Dimerization properties of human BAD -: Identification of a BH-3 domain and analysis of its binding to mutant BCL-2 and BCL-XL proteins

Bad, an inducer of programmed cell death, was recently isolated from a mouse cDNA library by its ability to bind to the anti-apoptotic protein BCL-2. Sequence analysis suggested that Bad was a member of the BCL-2 gene family that encodes both inducers and inhibitors of programmed cell death. To further analyze the role of BAD in the network of homo- and heterodimers formed by the BCL-2 family, we have cloned the human homologue of BAD and assessed its biological activity and its interactions with wild type and mutant BCL-2 family proteins. Our results indicate that the human BAD protein, like its mouse homologue, is able to induce apoptosis when transfected into mammalian cells. Furthermore, in yeast two-hybrid assays as well as quantitative in vitro interaction assays, human Bad interacted with BCL-2 and BCL-X-L. Sequence alignments of human BAD revealed the presence of a BH-3 homology domain as seen in other BCL-2 family proteins, Peptides derived from this domain were able to completely inhibit the dimerization of BAD with BCL-X-L. Thus, as previously shown for BAX, BAK, BCL-2, and BCL-X-L, the BH3 domain of BAD is required for its dimerization with other BCL-2 family proteins. BAD was further analyzed for its ability to bind to various mutants of BCL-2 and BCL-X-L that have lost the ability to bind BAX and BAK, some of which retain biological activity and some of which do not. Surprisingly, all of the mutated BCL-2 and BCL-X-L proteins analyzed strongly interacted with human BAD. Our data thus indicate that mutations in BCL-2 and BCL-X-L can differentially affect the heterodimeric binding of different death-promoting proteins and have implications concerning the relationship between heterodimerization and biological activity.