Characterization of interactions between protein kinase ASK1 and its binding partners

Kosek Dalibor¹, Kylarova Salome^1,2, Petrvalska Olivia¹, 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 and cardiovascular diseases, thus being a promising therapeutic target against these pathologies. Enzymatic activity of ASK1 is tightly regulated by phosphorylation, oligomerization and protein-protein interactions. Formation of high molecular complexes, ASK1 signalosomes, was observed as an essential element for oxidative stress-induced cell death.

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. Thioredoxine (TRX) binds to the N-terminal domain of ASK1 and it also prevents its activation. If exposed to ROS, TRX dissociates from ASK1 through unknown mechanism. This leads to the subsequent binding of TRAF2/6 and full activation. However, the precise mechanisms of processes that lead to ASK1 activation are still unclear. We present here the first structural and biophysical characterization of interactions between 14-3-3, TRX and corresponding binding domains of ASK1 using AUC, SAXS, time-resolved tryptophan fluorescence and mass spectrometry.

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