Bcl-2 on the Endoplasmic Reticulum Regulates Bax Activity by Binding to BH3-only Proteins*

Michael J. Thomenius, Nancy S. Wang, Edmunds Z. Reineks, Zhengqi Wang, and Clark W. DistelhorstDagger

From the Departments of Medicine and Pharmacology, Comprehensive Cancer Center, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland, Ohio 44106

Received for publication, August 29, 2002, and in revised form, November 22, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bcl-2 family members have been shown to be key mediators of apoptosis as either pro- or anti-apoptotic factors. It is thought that both classes of Bcl-2 family members act at the level of the mitochondria to regulate apoptosis, although the founding anti-apoptotic family member, Bcl-2 is localized to the endoplasmic reticulum (ER), mitochondrial, and nuclear membranes. In order to better understand the effect of Bcl-2 localization on its activity, we have utilized a Bcl-2 mutant that localizes only to the ER membrane, designated Bcl-2Cb5. Bcl-2Cb5 was expressed in MDA-MB-468 cells, which protected against apoptosis induced by the kinase inhibitor, staurosporine. Data presented here show that Bcl-2Cb5 inhibits this process by blocking Bax activation and cytochrome c release. Furthermore, we show that Bcl-2Cb5 can inhibit the activation of a constitutively mitochondrial mutant of Bax, indicating that an intermediate between Bcl-2 on the ER and Bax on the mitochondria must exist. We demonstrate that this intermediate is likely a BH3-only subfamily member. Data presented here show that Bcl-2Cb5 can sequester a constitutively active form of Bad (Bad3A) from the mitochondria and prevent it from activating Bax. These data suggest that Bcl-2 indirectly protects mitochondrial membranes from Bax, via BH3-only proteins.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bcl-2 family members comprise a group of proteins that regulate apoptosis. Bcl-2 family members can be grouped into three categories, the anti-apoptotic members including Bcl-2, Bcl-xL, and Mcl-1, the multidomain pro-apoptotic members such as Bax and Bak, and the BH3 domain only proteins such as Bim, Bid, Bad, and Bik (1). This family of proteins converges on the mitochondria to regulate events like cytochrome c release or mitochondrial membrane depolarization that ultimately determine cell fate.

The Bax subfamily has been implicated as the "gateway" to apoptosis (2). These proteins are required to initiate most forms of apoptosis (2, 3). During apoptosis, Bax translocates to the mitochondria and oligomerizes, causing cytochrome c release from the mitochondria (4-6). Translocation and oligomerization of Bax are preceded by a conformational change (7). Little is known about what induces this conformational change, although it has been shown that certain members of the BH3 subfamily can bind to and activate Bax and Bak (6, 8). Some evidence suggests that changes in mitochondrial membrane potential induce the activation of Bax (9). A prominent theory for Bax activation suggests that anti-apoptotic Bcl-2 family members inhibit Bax conformational change by direct interaction (10), although some studies suggest that Bcl-2 and Bcl-xL can inhibit apoptosis without directly interacting with Bax (11, 12).

The BH3 subfamily members are characterized by having only one of the four Bcl-2 homology domains. These proteins induce cell death at a point upstream of mitochondria and Bax subfamily activation (13, 3). BH3 proteins are either induced transcriptionally (14) or activated when an apoptotic signal is received (15-17). They are thought to induce apoptosis by binding to anti-apoptotic Bcl-2 family members and inhibiting their activity (18, 19) or by binding pro-apoptotic family members and inducing their activity (8, 20).

Anti-apoptotic Bcl-2 family members function at least in part by inhibiting cytochrome c release from the mitochondria. They perform this task by preventing translocation and/or activation of Bax-like proteins on the mitochondria (21, 22), however the mechanism of this inhibition is not entirely clear. In addition, it is not certain that the role of anti-apoptotic Bcl-2 family members is limited to the mitochondria. Much emphasis has been placed on Bcl-2 function on the mitochondria, although it has been reported that wild type Bcl-2 localizes to the mitochondria, endoplasmic reticulum (ER),1 and nuclear membranes (23) and there is growing evidence that the ER is important in apoptosis. Significant findings have been gathered suggesting a role for Bcl-2 family members in regulating ER calcium during apoptosis (24-28). Recent evidence suggests that pro-apoptotic Bcl-2 family members like Bik (29) or Bax and Bak (27) can reside on the ER to regulate apoptosis. Also, many apoptosis-regulating proteins such as caspase-12 (30) and Bap-31 (31) reside on or in the ER. It has also been reported that Bcl-2 targeted solely to the ER is functional (32). This ER-targeted form of Bcl-2, Bcl-2Cb5 is protective against many forms of apoptosis and has been shown to inhibit caspase activation and cytochrome c release (33). Previous work has shown that Bcl-2Cb5 can inhibit apoptosis induced by overexpression of Bax (34), suggesting that Bcl-2 can still inhibit the actions of pro-apoptotic family members when localized to the ER. These data demonstrate that Bcl-2, localized to the ER can inhibit mitochondrial events of apoptosis, which point to the presence of an intermediate between Bcl-2 and the mitochondria.

