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. 2008 Nov 25;105(47):18081-7.
doi: 10.1073/pnas.0808691105. Epub 2008 Nov 3.

Apoptosis is triggered when prosurvival Bcl-2 proteins cannot restrain Bax

Jamie I Fletcher  1 , Sarina Meusburger, Christine J Hawkins, David T Riglar, Erinna F Lee, W Douglas Fairlie, David C S Huang, Jerry M Adams
Affiliations

Apoptosis is triggered when prosurvival Bcl-2 proteins cannot restrain Bax

Jamie I Fletcher et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A. 2009 Feb 3;106(5):1678

Abstract

A central issue in the control of apoptosis is whether its essential mediators Bax and Bak must be restrained by Bcl-2-like prosurvival relatives to prevent their damaging mitochondria and unleashing apoptosis. The issue is particularly vexed for Bax, which is largely a cytosolic monomer in unstressed cells. To determine whether Bax regulation requires its binding by prosurvival relatives, we replaced a conserved aspartate in its BH3 interaction domain with arginine. Bax D68R functioned and behaved like wild-type Bax in localization and activation but had greatly impaired binding to the prosurvival family members. Nevertheless, Bcl-x(L) remained able to block apoptosis induced by Bax D68R. Whereas cells with sufficient Bcl-x(L) tolerated expression of Bax D68R, it provoked apoptosis when Bcl-x(L) was absent, downregulated, or inactivated. Moreover, Bax D68R rendered membrane bound by a C-terminal anchor mutation overwhelmed endogenous Bcl-x(L) and killed cells. These unexpected results suggest that engagement of Bax by its prosurvival relatives is a major barrier to its full activation. We propose that the Bcl-2-like proteins must capture the small proportion of Bax molecules with an exposed BH3 domain, probably on the mitochondrial membrane, to prevent Bax-imposed cell death, but that Bcl-x(L) also controls Bax by other mechanisms.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Designing a deregulated Bax mutant. (A) Sequence alignments illustrate key conserved residues. The BH3 domains from selected proapoptotic Bcl-2 family members (Left) show the invariant aspartate (red D) and the conserved hydrophobic residues in bold, whereas the sequences from the hydrophobic receptor grooves of the mammalian prosurvival relatives (Right) show the invariant arginine (R) in blue. (B) The D68R mutation compromises Bax binding to prosurvival proteins. Lysates prepared from 293T cells overexpressing HA-tagged human Bax or the D68R mutant, and FLAG-tagged prosurvival Bcl-2 proteins were immunoprecipitated with mouse monoclonal antibodies recognizing the HA, FLAG (FL), or an irrelevant control (C) tag. The immunoprecipitates were subjected to SDS-PAGE, transferred onto membranes, and the blots probed with rat anti-HA or -FLAG antibodies.
Fig. 2.
Fig. 2.
Bax D68R is functional but deregulated. (A) Like Bax, the D68R mutant is predominantly cytosolic but becomes membrane integrated after an apoptotic stimulus. Untreated or etoposide-treated (10 μM for 24 h) bax−/−bak−/− MEF stably expressing FLAG-tagged Bax or Bax D68R were separated into cytosolic (c), membrane-associated (a), and the carbonate-resistant membrane-integrated (i) fractions (see Materials and Methods). HSP70, cytosolic marker; VDAC1, integral mitochondrial outer membrane protein; GAPDH, loading control. *VDAC1 bands carried over from a previous blot. (B) Bax D68R retains full apoptotic function. The viability of reconstituted bax−/−bak−/− MEF (described in A) after etoposide treatment (0–10 μM) for 24 h was assessed by propidium iodide exclusion using flow cytometry. (C) Selectivity of BH3-only ligands for their prosurvival targets (see text). (D) Neutralization of Bcl-xL, Bcl-w, or both activates Bax D68R but not WT Bax. Viability of reconstituted bax−/−bak−/− MEF was determined 24 h after infection with retroviruses expressing the indicated BH3-only proteins. Data in B and D represent means ± 1 SEM of 3 or more independent experiments.
