DNA-PK target identification reveals novel links between DNA repair signaling and cytoskeletal regulation

PLoS One. 2013 Nov 25;8(11):e80313. doi: 10.1371/journal.pone.0080313. eCollection 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Line, Tumor
  • Cytoskeleton / metabolism*
  • DNA Damage
  • DNA Repair*
  • DNA-Activated Protein Kinase / metabolism
  • DNA-Activated Protein Kinase / physiology*
  • Humans
  • Molecular Sequence Data
  • Phosphorylation
  • Sequence Alignment
  • Signal Transduction*
  • Vimentin / metabolism

Substances

  • Vimentin
  • DNA-Activated Protein Kinase

Grant support

This work was supported by Institut Curie, CNRS, INSERM, and the Agence Nationale de la Recherche grant ANR-08-Biot-009-02 and DNA Therapeutics SA. E. Kotula and M.Quanz were recipients of a Ph.D. fellowship cofinanced by DNA Therapeutics SA and the Agence Nationale de la Recherche ANRT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.