title: DNA-PK target identification reveals novel links between DNA repair signaling and cytoskeletal regulation.
abstract: The DNA-dependent protein kinase (DNA-PK) may function as a key signaling kinase in various cellular pathways other than DNA repair. Using a two-dimensional gel electrophoresis approach and stable DNA double-strand break-mimicking molecules (Dbait32Hc) to activate DNA-PK in the nucleus and cytoplasm, we identified 26 proteins that were highly phosphorylated following DNA-PK activation. Most of these proteins are involved in protein stability and degradation, cell signaling and the cytoskeleton. We investigated the relationship between DNA-PK and the cytoskeleton and found that the intermediate filament (IF) vimentin was a target of DNA-PK in vitro and in cells. Vimentin was phosphorylated at Ser459, by DNA-PK, in cells transfected with Dbait32Hc. We produced specific antibodies and showed that Ser459-P-vimentin was mostly located at cell protrusions. In migratory cells, the vimentin phosphorylation induced by Dbait32Hc was associated with a lower cellular adhesion and migration capacity. Thus, this approach led to the identification of downstream cytoplasmic targets of DNA-PK and revealed a connection between DNA damage signaling and the cytoskeleton.
authors.affiliation: Institut Curie, Centre National de Recherche Scientifique (CNRS) UMR3347, Institut National de la Santé et de Recherche Médicale (INSERM) U1021, Université Paris-Sud 11, Centre Universitaire, Orsay, France ; DNA Therapeutics, Evry, France.
authors.forename: Ewa
authors.initials: E
authors.lastname: Kotula
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Faigle
authors.forename: Nathalie
authors.initials: N
authors.lastname: Berthault
authors.forename: Florent
authors.initials: F
authors.lastname: Dingli
authors.forename: Damarys
authors.initials: D
authors.lastname: Loew
authors.forename: Jian-Sheng
authors.initials: JS
authors.lastname: Sun
authors.forename: Marie
authors.initials: M
authors.lastname: Dutreix
authors.forename: Maria
authors.initials: M
authors.lastname: Quanz
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D003599:Cytoskeleton / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016415:Sequence Alignment; D015398:Signal Transduction; D014746:Vimentin / Q000378:metabolism
chemical_list: D014746:Vimentin; D051747:DNA-Activated Protein Kinase