior to induction of inactivation and degradation of the complex. SN - 1520-4995 UR - https://www.unboundmedicine.com/medline/citation/20364819/Mechanism_of_oxidative_stress_induced_ASK1_catalyzed_MKK6_phosphorylation_ L2 - https://dx.doi.org/10.1021/bi100010j DB - PRIME DP - Unbound Medicine ER -