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. 2016 Apr 15;30(8):973-88.
doi: 10.1101/gad.276725.115. Epub 2016 Apr 7.

Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane

Katelyn L O'Neill  1 , Kai Huang  1 , Jingjing Zhang  2 , Yi Chen  1 , Xu Luo  1
Affiliations

Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane

Katelyn L O'Neill et al. Genes Dev. .

Abstract

The mechanism of Bax/Bak activation remains a central question in mitochondria-dependent apoptotic signaling. While it is established that all proapoptotic Bcl-2 homology 3 (BH3)-only proteins bind and neutralize the anti-apoptotic Bcl-2 family proteins, how this neutralization leads to Bax/Bak activation has been actively debated. Here, genome editing was used to generate cells deficient for all eight proapoptotic BH3-only proteins (OctaKO) and those that lack the entire Bcl-2 family (Bcl-2 allKO). Although the OctaKO cells were resistant to most apoptotic stimuli tested, they underwent Bax/Bak-dependent and p53/Rb-independent apoptosis efficiently when both Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins, were inactivated or eliminated. Strikingly, when expressed in the Bcl-2 allKO cells, both Bax and Bak spontaneously associated with the outer mitochondrial membrane (OMM) through their respective helix 9, and this association triggered their homo-oligomerization/activation. Together, these results strongly suggest that the OMM, not BH3-only proteins or p53/Rb, is the long-sought-after direct activator of Bax/Bak following BH3-only-mediated neutralization of anti-apoptotic Bcl-2 proteins.

Keywords: BH3-only; Bax/Bak activation; anti-apoptotic Bcl-2 proteins; outer mitochondrial membrane.

