Minimal BH3 Peptides Promote Cell Death by Antagonizing Anti-apoptotic Proteins*

Carole Moreau, Pierre-François Cartron {ddagger}, Abigail Hunt §, Khaled Meflah, Douglas R. Green ¶, Gerard Evan §, François M. Vallette and Philippe Juin ||

From the § University of California, San Francisco Cancer Center, San Francisco, California 94123-0128, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, INSERM U419, 9 Quai Moncousu, 44035 Nantes, France

Received for publication, September 16, 2002 , and in revised form, February 10, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pro-apoptotic "BH3 domain-only" proteins of the Bcl-2 family (e.g. Bid and Bad) transduce multiple death signals to the mitochondrion. They interact with the anti-apoptotic Bcl-2 family members and induce apoptosis by a mechanism that requires the presence of at least one of the multidomain pro-apoptotic proteins Bax or Bak. Although the BH3 domain of Bid can promote the pro-apoptotic assembly and function of Bax/Bak by itself, other BH3 domains do not function as such. The latter point raises the question of whether, and how, these BH3 domains induce apoptosis. We show here that a peptide comprising the minimal BH3 domain from Bax induces apoptosis but is unable to stimulate the apoptotic activity of microinjected recombinant Bax. This relies on the inability of the peptide to directly induce Bax translocation to mitochondria or a change in its conformation. This peptide nevertheless interferes with Bax/Bcl-xL interactions in vitro and stimulates the apoptotic activity of Bax when combined with Bcl-xL. Similarly, a peptide derived from the BH3 domain of Bad stimulates Bax activity only in the presence of Bcl-xL. Thus, BH3 domains do not necessarily activate multidomain pro-apoptotic proteins directly but promote apoptosis by releasing active multidomain pro-apoptotic proteins from their anti-apoptotic counterparts.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis is a highly regulated process of cell demise triggered by internal or external stimuli. Most apoptotic signaling pathways converge on the mitochondrion and lead to a change in the mitochondrial outer membrane permeability (1). As a result, diverse apoptogenic proteins, such as cytochrome c (which allows the activation of a caspase 9/caspase-3 cascade via the cytosolic adapter Apaf-1), Smac/DIABLO, "apoptosis-inducing factor," endonuclease G, and HtrA2, are released from this organelle. The response of the mitochondrion to upstream stimuli is a critical control point in the regulation of apoptosis. It is crucial, therefore, to understand how this organelle integrates a great variety of death signals.

Bcl-2 family members are major regulators of mitochondrial integrity (2). Anti-apoptotic members such as Bcl-2 and Bcl-xL display sequence conservation throughout all four Bcl-2 homology domains (BH1–4), whereas the pro-apoptotic Bax and Bak possess homology in BH1–3 domains. These multidomain pro-apoptotic proteins have the innate ability to alter mitochondrial integrity, possibly via their ability to induce channels in the mitochondrial membrane (3). The ratio between anti-apoptotic and multidomain pro-apoptotic Bcl-2 family members helps determine the cellular susceptibility to death stimulation (4). NMR structural analysis of the Bcl-xL-BakBH3 peptide complex has revealed that the BH3 domain of Bak binds to a hydrophobic cleft formed by the BH1, BH2, and BH3 domains of Bcl-xL (5). Anti-apoptotic and multidomain pro-apoptotic Bcl-2 family members may thus engage, by a BH3 domain-dependent mechanism, in the formation of heterodimers in which they mutually antagonize each other's function (6).

The Bcl-2 family includes a third subgroup of pro-apoptotic members that display sequence homology only with the BH3 domain (7). These BH3 only proteins seem to act as the afferent effectors of various pro-apoptotic and anti-apoptotic signals (8). For instance, both Noxa and Puma are transcriptionally regulated by p53 (9, 10, 11); activation of Bid by caspase 8-mediated cleavage recruits the mitochondrial apoptotic pathway into death receptor signaling (12, 13), whereas the activity of Bad is negatively regulated by the Akt/protein kinase B survival signaling pathway (14). Murine cells lacking both multidomain Bax and Bak display long term resistance to mitochondrial damage and cell death induced by all BH3-only proteins tested (15, 16, 17). Thus, in mammals, activation of BH3 only proteins integrate diverse apoptotic stimuli into one single pathway by triggering Bax/Bak-mediated mitochondrial dysfunction.

All BH3-only proteins have the ability to bind to and to functionally antagonize anti-apoptotic Bcl-2 family members (8). The BH3 domain-dependent mechanism by which Bax/Bak pro-apoptotic function is recruited remains poorly characterized. Indeed, multidomain proteins, in viable cells, reside as inactive proteins located either at the mitochondria (Bak) or in the cytosol (Bax) (18). The direct binding of Bid to either Bax or Bak is sufficient to activate those proteins and allow their pro-apoptotic assembly in the mitochondrial membrane (19, 20, 21). Very recent evidence (22, 23) using synthetic peptides has indicated that the BidBH3 domain itself functions as a specific death ligand. These studies nevertheless revealed evidence for another functional subset of BH3 domains, which lack the ability to activate directly Bax/Bak but retain the ability to bind to anti-apoptotic Bcl-2 family members (22). It has remained unclear whether such BH3 domains can induce apoptosis and, if so, by which mechanism.

To explore this question further, we have analyzed in this study the apoptotic effects of a minimal BH3 domain synthetic peptide, comprising the critical 16 residues of the defined Bax BH3 domain (BaxBH3 (57–72)). These 16 residues contain sufficient information to bind to (5), and functionally antagonize (24), Bcl-xL and to induce specifically Bax/Bak-mediated apoptosis (24). We show here that, despite these properties, BaxBH3 cannot efficiently induce Bax translocation to mitochondria, evoke a change in Bax conformation, nor stimulate the apoptotic activity of recombinant Bax, whereas it can stimulate that of a mutant form of Bax that exhibits an increased ability to homodimerize. BaxBH3 interferes with Bax/Bcl-xL interactions in cell-free experiments and stimulates the apoptotic activity of Bax combined with Bcl-xL. Similarly, a peptide derived from the BH3 domain of Bad was also unable to stimulate Bax activity by itself but cooperated with Bcl-xL to activate it. Taken together, these data support a model in which BH3 peptides that mainly act as antagonists of anti-apoptotic Bcl-2 family members can induce apoptosis by interfering with the interaction between multidomain pro- and anti-apoptotic proteins.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies—The following antibodies were used: monoclonal anti-cytochrome c antibody 6H2B4 from Pharmingen; polyclonal anti-Bax BaxNT antibody from Upstate Biotechnology, Inc.; and monoclonal anti-Bax 2D2 and 6A7 antibodies from R & D Systems. The polyclonal anti-Bax TL41 antibody was raised against the BH3 domain of Bax (residues 57–72) (24). Horseradish peroxidase-conjugated antibodies and enhanced chemiluminescence reagents were obtained from Amersham Biosciences. Fluorescent Alexa 568TM-conjugated secondary antibodies were obtained from Molecular Probes. Unless indicated, all other reagents used in this study were obtained from Sigma.

Peptides and Recombinant Proteins—The high pressure liquid chromatography-purified BaxBH3 (KKLSECLKRIGDELDS), BaxBH3 L63A (KKLSECAKRIGDELDS), and BadBH3 (QRYGRELRRMSDEFVD) peptides were obtained from Genosys (Cambridge, UK).

Histidine-tagged human Bax{alpha}, Bax{Delta}ART, and Bcl-xL were obtained by subcloning of the coding regions into pDEST17 plasmid (Gateway, Invitrogen) as described previously (25). His-tagged proteins were expressed in the Escherichia coli strain XL-1 blue and purified by chromatography on nickel-Sepharose acid resin according to the manufacturer's instructions. Recombinant proteins were dialyzed against PBS1 and stored at -80 °C until used. The purification of GST, GST-Bcl-xL, and tBid from bacterial lysates has been described previously (24, 26).

