MCL-1 inhibits BAX in the absence of MCL-1/BAX Interaction

The BCL-2 family of proteins plays a major role in the control of apoptosis as the primary regulator of mitochondrial permeability. The pro-apoptotic BCL-2 homologues BAX and BAK are activated following the induction of apoptosis and induce cytochrome c release from mitochondria. A second class of BCL-2 homologues, the BH3-only proteins, is required for the activation of BAX and BAK. The activity of both BAX/BAK and BH3-only proteins is opposed by anti-apoptotic BCL-2 homologues such as BCL-2 and MCL-1. Here we show that anti-apoptotic MCL-1 inhibits the function of BAX downstream of its initial activation and translocation to mitochondria. Although MCL-1 interacted with BAK and inhibited its activation, the activity of MCL-1 against BAX was independent of an interaction between the two proteins. However, the anti-apoptotic function of MCL-1 required the presence of BAX. These results suggest that the pro-survival activity of MCL-1 proceeds via inhibition of BAX function at mitochondria, downstream of its activation and translocation to this organelle.     

Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis

Mitochondria are functionally and physically associated with heterotypic membranes, yet little is known about how these interactions impact mitochondrial outer-membrane permeabilization (MOMP) and apoptosis. We observed that dissociation of heterotypic membranes from mitochondria inhibited BAK/BAX-dependent cytochrome c (cyto c) release. Biochemical purification of neutral sphingomyelinases that correlated with MOMP sensitization suggested that sphingolipid metabolism coordinates BAK/BAX activation. Using purified lipids and enzymes, sensitivity to MOMP was achieved by in vitro reconstitution of the sphingolipid metabolic pathway. Sphingolipid metabolism inhibitors blocked MOMP from heavy membrane preparations but failed to influence MOMP in the presence of sphingolipid-reconstituted, purified mitochondria. Furthermore, the sphingolipid products, sphingosine-1-PO(4) and hexadecenal, cooperated specifically with BAK and BAX, respectively. Sphingolipid metabolism was also required for cellular responses to apoptosis. Our studies suggest that BAK/BAX activation and apoptosis are coordinated through BH3-only proteins and a specific lipid milieu that is maintained by heterotypic membrane-mitochondrial interactions.     

Pro-apoptotic complexes of BAX and BAK on the outer mitochondrial membrane

In multicellular organisms the regulated cell death apoptosis is critically important for both ontogeny and homeostasis. Mitochondria are indispensable for stress-induced apoptosis. The BCL-2 protein family controls mitochondrial apoptosis and initiates cell death through the pro-apoptotic activities of BAX and BAK at the outer mitochondrial membrane (OMM). Cellular survival is ensured by the retrotranslocation of mitochondrial BAX and BAK into the cytosol by anti-apoptotic BCL-2 proteins. BAX/BAK-dependent OMM permeabilization releases the mitochondrial cytochrome c (cyt c), which initiates activation of caspase-9. The caspase cascade leads to cell shrinkage, plasma membrane blebbing, chromatin condensation, and apoptotic body formation. Although it is clear that ultimately complexes of active BAX and BAK commit the cell to apoptosis, the nature of these complexes is still enigmatic. Excessive research has described a range of complexes, varying from a few molecules to several 10,000, in different systems. BAX/BAK complexes potentially form ring-like structures that could expose the inner mitochondrial membrane. It has been suggested that these pores allow the efflux of small proteins and even mitochondrial DNA. Here we summarize the current state of knowledge for mitochondrial BAX/BAK complexes and the interactions between these proteins and the membrane.     

Proapoptotic BAX and BAK control multiple initiator caspases

BAX and BAK operate at both the mitochondria and endoplasmic reticulum (ER) to regulate the intrinsic apoptotic pathway. An unresolved issue is whether any caspases can be activated in response to intrinsic apoptotic signals in the absence of BAX and BAK. Following organelle-specific intrinsic stress signals, including DNA damage and ER stress, we detected no activation of CARD-containing caspases (initiator CASP)-1, -2, -9, -11 and -12 in Bax(-/-)Bak(-/-) doubly deficient (DKO) cells. BCL-2 overexpression in these DKO cells provided no further protection to their already strong protection from DNA damage and ER stress. Moreover, there was no activation of effector CASP-3 and -7 in DKO cells, consistent with the lack of initiator caspase activity and disfavouring a BAX, BAK-independent intrinsic apoptotic pathway to activate initiator caspases. Thus, BAX and BAK confer an essential gateway for the activation of caspases in the intrinsic apoptotic pathway.     

Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c

We review data supporting a model in which activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux. The BH3 domain of tBID is not required for targeting but remains on the mitochondrial surface where it is required to trigger BAK to release cytochrome c. tBID functions not as a pore-forming protein but as a membrane targeted and concentrated death ligand. tBID induces oligomerization of BAK, and both Bid and Bak knockout mice indicate the importance of this event in the release of cytochrome c. In parallel, the full pro-apoptotic member BAX, which is highly homologous to BAK, rapidly forms pores in liposomes that release intravesicular FITC-cytochrome c approximately 20A. A definable pore progressed from approximately 11A consisting of two BAX molecules to a approximately 22A pore comprised of four BAX molecules, which transported cytochrome c. Thus, an activation cascade of pro-apoptotic proteins from BID to BAK or BAX integrates the pathway from surface death receptors to the irreversible efflux of cytochrome c. Cell Death and Differentiation (2000) 7, 1166 - 1173     

A BAX/BAK and cyclophilin D-independent intrinsic apoptosis pathway

Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER) stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO) cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-X(L) overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD) expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.     

Two pathways for tBID-induced cytochrome c release from rat brain mitochondria: BAK- versus BAX-dependence

The mechanisms of truncated BID (tBID)-induced Cyt c release from non-synaptosomal brain mitochondria were examined. Addition of tBID to mitochondria induced partial Cyt c release which was inhibited by anti-BAK antibodies, implicating BAK. Immunoblotting showed the presence of BAK, but not BAX, in brain mitochondria. tBID did not release Cyt c from rat liver mitochondria, which lacked both BAX and BAK. This indicated that tBID did not act independently of BAX and BAK. tBID plus monomeric BAX produced twice as much Cyt c release as did tBID or oligomeric BAX alone. Neither tBID alone nor in combination with BAX induced mitochondrial swelling. In both cases Cyt c release was insensitive to cyclosporin A plus ADP, inhibitors of the mitochondrial permeability transition (mPT). Recombinant Bcl-xL inhibited Cyt c release induced by tBID alone or in combination with monomeric BAX. Koenig's polyanion, an inhibitor of VDAC, suppressed tBID-induced Cyt c release from brain mitochondria mediated by BAK but not by BAX. Thus, tBID can induce mPT-independent Cyt c release from brain mitochondria by interacting with exogenous BAX and/or with endogenous BAK that may involve VDAC. In contrast, neither adenylate kinase nor Smac/DIABLO was released from isolated rat brain mitochondria via BAK or BAX.     

Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis

During apoptosis, pro‐apoptotic BAX and BAK are activated, causing mitochondrial outer membrane permeabilisation (MOMP), caspase activation and cell death. However, even in the absence of caspase activity, cells usually die following MOMP. Such caspase‐independent cell death is accompanied by inflammation that requires mitochondrial DNA (mtDNA) activation of cGAS‐STING signalling. Because the mitochondrial inner membrane is thought to remain intact during apoptosis, we sought to address how matrix mtDNA could activate the cytosolic cGAS‐STING signalling pathway. Using super‐resolution imaging, we show that mtDNA is efficiently released from mitochondria following MOMP. In a temporal manner, we find that following MOMP, BAX/BAK‐mediated mitochondrial outer membrane pores gradually widen. This allows extrusion of the mitochondrial inner membrane into the cytosol whereupon it permeablises allowing mtDNA release. Our data demonstrate that mitochondrial inner membrane permeabilisation (MIMP) can occur during cell death following BAX/BAK‐dependent MOMP. Importantly, by enabling the cytosolic release of mtDNA, inner membrane permeabilisation underpins the immunogenic effects of caspase‐independent cell death.     

tBID Homooligomerizes in the mitochondrial membrane to induce apoptosis.

Activation of the tumor necrosis factor R1/Fas receptor results in the cleavage of cytosolic BID to truncated tBID. tBID translocates to the mitochondria to induce the oligomerization of BAX or BAK, resulting in the release of cytochrome c (Cyt c). Here we demonstrate that in tumor necrosis factor alpha-activated FL5.12 cells, tBID becomes part of a 45-kDa cross-linkable mitochondrial complex that does not include BAX or BAK. Using fluorescence resonance energy transfer analysis and co-immunoprecipitation, we demonstrate that tBID-tBID interactions occur in the mitochondria of living cells. Cross-linking experiments using a tBID-GST chimera indicated that tBID forms homotrimers in the mitochondrial membrane. To test the functional consequence of tBID oligomerization, we expressed a chimeric FKBP-tBID molecule. Enforced dimerization of FKBP-tBID by the bivalent ligand FK1012 resulted in Cyt c release, caspase activation, and apoptosis. Surprisingly, enforced dimerization of tBID did not result in the dimerization of either BAX or BAK. Moreover, a tBID BH3 mutant (G94E), which does not interact with or induce the dimerization of either BAX or BAK, formed the 45-kDa complex and induced both Cyt c release and apoptosis. Thus, tBID oligomerization may represent an alternative mechanism for inducing mitochondrial dysfunction and apoptosis.     

