title: Homodimerization of the death-associated protein kinase catalytic domain: development of a new small molecule fluorescent reporter.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratoire d'Innovation Thérapeutique, Unité Mixte de Recherche 7200, Centre National de la Recherche Scientifique, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France.
authors.forename: Michael
authors.initials: M
authors.lastname: Zimmermann
authors.forename: Cédric
authors.initials: C
authors.lastname: Atmanene
authors.forename: Qingyan
authors.initials: Q
authors.lastname: Xu
authors.forename: Laetitia
authors.initials: L
authors.lastname: Fouillen
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
authors.forename: Dominique
authors.initials: D
authors.lastname: Bonnet
authors.forename: Claire
authors.initials: C
authors.lastname: Marsol
authors.forename: Marcel
authors.initials: M
authors.lastname: Hibert
authors.forename: Sarah
authors.initials: S
authors.lastname: Sanglier-Cianferani
authors.forename: Claire
authors.initials: C
authors.lastname: Pigault
authors.forename: Laurie K
authors.initials: LK
authors.lastname: McNamara
authors.forename: D Martin
authors.initials: DM
authors.lastname: Watterson
authors.forename: Jacques
authors.initials: J
authors.lastname: Haiech
authors.forename: Marie-Claude
authors.initials: MC
authors.lastname: Kilhoffer
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D064550:Death-Associated Protein Kinases; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
chemical_list: D051017:Apoptosis Regulatory Proteins; D005456:Fluorescent Dyes; D010455:Peptides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases