Mechanism of Bax/Bak Activation in Apoptotic Signaling

The Bcl-2 family proteins, including the anti-apoptotic members, pro-apoptotic effectors Bax/Bak, and the BH3-only proteins, are key components of the mitochondrial apoptotic pathway. The BH3-only protein Bid is critical for the mitochondrial pathway of apoptosis after TRAIL-induced death receptor activation. However, the mechanism of Bid activation during TRAIL-induced apoptosis is unclear. By putting back wild-type and mutants of Bid in Bid deficient (Bid KO) and Bid/Bax/Bak TKO colon cancer cells, we demonstrated that cleavage by caspase 8 and mitochondrial targeting are critical events for Bid activation. One of the biggest mysteries in apoptosis is how Bax/Bak was activated by BH3-only proteins during apoptosis. It has been widely accepted that mitochondria-dependent apoptosis initiates when select BH3-only proteins (direct activators) directly engage and activate Bax/Bak. By generating knock-out cell lines, we found that the reported direct activators, including Bid, Bim, Puma, and p53, are not required for Bax/Bak activation during apoptosis once the anti-apoptotic Bcl-2 proteins are neutralized. We then looked further into the mechanism of Bax/Bak activation during apoptosis. Through the reconstitution of cells lacking all eight pro-apoptotic BH3-only proteins, we demonstrate that all BH3-only proteins primarily target the anti-apoptotic Bcl-2 proteins Bcl-xL/Mcl-1, whose simultaneous suppression enables membrane-mediated spontaneous activation of Bax/Bak. BH3-only proteins’ apoptotic activities correlate with affinities for Bcl-xL/Mcl-1 instead of abilities to directly activate Bax/Bak. Further, Bid and Bim do not distinguish Bax from Bak or accelerate Bax/Bak activation following inactivation of Bcl-xL/Mcl-1. Remarkably, death ligand-induced apoptosis in cells lacking BH3-only proteins and Mcl-1 is fully restored by Bid mutants capable of neutralizing Bcl-xL, but not direct activation of Bax/Bak. Taken together, our findings provide a “Membrane-mediated Permissive” model, in which the BH3-only proteins only indirectly activate Bax/Bak by neutralizing the anti-apoptotic Bcl-2 proteins, and thus allowing Bax/Bak to undergo unimpeded, spontaneous activation in the mitochondrial outer membrane milieu, leading to apoptosis initiation