TY - JOUR
T1 - Bax/bak activation in the absence of Bid, Bim, Puma, and p53
AU - Zhang, J.
AU - Huang, K.
AU - O’neill, K. L.
AU - Pang, X.
AU - Luo, X.
N1 - Funding Information:
We are thankful for the mouse genome facility and DNA sequencing facility at UNMC, especially Dr Channabasavaiah Gurumurthy and Rolen Quadros, for their advice on the design of gene editing plasmids. We also thank Victoria Smith, Samantha Wall, and Dr Philip Hexley for their work in cell sorting and analysis at the UNMC Flow Cytometry Research Facility. This work was supported in part by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P30 GM106397 and by the Fred & Pamela Buffett Cancer Center Support Grant (P30CA036727).
Funding Information:
Acknowledgements. We are thankful for the mouse genome facility and DNA sequencing facility at UNMC, especially Dr Channabasavaiah Gurumurthy and Rolen Quadros, for their advice on the design of gene editing plasmids. We also thank Victoria Smith, Samantha Wall, and Dr Philip Hexley for their work in cell sorting and analysis at the UNMC Flow Cytometry Research Facility. This work was supported in part by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P30 GM106397 and by the Fred & Pamela Buffett Cancer Center Support Grant (P30CA036727).
Publisher Copyright:
© The Author(s) 2016.
PY - 2016
Y1 - 2016
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=85007425923&partnerID=8YFLogxK
U2 - 10.1038/cddis.2016.167
DO - 10.1038/cddis.2016.167
M3 - Article
C2 - 27310874
AN - SCOPUS:85007425923
VL - 7
JO - Cell Death and Disease
JF - Cell Death and Disease
SN - 2041-4889
IS - 6
M1 - e2266
ER -