Analysis of the Bax-Bak interaction during SAC-induced apoptosis

Abstract

Taxol binds to β-tubulin preventing mitotic spindle formation and microtubule attachment to kinetochores. The presences of non-attached kinetochores activates the spindle assembly checkpoint (SAC) resulting in chronic activation of CDK1, mitotic arrest and apoptosis. Taxol- induced apoptosis occurs via the mitochondrial apoptosis pathway. The activation of the key regulators of apoptosis, Bak and Bax, results in the mitochondrial outer membrane permeabilization (MOMP) followed by the release of apoptotic factors such as cytochrome c. Recent studies from our lab demonstrated that Taxol- induced apoptosis is Bak and Bax dependent. However, the mechanism by which Bax accumulates at the mitochondrial outer membrane and whether Bak plays a role in this mechanism during SAC activation remain unknown. Therefore, I examined how Bak mediates the recruitment of the pro-apoptotic Bax into the mitochondrial outer membrane. I confirmed that Bak is required for Bax recruitment to mitochondria and subsequent cell death. By isolating the mitochondria from Taxol-arrested cells and Bak immunoprecipitation, I have demonstrated that Bak forms a complex with Bax and MPTP proteins VDAC2 and ANT2 which suggest the contribution of the pro apoptotic proteins in the pore formation during SAC- induced apoptosis. Also, a mitochondrial Bak IP mass spectrometry screen identified a 41 novel Bak interacting proteins. Finally, I examined the interaction between recombinant Bak and Bax to identify the key amino acid residues involved in the binding of the two proteins. My data confirmed the direct interaction between Bak and Bax at the canonical binding site (BH3: hydrophobic groove). My results also identified a novel amino acid responsible of Bak:Bax interaction as Bak Y89. A mutant Bak (Y89F) completely abolished the interaction between both proteins. In conclusion, this study confirmed the important role of pro-apoptotic Bak in recruiting Bax to the mitochondria of SAC activated cells. As Bak and Bax interaction is crucial in Taxol-induced apoptosis, the key amino acid residue that regulates Bak and Bax interaction has been identified.