DNA-dependent protein kinase catalytic subunit: a target for an ICE-like protease in apoptosis.
Radiosensitive cell lines derived from X-ray cross complementing group 5 (XRCC5), SCID mice and a human glioma cell line lack components of the DNA-dependent protein kinase, DNA-PK, suggesting that DNA-PK plays an important role in DNA double-strand break repair. Another enzyme implicated in DNA repair, poly(ADP-ribose) polymerase, is cleaved and inactivated during apoptosis, suggesting that some DNA repair proteins may be selectively targeted for destruction during apoptosis. Here we demonstrate that DNA-PKcs, the catalytic subunit of DNA-PK, is preferentially degraded after the exposure of different cell types to a variety of agents known to cause apoptosis. However, Ku, the DNA-binding component of the enzyme, remains intact. Degradation of DNA-PKcs was accompanied by loss of DNA-PK activity. One cell line resistant to etoposide-induced apoptosis failed to show degradation of DNA-PKcs. Protease inhibitor data implicated an ICE-like protease in the cleavage of DNA-PKcs, and it was subsequently shown that the cysteine protease CPP32, but not Mch2alpha, ICE or TX, cleaved purified DNA-PKcs into three fragments of comparable size with those observed in cells undergoing apoptosis. Cleavage sites in DNA-PKcs, determined by antibody mapping and microsequencing, were shown to be the same for CPP32 cleavage and for cleavage catalyzed by extracts from cells undergoing apoptosis. These observations suggest that DNA-PKcs is a critical target for proteolysis by an ICE-like protease during apoptosis.
Song
Q
Q
Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, Bancroft Centre, Australia.
Lees-Miller
S P
SP
Kumar
S
S
Zhang
Z
Z
Chan
D W
DW
Smith
G C
GC
Jackson
S P
SP
Alnemri
E S
ES
Litwack
G
G
Khanna
K K
KK
Lavin
M F
MF
D000818:Animals; D000906:Antibodies / Q000737:chemistry; D017209:Apoptosis; D001483:Base Sequence; D020170:Caspase 1; D002384:Catalysis; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D017931:DNA Primers; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D005047:Etoposide / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D016513:Mice, SCID; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D000906:Antibodies; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D005047:Etoposide; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D003546:Cysteine Endopeptidases; D020170:Caspase 1