Preparation of the 14-3-3:ASK1 kinase complex for structural studies

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

¹ Faculty of Science, Charles University in Prague, 12843 Prague, Czech Republic
² Institute of Physiology, Academy of Science of Czech Republic, 14220 Prague, Czech Rep.
dal.kosek@gmail.com

ASK1 (Apoptosis signal-regulating kinase, MAP3K5) plays a critical role in the regulation of the apoptosis triggered by cellular oxidative stress, immune response or anticancer drugs as well as the development of several diseases [1]. ASK1 is a serine/threonine protein kinase from MAP3K family involved in the activation of p38 and JNK signal pathways by direct phosphorylation of MEKK4 and MEKK6. Its activity is strictly controlled by diverse mechanisms such as phosphorylation, oligomerization and protein-protein interactions. The 14-3-3 protein has been identified as one of the most important regulators of ASK1 enzymatic activity [2]. It binds to phosphorserine 967 and suppresses the enzymatic activity, and thus the signaling potential. Molecular mechanism of this regulatory interaction is still unknown. Here, we report the preparation and basic biophysical characterization of the 14-3-3:ASK1 complex for subsequent structural studies to understand and explain this interaction. We optimized expression, purification and phosphorylation protocols for the preparation of human recombinant ASK1 catalytic domain. Purification protocol for the preparation of the 14-3-3 protein had been developed previously. The result of ASK1 phosphorylation at Ser967 in vitro was verified by MALDI-TOF mass spectrometry. Interactions between ASK1 and the 14-3-3 protein were further studied using native electrophoresis and analytical ultracentrifugation.

1. A. Matsuzawa, H. Ichijo, Biochim. Biophys. Acta 1780, (2008), 1325-1336.

2. L. M. Cockrell, M. C. Puckett, E. H. Goldman, F. R. Khuri, H. Fu, Oncogene 29, (2010), 822-830.

This work was funded by Grant IAA501110801 of the Grant Agency of the Academy of Sciences of the Czech Republic, by Research Project MSM0021620857 and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.