Role of 14-3-3 protein in the regulation of Death-associated protein kinase 2 (DAPK2)

M. Horvath^{1, 2}, O. Petrvalska², V. Obsilova², T. Obsil^{1, 2}

¹Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 128 43 Prague 2, Czech Republic

²Department of Structural Biology of Signaling Proteins, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, 252 50 Vestec, Czech Republic

horvath.mm@gmail.com

Apoptosis and autophagy are tightly regulated biological processes, acting in synchro to secure proper development or survival of cells and multicellular organisms ¹. Death-associated protein kinase (DAPK) family of proteins plays an important role in commitment stage of apoptosis and progression of autophagy. Human DAPK family consists of five serine/threonine kinases with high homology in their catalytic domains located on N-terminus but vary in remaining regions ².370 amino acid long DAPK2 is the shortest member of the DAPK family that consists of three domains: catalytic domain (23-285), autoregulatory domain (288-330) and dimerization domain (331-370). DAPK2 is activated by Ca²⁺/Calmodulin (Ca²⁺/CaM) binding to the autoregulatory domain or by phosphorylation at S299 and inhibited by the phosphorylation at S318 and T369 ³. Phosphorylation at S318 triggers conformational change that prevents both: Ca²⁺/Calmodulin from binding and substrate from entering the active site, as visible from the crystal structure of DAPK2 ⁴. Phosphorylation at T369 is recognized by the 14-3-3 protein which disrupts dimerization and decreases the activity of DAPK2 [5,6]. Primary focus of this study is to characterize the 14-3-3/DAPK2 complex and propose a molecular mechanism of 14-3-3 role in negative regulation of DAPK2. Based on our low-resolution structural data combined with biophysical characterisation of the complex and functional studies, we propose a mechanism where 14-3-3 acts as a scaffold for autoinhibitory conformation of DAPK2 and prevents dephosphorylation of regulatory phospho-sites. In this conformation, the 14-3-3 protein not only interacts with C-terminal regulatory domains of DAPK2 but also with the kinase domain, hence explaining the observed inhibitory effect of 14-3-3 on DAPK2 kinase activity. Additionally, the 14-3-3/DAPK2 complex can be stabilized by small molecule compound Fusicoccin. Thus, this study not only provides mechanistic insight into DAPK2 regulation but also suggests an alternative way how to inhibit DAPK2 during cancer treatment.

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