Elangovan B, Chinnadurai G
Institute for Molecular Virology, St. Louis University Medical Center, St. Louis, Missouri 63110, USA.
J Biol Chem. 1997 Sep 26;272(39):24494-8. doi: 10.1074/jbc.272.39.24494.
Bik is a potent pro-apoptotic protein, which complexes with various anti-apoptotic proteins such as Bcl-2, Bcl-xL, 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-xL. 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-xL. 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-xL 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-xL. 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 Bcl-xL, 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-xL.
Bik是一种强效的促凋亡蛋白，它能与多种抗凋亡蛋白形成复合物，如Bcl-2、Bcl-xL、19 kDa腺病毒E1B和EBV-BHRF1。Bik促进细胞死亡的机制尚不清楚。它与其他促凋亡蛋白Bax、Bak、Bid和Hrk以及某些抗凋亡蛋白如Bcl-2和Bcl-xL共享一个保守结构域BH3。Bik的BH3结构域内的突变消除了其诱导细胞死亡以及与抗凋亡蛋白形成复合物的能力。这一结果与以下假设一致，即Bik可能通过与内源性细胞抗凋亡蛋白如Bcl-2和Bcl-xL形成复合物并拮抗其活性来促进细胞死亡。为了阐明蛋白质复合物形成与细胞死亡诱导之间的关系，我们从N端和C端缺失突变体文库中鉴定出了Bik与Bcl-2和Bcl-xL相互作用以及诱导有效细胞死亡所需的最小序列。酵母双杂交分析和哺乳动物细胞中表达蛋白的免疫沉淀分析表明，Bik的一个包含BH3结构域的52个氨基酸区域（第43至94位氨基酸）足以与Bcl-2和Bcl-xL高效异源二聚化。蛋白质相互作用研究进一步揭示，一个包含BH3结构域（第57至74位残基）的18个氨基酸区域构成了核心异源二聚化结构域。功能分析表明，一个表达第43至120位残基的Bik缺失突变体，它能与抗凋亡蛋白Bcl-2和Bcl-xL高效异源二聚化，但在引发细胞死亡方面存在缺陷。相反，一个表达额外C端序列（第43至134位氨基酸）的突变体与存活蛋白相互作用并引发有效细胞死亡。我们的结果表明，对于Bik介导的细胞死亡，仅由BH3结构域编码的异源二聚化活性是不够的，并提出了Bik可能独立于与存活蛋白如Bcl-2和Bcl-xL的异源二聚化而诱导细胞死亡的可能性。
