Bax/Bak activation in the absence of Bid, Bim, Puma, and p53.
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.
Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-7696, USA.|Xiangya Hospital, Central South University, Changsha 410008, China.
J
J
Zhang
Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-7696, USA.
K
K
Huang
Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-7696, USA.
K L
KL
O'Neill
Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-7696, USA.
X
X
Pang
Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-7696, USA.
X
X
Luo
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000172:deficiency / Q000235:genetics*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000172:deficiency / Q000235:genetics*; D000072224:Bcl-2-Like Protein 11 / Q000172:deficiency / Q000235:genetics*; D001713:Biphenyl Compounds / Q000494:pharmacology; D005786:Gene Expression Regulation; D055786:Gene Knockout Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009596:Nitrophenols / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000235:genetics*; D034741:RNA, Small Interfering / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency / Q000235:genetics*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics* / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics* / Q000378:metabolism; D051020:bcl-X Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
C501332:ABT-737; D051017:Apoptosis Regulatory Proteins; C494772:BAK1 protein, human; C494826:BAX protein, human; C438378:BBC3 protein, human; C494811:BCL2L1 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D000072224:Bcl-2-Like Protein 11; D001713:Biphenyl Compounds; C579095:MCL1 protein, human; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009596:Nitrophenols; D010879:Piperazines; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D013449:Sulfonamides; D016159:Tumor Suppressor Protein p53; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D049452:Green Fluorescent Proteins