Biophysical study of the complex between the 14-3-3 protein and the kinase domain of ASK1

Dalibor Kosek^1,2, Lenka Rezabkova^1,2, Veronika Obsilova² and Tomas Obsil^1,2

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

ASK1 (Apoptosis signal-regulating kinase, MAP3K5) plays a critical role in the regulation of the apoptosis triggered by the cellular oxidative stress, immune response or anticancer agents. So far it has been connected with the development of several neurodegenerative or cardiovascular diseases, diabetes and cancer. It is a homodimeric serine/threonine kinase from MAP3K family and its activity is tightly regulated by phosphorylation, oligomerization and protein-protein interaction. Once activated, ASK1 phosphorylates MAP2K4/7 or MAP2K3/6 that in turn activates JNK or p38 signaling pathways triggering apoptosis. The 14-3-3 protein has been identified as one of its most important physiological regulators. It binds to the Ser967 at the C-terminus of the kinase domain and holds ASK1 in an inactive state preventing the iniciation of the signaling. It has been shown that ASK1 is activated after dephosphorylation and dissociation of 14-3-3 in the presence of reactive oxygen species but the molecular mechanism of this interaction is still unknown.

Here we report the inicial biophysical characterization of the complex between 14-3-3 protein and kinase domain of ASK1. We optimized expression, purification and phosphorylation protocols for the preparation of recombinant enzymatically active human ASK1 catalytic domain. Purification protocol for the preparation of the 14-3-3 protein had been developed previously. Interaction with the 14-3-3 protein was verified using native electrophoresis and analytical ultracentrifugation. Determination of stochiometry and apparent K_d of the complex were performed using sedimentation velocity analysis.

This work was supported by the Grant Agency of Charles University in Prague (Grant 568912)