Sedimentation analysis of the interaction between ASK1 and its binding partners

Kosek Dalibor¹, Kylarova Salome^1,2, Psenakova Katarina^1,2, Rezabkova Lenka¹, Obsilova Veronika², Obsil Tomas¹

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

²Institute of Physiology, Academy of Science of Czech Republic, 14220 Prague, Czech Republic

kosek@natur.cuni.cz

Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, 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. ASK1 plays a key role in the pathogenesis of multiple diseases including cancer, neurodegeneration, cardiovascular diseases and diabetes, thus being a promising therapeutic target against these pathologies. The enzymatic activity of ASK1 is tightly regulated by phosphorylation, oligomerization and protein-protein interactions. The formation of high molecular complexes, ASK1 signalosomes, was observed as an essential element for oxidative stress-induced cell death.

Thioredoxin  (TRX)  is an oxidoreductase which was found to bind the N-terminal domain of ASK1 in its inactive state and it prevents its activation. If exposed to reactive oxygen species (ROS), TRX dissociates from ASK1 through unknown mechanism. This leads to the subsequent binding of TRAF2/6 and full activation. The 14-3-3 protein was identified as one of the most important physiological regulators of ASK1. It binds to the phosphorylated Ser967 at the C-terminus of the kinase domain ASK1 and  maintains its inactive state, thus preventing the signaling initiation. It has been previously shown that ASK1 is activated after dephosphorylation of  Ser967 and dissociation of 14-3-3 in the presence of ROS but mechanism of inhibition is also unclear  possibly similar to other 14-3-3 regulated comlexes.

Here we present  analysis of interactions of TRX or 14-3-3 protein with corresponding binding domains of ASK1 using AUC. We revealed possible binding interface between TRX and ASK1 and importance of different cystein residues in the binding mechanis [1]. We also characterized the complex with 14-3-3 reveling its weak and transient nature.

1.         Kosek D., Kylarova S., Psenakova K., Rezabkova L., Herman P., Vecer J., Obsilova V., Obsil T.:    J. Biol. Chem., 289(35), 24463-74 (2014)

This work was supported by the Czech Science Foundation (Project #14-10061S) and the Grant Agency of Charles University in Prague (568912).