Bik is a potent pro-apoptotic protein, which complexes with various anti-apoptotic proteins such as Bcl-2, Bcl-x(L), 19-kDa adenovirus E1B, and EBV-BHRF1, The mechanism by which Bik promotes cell death is not known, It shares a conserved domain, BH3, with other pro-apoptotic proteins, Bar, Bah, Bid, and Hrk, and certain anti-apoptosis proteins such as Bcl-2 and Bcl-x(L). Mutations within the BH3 domain of Bik abrogate its ability to induce cell death and to complex with anti-apoptosis proteins, This result is consistent with the hypothesis that Bik may promote cell death by complexing with and antagonizing the activity of endogenous cellular anti-apoptosis proteins such as Bcl-2 and Bcl-x(L). To elucidate the relationship between protein complex formation and induction of cell death, we have identified the minimal sequences of Bik, from a library of N-terminal and C-terminal deletion mutants, required for interaction with Bcl-2 and Bcl-x, and for inducing efficient cell death, Two-hybrid analysis ill yeast and immunoprecipitation analysis of proteins expressed in mammalian cells indicate that a 52-amino acid region (amino acids 43-94) of Bik, encompassing the BH3 domain, is sufficient for efficient heterodimerization with Bcl-2 and Bcl-x(L) Protein interaction studies further reveal that an 18-amino acid region, encompassing the BH3 domain (residues 57-74), constitutes the core heterodimerization domain. Functional analysis indicates that a Bik deletion mutant expressing residues 43-120, which efficiently heterodimerizes with the anti-apoptosis proteins Bcl-2 and Bel-x(L), is defective in eliciting cell death. In contrast, a mutant expressing additional C-terminal sequences (amino acids 43-134) interacts with the survival proteins and elicits efficient cell death. Our results suggest that for Bik-mediated cell death, the heterodimerization activity encoded by the BH3 domain alone is insufficient and raise the possibility-that Bik may induce cell death autonomous of heterodimerization with survival proteins such as Bcl-2 and Bcl-x(L).
