The proapoptotic BH3-only protein BAD transduces cell death signals independently of its interaction with Bcl-2

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.