Microinjection Experiments—Rat-1 fibroblasts, grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS and 0.1 mM glutamine, were seeded on sterile coverslips the day prior to microinjection. Microinjection was performed as described previously (27) using an InjectMan NI2 micromanipulator and a FemtoJet injector from Eppendorf (Germany). Identical, standardized conditions of pressure (100 hPa) and time (0.1 s) were used in all experiments. Peptides and/or recombinant proteins were diluted in PBS together with dextran 70 kDa-conjugated lysine-fixable Oregon Green® (Molecular Probes, 0.5% final concentration) as a co-injection marker. Typically, 100 cells were microinjected for each condition in each experiment. The percentage of positive (i.e. fluorescent) cells exhibiting morphological features of apoptosis following microinjection was evaluated as described previously (27) using an inverted fluorescence microscope (DMIRE2, Leica, France).

Immunocytochemistry—Immunocytochemical staining of microinjected cells was performed as described previously (24). Both the primary polyclonal anti-Bax NT antibody and the anti-rabbit secondary antibodies were used at a 1:200 dilution. Images presented in this study were collected on a Leica TCS NT confocal microscope with a 100 x 1.3 NA Fluotar objective (Leica, France).

Cell-free Assays—Mitochondria were isolated from normal rat liver as described previously (28). For mitochondrial targeting assays, [35S]Met-labeled proteins (Amersham Biosciences) were synthesized from cDNAs using the TNT-coupled Transcription/Translation system (Promega, France) and quantified as described previously (28). Radiolabeled Bax (4 fmol) was incubated with isolated mitochondria (2.5 mg proteins/ml) and with the indicated concentration of recombinant tBid or BaxBH3 at 30 °C for 1 h in 40 µl of standard import buffer (250 mM sucrose, 80 mM KCl, 10 mM MgCl2,10mM malic acid, 8 mM succinic acid, 1 mM ATP-Mg2+, 20 mM MOPS, pH7.5). When indicated, 35S-Bax was preincubated with unlabeled GST or GST-Bcl-xL (2 fmol) for 30 min prior to incubation with mitochondria in the presence or in the absence of BaxBH3 (200 nM). Radiolabeled proteins bound to the mitochondria were recovered by centrifugation of the incubation mixture at 8,000 x g for 10 min at 4 °C, separated by SDS-PAGE, and analyzed by scanning with a PhosphorImager (Amersham Biosciences) followed by quantification with IPLab gel program (Signal Analytics).

For cytochrome c release assays, recombinant Bax{alpha} (5 nM) was preincubated with either GST or GST-BclxL (2.5 nM) for 30 min prior to its incubation with mitochondria (1 mg of proteins/ml) in the absence or in the presence of BaxBH3 (200 nM) for an additional hour in 100 µl of import buffer. Mitochondria were then recovered, and the amount of mitochondrial cytochrome c was analyzed by Western blotting and quantified with IPLab gel program (Signal Analytics).

Protein Binding Experiments—In vitro His-protein binding assays were performed as described previously (25). Briefly, each radiolabeled protein (4 or 8 fmol as indicated) was incubated with an equimolar concentration of either His-tagged Bcl-xL or Bax{alpha} immobilized on nickel-Sepharose in 50 µl of binding buffer (142 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.4), 0.5 mM dithiothreitol, 1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, and a mixture of other protease inhibitors) at 4 °C for 2 h. BaxBH3 or BaxBH3 L63A (200 nM final concentration) was then added, and the mixture was incubated for an additional hour. Protein complexes were then centrifuged at 13,000 x g for 5 min at 4 °C, washed three times in binding buffer, eluted in elution buffer (50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, and 250 mM imidazole), separated by SDS-PAGE, and analyzed with a PhosphorImager as described above. For mild proteolysis experiments, both the pellet and the supernatant from the first centrifugation at 13,000 x g were treated with trypsin (1 mg/ml) for 15 min at 4 °C. Proteolysis was then stopped by the addition of 10 mg/ml soybean trypsin inhibitor prior to analysis as described above. To assess directly the sensitivity of radiolabeled Bax to trypsin treatment in the absence of His-tagged Bcl-xL, the radiolabeled protein (8 fmol) was incubated with tBid (8 fmol, 0.2 nM final concentration), the indicated peptides (200 nM), or was left untreated in 40 µl of binding buffer for 1 h at 30 °C prior to trypsin treatment as described above.

For immunoprecipitation experiments, soluble His-tagged Bcl-xL-radiolabeled Bax complexes were prepared as follows. Radiolabeled Bax (20 fmol) was incubated with 20 fmol of His tagged Bcl-xL immobilized on nickel-Sepharose in 250 µl of binding buffer at 4 °C for 2 h; protein complexes were then centrifuged at 13,000 x g for 5 min at 4 °C, washed three times in binding buffer, and then eluted in 20 µl of elution buffer. SDS-PAGE analysis and quantification using a PhosphorImager as described above showed that the resulting soluble complexes contained in average 4 fmol of radiolabeled Bax. These soluble complexes, or equivalent amounts of free radiolabeled Bax (4 fmol), were incubated with an equimolar amount of tBid (0.2 nM final concentration) or with the indicated peptides (200 nM) for 1 h at 30 °C in 40 µl of standard import buffer. The anti-Bax 2D2, 6A7, or TL41 antibodies were then added (1:10 final dilution) for an additional 1-h incubation at 4 °C. Antibody-protein complexes were then isolated by incubation with either Zysorbin-G (for the 6A7 and 2D2 antibodies) or Zysorbin (for the TL41 antibody) followed by centrifugation and washes according to the manufacturer's instructions (Zymed Laboratories Inc.). SDS-PAGE analysis and quantification of the immunoprecipitated and radiolabeled proteins using a PhosphorImager were performed as described above.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BaxBH3 Does Not Stimulate the Cytotoxic Activity of Wild Type Bax—In order to investigate the mechanism of action of BH3 domains, we used a synthetic peptide comprising the known BH3 domain of Bax (residues 57–72), BaxBH3. This peptide interacts with (5), and functionally antagonizes (24), Bcl-xL. Moreover, upon microinjection into fibroblasts, it induces apoptosis by a mechanism that requires either Bax or Bak (24). One possible reason for this is that this peptide directly activates the multidomain proteins, essentially acting as a death ligand (22). This notion appears consistent with its reported ability to interact physically with full-length Bax (29).

We reasoned that if BaxBH3 can directly activate Bax, it should be more efficient in inducing apoptosis in cells with increased Bax levels; on the other hand, we would expect to see no increased sensitivity to BaxBH3 in these cells if BaxBH3 were unable to stimulate Bax function directly. We therefore made use of a recombinant full-length Bax (Bax{alpha}) and assessed its effect on whole intact cells in the presence or in the absence of the BaxBH3 peptide. The recombinant protein used was prepared in the absence of detergent, in order to prevent the artifactual activation of Bax reported to occur during the purification procedure (30). This purified Bax{alpha} protein did not associate with Bax{alpha} in cell-free experiments ((25) see also Fig. 6). It is therefore unlikely to adopt a homodimeric structure. As shown in Fig. 1 (top left panel), microinjection of recombinant Bax{alpha} (0.5 nM) induced limited apoptosis even 4 h following injection. In order to check that this recombinant protein can be activated by certain stimuli, we co-injected Bax{alpha} with a recombinant protein equivalent to the caspase 8 cleavage product of Bid (tBid, 0.5 nM) (26). This led to far more widespread apoptosis than that observed upon microinjection of either Bax{alpha} or tBid alone (Fig. 1, top right panel). Apoptotic synergy between these two proteins was evident as early as 2 h after microinjection, strongly supporting the view that recombinant Bax{alpha} by itself is poorly apoptogenic but can be activated by signals such as Bid activation. Of note, the fact that subnanomolar quantities of tBid and Bax{alpha} efficiently induced apoptosis is consistent with a previous report (31) showing that similar concentrations of tBid elicit significant mitochondrial dysfunction and cell death.



