BCL-2 selectively interacts with the BID-induced open conformer of BAK, inhibiting BAK auto-oligomerization.
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.
Ruffolo
Salvatore C
SC
Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada.
Shore
Gordon C
GC
D000595:Amino Acid Sequence; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D050998:bcl-2 Homologous Antagonist-Killer Protein
D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins; D008565:Membrane Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050998:bcl-2 Homologous Antagonist-Killer Protein