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Figures

Figure 1.
Figure 1.
Generation of OctaKO cells. (A) Diagram of strategy for the generation of OctaKO cells. (B) Genomic sequences for Hrk at the targeted region (underlined). The dotted line indicates the deletion, and an asterisk indicates the 1-base-pair insertion. (C) Genomic sequences for Bmf at the targeted region (underlined). An asterisk indicates the mutation. (D) Western blot analysis of the three OctaKO clones. (E) The wild-type (WT) and mutant clones of HCT116 were treated with 25 ng/mL TRAIL for 5 h or 3 µM thapsigargin (TG) for 24 h or were serum-starved for 48 h. Following each treatment, cell lysates were Western-blotted against an anti-PARP antibody. A representative of three independent experiments is shown. Following each treatment, cell lysates were harvested for Western blot with an anti-PARP antibody. (F) Following each treatment as described in E, cells were stained by Annexin V and analyzed by flow cytometry. The results are the mean ± SEM of at least three independent experiments.
Figure 2.
Figure 2.
Suppression of Bcl-xL and Mcl-1 efficiently induces apoptosis in OctaKO cells. (A) Cell lysates were harvested following siRNA transfection as in B and Western-blotted with anti-PARP antibody. (B) Cells were treated with either 500 J/M2 UV, 2.5 µM ABT-737, or both. Sixteen hours later, cell lysates were harvested and Western-blotted with anti-PARP antibody. (C) Cells treated by the combination of UV and ABT-737 for 16 h were stained with Annexin V and analyzed by flow cytometry. Each data point is an average of at least three independent experiments. (D) Strategy for the generation of OctaKO/Mcl-1 knockout (KO) clones. (E) Strategy for the generation of Mcl-1 knockout HCT116 cells. (F) Western blot of the cell lysates of the indicated cell lines with the indicated antibodies. An asterisk indicates a nonspecific protein. (G) Cells were treated with 25 ng/mL TRAIL for 5 h, 3 µM thapsigargin (TG) for 24 h, or 2.5 µM ABT-737 for 2 h and harvested for Western blot with anti-PARP antibody. (H) ABT-737 (2.5 µM) was added to the indicated cell lines, which were harvested at the indicated time points for Western blot with anti-PARP antibody.
Figure 3.
Figure 3.
BH3-only and p53/Rb-independent apoptosis following inactivation of Bcl-xL and Mcl-1. (A) Diagram for the generation of Octa/p53/Rb1 knockout (KO) cells. (B) Western blot of the indicated cell lines. An asterisk indicates a nonspecific protein. (C) Cells were harvested following siRNA transfection for Western blot with anti-PARP antibody. (D) Cells were treated with 25 ng/mL TRAIL for 5 h, 3 µM thapsigargin (TG) for 24 h, 2.5 µM ABT-737 for 16 h, 500 J/M2 UV, or a combination of 500 J/M2 UV and 2.5 µM ABT-737 for 16 h. Following the indicated treatments, cell lysates were generated for Western blot with anti-PARP antibody. (E) Cells were treated by TRAIL, thapsigargin, or the combination of UV and ABT-737 as described in D and were stained with Annexin V followed by flow cytometry analysis. The results are the mean ± SEM of at least three independent experiments.
Figure 4.
Figure 4.
Suppression of anti-apoptotic Bcl-2 proteins triggers mitochondrial translocation of Bax and apoptosis in the OctaKO cells. (A) Diagram for generating GFP-Bax put-back Octa/Mcl-1/Bax/Bak knockout (KO) cells. (B) Expression level of GFP-Bax in the indicated cells. Cell lysates were Western-blotted against an anti-Bax antibody. An asterisk indicates a nonspecific protein. (C,D) The sorted pools were treated with 2.5 µM ABT-737. Six hours later, cells were harvested for Western blot against PARP (C) or stained with MitoTracker (D) and photographed under a fluorescence microscope. (E) Transient transfection of plasmids expressing GFP-Bad or its BH3 domain mutant BadL114E in the indicated cell lines. Twenty hours after transfection, cells were harvested for Western blot analysis. (F) Transient expression of Flag-Bad and its BH3 mutant (F-BadL114E) in the indicated cells followed by Western blot analysis. (G) Octa/Mcl-1/Bax/Bak knockout/GFP-Bax cells were cotransfected with an RFP-expressing plasmid (pDsRed) and a plasmid expressing the GFP vector, GFP-Bad, or GFP-BadL114E. Cells were photographed under a fluorescence microscope. (H) Quantification of the cells that displayed GFP-Bax mitochondrial translocation among the transfected cells as described in the Materials and Methods. The values are the average of two independent experiments with error of the mean.
Figure 5.
Figure 5.
Generation of Bcl-2 allKO cells and the constitutive activities of Bax/Bak. (A) Diagram of strategy for the generation of Bcl-2 allKO cells. (B) Western blot analysis of the wild-type (WT), OctaKO clone (cln) B, and two Bcl-2 allKO clones for Bcl-2 family proteins. The asterisk indicates a nonspecific protein. (C) Genomic sequences for A1 at the targeted region (underlined). The dotted line indicates a deletion, and the asterisk indicates an insertion. (D) The wild-type and mutant clones of HCT116 were treated with 25 ng/mL TRAIL for 5 h or a combination of 500 J/M2 UV and 2.5 µM ABT-737 for 16 h. Following each treatment, cell lysates were Western-blotted against PARP. A representative of two independent experiments is shown. (E,F) Bcl-2 allKO clone B grown in 35-mm plates were transiently transfected with plasmids expressing GFP fusion proteins of wild-type Bax, Bak with or without z-VAD-fmk, and their respective BH3 mutants. Sixteen hours after transfection, whole-cell lysates were harvested for Western blotting against GFP or PARP. (G) DKO and Bcl-2 allKO cells carrying the tetracycline (Tet)-inducible expression system for GFP, G-Bax, or G-Bak were induced by the addition of 2 µg/mL doxycycline (Dox). Six hours after induction, cells were harvested for Western blot analysis. (H,I) Bcl-2 allKO cells carrying the Tet-inducible expression system for Bax (H) or Bak (I) were induced by 2 µg/mL Dox. At the indicated time points after induction, cells were harvested for Western blot analysis.
Figure 6.
Figure 6.
Mitochondrial association-dependent spontaneous homo-oligomerization of Bax/Bak in Bcl-2 allKO cells. (A) Spontaneous mitochondrial targeting of Bax and Bak in Bcl-2 allKO cells. Bcl-2 allKO and Bax/Bak DKO cells carrying the Tet-on system expressing GFP, G-Bax, or G-Bak were treated with 2 µg/mL Dox for 8 h before being fixed and stained for TOM20. z-VAD (50 µM) was added to the Bcl-2 allKO cells expressing G-Bax or G-Bak to prevent apoptosis and allow detection of the protein. (B) Bcl-2 allKO cells carrying the Tet-on system expressing G-Bax or G-Bak were induced with 2 µg/mL Dox in the presence of 50 µM z-VAD. The same cells were infected with a retrovirus constitutively expressing Bcl-xL and were induced with 2 µg/mL Dox. Sixteen hours after Dox induction, cells were solubilized in buffer A (20 mM HEPES-KOH, 10 mM KCL, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol [DTT], 0.1 PMSF, 5 mg/mL pepstatin A, 10 mg/mL leupeptin) with 2% CHAPS. Cell lysates were fractionated on a Superdex 200 column. Each of the indicated 1-mL fractions was Western-blotted against GFP. (C) Diagram of Bcl-xL, Bax, Bak, C-terminal truncation mutants of Bax/Bak, and the Bax/Bak-Bcl-xL chimeric proteins. (D) Bcl-2 allKO cells carrying the Tet-on system expressing the GFP fusion proteins of the Bax/Bak wild type and mutants listed in C were treated with 2 µg/mL Dox in the presence of 50 µM z-VAD for 8 h before being fixed and stained for TOM20. (E) The same cells as in D were treated with 2 µg/mL Dox for 16 h, lysed in buffer A with 2% CHAPS, and subjected to gel filtration analysis as described in B. (F) Bcl-2 allKO cells used in D and E were induced by Dox in the presence or absence of z-VAD for the indicated time before being harvested and subjected to Western blot against GFP. (G) DKO cells carrying the Tet-on-inducible expression system for the wild-type and mutant Bax/Bak were induced by Dox for the indicated times, lysed in buffer A with 2% CHAPS, and subjected to gel filtration analysis as described in B.
Figure 7.
Figure 7.
The membrane-induced spontaneous activation model for Bax/Bak. Following a BH3-only-mediated inactivation of anti-apoptotic Bcl-2 proteins, Bax spontaneously associated with the OMM through α9, and the OMM mediates homo-oligomerization of both Bax and Bak.
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