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FIG. 6.
Effect of either BaxBH3 or BadBH3 on Bax/Bcl-xL interactions in vitro. A, left panel, IVTR Bax{alpha}, Bax{Delta}ART, or Bcl-xL (4 fmol) were preincubated with purified His-tagged Bax{alpha} before addition of the indicated peptides (200 nM final concentration). The percentage of 35S-labeled proteins bound to His-tagged Bax{alpha} was estimated after SDS-PAGE and fluorography using the initial input (4 fmol) as 100% as described under "Materials and Methods." Data are means (±S.E.) of three independent experiments. Top panel, autoradiogram illustrating one representative experiment. 1 fmol of each radiolabeled protein (i25%) was loaded where indicated. Right panel, IVTR Bax{alpha} (4 fmol in the absence or in the presence of 4 fmol of unlabeled tBid) or Bax{Delta}ART (4 fmol) was preincubated with purified His-tagged Bcl-xL before addition of the indicated peptides (200 nM final concentration). The percentage of 35S-labeled proteins bound to His-tagged Bcl-xL was evaluated as described above. Data are means (±S.E.) of three independent experiments. Top panel, autoradiogram illustrating one representative experiment. B, top panel, IVTR Bax{alpha} (8 fmol) preincubated with either tBid (0.2 nM) or with the indicated peptides (200 nM) was treated or not with trypsin as described under "Materials and Methods." The amount of protease-resistant 35S-labeled proteins was evaluated and expressed as a fraction of the initial amount of radiolabeled Bax. Data are means (±S.E.) of three independent experiments. Top panel, autoradiogram illustrating one representative experiment. Bottom panel, IVTR Bax{alpha} (8 fmol) was preincubated with purified His-tagged Bcl-xL in the presence of the indicated peptides (200 nM) as in A (right panel). His-bound complexes (pellet, P) and free proteins (supernatant, S) were then treated with trypsin as described under "Materials and Methods." The amount of protease-resistant 35S-labeled proteins was then evaluated and expressed as a fraction of the initial amount (8 fmol) of radiolabeled Bax. Data are means (±S.E.) of four independent experiments. Top panel, autoradiogram illustrating one representative experiment. 2 fmol of untreated radiolabeled Bax (25%) and 8 fmol of trypsin-treated radiolabeled Bax (t100%) were loaded where indicated. C, IVTR Bax{alpha} (4 fmol, left panel) or an equivalent amount of His-tagged Bcl-xL-bound IVTR Bax (prepared as described under "Materials and Methods," right panel) were incubated, as indicated, with either tBid (0.2 nM), BaxBH3 (200 nM), BaxBH3 L63A (200 nM), BadBH3 (200 nM), or left untreated prior to immunoprecipitation with the indicated antibodies. Immunoprecipitated 35S-labeled proteins were analyzed and quantified as described under "Materials and Methods." Data are means (±S.E.) of three independent experiments. Insets, autoradiograms illustrating one representative experiment for each condition. 1 fmol of the initial, non-immunoprecipitated radiolabeled Bax (25%) was loaded in each gel for illustrative purposes.

 


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FIG. 1.
Effect of BaxBH3 on the pro-apoptotic activity of Bax{alpha} His-tagged recombinant Bax{alpha} (0.5 nM), tBid (0.5 nM alone or together with 0.5 nM Bax{alpha}), BaxBH3 (0.5 nM alone or together with 0.5 nM Bax{alpha}), and BaxBH3 L63A (0.5 nM alone or together with 0.5 nM Bax{alpha}) were mixed with Oregon Green dextran (0.5% w/v) in PBS and microinjected into Rat-1 fibroblasts, grown in 10% FCS. Cells were then incubated at 37 °C, and death of microinjected cells was assessed morphologically by fluorescence microscopy at the indicated times. Data are means (±S.E.) of at least three independent experiments.

 

We then analyzed whether BaxBH3 stimulated the pro-apoptotic function of Bax{alpha}. Microinjection of 0.5 nM BaxBH3 induced apoptosis to a certain degree (Fig. 1, bottom left panel), whereas microinjection of the mutant BaxBH3 L63A (0.5 nM), which does not interact with Bcl-xL, had only a limited effect on cell viability (Fig. 1, bottom right panel). In sharp contrast to what we observed for tBid, co-injection of Bax{alpha} and BaxBH3 did not result in an increased apoptosis in the microinjected cells (Fig. 1, bottom left panel). A similar lack of effect on Bax{alpha} activity was observed with the mutant BaxBH3 L63A peptide (Fig. 1, bottom right panel). Thus, BaxBH3 induces apoptosis, but it does not impact on the apoptotic activity of the native form of Bax.

BaxBH3 Neither Stimulates Bax Association with Mitochondria nor Significantly Modifies Its Conformation—Bax in its native conformation is only poorly targeted to mitochondria, and it acquires the ability to interact with its mitochondrial site of action only in response to certain death stimuli, including Bid activation (32). We therefore asked whether the lack of effect of BaxBH3 on Bax{alpha} apoptotic function depended upon its inability to stimulate Bax{alpha} targeting to mitochondria. For this purpose, we used an in vitro system employing the 35S-labeled transcription-translation product of Bax cDNA in rabbit reticulocyte lysate together with purified mitochondria from rat liver. Previous analysis of Bax association with mitochondria in such a system has confirmed that Bax targeting to mitochondria is mostly inefficient but that it can be triggered by addition of cytosolic extracts from apoptotic cells (28, 33) or induced by specific mutations that serve to increase Bax apoptotic activity (25, 34). As a positive control, we checked that the addition of low concentrations (1–10 nM) of tBid stimulated the targeting of in vitro translated, radiolabeled (IVTR) Bax{alpha} to isolated mitochondria in a dose-dependent manner (Fig. 2A). In sharp contrast, the addition of BaxBH3 at 10 nM did not stimulate Bax{alpha} association to mitochondria (Fig. 2A). Increased concentrations of the peptide, up to 10 µM, failed to exhibit any effect on Bax{alpha} targeting (not shown, see also below in Fig. 5). We conclude that BaxBH3 is unable to stimulate Bax{alpha} association to mitochondria.



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FIG. 2.
Effect of BaxBH3 on Bax mitochondrial targeting and conformation. A, effect of BaxBH3 on Bax targeting to mitochondria. 4 fmol of IVTR Bax{alpha} were incubated with mitochondria in the absence or in the presence of the indicated concentrations of recombinant tBid or BaxBH3. The amount of radiolabeled Bax associated with the mitochondria was then analyzed by SDS-PAGE and autoradiography. Quantitative values were obtained as described under "Materials and Methods" and were normalized to the amount of Bax associated to mitochondria in the absence of tBid and BaxBH3. Data are means (±S.E.) of at least three independent experiments. Top panel, autoradiogram illustrating one representative experiment. B, effect of microinjected BaxBH3 on endogenous Bax conformation. BaxBH3 or tBid (0.5 nM) mixed with Oregon Green dextran (0.5% w/v) in PBS was microinjected into Rat-1 fibroblasts incubated in the presence of benzyloxycarbonyl-VAD-fluoromethyl ketone (50 µM). Cells were then incubated for 3 h at 37 °C prior to fixation and immunostained with anti-Bax NT antibody as described under "Material and Methods." Cells were then analyzed by fluorescence microscopy. The percentage of cells microinjected with either tBid or BaxBH3 that exhibits an increase in BaxNT staining was then analyzed. Data are means (±S.E.) of three independent microinjection experiments. Top panel, one representative example of Rat-1 fibroblasts microinjected with tBid. Green fluorescence allows identification of the microinjected cells, and red fluorescence shows BaxNT immunostaining of the same cells. White arrows indicate cells exhibiting a typical increase in Bax NT staining.

