Structural characterization of protein kinase ASK1 and its interaction with thioredoxin

K. Psenakova1, V. Veverka2, R. Hexnerova2, P. Srb2, V. Obsilova3, T. Obsil1

1Faculty of Science, Charles University in Prague, 12843 Prague, Czech Republic

2Institute of Organic Chemistry and Biochemistry, CAS, 16610 Prague, Czech Republic

3Institute of Physiology, CAS, 14220 Prague, Czech Republic

 

 Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase (MAPK) cascade, an essential part of the cell defence system against stressors. The function of ASK1 is associated with the activation of apoptosis in various cells, the regulation of ASK1 depends on several stimuli, including oxidative stress (presence of ROS) and therefore, ASK1 plays a key role in the pathogenesis of many diseases including cancer, neurodegeneration and cardiovascular diseases. The activity of ASK1 is regulated by its interaction with multiple proteins; current research is focused on two physiological inhibitors, mammalian thioredoxin (TRX) and the 14-3-3 protein [1]. ASK1 is under normal conditions in an inactive complex with bound TRX and 14-3-3. As a response to oxidative stress condition, TRX and 14-3-3 dissociate and ASK1 become active. However, the molecular mechanism of the ASK1 activation is still not fully understood, as there are almost no structural data available. Therefore, the aim of this study was the structural characterization of the TRX-binding domain of ASK1 (ASK1-TBD) and ASK1-TBD:TRX complex formation.

 

We have previously shown that ASK1-TBD forms with TRX well defined and stable complex under reducing conditions. Site-directed mutagenesis revealed that formation of disulfide bond between Cys32 and Cys35 in a TRX molecule is the main factor responsible for complex dissociation under oxidative stress. ASK1-TBD contains seven cysteine residues with the residue Cys250 being the only cysteine which is both solvent exposed and essential for TRX binding in reducing conditions. The oxidative stress also induces intramolecular disulfide bonds formation within ASK1-TBD and affects its structure in regions important for TRX1 binding [2,3].

In this study we present structural characterization of the regulation of ASK1 via structural model of ASK1-TBD in both reduced and oxidized conditions and ASK1-TBD:TRX complex based on sparse NMR data, crosslinking mass spectrometry and small-angle x-ray scattering (SAXS) data.

         

1.         Saitoh, M.; Nishitoh, H.; Fujii, M.; Takeda, K.; Tobiume, K.; Sawada, Y.; Kawabata, M.; Miyazono, K.; Ichijo, H.: EMBO J. 17, 2596 (1998).

2.         Kosek D.; Kylarova S.; Psenakova K.; Rezabkova L.; Herman P.; Vecer J.; Obsilova V.; Obsil T.: Journal of Biological Chemistry 279, 24463 (2014)

3.         Kylarova S.; Kosek D.; Petrvalska O.; Psenakova K.; Man P.; Vecer J.; Herman P.; Obsilova V.; Obsil T.: FEBS Journal 283, 3821 (2016)

 

This work was supported by the Grant Agency of the Charles University in Prague (No. 368216).