Recruitment of phosphorylated NPM1 to sites of DNA damage through RNF8-dependent ubiquitin conjugates

Cancer Res. 2010 Sep 1;70(17):6746-56. doi: 10.1158/0008-5472.CAN-10-0382. Epub 2010 Aug 16.

Abstract

Protein accumulation at DNA double-strand breaks (DSB) is essential for genome stability; however, the mechanisms governing these events are not fully understood. Here, we report a new role for the nucleophosmin protein NPM1 in these mechanisms. Thr199-phosphorylated NPM1 (pT199-NPM1) is recruited to nuclear DNA damage foci induced by ionizing radiation (IR). Foci formation is impaired by depletion of the E3 ubiquitin ligases RNF8 and RNF168 or the E2 Ubc13, and pT199-NPM1 binds to Lys63-linked ubiquitin polymers in vitro. Thus, phosphorylated NPM1 may interact with RNF8-dependent ubiquitin conjugates at sites of DNA damage. The interaction was found to rely on T199 phosphorylation, an acidic tract, and an adjacent ubiquitin-interacting motif-like domain. Depletion of the breast cancer suppressor BRCA1 or its partner, RAP80, enhanced IR-induced NPM1 foci and prolonged persistence of the foci, possibly implicating BRCA1 in pT199-NPM1 action and dynamics. Replacement of endogenous NPM1 with its nonphosphorylable T199A mutant prolonged persistence of IR-induced RAD51 foci accompanied by unrepaired DNA damage. Collectively, our findings suggest that phosphorylated NPM1 is a novel component in DSB repair that is recruited by ubiquitin conjugates downstream of RNF8 and RNF168.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • BRCA1 Protein / deficiency
  • BRCA1 Protein / metabolism
  • DNA Breaks, Double-Stranded*
  • DNA Repair / physiology
  • DNA-Binding Proteins / metabolism*
  • HeLa Cells
  • Humans
  • Leupeptins / pharmacology
  • Nuclear Proteins / metabolism*
  • Nucleophosmin
  • Phosphorylation
  • Proteasome Endopeptidase Complex / metabolism
  • Proteasome Inhibitors
  • Ubiquitin / metabolism*
  • Ubiquitin-Protein Ligases

Substances

  • BRCA1 Protein
  • BRCA1 protein, human
  • DNA-Binding Proteins
  • Leupeptins
  • NPM1 protein, human
  • Nuclear Proteins
  • Proteasome Inhibitors
  • RNF8 protein, human
  • Ubiquitin
  • Nucleophosmin
  • Ubiquitin-Protein Ligases
  • Proteasome Endopeptidase Complex
  • ATP dependent 26S protease
  • benzyloxycarbonylleucyl-leucyl-leucine aldehyde