 


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FIG. 5.
Effect of either BaxBH3 or BadBH3 on Bax pro-apoptotic activity in the presence of Bcl-xL. A, the indicated combination of Bax{alpha} (0.5 nM), GST-Bcl-xL (0.25 nM), GST(0.25 nM), and BaxBH3 (0.5 nM) were mixed with Oregon Green dextran (0.5% w/v) in PBS and microinjected into Rat-1 fibroblasts, grown in 10% FCS. Cells were then incubated at 37 °C, and cell death in the microinjected population was assayed as in Fig. 1. Data are means (±S.E.) of three independent experiments. B, experiments were performed as in A using BadBH3 instead of BaxBH3.

 

The ability of certain stimuli, such as Bid activation, to induce translocation of Bax to mitochondria has been ascribed to their ability to induce a change in Bax conformation. Such change in Bax is accompanied by the exposure of an epitope located in its N terminus, which can be detected in whole cells by immunohistochemical analysis with a specific antibody (BaxNT (35)). To understand better the inability of BaxBH3 to induce Bax translocation, we asked whether BaxBH3 or tBid, when microinjected into Rat-1 cells, induced exposure of this epitope in endogenous Bax. We used subnanomolar doses of both tBid and BaxBH3, which we had shown by themselves induce only modest apoptosis 3 h following microinjection (see Fig. 1). Microinjection was performed in the presence of the broad range caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone (50 µM) to avoid caspase-induced changes in Bax conformation (32). Cells were fixed 3 h after microinjection with either tBid or BaxBH3 (0.5 nM) and immunostained using the conformation-specific BaxNT antibody as described previously (24). As shown in Fig. 2B, a significant portion of Rat-1 fibroblasts microinjected with tBid (0.5 nM) exhibited a marked increase in Bax NT immunostaining. This increased staining appeared as a punctate, extranuclear pattern consistent with a mitochondrial localization for the modified Bax molecules (see color panel in Fig. 2B). Careful quantification indicated that ~25% of cells exhibited the change in Bax NT immunostaining 3 h after tBid microinjection (Fig. 2B). In sharp contrast, less than 10% of cells exhibited a similar feature 3 h after microinjection with BaxBH3 (0.5 nM). Thus, introduction of BaxBH3 into intact cells did not modify Bax conformation as efficiently as an equimolar amount of tBid. This suggests that BaxBH3 requires additional endogenous factors to induce efficiently a change in Bax conformation. Taken together, those results indicate that BaxBH3 is unable to induce a significant change in Bax conformation and to stimulate its association to mitochondria.

BaxBH3 Stimulates the Cytotoxic Activity of a Recombinant Bax Mutant Lacking Its N-terminal End—Although it is unable to stimulate Bax{alpha} directly, BaxBH3 nevertheless recruits Bax function in cells (24). One possible explanation for these paradoxical results is that BaxBH3 can functionally cooperate with Bax, provided Bax is in receipt of a sufficient threshold of activation by other signals. To address this question directly, we tested the effect of BaxBH3 on the pro-apoptotic function of a recombinant mutant form of Bax, Bax{Delta}ART. This recombinant protein consists of a Bax protein lacking its N-terminal 19 residues. This extreme N terminus of Bax normally regulates the apoptotic function of Bax negatively, and its deletion consequently results in an increased ability of Bax to homodimerize, interact with mitochondria, and promote apoptosis (25, 32, 36). Microinjection of recombinant Bax{Delta}ART (0.5 nM) induced little apoptosis during the first 3 h following delivery (Fig. 3). However, by 4 h, a significant proportion of the microinjected cells exhibited morphological features of apoptosis. At this time, the extent of cell death observed in the microinjected population was 2-fold higher than that observed in control cells injected with the same amount of Bax{alpha} (compare Fig. 3 to Fig. 1A). This increased apoptosis in cells injected with Bax{Delta}ART, as opposed to cells injected with Bax{alpha}, was also observed at later time points (data not shown), indicating that, as expected, Bax{Delta}ART is more apoptogenic than Bax{alpha}. Co-injection of amounts of BaxBH3 that were unable to stimulate Bax{alpha} apoptotic activity (0.5 nM, see above in Fig. 1B) with Bax{Delta}ART led to a significant increase in the rate and the extent of cell death as early as 2 h following injection (Fig. 3). In sharp contrast, the mutant BaxBH3 L63A peptide had no effect on Bax{Delta}ART apoptotic activity (Fig. 3). Therefore, BaxBH3 can specifically sensitize cells to the deleterious effect of an activated form of Bax. This indicates that the inability of BaxBH3 to sensitize cells to Bax{alpha} relies on its inability to trigger a change in the conformation of the protein and not on its inability to sensitize cells to Bax apoptotic activity under the conditions used.



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FIG. 3.
Effect of BaxBH3 on Bax{Delta}ART pro-apoptotic activity. Recombinant Bax{Delta}ART (0.5 nM) alone or in the presence of either BaxBH3 (0.5 nM) or BaxBH3 L63A (0.5 nM) was mixed with Oregon Green dextran (0.5% w/v) in PBS and microinjected into Rat-1 fibroblasts, grown in 10% FCS. Cells were then incubated at 37 °C, and cell death in the microinjected population was assayed as in Fig. 1. Data are means (±S.E.) of three independent experiments.

 

BaxBH3 Stimulates the Cytotoxic Activity of Wild Type Bax in the Presence of Bcl-xL—The observation that BaxBH3 can promote Bax-dependent apoptosis without providing a threshold for the activation of Bax{alpha} suggests that additional ratelimiting partners are required for BaxBH3 to promote Bax function. It is well established that BH3 peptides bind Bcl-xL, suggesting the possibility that BH3 peptides promote apoptosis by releasing Bax from dimers formed with its anti-apoptotic counterparts. Alternatively, additional factors, distinct from Bcl-xL itself, might instead be required for the peptide to activate Bax{alpha} indirectly. To distinguish between these two possibilities, we analyzed whether the presence of Bcl-xL suffices to allow our BH3 peptide to cooperate with Bax{alpha}.

We first analyzed the effect of BaxBH3 on mitochondrial targeting of Bax following its incubation with a recombinant GST-Bcl-xL fusion protein. As shown in Fig. 4A, IVTR Bax{alpha} preincubated with the control recombinant protein GST (2:1 ratio) interacted poorly with mitochondria whether or not BaxBH3 (200 nM) was present. IVTR Bax{alpha} preincubated with GST-Bcl-xL (2:1 ratio) also interacted poorly with mitochondria in the absence of BaxBH3. It should be noted that the recombinant Bcl-xL used here lacks its C-terminal end and is therefore unable to interact with mitochondria itself (24). Addition of 200 nM of BaxBH3 resulted in a significant increase in the mitochondrial targeting of IVTR Bax{alpha} preincubated with GST-Bcl-xL (Fig. 4A). Thus, the BH3 peptide can stimulate Bax{alpha} association with mitochondria provided Bcl-xL is present.



