Related Experiment Videos
DNA-dependent protein kinase is a target for a CPP32-like apoptotic protease
1Department of Molecular Biology, Cell Biology, and Biochemistry, Box G, Brown University, Providence, Rhode Island 02912, USA.
Abstract:
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.
Insights
The protease CPP32 cleaves DNA-dependent protein kinase catalytic subunit (DNA-PKcs) during apoptosis. This cleavage, induced by staurosporine, precedes DNA degradation and is not inhibited by Bcl-2.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a crucial role in DNA repair.
- Apoptosis, or programmed cell death, involves complex proteolytic events.
- Staurosporine (STS) is a well-established inducer of apoptosis in various cell types.
Purpose of the Study:
- To investigate the proteolytic processing of DNA-PKcs during STS-induced apoptosis.
- To identify the specific protease responsible for DNA-PKcs cleavage.
- To elucidate the relationship between DNA-PKcs proteolysis and apoptotic DNA degradation.
Main Methods:
- Treatment of HL-60 cells with staurosporine (STS).
- Analysis of DNA-PKcs cleavage using Western blotting and in vitro assays.
- Assessment of protease activity using specific inhibitors, including a CPP32 inhibitor.
Main Results:
- Specific proteolytic cleavage of DNA-PKcs was observed in STS-treated HL-60 cells.
- DNA-PKcs proteolysis correlated with or preceded apoptotic DNA fragmentation.
- The CPP32 protease, or a similar protease, was identified as responsible for DNA-PKcs cleavage.
Conclusions:
- CPP32-mediated proteolysis of DNA-PKcs is a key event during STS-induced apoptosis.
- This cleavage event occurs upstream of Bcl-2's protective function.
- Understanding this pathway provides insights into apoptosis regulation and DNA damage response.