Simonian P L, Grillot D A, Merino R, Nuñez G
Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 1996 Sep 13;271(37):22764-72. doi: 10.1074/jbc.271.37.22764.
Bax, a member of the Bcl-2 family of proteins, has been shown to promote apoptosis while other members of the family, including Bcl-XL and Bcl-2, inhibit cell death induced by a variety of stimuli. The mechanism by which Bax promotes cell death is poorly understood. In the present report, we assessed the ability of Bax to antagonize the death repressor activity of Bcl-XL during chemotherapy-induced apoptosis in the lymphoid cell line, FL5.12. Expression of wild-type Bax countered the repressor activity of Bcl-XL against cell death mediated by VP-16 and cisplatin. We performed site-directed mutagenesis of the BH1, BH2, and BH3 homology regions in Bax to determine the ability of wild-type and mutant Bax to heterodimerize with Bcl-XL and to antagonize the protective effect of Bcl-XL against chemotherapy-induced apoptosis. Bax proteins expressing alanine substitutions of the highly conserved amino acids glycine 108 in BH1, tryptophan 151 and 158 in BH2, and glycine 67 and aspartic acid 68 in BH3 retained their ability to promote chemotherapy-induced cell death that was inhibited by Bcl-XL and to form heterodimers with Bcl-XL. Bax proteins containing deletions of the most highly conserved amino acids in BH1 (Delta102-112) and BH2 (Delta151-159) maintained the ability of Bax to antagonize the death repressor activity of Bcl-XL and to associate with Bcl-XL. However, Bax with BH3 deleted did not form heterodimers with Bcl-XL, but retained its ability to counter the death repressor activity of Bcl-XL. These results demonstrate that the conserved BH3, but not BH1 or BH2, homology region of Bax is necessary for its interaction with Bcl-XL in mammalian cells. Furthermore, our results indicate that Bax does not require BH1, BH2, BH3, or heterodimerization with Bcl-XL to counter the death repressor activity of Bcl-XL. Therefore, Bax can antagonize Bcl-XL during VP-16 and, in a lesser degree, during cisplatin-induced cell death independent of its heterodimerization with Bcl-XL.
Bax是Bcl-2蛋白家族的成员之一，已被证明可促进细胞凋亡，而该家族的其他成员，包括Bcl-XL和Bcl-2，则可抑制多种刺激诱导的细胞死亡。目前对Bax促进细胞死亡的机制了解甚少。在本报告中，我们评估了在淋巴样细胞系FL5.12化疗诱导的细胞凋亡过程中，Bax拮抗Bcl-XL死亡抑制活性的能力。野生型Bax的表达抵消了Bcl-XL对依托泊苷（VP-16）和顺铂介导的细胞死亡的抑制活性。我们对Bax的BH1、BH2和BH3同源区域进行了定点诱变，以确定野生型和突变型Bax与Bcl-XL异源二聚化的能力，以及拮抗Bcl-XL对化疗诱导的细胞凋亡的保护作用。表达BH1中高度保守的氨基酸甘氨酸108、BH2中色氨酸151和158以及BH3中甘氨酸67和天冬氨酸68被丙氨酸替代的Bax蛋白，保留了其促进化疗诱导的细胞死亡的能力（该细胞死亡被Bcl-XL抑制），并能与Bcl-XL形成异源二聚体。包含BH1（Δ102 - 112）和BH2（Δ151 - 159）中最高度保守氨基酸缺失的Bax蛋白，维持了Bax拮抗Bcl-XL死亡抑制活性以及与Bcl-XL结合的能力。然而，缺失BH3的Bax不能与Bcl-XL形成异源二聚体，但保留了其对抗Bcl-XL死亡抑制活性的能力。这些结果表明，Bax保守的BH3同源区域（而非BH1或BH2）对于其在哺乳动物细胞中与Bcl-XL相互作用是必需的。此外，我们的结果表明，Bax不需要BH1、BH2、BH3或与Bcl-XL异源二聚化来对抗Bcl-XL的死亡抑制活性。因此，在依托泊苷诱导的细胞死亡过程中，Bax可以拮抗Bcl-XL，在顺铂诱导的细胞死亡过程中，Bax在较小程度上也可拮抗Bcl-XL，且这一过程与其与Bcl-XL的异源二聚化无关。
