Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies

Although the BCL-2 family constitutes a crucial checkpoint in apoptosis, the intricate interplay between these family members remains elusive. Here, we demonstrate that BIM and PUMA, similar to truncated BID (tBID), directly activate BAX–BAK to release cytochrome c. Conversely, anti-apoptotic BCL-2–BCL-XL–MCL-1 sequesters these 'activator' BH3-only molecules into stable complexes, thus preventing the activation of BAX–BAK. Extensive mutagenesis of BAX–BAK indicates that their activity is not kept in check by BCL-2–BCL-XL–MCL-1. Anti-apoptotic BCL-2 members are differentially inactivated by the remaining 'inactivator' BH3-only molecules including BAD, NOXA, BMF, BIK/BLK and HRK/DP5. BAD displaces tBID, BIM or PUMA from BCL-2–BCL-XL to activate BAX–BAK, whereas NOXA specifically antagonizes MCL-1. Coexpression of BAD and NOXA killed wild-type but not Bax, Bak doubly deficient cells or Puma deficient cells with Bim knockdown, indicating that activator BH3-only molecules function downstream of inactivator BH3-only molecules to activate BAX–BAK. Our data establish a hierarchical regulation of mitochondrion-dependent apoptosis by various BCL-2 subfamilies.