Characterization of the Activation Mechanism of Bax

Abstract

Mitochondrial outer membrane permeabilization (MOMP) is regulated by protein-protein and protein-membrane interactions between Bcl-2 family proteins. These interactions are governed by the concentrations and relative binding affinities of the proteins for each other. These affinities are altered by conformation changes of Bcl-2 family proteins resulting from interactions with each other and with membranes. How Bcl-2 proteins transition into and out of the conformations that controls their functions, and ultimately the fate of the cell, is not well understood. Here, kinetic analysis of the pore-forming Bcl-2 family member, Bax, revealed that Bax undergoes a conformational rearrangement through at least one structurally distinct intermediate that is a necessary precursor to pore formation. We discover that four cancer-associated Bax point mutants are trapped in the intermediate state, suggesting that transitions into and out of this intermediate can be modulated independently with consequences for the execution of apoptosis. Furthermore we report that the conformation changes Bax undergoes can be regulated by phosphorylation of Bax on residue S184 by the pro-survival kinase, Akt. Phosphorylation converts Bax into an anti-apoptotic protein that functions in a dominant-negative fashion. Bioinformatics revealed that in human cancers, higher levels of Bax are positively associated with high levels of PI3K/AKT pathway genes representing an added mechanism for cancer cells to evade apoptosis. Additionally we studied the interactions between Bax, the anti-apoptotic protein Bcl-XL, the sensitizer BH3 protein Bad and the BH3 activator protein Bid. We uncover a new mechanism of apoptosis regulation whereby Bad binds to one monomer of a Bcl-XL dimer eliciting an activating conformation change in a tBid bound to the other monomer of the Bcl-XL dimer. This allows Bad to function as a non-competitive inhibitor of Bcl-XL, and represents a novel mechanism that significantly enhances the potency of Bad to elicit apoptosis.