The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response.
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
Arjan B
AB
Department of Physiological Chemistry, University Medical Centre Utrecht, Utrecht, The Netherlands. a.b.brenkman@umcutrecht.nl
van den Broek
Niels J F
NJ
de Keizer
Peter L J
PL
van Gent
Dik C
DC
Burgering
Boudewijn M T
BM
D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D051379:Mice; D046912:Multiprotein Complexes / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D013312:Stress, Physiological; D053719:Tandem Mass Spectrometry; D014157:Transcription Factors / Q000378:metabolism*
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C108803:FOXO4 protein, human; D051858:Forkhead Transcription Factors; C525394:FoxO4 protein, mouse; D046912:Multiprotein Complexes; D014157:Transcription Factors; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen