BH3 domain of BAD is required for heterodimerization with BCL-X-L and pro-apoptotic activity

BAD interacts with anti-apoptotic molecules BCL-2 and BCL-X-L and promotes apoptosis. BAD is phosphorylated on serine residues in response to a survival factor, interleukin-3. Phosphorylated BAD cannot bind to BCL-X-L or BCL-2 at membrane sites and is found in the cytosol bound to 14-3-3. We report here that deletion mapping and site-directed mutagenesis identified a BH3 domain within BAD that proved necessary for both its heterodimerization with BCL-X-L and its death agonist activity. Substitution of the conserved Leu(151) with Ala in the BH3 amphipathic alpha-helix abrogated both functions. The BAD Leu(151) mutant was predominantly in the cytosol bound to 14-3-3. The BH3 domain of BCL-2 also proved important for BCL-2/BAD interaction. These results establish a critical role for a BH3 domain within BAD and provide evidence that BAD may function as a death ligand whose pro-apoptotic activity requires heterodimerization with BCL-X-L.