title: Targeting Bax interaction sites reveals that only homo-oligomerization sites are essential for its activation.
abstract: Bax is a proapoptotic Bcl-2 family member that has a central role in the initiation of mitochondria-dependent apoptosis. However, the mechanism of Bax activation during apoptosis remains unsettled. It is believed that the activation of Bax is mediated by either dissociation from prosurvival Bcl-2 family members, or direct association with BH3-only members. Several interaction sites on Bax that mediate its interactions with other Bcl-2 family members, as well as its proapoptotic activity, have been identified in previous studies by other groups. To rigorously investigate the functional role of these interaction sites, we knocked in their respective mutants using HCT116 colon cancer cells, in which apoptosis induced by several stimuli is strictly Bax-dependent. Bax-mediated apoptosis was intact upon knock-in (KI) of K21E and D33A, which were shown to block the interaction of Bax with BH3-only activators. Apoptosis was partially reduced by KI of D68R, which impairs the interaction of Bax with prosurvival members, and S184V, a constitutively mitochondria-targeting mutant. In contrast, apoptosis was largely suppressed by KI of L70A/D71A, which blocks homo-oligomerization of Bax and its binding to prosurvival Bcl-2 family proteins. Collectively, our results suggest that the activation of endogenous Bax in HCT116 cells is dependent on its homo-oligomerization sites, but not those previously shown to interact with BH3-only activators or prosurvival proteins only. We therefore postulate that critical interaction sites yet to be identified, or mechanisms other than protein-protein interactions, need to be pursued to delineate the mechanism of Bax activation during apoptosis.
authors: College of Life Sciences, Sichuan University, Chengdu, Sichuan 610064, China.
authors: R
authors: R
authors: Peng
authors: J-S
authors: JS
authors: Tong
authors: H
authors: H
authors: Li
authors: B
authors: B
authors: Yue
authors: F
authors: F
authors: Zou
authors: J
authors: J
authors: Yu
authors: L
authors: L
authors: Zhang
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D000072224:Bcl-2-Like Protein 11; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism; D004789:Enzyme Activation; D055879:Gene Knock-In Techniques; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C438378:BBC3 protein, human; C000606775:BCL2L11 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; D000072224:Bcl-2-Like Protein 11; D008565:Membrane Proteins; D033681:Mitochondrial Membrane Transport Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C581062:RTL10 protein, human; D051028:bcl-2-Associated X Protein
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