Simonian P L, Grillot D A, Andrews D W, Leber B, Nuñez G
Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 1996 Dec 13;271(50):32073-7. doi: 10.1074/jbc.271.50.32073.
Bax, a member of the Bcl-2 family of proteins, has been shown to accelerate apoptosis induced by growth factor withdrawal, gamma-irradiation, and the chemotherapeutic agent, etoposide. The mechanism by which Bax promotes apoptosis is poorly understood. Bax forms homodimers which have been suggested to act as accelerators or inducers of cell death. However, the requirement for homodimerization of Bax to promote cell death remains unclear. We performed site-directed mutagenesis of the BH1, BH2, and BH3 in Bax to determine the regions of Bax required for homodimerization and to define the role of Bax homodimers in cell death induced by chemotherapy drugs. Bax proteins expressing alanine substitutions of the highly conserved amino acids glycine 108 (G108) in BH1, tryptophan 158 (W158) in BH2, and glycine 67 and aspartic acid 68 (GD67-68) in BH3 as well as deletion of the most conserved amino acids in BH1 (Delta102-112) and BH2 (Delta151-159) and deletion of BH3 (Delta63-71) maintained their ability to accelerate chemotherapy-induced cell death. Immunoprecipitation studies revealed that Bax with deletions in BH1 and BH2 still associated with wild-type Bax while deletion of BH3 disrupted Bax homodimerization. These results demonstrate that Bax does not require the conserved regions of homology, BH1, BH2, or BH3, to accelerate chemotherapy-induced cell death. Furthermore, our results established BH3 as a region required for Bax homodimerization in mammalian cells and demonstrate that monomeric forms of Bax are active in accelerating cell death induced by chemotherapy agents.
Bax是Bcl-2蛋白家族的成员之一，已被证明可加速因生长因子撤除、γ射线照射及化疗药物依托泊苷所诱导的细胞凋亡。Bax促进细胞凋亡的机制尚不清楚。Bax可形成同型二聚体，有人认为其作为细胞死亡的促进剂或诱导剂发挥作用。然而，Bax同型二聚化在促进细胞死亡方面的必要性仍不明确。我们对Bax的BH1、BH2和BH3进行了定点诱变，以确定Bax同型二聚化所需的区域，并明确Bax同型二聚体在化疗药物诱导的细胞死亡中的作用。表达BH1中高度保守氨基酸甘氨酸108（G108）、BH2中色氨酸158（W158）、BH3中甘氨酸67和天冬氨酸68（GD67 - 68）的丙氨酸替代的Bax蛋白，以及BH1（Δ102 - 112）和BH2（Δ151 - 159）中最保守氨基酸的缺失和BH3（Δ63 - 71）的缺失，均保持了其加速化疗诱导细胞死亡的能力。免疫沉淀研究表明，BH1和BH2缺失的Bax仍与野生型Bax相关联，而BH3的缺失则破坏了Bax同型二聚化。这些结果表明，Bax加速化疗诱导的细胞死亡并不需要同源保守区域BH1、BH2或BH3。此外，我们的结果确定BH3是哺乳动物细胞中Bax同型二聚化所需的区域，并证明Bax的单体形式在加速化疗药物诱导的细胞死亡中具有活性。
