The DNA-dependent protein kinase catalytic subunit (p460) is cleaved during Fas-mediated apoptosis in Jurkat cells.
The DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase linked to DNA repair and V(D)J recombination. It is composed of a 460-kDa catalytic subunit (DNA-PKcs) and a 70/86-kDa heterodimeric regulatory component that is identical with the human autoantigen Ku. The regulatory subunit targets the catalytic subunit to the free ends of dsDNA breaks. Since apoptosis is associated with internucleosomal chromatin fragmentation and creation of dsDNA breaks, we examined whether the biochemical amounts of either DNA-PKcs or Ku changed during apoptosis mediated by the cell surface receptor Fas. We found that the catalytic subunit was cleaved into several smaller polypeptides early in apoptosis. In contrast to DNA-PKcs, Ku was neither cleaved nor decreased in amount during apoptosis. We then extended our in vivo results to a cellfree system. Cytosolic extracts derived from apoptotic cells were able to cleave DNA-PKcs into polypeptides of sizes identical with those seen in vivo, and this cleavage was inhibited by the cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. Furthermore, DNA-PKcs was cleaved in vitro by purified apopain (CPP32), but not IL-1beta-converting enzyme. Cleavage was also inhibited by the specific tetrapeptide DEVD (amino acids 2709-2712 of the DNA-PKcs sequence), suggesting a candidate position for protease action. Finally, we found that the catalytic activity of DNA-PKcs was decreased in apoptotic cells. We conclude that DNA-PKcs is subject to selective cleavage by proteases during apoptosis. Cleavage of DNA-PKcs may represent a mechanism for regulating the function of DNA-dependent kinase during programmed cell death.
McConnell
K R
KR
Department of Internal Medicine, Medical College of Georgia, Augusta 30912, USA.
Dynan
W S
WS
Hardin
J A
JA
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D002384:Catalysis; D002457:Cell Extracts / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003600:Cytosol / Q000502:physiology; D004247:DNA / Q000502:physiology*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D003609:Dactinomycin / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D019014:fas Receptor / Q000502:physiology*
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D019014:fas Receptor; D003609:Dactinomycin; D004247:DNA; D003513:Cycloheximide; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases