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DNA-dependent protein kinase catalytic subunit: a target for an ICE-like protease in apoptosis
Q Song1, S P Lees-Miller, S Kumar
1Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, Bancroft Centre, Australia.
Abstract:
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.
Insights
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is degraded during apoptosis, leading to reduced DNA repair activity. This degradation is mediated by the protease CPP32, highlighting a key mechanism in programmed cell death.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- DNA-dependent protein kinase (DNA-PK) is crucial for DNA double-strand break repair.
- Apoptosis involves the targeted degradation of cellular proteins, including DNA repair enzymes.
Purpose of the Study:
- To investigate the role of DNA-PKcs in apoptosis.
- To identify the specific proteases involved in DNA-PKcs degradation during apoptosis.
Main Methods:
- Exposure of various cell types to apoptosis-inducing agents.
- Analysis of DNA-PKcs and Ku protein levels and DNA-PK activity.
- Protease inhibitor studies and in vitro cleavage assays using purified DNA-PKcs and caspases.
Main Results:
- DNA-PKcs, but not Ku, is preferentially degraded during apoptosis, leading to loss of DNA-PK activity.
- A cell line resistant to apoptosis did not exhibit DNA-PKcs degradation.
- The cysteine protease CPP32 was identified as the primary enzyme cleaving DNA-PKcs during apoptosis.
Conclusions:
- DNA-PKcs is a specific substrate for CPP32 during apoptosis.
- Targeted degradation of DNA-PKcs by CPP32 contributes to the apoptotic process by inactivating DNA repair pathways.