Fig. 3.
Fig. 3.
Bax D68R is constitutively active in the absence of Bcl-xL. (A) Bax D68R kills MEF lacking Bcl-xL. The viability of MEF lacking the indicated prosurvival proteins was determined by propidium iodide exclusion 24 h after infection with retroviruses expressing either WT or D68R Bax. (B) Bax D68R prevents colony formation by Bcl-xL-deficient MEF. The bcl-x−/− MEF or a subclone stably expressing Bcl-xL were infected with retroviruses expressing Bax, Bax D68R, or an empty control vector and colony formation assessed 6 d later. (C) Only Bcl-xL counters apoptosis induced by Bax D68R in MEF lacking Bcl-xL. The viability of bcl-x−/− MEF stably expressing the indicated prosurvival proteins was determined 24 h after reinfection with a Bax or Bax D68R retrovirus. Data in A and C (except for Bcl-xL mt1) represent means ± 1 SEM of 3 or more independent experiments.
Fig. 4.
Fig. 4.
Bcl-xL antagonizes Bax D68R in yeast but binds only weakly to a Bax D68R BH3 peptide. (A) Bcl-xL counters growth suppression of yeast by Bax D68R. Yeast cotransformed with constructs encoding the indicated prosurvival proteins and Bax or Bax D68R (all full length), each under the control of an inducible (GAL) promoter were spotted onto repressing glucose (OFF) or inducing galactose (ON) plates as 5-fold serial dilutions. Images are representative of 2 independent experiments. (B) Relative affinity (IC50 in μM) of the C-terminally truncated prosurvival proteins (see Materials and Methods) for WT or D68R Bax BH3 peptides (34-mers), determined by solution competition assays. Data shown represent means ± 1 SD of 2 independent experiments. *Data from experiments using human Bcl-xL Δ45–84 ΔC24; comparable results were obtained with mouse Bcl-xL ΔC24.
Fig. 5.
Fig. 5.
Enhanced killing when Bax D68R is forced onto membranes. (A) MEF may possess sufficient endogenous Bcl-xL (blue) to counter the small fraction of membrane-bound Bax D68R (orange) (Fig. 2A). This capacity might be overwhelmed if the predominantly cytosolic Bax D68R is driven onto membranes. (B) Bax S184L is fully functional. The viability of reconstituted bax−/−bak−/− MEF (described in Fig. 2A) after etoposide treatment (0–10 μM) for 24 h was assessed by propidium iodide exclusion using flow cytometry. (C) Combining the deregulated D68R mutation with S184L enhances Bax-mediated apoptosis. Colony formation was assessed for parental bax−/−bak−/− MEF, or these MEF stably overexpressing Bcl-xL, after infection with retroviruses expressing WT Bax or mutant (D68R, S184L, or D68R/S184L) forms of Bax. Data represent means ± 1 SEM of 3 or more independent experiments. Results were compared using two-tailed unpaired Student's t tests. ns, P > 0.05.
Fig. 6.
Fig. 6.
Model for Bax regulation. (A) In healthy cells, Bax (orange) is predominantly cytosolic but is in equilibrium with minor populations on the mitochondrial membrane. Posttranslational modifications, physical stimuli, and certain BH3-only proteins (e.g., tBid) may enhance its translocation to membranes. However, any membrane-bound Bax conformers with the BH3 domain exposed (“primed Bax”) are sequestered by available prosurvival Bcl-2 proteins (blue), preventing full Bax activation and apoptosis. (B) Upon cellular stress, the capacity of the prosurvival proteins to bind Bax can be overwhelmed because of their inactivation by BH3-only proteins (red), or their depressed synthesis or degradation (prosurvival ghost). Left unchecked, the membrane-bound Bax can begin to self-associate and oligomerize. (C) Further translocation of Bax molecules, perhaps by direct recruitment from the cytosol, stimulates the Bax oligomerization that is thought to permeabilize the outer mitochondrial membrane, leading to activation of the caspases that dismantle the cell.
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