Contribution of BH3-domain and Transmembrane-domain to the Activity and Interaction of the Pore-forming Bcl-2 Proteins Bok, Bak, and Bax

Abstract

Central to intrinsic apoptosis signaling is the release of cytochrome c from mitochondria, which depends on the pro-apoptotic effector proteins Bax, Bak or Bok. These pore-forming effector proteins share four Bcl-2 homology (BH) domains, a functionally essential and conserved sequence of hydrophobic amino acids in their BH3-domain and a C-terminal transmembrane-domain whose specific function remains rather unknown. To elucidate the molecular basis of Bok-mediated apoptosis we analyzed apoptosis induction by transmembrane-domain deficient BokΔTM compared to the respective Bax and Bak proteins and proteins in which the first leucine in the BH3-stretch was mutated to glutamic acid. We show that deletion of the C-terminal transmembrane-domain reduces the pro-apoptotic function of each protein. Mutation of the first leucine in the BH3-domain (L78E) blocks activity of Bak, while mutation of the homologue residues in Bax or Bok (L63E and L70E respectively) does not affect apoptosis induction. Unexpectedly, combined mutation of the BH3-domain and deletion of the transmembrane-domain enhances the pro-apoptotic activity of Bok(L70E)ΔTM by abolishing the interaction with anti-apoptotic proteins, especially the primary Bok-inhibitory protein Mcl-1. These results therefore suggest a specific contribution of the transmembrane-domain to the pro-apoptotic function and interaction of Bok.

The BCL-2 selective inhibitor ABT-199 sensitizes soft tissue sarcomas to proteasome inhibition by a concerted mechanism requiring BAX and NOXA

“…The shift towards apoptosis still prevails even when considering the BZB-induced increase of MCL-1 expression, because also expression of NOXA is enhanced. In this scenario apoptosis is augmented irrespective of whether BOK interacts with anti-apoptotic BCL-2 proteins 13,14 or not 22,51 . Although interaction of BOK with anti-apoptotic BCL-2 proteins is debated, stabilization of BOK and MCL-1 might even be caused upstream of the proteasome by interaction of BOK with MCL-1.…”

“…Like BH3 -only proteins, the pro-apoptotic multidomain effector proteins specifically interact with anti-apoptotic BCL-2 proteins. Although interaction of BOK with anti-apoptotic proteins is debated 13 , 14 , the binding preferences of BAK and BAX are largely established. BAK preferentially binds to MCL-1 and BCL-xL 15 , while BAX binds to BCL-2 , BCL-xL, and BCL-w 16 .…”

“…How active BOK is prevented from apoptosis induction is rather unclear. Although inhibition of BOK by anti-apoptotic BCL-2 proteins, such as MCL-1, has been repeatedly shown 13 , 14 , 21 , a recent publication proposed that BOK does neither interact with anti-apoptotic BCL-2 proteins nor requires activation by BH3-only proteins 22 . Rather, BOK activity is controlled at the level of protein stability and is prevented from constitutive apoptosis induction by the AMFR/gp78 E3 ubiquitin ligase complex that targets BOK to proteasomal degradation.…”

“…Interestingly, proteasome inhibitors have also been reported to induce expression of the pro-apoptotic BH3-only protein NOXA 24 . NOXA specifically binds and inactivates the anti-apoptotic proteins MCL-1 and A1 25 that in turn predominantly counteract the effectors BAK 26 – 28 and allegedly BOK 13 , 14 .…”

Understanding and drugging the Bcl-2 transmembrane interactome for tumor treatment

“…Specifically, functional roles for Bcl TMDs have been previously described by our group and others 58,91,117,120 , highlighting the notion that interactions…”