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FIG. 4.
Effect of BaxBH3 on Bax interaction with mitochondria in the presence of Bcl-xL. A, effect of BaxBH3 on Bax mitochondrial targeting in the presence of Bcl-xL. 4 fmol of IVTR Bax{alpha} were preincubated with 2 fmol of either GST-Bcl-xL or GST as a negative control prior to incubation with mitochondria (2.5 mg/ml) in the absence or in the presence of BaxBH3 (200 nM). The amount of radiolabeled Bax{alpha} associated to mitochondria was evaluated as described in Fig. 2A. Data are means (±S.E.) of four independent experiments. Top panel, autoradiogram illustrating one representative experiment. B, effect of BaxBH3 on Bax-induced cytochrome c release in the presence of Bcl-xL. Recombinant Bax{alpha} (5 nM) preincubated with either GST-Bcl-xL or GST (2.5 nM) was added to isolated mitochondria (1 mg/ml) in the absence or in the presence of BaxBH3 (200 nM) for 1 h at 30 °C. The amount of cytochrome c in the mitochondrial fraction was then evaluated as described under "Materials and Methods." The amount of mitochondrial cytochrome c following incubation with Bax{alpha} in the absence of Bcl-xL and BaxBH3 was used at 100%.

 

We next investigated whether the BaxBH3 peptide, when combined with Bcl-xL, elicits an increased ability of Bax{alpha} to induce cytochrome c release from isolated mitochondria. Incubation of isolated mitochondria (1.5 mg of protein/ml) with recombinant Bax{alpha} (5 nM) plus GST (2.5 nM) for 1 h at 30 °C led to no significant cytochrome c release from the mitochondrial pellet (data not shown). As well, the presence of an excess of BaxBH3 peptide (200 nM) plus Bax{alpha}/GST did not significantly affect cytochrome c release (Fig. 4B). In contrast, the addition of BaxBH3 (200 nM) to a combination of Bax{alpha} and GST-Bcl-xL in a 2:1 ratio leads to a significant loss of cytochrome c from mitochondria (Fig. 4B). Incubation of the BH3 peptide in the presence of GST-Bcl-xL had no effect on mitochondrial cytochrome c (data not shown), indicating that induction of cytochrome c release by BaxBH3 under the conditions used depends on the presence of both Bax{alpha} and Bcl-xL. Thus, BaxBH3 stimulates Bax{alpha}-induced cytochrome c release provided Bcl-xL is present.

We next determined the ability of BaxBH3 to modulate Bax apoptotic activity in the presence GST-Bcl-xL. Bax{alpha} (0.5 nM) combined with GST-Bcl-xL (0.25 nM) was even less efficient in inducing apoptosis than Bax{alpha} alone (compare Fig. 5A to Fig. 1, top left panel). This is consistent with the protective effect of microinjected GST-Bcl-xL against UV-induced apoptosis reported previously (24). This protective effect was further confirmed by the observation that BaxBH3 (0.5 nM) combined with GST-Bcl-xL was less efficient in inducing apoptosis than the peptide alone (compare Fig. 5A to Fig. 1, bottom left panel). In sharp contrast, addition of BaxBH3 (0.5 nM) to a mixture of Bax (0.5 nM) and GST-Bcl-xL (0.25 nM) led to a significantly more efficient induction of apoptosis than that observed in cells injected with BaxBH3 and Bax{alpha} in the absence of GST-Bcl-xL (Fig. 5A). This enhanced apoptosis was not observed when GST alone (0.25 nM) was used instead of GST-Bcl-xL, or when the mutant-inactive peptide BaxBH3 L63A (0.5 nM) was used instead of BaxBH3 (data not shown). Our recombinant Bcl-xL protein therefore diminishes the apoptotic activity of either BaxBH3 or Bax{alpha} alone, but it allows BaxBH3 to stimulate Bax{alpha} pro-apoptotic activity. In order to investigate whether this cooperativity between Bcl-xL and BaxBH3 can extend to other BH3 peptides, we used another synthetic 16-mer peptide encompassing the BH3 domain of Bad. It has been shown recently (22) that this BH3 domain is unable to activate Bax directly. Consistent with this observation, we observed that microinjection of BadBH3 (0.5 nM) alone or together with Bax{alpha} (0.5 nM) induced similar levels of apoptosis (Fig. 5B). BadBH3 (0.5 nM) combined with GST-Bcl-xL was less efficient in inducing apoptosis than the peptide alone (Fig. 5B). However, the addition of BadBH3 (0.5 nM) to a mixture of Bax (0.5 nM) and GST-Bcl-xL (0.25 nM) enhanced significantly the induction of apoptosis as compared with that observed with BadBH3 and Bax{alpha} in the absence of GST-Bcl-xL (Fig. 5B). Taken altogether, these data show that the presence of Bcl-xL is sufficient to allow both BaxBH3 and BadBH3 to stimulate the apoptotic activity of Bax{alpha}.

BaxBH3 Releases Bax from Heterodimers Formed with Bcl-xL—The previous results are mostly consistent with a model in which BaxBH3 releases an activated Bax from heterodimerization with Bcl-xL. To explore this idea, we analyzed the interaction between Bax{alpha} and Bcl-xL in the presence or in the absence of BaxBH3. We used a cell-free assay in which tagged recombinant proteins were assayed for their abilities to pull-down IVTR proteins (25). As shown in Fig. 6A (left panel), recombinant Bax{alpha} was unable to associate with either IVTR Bax{alpha} or IVTR Bax{Delta}ART as reported previously (25). In sharp contrast, recombinant Bax{alpha} associated with IVTR Bcl-xL under the same conditions (Fig. 6A, left panel). This binding was inhibited by an excess of BaxBH3 peptide but not by the inactive peptide BaxBH3 L63A (Fig. 6A, left panel). Conversely, recombinant Bcl-xL significantly bound to IVTR Bax{alpha}, in a manner that could be specifically inhibited by BaxBH3 (Fig. 6A, right panel). Of note, the amount of Bax{alpha} bound to recombinant Bcl-xL could be further increased by preincubating IVTR Bax{alpha} with unlabeled tBid (Fig. 6A, right panel). This suggests that the affinity of Bax for Bcl-xL correlates with its ability to adopt an active configuration, a notion that was confirmed by the finding that Bax{Delta}ART bound more efficiently to Bcl-xL than Bax{alpha} under similar conditions (Fig. 6A, right panel). Thus, Bax{alpha} is unable to homodimerize, but it nonetheless associates with Bcl-xL, albeit with less efficiency than its activated forms. Furthermore, BaxBH3 displaces Bax from the heterodimers it forms with Bcl-xL.

