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The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response
Author(s) -
Brenkman Arjan B.,
Broek Niels J. F.,
Keizer Peter L. J.,
Gent Dik C.,
Burgering Boudewijn M. T.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.10-158717
Subject(s) - ku70 , ku80 , dna damage , biology , microbiology and biotechnology , dna repair , transcription factor , dna , gene , dna binding protein , genetics
In this study, we searched for proteins regulating the tumor suppressor and life‐span regulator FOXO4. Through an unbiased tandem‐affinity purification strategy combined with mass spectrometry, we identified the heterodimer Ku70/Ku80 (Ku), a DNA double‐strand break repair component. Using biochemical interaction studies, we found Ku70 to be necessary and sufficient for the interaction. FOXO4 mediates its tumor‐suppressive function in part through transcriptional regulation of the cell cycle arrest p27 kip1 gene. Immunoblotting, luciferase reporter assays, and flow cytometry showed that Ku70 inhibited FOXO4‐mediated p27 kip1 transcription and cell cycle arrest induction by >40%. In contrast, Ku70 RNAi but not control RNAi significantly increased p27 kip1 transcription. In addition, in contrast to wild‐type mouse embryonic stem (ES) cells, Ku70 −/− ES cells showed significantly increased FOXO activity, which was rescued by Ku70 reexpression. Immunofluorescence studies demonstrated that Ku70 sequestered FOXO4 in the nucleus. Interestingly, the Ku70‐FOXO4 interaction stoichiometry followed a nonlinear dose‐response curve by hydrogen peroxide‐generated oxidative stress. Low levels of oxidative stress increased interaction stoichiometry up to 75%, peaking at 50 µM, after which dissociation occurred. Because the Ku70 ortholog in the roundworm Caenorhabditis elegans was shown to regulate life span involving C. elegans FOXO, our findings suggest a conserved critical Ku70 role for FOXO function toward coordination of a survival program, regulated by the magnitude of oxidative damage.—Brenkman, A. B., van den Broek, N. J. F., de Keizer, P. L. J., van Gent, D. C., Burgering, B. M. T. The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response. FASEB J . 24, 4271–4280 (2010). www.fasebj.org