Abstract
The BH3-only protein BAD binds to Bcl-2 family proteins through its BH3 domain. Recent studies suggest that BAD binds to both Bcl-2 and Bcl-XL, however mediates its pro-apoptotic functions through inhibition of Bcl-XL, but not Bcl-2. In this paper we addressed this issue using a BAD mutant within the BH3 domain, by substitution of Asp 119 with Gly (BADD119G), which selectively abrogates an ability to interact with Bcl-2. Confocal microscopy revealed that mutation of BAD at D119 does not affect BAD targeting to the mitochondrial membrane in serum-starved COS-7 cells. However, co-precipitation assays indicated that, whereas wild-type BAD (BADwt) directly interacts with Bcl-2 and Bcl-XL, BADD119G interacts only with Bcl-XL. Nevertheless both BADwt and BADD119G could introduce apoptosis and diminish the anti-apoptotic effect of Bcl-2 and Bcl-XL in a similar manner in a co-transfection assay. These data thus suggest that Asp119 is a crucial site within the BH3 domain of BAD for interaction of BAD with Bcl-2, but is dispensable for the interaction of BAD with Bcl-XL, for its targeting to mitochondria, and most importantly, for its pro-apoptotic functions. Thus, we confirm that neutralization of Bcl-2 function is marginal for BAD-mediated apoptosis.
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Abbreviations
- GFP:
-
green fluorescent protein
- GST:
-
glutathione S-transferase
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Acknowledgements
We thank Drs. JC Reed (Burnham Institute) and Y Tsujimoto for providing cDNAs, and Ms Miyuki Itoh for helping with construction of plasmids and generation of mutant BAD. Supported by Grants-in-Aid for Cancer Research and for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (M Aadachi, K Imai).
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Adachi, M., Imai, K. The proapoptotic BH3-only protein BAD transduces cell death signals independently of its interaction with Bcl-2. Cell Death Differ 9, 1240–1247 (2002). https://doi.org/10.1038/sj.cdd.4401097
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DOI: https://doi.org/10.1038/sj.cdd.4401097
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