Structural Insight into the 14-3-3 Protein-Dependent Inhibition of Protein Kinase ASK1

Olivia Petrvalska1,2, Dalibor Kosek1,2, Zdenek Kukacka3, Zdenek Tosner1, Jaroslav Vecer4, Petr Herman4, Veronika Obsilova2,*, Tomas Obsil1,2,*

1Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, 12843 Prague,

 2Institute of Physiology, The Czech Academy of Sciences, 14220 Prague,

 3Institute of Microbiology, The Czech Academy of Sciences, 14220 Prague,

 4Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, 12116 Prague, Czech Republic


Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, regulates diverse physiological processes such as apoptosis, cytokine secretion or cell differentiation. The activity of ASK1, which is triggered by various stress stimuli, is regulated through homo-oligomerization and interaction with several proteins including the 14-3-3 protein which binds to the phosphorylated motif located at the C-terminus of the kinase domain of ASK1 and suppresses its catalytic activity through unknown mechanism. In this study, we performed biophysical and structural analysis of the complex between the kinase domain of ASK1 phosphorylated at S966 (pASK1-CD) and the 14-3-3z protein using AUC, SAXS and chemical cross-linking. Our results show that the complex between 14-3-3z and pASK1-CD is dynamic and conformationally heterogeneous with both proteins sampling several mutual orientations. In addition, structural analysis together with results of phosphorus NMR and time-resolved tryptophan fluorescence measurements suggest that the 14-3-3z dimer interacts with regions from the C-lobe of the kinase domain of ASK1 and induces conformational change in its active site. Thus, our study provides new insight into the interaction between the kinase domain of ASK1 and 14-3-3z and offers a plausible structural explanation for the 14-3-3 protein-dependent inhibition of ASK1 kinase activity.

This work was supported by the Czech Science Foundation (Project 14-10061S) and The Czech Academy of Sciences (Research Projects RVO: 67985823 of the Institute of Physiology).