Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jun 16;7(6):e2266.
doi: 10.1038/cddis.2016.167.

Bax/Bak activation in the absence of Bid, Bim, Puma, and p53

J Zhang  1   2 , K Huang  1 , K L O'Neill  1 , X Pang  1 , X Luo  1
Affiliations

Bax/Bak activation in the absence of Bid, Bim, Puma, and p53

J Zhang et al. Cell Death Dis. .

Abstract

How BH3-only proteins activate Bax/Bak, the two gateway proteins of the mitochondria-dependent apoptotic pathway, remains incompletely understood. Although all pro-apoptotic BH3-only proteins are known to bind/neutralize the anti-apoptotic Bcl-2 proteins, the three most potent ones, Bid (tBid), Bim, and Puma, possess an additional activity of directly activating Bax/Bak in vitro. This latter activity has been proposed to be responsible for triggering Bax/Bak activation following apoptotic stimulation. To test this hypothesis, we generated Bid(-/)(-)Bim(-/)(-)Puma(-/)(-) (TKO), TKO/Bax(-/)(-)/Bak(-/)(-) (PentaKO), and PentaKO/Mcl-1(-/-) (HexaKO) HCT116 cells through gene editing. Surprisingly, although the TKO cells were resistant to several apoptotic stimuli, robust apoptosis was induced upon the simultaneous inactivation of Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins known to suppress Bax/Bak activation and activity. Importantly, such apoptotic activity was completely abolished in the PentaKO cells. In addition, ABT-737, a BH3 mimetic that inhibits Bcl-xL/Bcl-w/Bcl-2, induced Bax activation in HexaKO cells reconstituted with endogenous level of GFP-Bax. Further, by generating TKO/p53(-/-) (QKO) cells, we demonstrated that p53, a tumor suppressor postulated to directly activate Bax, is not required for Bid/Bim/Puma-independent Bax/Bak activation. Together, these results strongly suggest that the direct activation activities of Bid (tBid), Bim, Puma, and p53 are not essential for activating Bax/Bak once the anti-apoptotic Bcl-2 proteins are neutralized.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Generation of the TKO and PentaKO HCT116 cells. (a) Diagram of the strategy for the generation of Bid−/− Bim−/−Puma−/− HCT116 cells. (b) Western blot of the cell lysates from the three TKO clones. * indicates a non-specific protein. (c) Diagram of the strategy for the generation of TKO/Bax−/−Bak−/− (PentaKO) HCT116 cells. (d) Whole cell lysates of the indicated cell lines were Western blotted against the indicated antibodies
Figure 2
Figure 2
Loss of Bid, Bim, and Puma suppresses apoptosis induced by multiple stimuli. (a) The wild-type and the mutant clones of HCT116 were treated with TRAIL (25 ng/ml) for 5 h or with Thapsigargin (3 μM) for 24 h or 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. (b) The indicated cells were treated by the same apoptotic stimuli as in (a), and were stained with Annexin V followed by flow cytometry analysis. Each data point is mean +/− S.E.M. from at least three independent experiments
Figure 3
Figure 3
Bax/Bak-dependent apoptosis in the absence of Bid, Bim, and Puma in HCT116 cells following suppression of Bcl-xL and Mcl-1. (a) Cell lysates were harvested following transfection by the indicated siRNA oligos as described in Materials and Methods, and western blotted with anti-PARP antibody. (b) Cells were treated by either UV (500J/M2) or ABT-737 (2.5 μM) or both. Sixteen hours later, cell lysates were harvested and western blotted with anti-PARP antibody. (c) Cells were treated by the combination of UV and ABT-737 for 16 h, the same as in (b), and were stained with Annexin V followed by flow cytometry analysis. The results are the mean +/− S.E.M. of at least three independent experiments
Figure 4
Figure 4
ABT-737 induces Bax activation in the absence of Bid, Bim, Puma, Bak, and Mcl-1. (a) Diagram for generating HexaKO cells. (b) Western blot of the cell lysates from the wild-type and HexaKO cells. * indicates a non-specific protein. (c) Expression level of GFP-Bax in the indicated cells. Cell lysates were western blotted against an anti-Bax or anti-GFP antibody. (d) HexaKO cells reconstituted with GFP or GFP-Bax were treated with ABT-737 (2.5 μM) for 6 h. The cell lysates were western blotted with anti-PARP antibody. (e) HexaKO cells were treated with ABT-737 (2.5 μM) in the presence of z-VAD (50 μM) for six hours before they were harvested. These cells were lysed in buffer A with 2% CHAPS and loaded onto a Superdex 200 column for gel-filtration analysis. Fractions were western blotted against an anti-GFP antibody. (f) HexaKO cells were treated with ABT-737 in the presence of Z-VAD for 6 h and then stained with MitoTracker and photographed under a fluorescence microscope
Figure 5
Figure 5
Suppression of Bcl-xL and Mcl-1 causes apoptosis in the absence of Bid, Bim, Puma, and p53. (a) Diagram for the generation of Bid/Bim/Puma/p53 QKO cells. (b) Western blot of the indicated cell lines. * indicates a non-specific protein. (c) The indicated cells were treated with TRAIL (25 ng/ml) for 5 h or with Thapsigargin (3 μM) for 24 h or serum starved for 48 h or treated with combination of UV (500 J/M2) and ABT-737 (2.5 μM) for 16 h. Following the indicated treatments, cell lysates were generated for western blot with anti-PARP antibody. (d) Cells were harvested following siRNA transfection for western blot with anti-PARP antibody. A representative of three independent experiments is shown
Figure 6
Figure 6
The Linear Model for Bax/Bak activation during apoptosis. In this model, the primary targets of BH3-only proteins are Bcl-xL and Mcl-1, whose primary function is to suppress Bax/Bak activation. Following apoptotic stimulation, the activated BH3-only proteins neutralize/inactivate both Bcl-xL and Mcl-1, allowing Bax and Bak to become activated without the direct activation by BH3-only proteins and p53
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Fuchs Y, Steller H. Programmed cell death in animal development and disease. Cell 2011; 147: 742–758. - PMC - PubMed
    1. Tait SW, Green DR. Mitochondria and cell death: outer membrane permeabilization and beyond. Nat Rev Mol Cell Biol 2010; 11: 621–632. - PubMed
    1. Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 200; 9: 47–59. - PubMed
    1. Danial NN, Korsmeyer SJ. Cell death: critical control points. Cell 2004; 116: 205–219. - PubMed
    1. Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR. The BCL-2 family reunion. Mol Cell 2010; 37: 299–310. - PMC - PubMed

Publication types

MeSH terms

Substances