BCL-2-Related Protein Expression in Apoptosis: Oxidative Stress Versus Serum Deprivation in PC12 Cells

Abstract: Expression of the BCL-2 protein family members, BAX, BAK, BAD, BCL-xL, BCL-xS, and BCL-2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H₂O₂ treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H₂O₂-induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose-dependent increase in BAX, (b) a dose-independent increase in BAK, and (c) a dose-dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time-dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis-sparing medium (i.e., NGF-supplemented serum-free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL-xL expression was increased by H₂O₂ but unaffected by serum deprivation or long-term NGF treatment. Neither BCL-2 nor BCL-xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL-xL is altered in a stimulus-dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL-2-related proteins induced by H₂O₂ exposure and NGF rescue could reflect activation in part of a common antioxidant pathway.     

BCL2 and related prosurvival proteins require BAK1 and BAX to affect autophagy

It is widely thought that prosurvival BCL2 family members not only inhibit apoptosis, but also block autophagy by directly binding to BECN1/Beclin 1. To distinguish whether BCL2, BCL2L1/BCL-XL, or MCL1 influence autophagy directly, or indirectly, through their effects on apoptosis, we compared normal cells to those lacking BAX and BAK1. In cells able to undergo mitochondria-mediated apoptosis, inhibiting the endogenous prosurvival BCL2 family members induces both autophagy and cell death, but when BAX and BAK1 are deleted, neither inhibiting nor overexpressing BCL2, BCL2L1, or MCL1 causes any detectable effect on LC3B lipidation, LC3B turnover, or autolysosome formation. These results show that prosurvival BCL2 family members influence autophagy only indirectly, by inhibiting activation of BAX and BAK1.     

G0 function of BCL2 and BCL-xL requires BAX, BAK, and p27 phosphorylation by Mirk, revealing a novel role of BAX and BAK in quiescence regulation

BCL2 and BCL-x(L) facilitate G(0) quiescence by decreasing RNA content and cell size and up-regulating p27 protein, but the precise mechanism is not understood. We investigated the relationship between cell cycle regulation and the anti-apoptosis function of BCL2 and BCL-x(L). Neither caspase inhibition nor abrogation of mitochondria-dependent apoptosis by BAX and BAK deletion fully recapitulated the G(0) effects of BCL2 or BCL-x(L), suggesting that mechanisms in addition to anti-apoptosis are involved in the cell cycle arrest function of BCL2 or BCL-x(L). We found that BCL2 and BCL-x(L) expression in bax(-/-) bak(-/-) cells did not confer cell cycle effects, consistent with the G(0) function of BCL2 and BCL-x(L) being mediated through BAX or BAK. Stabilization of p27 in G(0) in BCL2 or BCL-x(L) cells was due to phosphorylation of p27 at Ser(10) by the kinase Mirk. In bax(-/-) bak(-/-) cells, total p27 and p27 phosphorylated at Ser(10) were elevated. Re-expression of BAX in bax(-/-) bak(-/-) cells and silencing of BAX and BAK in wild type cells confirmed that endogenous BAX and BAK modulated p27. These data revealed a novel role for BAX and BAK in the regulation of G(0) quiescence.     

BCL2 and related prosurvival proteins require BAK1 and BAX to affect autophagy

It is widely thought that prosurvival BCL2 family members not only inhibit apoptosis, but also block autophagy by directly binding to BECN1/Beclin 1. To distinguish whether BCL2, BCL2L1/BCL-XL, or MCL1 influence autophagy directly, or indirectly, through their effects on apoptosis, we compared normal cells to those lacking BAX and BAK1. In cells able to undergo mitochondria-mediated apoptosis, inhibiting the endogenous prosurvival BCL2 family members induces both autophagy and cell death, but when BAX and BAK1 are deleted, neither inhibiting nor overexpressing BCL2, BCL2L1, or MCL1 causes any detectable effect on LC3B lipidation, LC3B turnover, or autolysosome formation. These results show that prosurvival BCL2 family members influence autophagy only indirectly, by inhibiting activation of BAX and BAK1.     

