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Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xL and promotes apoptosis
K Komatsu1, T Miyashita, H Hang
1Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Department of Pharmacology and Therapeutics, University of South Florida College of Medicine, 12902 Magnolia Drive, Tampa, Florida 33612, USA.
Abstract:
DNA damage induces apoptosis through a signalling pathway that can be suppressed by the BCL-2 protein, but the mechanism by which DNA damage does this is unknown. Here, using yeast two-hybrid and co-immunoprecipitation studies, we show that RAD9, a human protein involved in the control of a cell-cycle checkpoint, interacts with the anti-apoptotic Bcl-2-family proteins BCL-2 and BCL-x L, but not with the pro-apoptotic BAX and BAD. When overexpressed in mammalian cells, RAD9 induces apoptosis that can be blocked by BCL-2 or BCL-x L. Conversely, antisense RAD9 RNA suppresses cell death induced by methyl methanesulphonate. These findings indicate that RAD9 may have a new role in regulating apoptosis after DNA damage, in addition to its previously described checkpoint-control and other radioresistance-promoting functions.
Insights
Researchers discovered that RAD9 protein interacts with anti-apoptotic proteins BCL-2 and BCL-x L, revealing a new role in regulating DNA damage-induced apoptosis.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- DNA damage triggers apoptosis, a programmed cell death process.
- The BCL-2 protein family regulates apoptosis, but the precise mechanism linking DNA damage to this pathway remains unclear.
- RAD9 is a known human protein involved in cell-cycle checkpoint control.
Purpose of the Study:
- To elucidate the mechanism by which DNA damage influences apoptosis.
- To investigate the interaction of RAD9 with BCL-2 family proteins in the context of DNA damage-induced apoptosis.
Main Methods:
- Yeast two-hybrid assays to identify protein interactions.
- Co-immunoprecipitation studies to confirm protein binding.
- Overexpression of RAD9 in mammalian cells.
- Antisense RAD9 RNA to inhibit gene expression.
Main Results:
- RAD9 was found to interact with anti-apoptotic proteins BCL-2 and BCL-x L, but not with pro-apoptotic proteins BAX and BAD.
- Overexpression of RAD9 induced apoptosis in mammalian cells, which was inhibited by BCL-2 or BCL-x L.
- Antisense RAD9 RNA suppressed methyl methanesulphonate-induced cell death.
Conclusions:
- RAD9 plays a novel role in the regulation of apoptosis following DNA damage.
- This function is independent of its previously known roles in cell-cycle checkpoint control and radioresistance.