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  • BCL-2 selectively interacts with the BID-induced open conformer of BAK, inhibiting BAK auto-oligomerization.

BCL-2 selectively interacts with the BID-induced open conformer of BAK, inhibiting BAK auto-oligomerization.

The Journal of biological chemistry (2003-05-02)
Salvatore C Ruffolo, Gordon C Shore
ABSTRACT

Caspase-8 cleaves BID to tBID, which targets mitochondria and induces oligomerization of BAX and BAK within the outer membrane, resulting in release of cytochrome c from the organelle. Here, we have initiated these steps in isolated mitochondria derived from control and BCL-2-overexpressing cells using synthetic BH3 peptides and subsequently analyzed the BCL members by chemical cross-linking. The results show that the BH3 domain of BID interacts with and induces an "open" conformation of BAK, exposing the BAK N terminus. This open (activated) conformer of BAK potently induces oligomerization of non-activated ("closed") conformers, causing a cascade of BAK auto-oligomerization. Induction of the open conformation of BAK occurs even in the presence of excess BCL-2, but BCL-2 selectively interacts with this open conformer and blocks BAK oligomerization and cytochrome c release, dependent on the ratio of BID BH3 and BCL-2. This mechanism of inhibition by BCL-2 also occurs in intact cells stimulated with Fas or expressing tBID. Although BID BH3 interacts with both BCL-2 and BAK, the results indicate that when BCL-2 is in excess it can sequester the BID BH3-induced activated conformer of BAK, effectively blocking downstream events. This model suggests that the primary mechanism for BCL-2 blockade targets activated BAK rather than sequestering tBID.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
LC-SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate))
Sigma-Aldrich
Anti-Fas Antibody (human, activating), clone CH11, clone CH11, Upstate®, from mouse