Homodimerization of the death-associated protein kinase catalytic domain: development of a new small molecule fluorescent reporter.

• Main Authors: Michael Zimmermann, Cédric Atmanene, Qingyan Xu, Laetitia Fouillen, Alain Van Dorsselaer, Dominique Bonnet, Claire Marsol, Marcel Hibert, Sarah Sanglier-Cianferani, Claire Pigault, Laurie K McNamara, D Martin Watterson, Jacques Haiech, Marie-Claude Kilhoffer
• Format: Article
• Language: English
• Published: Public Library of Science (PLoS) 2010-11-01
• Series: PLoS ONE
• Online Access: http://europepmc.org/articles/PMC2994711?pdf=render

Death-Associated Protein Kinase (DAPK) is a member of the Ca2+/calmodulin regulated serine/threonine protein kinases. Its biological function has been associated with induced cell death, and in vivo use of selective small molecule inhibitors of DAPK catalytic activity has demonstrated that it is a potential therapeutic target for treatment of brain injuries and neurodegenerative diseases.In the in vitro study presented here, we describe the homodimerization of DAPK catalytic domain and the crucial role played by its basic loop structure that is part of the molecular fingerprint of death protein kinases. Nanoelectrospray ionization mass spectrometry of DAPK catalytic domain and a basic loop mutant DAPK protein performed under a variety of conditions was used to detect the monomer-dimer interchange. A chemical biological approach was used to find a fluorescent probe that allowed us to follow the oligomerization state of the protein in solution.The use of this combined biophysical and chemical biology approach facilitated the elucidation of a monomer-dimer equilibrium in which the basic loop plays a key role, as well as an apparent allosteric conformational change reported by the fluorescent probe that is independent of the basic loop structure.