The Bcl-2-associated death promoter (BAD) lowers the threshold at which the Bcl-2-interacting domain death agonist (BID) triggers mitochondria disintegration

The Bcl-2-associated death promoter (BAD) protein, like many other BH3-only proteins, is known to promote apoptosis through the intrinsic mitochondrial pathway. Unlike the BH3-interacting domain death agonist (BID) protein, BAD cannot directly trigger apoptosis but, instead, lowers the threshold at which apoptosis is induced. In many mathematical models of apoptosis, BAD is neglected or abstracted. The work presented here considers the incorporation of BAD and its various modifications in a model of the tBID-induction of BAK (Bcl-2 homologous antagonist killer) or the tBID-induction of BAX (Bcl-2-associated X protein). Steady state equations are used to develop an explicit formula describing the total concentration level of tBID, guaranteed to trigger apoptosis, as a bilinear function of the total BAD concentration level and the total anti-apoptotic protein concentration level (usually Bcl-2 or Bcl-x_L). In particular, the formula explains how the pro-apoptotic protein BAD lowers the threshold at which tBID induces BAK/BAX activation—reducing the level of total Bcl-2/Bcl-x_L available to inhibit tBID signaling in the mitochondria. Attention is then turned to the experimental data surrounding BAD phosphorylation, a process known to inhibit the pro-apoptotic effects of BAD. To address this data, the phosphorylation process is modeled following two separate kinetics in which either free unbound BAD is the assumed substrate or Bcl-x_L/Bcl-2-bound BAD is the assumed substrate. Bifurcation analysis and further analysis of the bilinear equation validate experiments, which suggest that BAD phosphorylation prevents irreversible BAK/BAX-mediated apoptosis, even when phosphorylation-induced dissociation of Bcl-x_L/Bcl-2-bound BAD is blocked. It is also shown that a cooperative, even synergistic, removal of mitochondrial BAD is seen when both types of phosphorylation are assumed possible. The presented work, however, reveals that the balance between BAD phosphorylation and dephosphorylation modulates the degree to which BAD influences the signaling from tBID to BAK/BAX. Our model shows that both the mode(s) of phosphorylation and the BAD dephosphorylation rate become important factors in determining whether BAD influences the activation of the BAK/BAX signal or not. Such potential variations in the pro-apoptotic effects of BAD are used to explain some of the inconsistent experimental data surrounding BAD phosphorylation. Nonetheless, our model serves to evaluate BAD and its sensitizing effects on the tBID-induction of BAK/BAX and thus aid in predicting when the incorporation of BAD in an apoptosis signaling model is important and when it is not.     

Consequences of the combined loss of BOK and BAK or BOK and BAX

The multi-BCL-2 homology domain pro-apoptotic BCL-2 family members BAK and BAX have critical roles in apoptosis. They are essential for mitochondrial outer-membrane permeabilization, leading to the release of apoptogenic factors such as cytochrome-c, which promote activation of the caspase cascade and cellular demolition. The BOK protein has extensive amino-acid sequence similarity to BAK and BAX and is expressed in diverse cell types, particularly those of the female reproductive tissues. The BOK-deficient mice have no readily discernible abnormalities, and its function therefore remains unresolved. We hypothesized that BOK may exert functions that overlap with those of BAK and/or BAX and examined this by generating Bok^−/−Bak^−/− and Bok^−/−Bax^−/− mice. Combined loss of BOK and BAK did not elicit any noticeable defects, although it remains possible that BOK and BAK have critical roles in developmental cell death that overlap with those of BAX. In most tissues examined, loss of BOK did not exacerbate the abnormalities caused by loss of BAX, such as defects in spermatogenesis or the increase in neuronal populations in the brain and retina. Notably, however, old Bok^−/−Bax^−/− females had abnormally increased numbers of oocytes from different stages of development, indicating that BOK may have a pro-apoptotic function overlapping with that of BAX in age-related follicular atresia.     

Platinum resistant cancer cells conserve sensitivity to BH3 domains and obatoclax induced mitochondrial apoptosis

Resistance to cisplatin chemotherapy remains a major hurdle preventing effective treatment of many solid cancers. BAX and BAK are pivotal regulators of the mitochondrial apoptosis pathway, however little is known regarding their regulation in cisplatin resistant cells. Cisplatin induces DNA damage in both sensitive and resistant cells, however the latter exhibits a failure to initiate N-terminal exposure of mitochondrial BAK or mitochondrial SMAC release. Both phenotypes are highly sensitive to mitochondrial permeabilisation induced by exogenous BH3 domain peptides derived from BID, BIM, NOXA (which targets MCL-1 and A1), and there is no significant change in their prosurvival BCL2 protein expression profiles. Obatoclax, a small molecule inhibitor of pro-survival BCL-2 family proteins including MCL-1, decreases cell viability irrespective of platinum resistance status across a panel of cell lines selected for oxaliplatin resistance. In summary, selection for platinum resistance is associated with a block of mitochondrial death signalling upstream of BAX/BAK activation. Conservation of sensitivity to BH3 domain induced apoptosis can be exploited by agents such as obatoclax, which directly target the mitochondria and BCL-2 family.     

