DNA-dependent protein kinase is a target for a CPP32-like apoptotic protease.
We demonstrate that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is specifically, proteolytically cleaved in HL-60 cells treated with staurosporine (STS), a potent inducer of apoptosis. The proteolysis of DNA-PKcs correlated with or preceded apoptotic chromosomal DNA degradation. Cell-free extracts prepared from STS-treated HL-60 cells recapitulated the proteolysis of DNA-PKcs in an in vitro assay using purified DNA-PK as the substrate. Western blot analyses of the apoptotic cell extract showed that the 32-kDa precursor of CPP32 is expressed in HL-60 cells and processed following STS treatment. In addition, whereas the DNA-PKcs protease activity was not inhibitable by many conventional protease inhibitors, it was inhibitable by a highly selective peptide-derived inhibitor of CPP32. These data strongly suggest that CPP32, or a CPP32-like protease, is responsible for DNA-PKcs proteolysis. Finally, our results demonstrated that the cleavage of DNA-PKcs in vitro proceeded in the presence of Bcl-2, indicating that the function provided by Bcl-2 lies upstream the proteolysis of DNA-PKcs.
Han
Z
Z
Department of Molecular Biology, Cell Biology, and Biochemistry, Box G, Brown University, Providence, Rhode Island 02912, USA.
Malik
N
N
Carter
T
T
Reeves
W H
WH
Wyche
J H
JH
Hendrickson
E A
EA
D000477:Alkylating Agents / Q000494:pharmacology; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D018922:HL-60 Cells; D006868:Hydrolysis; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013379:Substrate Specificity
D000477:Alkylating Agents; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007461:Iodoacetates; D019253:Proto-Oncogene Proteins c-bcl-2; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D005033:Ethylmaleimide; D019807:Iodoacetic Acid