Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that activates c-Jun N-terminal kinase and p38 MAP kinase pathways in response to various stress stimuli, including oxidative stress, endoplasmic reticulum stress, and calcium ion influx. The function of ASK1 is associated with the activation of apoptosis in various cells and plays a key role in the pathogenesis of multiple diseases including cancer, neurodegeneration and cardiovascular diseases. The kinase activity of ASK1 is regulated by many factors, including binding of thioredoxin 1 (TRX1) and the 14-3-3 protein that both function as inhibitors of ASK1 . However, the mechanisms by which these binding interactions inhibit ASK1 are still unclear. We have previously shown that the TRX1 binding domain of N-terminal ASK1 (ASK1-TBD) is a compact monomeric and rigid domain that under reducing conditions forms with TRX1 a stable and well defined complex with 1:1 molar stoichiometry. We have also showed that the complex formation does not involve the formation of intermolecular disulfide bonds and that residues from the catalytic WCGPC motif of TRX1 are essential for complex stability with Trp31 being directly involved in the binding interaction .
The aim of this study was to investigate the role of individual cysteine residues from TRX1 as well as from ASK1-TBD in the interaction between these two proteins. Sedimentation velocity analysis together with the site-directed mutagenesis revealed that from five cysteine residues in human TRX1 molecule, the residue Cys32 is crucial for TRX1 binding to ASK1-TBD in reducing conditions. Formation of disulfide bond between Cys32 and Cys35 is the main factor responsible for complex dissociation under oxidative stress. The sequence of ASK1-TBD contains seven Cys residues from which six seem to be well accessible at the surface of the ASK1-TBD molecule. Oxidation of ASK1-TBD leads to the formation of one intramolecular disulfide bond between cysteines Cys225 and Cys226. Site-directed mutagenesis of ASK1-TBD Cys residues suggested that some of these residues play an important role in the stability of the ASK1-TBD:TRX1 complex.
This work was supported by the Czech Science Foundation (Project 14-10061S) and The Czech Academy of Sciences (Research Projects RVO: 67985823 of the Institute of Physiology).