We then investigated whether the binding of Bax{alpha} to Bcl-xL, or its release from Bcl-xL upon treatment with BaxBH3, can significantly contribute to a change in its conformation and to its activation. In a first series of experiments, we analyzed the trypsin sensitivity of Bax following its association with Bcl-xL in cell-free pull-down assays. Previous experiments (28) have shown that incubation of Bax with apoptotic cytosols increases its resistance to mild proteolysis while concomitantly increasing its targeting to mitochondria. Moreover, mutants of Bax that exhibit an increased ability to homodimerize and to translocate to mitochondria also exhibit increased resistance to mild proteolysis (25). Consistent with the notion that a change in Bax sensitivity to proteolysis accompanies its activation, we observed that tBid-treated IVTR Bax{alpha} was highly resistant to mild proteolysis with trypsin, whereas untreated IVTR Bax{alpha} was not (Fig. 6B, top panel). In sharp contrast, Bax{alpha} sensitivity to proteolysis was neither affected by the presence of BaxBH3 nor by that of BaxBH3L63 (Fig. 6B, top panel). In a pull-down experiment using recombinant His-tagged Bcl-xL as described above, most Bcl-xL-bound IVTR Bax{alpha} exhibited resistance to trypsin treatment, whereas there was no detectable trypsin-resistant Bax{alpha} in the unbound fraction (compare Fig. 6B, bottom panel, to Fig. 6A). Addition of BaxBH3 resulted in a complete loss of Bcl-xL-bound, protease-resistant Bax{alpha} and the concomitant appearance of protease-resistant Bax{alpha} in the unbound fraction (Fig. 6B, bottom panel). In control experiments, no effect of the mutant BaxBH3 L63A was observed. Thus, Bcl-xL binding to Bax{alpha} is sufficient to modify its sensitivity to proteolysis, whereas the action of BaxBH3 is to trigger the release of a modified Bax from Bcl-xL. In a second series of experiments, we analyzed the ability of three epitope-specific antibodies to immunoreact with Bax in the presence of Bcl-xL, BH3 peptides, or both. The specific anti-Bax antibodies used were as follows: (i) the 2D2 monoclonal antibody that was raised against a peptide encompassing residues 3–16 of human Bax, an epitope which is exposed regardless of Bax conformation (37); (ii) the 6A7 monoclonal antibody that was raised against a peptide encompassing residues 12–24, which only binds to active Bax (38); (iii) the TL41 polyclonal antibody that was produced against a peptide sequence (residues 57–72), which represents the minimal Bax BH3 domain (24). In non-denaturing conditions, only the 2D2 antibody could immunoprecipitate IVTR Bax{alpha} that was left untreated (data not shown) or that was treated with the control peptide BaxBH3 L63A (Fig. 6C, left panel). In sharp contrast, all three antibodies could immunoprecipitate t-Bid treated IVTR Bax{alpha} (Fig. 6C, left panel). This indicates that the epitope for the 6A7 antibody, and the epitope for the TL41 antibody within the Bax BH3 domain, are normally hidden in native Bax but become exposed following activation of Bax by tBid. Treatment of IVTR Bax{alpha} with either BaxBH3 or BadBH3 had no effect on the immunoreactivity of the radiolabeled protein with either the 6A7 or the TL41 antibodies (Fig. 6C, left panel), further supporting the notion that these peptides cannot activate Bax by themselves. As shown in Fig. 6C (right panel), radiolabeled Bax complexed to Bcl-xL could be immunoprecipitated by the 2D2 antibody. This observation is consistent with the ability of an antibody raised against the equivalent peptide sequence to immunoreact with Bcl-xL-bound murine Bax reported previously (38). In sharp contrast, neither the 6A7 nor the TL41 conformation-specific antibodies could immunoprecipitate Bcl-xL-bound IVTR Bax{alpha} in native conditions (Fig. 6C), although they could do so in denaturing conditions (0.1% SDS, 0.1% Triton X-100, data not shown). Thus, the inability of these antibodies to immunoreact with Bax in non-denaturing conditions likely results from the inaccessibility of the corresponding epitopes in the Bcl-xL-bound protein. Moreover, when Bcl-xL-Bax complexes were treated with either BaxBH3 or BadBH3, but not with the mutant peptide BaxBH3 L63A, Bax{alpha} was significantly immunoprecipitated by the 2D2, the 6A7, and the TL41 antibodies (Fig. 6C, right panel). Similarly, IVTR Bax{alpha} directly released from nickel-Sepharose-bound His-tagged Bcl-xL by wild type BH3 peptides (as in Fig. 6B) also immunoreacted with all three antibodies (data not shown). Thus, both epitopes for the 6A7 antibody and the TL41 antibody become accessible when Bax molecules are specifically released from Bcl-xL by BH3 peptides. Taken altogether, these results indicate that, when it is directly activated by tBid, Bax becomes resistant to proteolysis, whereas its BH3 domain and a region in its N-terminal end (the 6A7 epitope) become exposed. Binding to Bcl-xL is sufficient to modify the sensitivity to proteolysis of Bax, but the release of Bax from Bcl-xL, induced by BH3 peptides, is necessary to produce an active Bax, as probed by the accessibility of the resulting protein to conformation-specific antibodies.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The underlying mechanism by which BH3-only proteins recruit Bax and Bak to induce apoptosis is a key to the way mitochondria integrate multiple stimuli into one single apoptotic pathway. In many healthy cells, Bax and Bak appear to be expressed in an inactive conformation, unable to self-associate. Thus, to exert their apoptotic function, these proteins need to undergo conformational changes. Emerging evidence indicates that some BH3-only proteins, such as Bid, can function as death agonists that activate directly Bax and Bak. It is formally possible that certain domains in those death agonists, distinct from the BH3 one, could contribute to their ability to promote the pro-apoptotic changes in Bax or Bak. However, the ability of these proteins to activate Bax/Bak seems to rely in great part on some specific information contained in their BH3 domain. Indeed, short synthetic peptides encompassing certain BH3 domains are sufficient to trigger Bax/Bak activation (22).

In this study, we used a synthetic peptide representing the BH3 domain of Bax (BaxBH3), which composes the minimal sequence required to antagonize anti-apoptotic family members. We report here that BaxBH3 has no significant effect on either the apoptotic activity, the mitochondrial targeting, and/or the conformation of Bax{alpha} (Figs. 1 and 2). We found that another synthetic peptide representing the minimal BH3 domain of Bad is also unable to stimulate Bax activity (Fig. 5B). Thus, BH3 peptides that contain sufficient information to occupy the surface pocket of the anti-apoptotic Bcl-2 family members do not all necessarily support direct activation of Bax/Bak. Consistent with this, BH3 peptides derived from Bad, Bik, or Noxa were shown to lack the ability to activate Bax/Bak (22), whereas small molecule non-peptidic ligands of Bcl-xL are reportedly unable to induce Bax insertion into mitochondrial membranes (6). These observations show that another subset of BH3 domains exists, which can function as survival antagonists but not as death agonists for Bax/Bak.

The lack of effect of BaxBH3 on Bax{alpha} activity sharply contrasts with the ability of BaxBH3 to promote the apoptotic function of Bax{Delta}ART, an N-terminal deletion of Bax that displays an enhanced ability to homodimerize and to induce apoptosis (Fig. 3). The fact that the activity of Bax{Delta}ART can be further enhanced by BaxBH3 indicates that BaxBH3 can functionally cooperate with Bax, so long as Bax is provided with a sufficient trigger to lower its threshold of activation. Because BaxBH3 prevents Bax{Delta}ART binding to Bcl-xL (Fig. 6A), BaxBH3 is likely to enhance the apoptotic activity of Bax{Delta}ART by preventing the inhibitory association of this activated form of Bax with endogenous Bcl-xL and possibly Bcl-2. One interesting implication from this is that death agonists and survival antagonists (typified by our BH3 peptide) may activate cell death by mechanistically distinct, yet cooperative pathways. In agreement with this, we have observed that BH3 peptides can sensitize cells to apoptosis induction by microinjected tBid.2

Even though BaxBH3 is unable to activate Bax directly, the fact remains that it induces apoptosis (Fig. 1; see also Refs. 39 and 40). Our experiments therefore constitute proof of concept that the ability to function as a death ligand is not absolutely required for a BH3 domain to promote apoptosis. The observation that the BH3-only protein BimL lacks the ability to activate Bax directly but retains the ability to interact with Bcl-xL and induce apoptosis is consistent with this view (41). As BaxBH3 specifically induces apoptosis by a mechanism requiring the presence of either Bax or Bak (24), these experiments also imply that the ability of this peptide to promote Bax/Bak-mediated apoptosis must involve some other intermediaries. One possibility is that survival antagonists such as BaxBH3 promote the release from Bcl-xL/Bcl-2 of some death agonists such as Bid (17). It should be noted, however, that a mutant of Bcl-xL that cannot interact with Bax or Bak but interacts with Bid is less efficient than wild type Bcl-xL in preventing Bax-mediated mitochondrial dysfunction (42). Thus anti-apoptotic Bcl-2 family members may act on Bax/Bak directly, and their functional antagonists may promote Bax/Bak activity by suppressing this interaction. We found that addition of Bcl-xL was sufficient to allow BaxBH3 to stimulate the ability of Bax to interact with mitochondria, to induce cytochrome c release (Fig. 4), and to induce apoptosis upon microinjection (Fig. 5A). Similarly, we found that BadBH3 had no impact on the apoptotic activity of microinjected Bax by itself but that it could stimulate it when combined with Bcl-xL (Fig. 5B). Thus, induction of Bax-mediated apoptosis by a BH3 domain does not necessarily involve any other partner than Bcl-xL itself. This strongly favors a model in which survival antagonists promote apoptosis by inducing the release of Bax/Bak from heterodimers they engage with Bcl-xL/Bcl-2.