Obatoclax induces Atg7-dependent autophagy independent of beclin-1 and BAX/BAK

Direct pharmacological targeting of the anti-apoptotic B-cell lymphoma-2 (BCL-2) family is an attractive therapeutic strategy for treating cancer. Obatoclax is a pan-BCL-2 family inhibitor currently in clinical development. Here we show that, although obatoclax can induce mitochondrial apoptosis dependent on BCL-2 associated x protein/BCL-2 antagonist killer (BAX/BAK) consistent with its on-target pharmacodynamics, simultaneous silencing of both BAX and BAK did not abolish acute toxicity or loss of clonogenicity. This is despite complete inhibition of apoptosis. Obatoclax dramatically reduced viability without inducing loss of plasma membrane integrity. This was associated with rapid processing of light chain-3 (LC3) and reduction of S6 kinase phosphorylation, consistent with autophagy. Dramatic ultrastructural vacuolation, not typical of autophagy, was also induced. Silencing of beclin-1 failed to prevent LC3 processing, whereas knockout of autophagy-related (Atg)7 abolished LC3 processing but failed to prevent obatoclax-induced loss of clonogenicity or ultrastructural changes. siRNA silencing of Atg7 in BAX/BAK knockout mouse embryonic fibroblasts did not prevent obatoclax-induced loss of viability. Cells selected for obatoclax resistance evaded apoptosis independent of changes in BCL-2 family expression and displayed reduced LC3 processing. In summary, obatoclax exhibits BAX- and BAK-dependent and -independent mechanisms of toxicity and activation of autophagy. Mechanisms other than autophagy and apoptosis are blocked in obatoclax resistant cells and contribute significantly to obatoclax''s anticancer efficacy.     

Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX

Interactions between the BCL-2 family proteins determine the cell's fate to live or die. How they interact with each other to regulate apoptosis remains as an unsettled central issue. So far, the antiapoptotic BCL-2 proteins are thought to interact with BAX weakly, but the physiological significance of this interaction has been vague. Herein, we show that recombinant BCL-2 and BCL-w interact potently with a BCL-2 homology (BH) 3 domain-containing peptide derived from BAX, exhibiting the dissociation constants of 15 and 23 nM, respectively. To clarify the basis for this strong interaction, we determined the three-dimensional structure of a complex of BCL-2 with a BAX peptide spanning its BH3 domain. It revealed that their interactions extended beyond the canonical BH3 domain and involved three nonconserved charged residues of BAX. A novel BAX variant, containing the alanine substitution of these three residues, had greatly impaired affinity for BCL-2 and BCL-w, but was otherwise indistinguishable from wild-type BAX. Critically, the apoptotic activity of the BAX variant could not be restrained by BCL-2 and BCL-w, pointing that the observed tight interactions are critical for regulating BAX activation. We also comprehensively quantified the binding affinities between the three BCL-2 subfamily proteins. Collectively, the data show that due to the high affinity of BAX for BCL-2, BCL-w and A1, and of BAK for BCL-X(L), MCL-1 and A1, only a subset of BH3-only proteins, commonly including BIM, BID and PUMA, could be expected to free BAX or BAK from the antiapoptotic BCL-2 proteins to elicit apoptosis.     

BAX and BAK1 are dispensable for ABT-737-induced dissociation of the BCL2-BECN1 complex and autophagy

Disruption of the complex of BECN1 with BCL2 or BCL2L1/BCL-X_L is an essential switch that turns on cellular autophagy in response to environmental stress or treatment with BH3 peptidomimetics. Recently, it has been proposed that BCL2 and BCL2L1/BCL-X_L may inhibit autophagy indirectly through a mechanism dependent on the proapoptotic BCL2 family members, BAX and BAK1. Here we report that the BH3 mimetic, ABT-737, induces autophagy in parallel with disruption of BCL2-BECN1 binding in 2 different apoptosis-deficient cell types lacking BAX and BAK1, namely in mouse embryonic fibroblasts cells and in human colon cancer HCT116 cells. We conclude that the BH3 mimetic ABT-737 induces autophagy through a BAX and BAK1-independent mechanism that likely involves disruption of BECN1 binding to antiapoptotic BCL2 family members.