Functional Dissection of the Pro-apoptotic Protein Bik

Abstract

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, Bax, Bak, 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 complex-ing 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 L and for inducing efficient cell death. Two-hybrid analysis in 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 het-erodimerization domain. Functional analysis indicates that a Bik deletion mutant expressing residues 43-120, which efficiently heterodimerizes with the anti-apopto-sis proteins Bcl-2 and Bcl-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 het-erodimerization activity encoded by the BH3 domain alone is insufficient and raise the possibility that Bik may induce cell death autonomous of heterodimeriza-tion with survival proteins such as Bcl-2 and Bcl-x L. Apoptosis is a normal physiological process required for selective elimination of cells in multicellular organisms. This process is either positively or negatively modulated by a number of external and internal factors. The bcl-2 proto-oncogene is a well known inhibitor of apoptosis (reviewed in Refs. 1 and 2). Recently, a number of vertebrate cellular and viral proteins structurally related to Bcl-2 have been identified. The various Bcl-2 homologs have been shown to possess either anti-apo-ptotic or pro-apoptotic activity. The pro-apoptotic homologs include Bax (3), Bak (4-6), Bik (7, 8), Bid (9), and Hrk (10), which function as dominant death inducers when overexpressed. The mechanism(s) by which anti-apoptotic and pro-apoptotic proteins elicit their respective activities are not known. A common feature of the various Bcl-2 family members is that they form homo-and heterodimeric complexes (3, 11, 12). Based on this observation, it has been proposed that anti-apoptotic proteins such as Bcl-2 promote cell survival by complexing with pro-apoptotic proteins such as Bax, thereby antagonizing the death-promoting activity of these proteins (3, 11). However, it has recently been observed that certain mutants of Bcl-x L , defective in complex formation with Bax and Bak, are still able to promote cell survival (13). This raises the possibility that at least certain anti-apoptotic members of the Bcl-2 family of proteins may promote cell survival independently of their interaction with known pro-apoptotic proteins such as Bax and Bak. The pro-apoptotic proteins Bax and Bak are closely related to Bcl-2 whereas Bik is more distantly related. Bax and Bak contain three conserved domains, BH1, BH2, and BH3, similar to Bcl-2, in addition to the characteristic C-terminal transmem-brane domain common to all Bcl-2 family proteins. In contrast, Bik shares only the BH3 and the C-terminal transmembrane domains with other Bcl-2 family proteins. The presence of the BH3 domain in Bik, which is otherwise unrelated to Bax and Bak, suggests a critical role for the BH3 domain in the common cell death-inducing activity of the three different proteins. The dual role of the BH3 domain in inducing cell death and mediating heterodimerization with survival proteins was elucidated by detailed mutagenesis of Bak, where a minigene encoding a 51-amino acid region, encompassing the BH3 domain, was found to be sufficient for inducing cell death and for het-erodimerization with Bcl-x L (14). Further, mutations within the BH3 domain of Bak, Bax, and Bik were found to abrogate the ability of these proteins to heterodimerize with the survival proteins such as Bcl-2 and Bcl-x L and to eliminate their apo-ptotic activity (7, 14). Like Bik, two recently identified pro-apoptotic proteins, Bid (9) and Hrk (10), that complex with Bcl-2 and Bcl-x L share only the BH3 domain with other Bcl-2 family members. Thus, the two activities (i.e. heterodimeriza-tion with survival proteins and induction of cell death) of Bax, Bak, Bik, Bid, and Hrk mediated by the BH3 domain appear to be linked. These results are consistent with the hypothesis that these pro-apoptotic proteins promote cell death by complexing with endogenous survival proteins such as Bcl-2 and Bcl-x L. The results presented in this report suggest that complex formation of Bik with Bcl-2 and Bcl-x L alone is insufficient for promoting cell death. Efficient Bik-mediated cell death requires additional sequence elements, in addition to the conserved BH3 domain.