Most of the arguments against the aforementioned model arise from the observation that Bax/Bak and their anti-apoptotic counterparts exhibit only modest interaction in some cell types unless apoptosis is triggered (38, 43). We confirm that Bax binds to Bcl-xL with greater efficacy in its active conformation; its association to Bcl-xL is indeed stabilized by the addition of tBid or by a deletion in its N-terminal regulating domain (Fig. 6). However, we have also observed that, despite its inability to self-associate, Bax{alpha} can bind to Bcl-xL (Fig. 6). This observation is consistent with whole cells studies in which Bax fused to a fluorescent protein, although inefficient at inducing apoptosis by itself, nonetheless exhibited significant interaction with Bcl-2 and Bcl-xL as assayed by fluorescence resonance energy transfer (6, 44).

Bax{alpha}/Bcl-xL interactions were significantly inhibited by BaxBH3 (Fig. 4). Similarly, in vivo interactions between Bax and Bcl-xL were disrupted by small molecule ligands of the BH3-binding pocket of Bcl-xL (6). Thus, binding of the BH3 domain of Bax within the hydrophobic pocket of Bcl-xL is likely to be essential for the formation of a stable heterodimer. Residues within the BH3 domain of Bax that are critical for dimer formation are oriented toward the hydrophobic core of wild type Bax (46). Thus, major conformational modifications in Bax should accompany its BH3-dependent binding to Bcl-xL. Our observation that, upon its binding to Bcl-xL, Bax acquires resistance to mild proteolysis is consistent with this idea (Fig. 6). It is striking to note that protease resistance is a property that Bax also displays when it is activated by apoptotic cytosols (28), tBid (this study), or by site-directed mutagenesis (25). Thus, although the very nature of the protease-resistant, Bcl-xL-bound form of Bax requires further characterization, it is tempting to speculate that it may be close, on some aspects, to that of an active Bax. Microinjection experiments nevertheless suggest that, as long as it is bound to Bcl-xL, Bax does not exert its apoptotic activity (Fig. 5). Our immunoprecipitation experiments using conformation-specific antibodies further confirms this by showing that Bcl-xL-bound Bax does not fulfill all the criteria of an active Bax (Fig. 6). Indeed, tBid-activated Bax, but not native or Bcl-xL-bound Bax, can immunoreact with the well documented conformation-specific 6A7 antibody and with a polyclonal antibody raised against the minimal Bax BH3 domain (TL41). It was initially proposed that the epitope for the 6A7 antibody might be in the vicinity of the BH3 dimerization domain of Bax (38). Our observation that exposure of the 6A7 epitope occurs concomitantly with the exposure of an epitope within the BH3 domain of Bax is certainly consistent with this view. It also indicates that the BH3 domain of Bax is hidden within the native protein but is exposed when Bax is active. Dimerization of Bax with Bcl-xL may prevent both the 6A7 and the TL41 epitopes from being exposed, or alternatively, it may mask these epitopes on Bax (38). When specifically released from Bcl-xL by BH3 peptides, however, Bax recapitulates the features of tBid-activated Bax, as it becomes accessible to both conformation-specific antibodies and retains its resistance to mild proteolysis. This is most consistent with a model in which Bcl-xL binds to inactive Bax, modifies it, and then releases active Bax in response to a BH3 peptide. One provocative implication from this is that Bcl-xL should cooperate with ligands of its BH3 domain-binding pocket to induce Bax-mediated apoptosis, which may explain why cells overexpressing Bcl-xL are more sensitive to induction of apoptosis by a BH3 mimetic (47).

In summary, we have analyzed in this study the apoptotic function of a synthetic BH3 peptide comprising sufficient information to antagonize functionally anti-apoptotic Bcl-2 family members, and we found that it does not function as a death agonist of Bax. This BH3 peptide can nonetheless promote Bax-dependent apoptosis by interfering with the ability of Bax to interact with, and be suppressed by, Bcl-xL. There is a growing list of small molecules that have been selected for their ability to occupy the BH3-binding pocket of anti-apoptotic Bcl-2 family members and that act as true BH3 mimetics (6, 45, 47). Our results indicate that such molecules may efficiently induce apoptosis in cells expressing high levels of the anti-apoptotic Bcl-2 family members by triggering the release of active multidomain pro-apoptotic proteins from these survival proteins.


    FOOTNOTES
 
* This work was supported in part by Association pour la Recherche Contre le Cancer Grant 4455 (to P. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by a fellowship from the Ligue Départementale Contre le Cancer Doubs/Montbeliard. Back

|| To whom correspondence should be addressed: INSERM U419, 9 Quai Moncousu, 44035 Nantes, France. Tel.: 33-24-008-4083; Fax: 33-24-008-4082; E-mail: pjuin{at}nantes.inserm.fr.

1 The abbreviations used are: PBS, phosphate-buffered saline; FCS, fetal calf serum; MOPS, 4-morpholinepropanesulfonic acid; IVTR, in vitro translated radiolabeled; GST, glutathione S-transferase. Back

2 C. Moreau, P.-F. Cartron, A. Hunt, K. Meflah, D. R. Green, G. Evan, F. M. Vallette, and P. Juin, unpublished data. Back


