Underphosphorylated BAD interacts with diverse antiapoptotic Bcl-2 family proteins to regulate apoptosis

Survival factors activate kinases which, in turn, phosphorylate the proapop totic Bcl-xl/Bcl-2-associated death promoter homolog (BAD) protein at key s erine residues. Phosphorylated BAD interacts with 14-3-3 proteins, and over expression of 14-3-3 attenuates BAD-mediated apoptosis. Although BAD is kno wn to interact with Bcl-2, Bcl-w, and Bcl-xL, the exact relationship betwee n BAD and anti- or proapoptotic Bcl-2 proteins has not been analyzed system atically. Using the yeast two-hybrid protein interaction assay, we found th at BAD interacted negligibly with proapoptotic Bcl-2 proteins. Even though wild type BAD only interacted with selected numbers of antiapoptotic protei ns, underphosphorylated mutant BAD interacted with all antiapoptotic Bcl-2 proteins tested (Bcl-2, Bcl-w, Bcl-xL, Bfl-1/A1, Mcl-1, Ced-9, and BHRF-1). Using nonphosphorylated recombinant BAD expressed in bacteria, direct inte ractions between BAD and diverse antiapoptotic Bcl-2 members were also obse rved. Furthermore, apoptosis induced by BAD was blocked by coexpression wit h Bcl-2, Bcl-w, and Bfl-1. Comparison of BAD orthologs from zebrafish to hu man indicated the conservation of a 14-3-3 binding site and the BH3 domain during evolution. Thus, highly conserved BAD interacts with diverse antiapo ptotic Bcl-2 members to regulate apoptosis.