DNA damage-induced apoptosis requires the DNA-dependent protein kinase, and is mediated by the latent population of p53.
Mouse embryo fibroblasts (MEFs) expressing the adenovirus E1A protein undergo apoptosis upon exposure to ionizing radiation. We show here that immediately following gamma-irradiation, latent p53 formed a complex with the catalytic subunit of the DNA-dependent protein kinase (DNA-PK(CS)). The complex formation was DNase sensitive, suggesting that the proteins came together on the DNA, conceivably at strand breaks. This association was accompanied by phosphorylation of pre-existing, latent p53 at Ser18 (corresponding to Ser15 in human p53), which was not found in DNA-PK(CS)(-/-) cells. Most significantly, DNA damage-induced apoptosis was abolished in both DNA-PK(CS)(-/-) and p53(-/-) cells. In addition, blocking synthesis of inducible p53 by cycloheximide did not abrogate apoptosis, suggesting that the latent population of p53 is sufficient for executing the apoptotic program. Finally, E1A-expressing MEFs from a p53 "knock-in" mouse where Ser18 was mutated to an alanine had an attenuated apoptotic response, indicating that phosphorylation of this site by DNA-PK is a contributing factor for apoptosis.
Woo
Richard A
RA
Cancer Biology Research Group and Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
Jack
Melissa T
MT
Xu
Yang
Y
Burma
Sandeep
S
Chen
David J
DJ
Lee
Patrick W K
PW
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002458:Cell Fractionation; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004249:DNA Damage; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects*; D005720:Gamma Rays; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009687:Nuclear Proteins; D017354:Point Mutation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011500:Protein Synthesis Inhibitors; D016159:Tumor Suppressor Protein p53; D003513:Cycloheximide; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; C497264:Prkdc protein, mouse; D017346:Protein Serine-Threonine Kinases