    ACKNOWLEDGMENTS
 
We thank Dr. L. Oliver for critical reading of this manuscript.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Green, D. R. (2000) Cell 102, 1–4[Medline] [Order article via Infotrieve]
  2. Adams, J. M., and Cory, S. (1998) Science 281, 1322–1326[Abstract/Free Full Text]
  3. Degterev, A., Boyce, M., and Yuan, J. (2001) J. Cell Biol. 155, 695–698[Abstract/Free Full Text]
  4. Oltvai, Z., Milliman, C., and Korsmeyer, S. (1993) Cell 74, 609–619[Medline] [Order article via Infotrieve]
  5. Sattler, M., Liang, H., Nettesheim, D., Meadows, R. P., Harlan, J. E., Eberstadt, M., Yoon, H. S., Shuker, S. B., Chang, B. S., Minn, A. J., Thompson, C. B., and Fesik, S. W. (1997) Science 275, 983–986[Abstract/Free Full Text]
  6. Degterev, A., Lugovskoy, A., Cardone, M., Mulley, B., Wagner, G., Mitchison, T., and Yuan, J. (2001) Nat. Cell Biol. 3, 173–182[CrossRef][Medline] [Order article via Infotrieve]
  7. Kelekar, A., and Thompson, C. (1998) Trends Cell Biol. 8, 324–330[CrossRef][Medline] [Order article via Infotrieve]
  8. Hunt, A., and Evan, G. (2001) Science 293, 1784–1785[Free Full Text]
  9. Oda, E., Ohki, R., Murasawa, H., Nemoto, J., Shibue, T., Yamashita, T., Tokino, T., Taniguchi, T., and Tanaka, N. (2000) Science 288, 1053–1058[Abstract/Free Full Text]
  10. Yu, J., Zhang, L., Hwang, P., Kinzler, K., and Vogelstein, B. (2001) Mol. Cell 7, 673–682[Medline] [Order article via Infotrieve]
  11. Nakano, K., and Vousden, K. H. (2001) Mol. Cell 7, 683–694[Medline] [Order article via Infotrieve]
  12. Li, H., Zhu, H., Xu, C. J., and Yuan, J. (1998) Cell 94, 491–501[Medline] [Order article via Infotrieve]
  13. Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. (1998) Cell 94, 481–490[Medline] [Order article via Infotrieve]
  14. Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M. E. (1997) Cell 91, 231–241[Medline] [Order article via Infotrieve]
  15. Wei, M. C., Zong, W. X., Cheng, E. H., Lindsten, T., Panoutsakopoulou, V., Ross, A. J., Roth, K. A., MacGregor, G. R., Thompson, C. B., and Korsmeyer, S. J. (2001) Science 292, 727–730[Abstract/Free Full Text]
  16. Zong, W. X., Lindsten, T., Ross, A. J., MacGregor, G. R., and Thompson, C. B. (2001) Genes Dev. 15, 1481–1486[Abstract/Free Full Text]
  17. Cheng, E. H., Wei, M. C., Weiler, S., Flavell, R. A., Mak, T. W., Lindsten, T., and Korsmeyer, S. J. (2001) Mol. Cell 8, 705–711[CrossRef][Medline] [Order article via Infotrieve]
  18. Wolter, K. G., Hsu, Y. T., Smith, C. L., Nechushtan, A., Xi, X. G., and Youle, R. J. (1997) J. Cell Biol. 139, 1281–1292[Abstract/Free Full Text]
  19. Wang, K., Yin, X. M., Chao, D. T., Milliman, C. L., and Korsmeyer, S. J. (1996) Genes Dev. 10, 2859–2869[Abstract]
  20. Desagher, S., Osen-Sand, A., Nichols, A., Eskes, R., Montessuit, S., Lauper, S., Maundrell, K., Antonsson, B., and Martinou, J. C. (1999) J. Cell Biol. 144, 891–901[Abstract/Free Full Text]
  21. Korsmeyer, S. J., Wei, M. C., Saito, M., Weiler, S., Oh, K. J., and Schlesinger, P. H. (2000) Cell Death Differ. 7, 1166–1173[CrossRef][Medline] [Order article via Infotrieve]
  22. Letai, A., Bassik, M., Walensky, L., Sorcinelli, M., Weiler, S., and Korsmeyer, S. (2002) Cancer Cell 2, 183–192[CrossRef][Medline] [Order article via Infotrieve]
  23. Kuwana, T., Mackey, M. R., Perkins, G., Ellisman, M. H., Latterich, M., Schneiter, R., Green, D. R., and Newmeyer, D. D. (2002) Cell 111, 331–342[Medline] [Order article via Infotrieve]
  24. Juin, P., Hunt, A., Littlewood, T., Griffiths, B., Brown, L., Korsmeyer, S., and Evan, G. (2002) Mol. Cell. Biol. 22, 6158–6169[Abstract/Free Full Text]
  25. Cartron, P. F., Moreau, C., Oliver, L., Mayat, E., Meflah, K., and Vallette, F. M. (2002) FEBS Lett. 512, 95–100[CrossRef][Medline] [Order article via Infotrieve]
  26. von Ahsen, O., Renken, C., Perkins, G., Kluck, R. M., Bossy-Wetzel, E., and Newmeyer, D. D. (2000) J. Cell Biol. 150, 1027–1036[Abstract/Free Full Text]
  27. Juin, P., Hueber, A. O., Littlewood, T., and Evan, G. (1999) Genes Dev. 13, 1367–1381[Abstract/Free Full Text]
  28. Tremblais, K., Oliver, L., Juin, P., Le Cabellec, T. M., Meflah, K., and Vallette, F. M. (1999) Biochem. Biophys. Res. Commun. 260, 582–591[CrossRef][Medline] [Order article via Infotrieve]
  29. Cosulich, S. C., Worrall, V., Hedge, P. J., Green, S., and Clarke, P. R. (1997) Curr. Biol. 7, 913–920[Medline] [Order article via Infotrieve]
  30. Antonsson, B., Montessuit, S., Lauper, S., Eskes, R., and Martinou, J. C. (2000) Biochem. J. 345, 271–278[CrossRef][Medline] [Order article via Infotrieve]
  31. Madesh, M., Antonsson, B., Srinivasula, S. M., Alnemri, E. S., and Hajnoczky, G. (2002) J. Biol. Chem. 277, 5651–5659[Abstract/Free Full Text]
  32. Ruffolo, S. C., Breckenridge, D. G., Nguyen, M., Goping, I. S., Gross, A., Korsmeyer, S. J., Li, H., Yuan, J., and Shore, G. C. (2000) Cell Death Differ. 7, 1101–1108[CrossRef][Medline] [Order article via Infotrieve]
  33. Nomura, M., Shimizu, S., Ito, T., Narita, M., Matsuda, H., and Tsujimoto, Y. (1999) Cancer Res. 59, 5542–5548[Abstract/Free Full Text]
  34. Goping, I. S., Gross, A., Lavoie, J. N., Nguyen, M., Jemmerson, R., Roth, K., Korsmeyer, S. J., and Shore, G. C. (1998) J. Cell Biol. 143, 207–215[Abstract/Free Full Text]
  35. Perez, D., and White, E. (2000) Mol. Cell 6, 53–63[Medline] [Order article via Infotrieve]
  36. Cartron, P. F., Oliver, L., Martin, S., Moreau, C., LeCabellec, M. T., Jezequel, P., Meflah, K., and Vallette, F. M. (2002) Hum. Mol. Genet. 11, 675–687[Abstract/Free Full Text]
  37. Hsu, Y.-T., Wolter, K. G., and Youle, R. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3668–3672[Abstract/Free Full Text]
  38. Hsu, Y. T., and Youle, R. J. (1997) J. Biol. Chem. 272, 13829–13834[Abstract/Free Full Text]
  39. Shangary, S., and Johnson, D. E. (2002) Biochemistry 41, 9485–9495[CrossRef][Medline] [Order article via Infotrieve]
  40. Finnegan, N. M., Curtin, J. F., Prevost, G., Morgan, B., and Cotter, T. G. (2001) Br. J. Cancer 85, 115–121[CrossRef][Medline] [Order article via Infotrieve]
  41. Terradillos, O., Montessuit, S., Huang, D. C., and Martinou, J. C. (2002) FEBS Lett. 522, 29–34[CrossRef][Medline] [Order article via Infotrieve]
  42. Eskes, R., Desagher, S., Antonsson, B., and Martinou, J. C. (2000) Mol. Cell. Biol. 20, 929–935[Abstract/Free Full Text]
  43. Gross, A., Jockel, J., Wei, M. C., and Korsmeyer, S. J. (1998) EMBO J. 17, 3878–3885[Abstract/Free Full Text]
  44. Mahajan, N. P., Linder, K., Berry, G., Gordon, G. W., Heim, R., and Herman, B. (1998) Nat. Biotechnol. 16, 547–552[Medline] [Order article via Infotrieve]
  45. Enyedy, I. J., Ling, Y., Nacro, K., Tomita, Y., Wu, X., Cao, Y., Guo, R., Li, B., Zhu, X., Huang, Y., Long, Y. Q., Roller, P. P., Yang, D., and Wang, S. (2001) J. Med. Chem. 44, 4313–4324[CrossRef][Medline] [Order article via Infotrieve]
  46. Suzuki, M., Youle, R. J., and Tjandra, N. (2000) Cell 103, 645–654[Medline] [Order article via Infotrieve]
  47. Tzung, S. P., Kim, K. M., Basanez, G., Giedt, C. D., Simon, J., Zimmerberg, J., Zhang, K. Y., and Hockenbery, D. M. (2001) Nat. Cell Biol. 3, 183–191[CrossRef][Medline] [Order article via Infotrieve]