Thioredoxin promotes ASK1 ubiquitination and degradation to inhibit ASK1-mediated apoptosis in a redox activity-independent manner.
It has been shown that thioredoxin (Trx) in a reduced form binds to and inhibits apoptosis signal-regulating kinase 1 (ASK1). Apoptotic stimuli such as tumor necrosis factor (TNF) and reactive oxygen species (ROS) activate ASK1 in part by oxidizing Trx (forming intramolecular disulfide between C32 and C35) to release Trx from ASK1. In the present study, we examined if Trx affects ASK1 protein stability and whether the redox activity of Trx is critical in regulating ASK1 activity. First, we showed that overexpression of the wild-type Trx (Trx-WT) in endothelial cells induced ASK1 ubiquitination and degradation. Trx-induced ASK1 ubiquitination/degradation could be blocked by ASK1 activators TNF and TRAF2. We then tested the single-mutation of Trx at the catalytic site C32 or C35 (Trx-C32S or Trx-C35S) and the double-mutation (Trx-CS). The results showed that the single mutants (but not Trx-CS) retained the binding activity for ASK1 and the ability to induce ASK1 ubiquitination/degradation. Unlike Trx-WT, Trx-C32S and Trx-C35S mutants constitutively bind to ASK1 even in the presence of hydrogen peroxide in vitro and TNF in vivo. Finally, we showed that the single mutants (not Trx-WT) significantly (n=4 and P<0.05) inhibited ASK1-induced JNK activation, caspase 3 activity, and apoptosis in TNF/ROS-resistant manner. Our data suggest that association of Trx with ASK1 through a single Cysteine (C32 or C35) is necessary and sufficient for Trx activity in inducing ASK1 ubiquitination/degradation leading to inhibition of ASK1-induced apoptosis.
Liu
Yingmei
Y
Center for Cardiovascular Research, University of Rochester Medical Center, Rochester, NY 14642, USA.
Min
Wang
W
D000818:Animals; D017209:Apoptosis; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D048848:MAP Kinase Kinase Kinase 5; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D011506:Proteins / Q000502:physiology; D047992:TNF Receptor-Associated Factor 2; D013879:Thioredoxins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014452:Ubiquitins / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D016877:Oxidants; D011506:Proteins; D047992:TNF Receptor-Associated Factor 2; D014409:Tumor Necrosis Factor-alpha; D014452:Ubiquitins; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D048848:MAP Kinase Kinase Kinase 5; D020930:MAP Kinase Kinase Kinases; C482858:MAP3K5 protein, human