In the work reported here, we investigated the mechanism by which Bcl-2 on the ER membrane can inhibit mitochondrial events of apoptosis. We utilized the cytochrome b5 (Cb5) targeting sequence to target Bcl-2 to the ER, to examine changes in activation and subcellular distribution of pro-apoptotic Bcl-2 family members. We report that Bcl-2Cb5 can inhibit the conformational change of Bax from the ER, suggesting that Bcl-2 inhibits Bax by a mechanism other than direct interaction. Also, evidence presented here indicates that Bax activation, induced by a constitutively active form of Bad (Bad3A) is inhibited by Bcl-2 on the ER. We demonstrate that this inhibition is due to a direct interaction between Bad3A and Bcl-2Cb5.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA Constructs-- The Bcl-2Cb5 chimera was generated in both FLAG (FL) and GFP tag vectors as described in Ref. 34. pCMV-tag2B, pCMV-tag3A (Stratagene), and pEMD-C1 parental vectors were used. Myc-tagged Bax was generated by subcloning the murine Bax cDNA into the EcoRI and XhoI sites of the pCMV-tag3A vector. Mutations in FL-Bcl-2Cb5 and Myc-Bax were generated using the QuickChangeTM site-directed mutagenesis kit (Stratagene). HA-Bad3A was generated as described (35) in pCDNA3 vector (Stratagene).

Cell Culture-- MDA-MB-468 cells were obtained from American Type Culture Collection. They were cultured in IMEM (BIOSOURCE) with 10% fetal bovine serum, 1% L-glutamine, and 1% non-essential amino acids. MCF7 cells were obtained from American Type Culture Collection. They were cultured in minimal essential medium (MEM) (Invitrogen) with 10% fetal bovine serum, 1% L-glutamine, 1 mM sodium pyruvate. All cells were grown in 7% CO2 at 37 °C.

Transfection-- All transfections were performed with the FuGENE 6 transfection reagent (Roche Molecular Biochemicals) following the manufacturer's protocols. A FuGENE 6 to DNA ratio of 3 µl:2 µg was maintained for all experiments. The FuGENE was diluted in Optimem (Invitrogen) prior to reaction with DNA. Stable transfectants of pCMV-tag2B, FL-Bcl-2Cb5, and FL-Bcl-2 vectors into MDA-MB-468 were generated by transient transfection followed by geneticin selection. Following transfection, cells were grown in 0.6 mg/ml geneticin and kept under selective pressure while in culture.

Transient transfections of MCF7 cells were performed using FuGENE 6. Cells for co-immunoprecipitation and cross-linking experiments were grown on 100-mm dishes to 50-60% confluency. They were transfected with 8.4 µl of FuGENE 6, 280 µl of Optimem, and 5.6 µg of DNA and left overnight. Cells for microscopy were grown on 4-chamber or 2-chamber Lab-Tek II Chambered Coverglass Slides (Nalge Nunc) to 50-60% confluency. They were transfected using 1.5 µl of FuGENE 6, 50 µl of Optimem, and 1 µg of DNA and incubated overnight.

Confocal and Epifluorescence Microscopy-- Fluorescence images were taken on a Zeiss Axiovert S100 epifluorescence microscope (Zeiss), with a Zeiss 63Xoil/1.4N.A. Plan Apochromat objective or a Zeiss 40Xoil/1.3N.A. Fluar objective. GFP fluorescence and Alexa 488 (Molecular Probes) fluorescence were detected with an XF23 filter cube (excitation = 485 nm, emission = 535 nm). Filter sets were obtained from Omega Optical. MitoTracker Red and Alexa 594 (Molecular Probes) fluorescence was detected with XF67 dichroic/emission filter and 575DF25 excitation filter (excitation = 575 nm, emission = 630 nm). Images were taken on a Hamamatsu ORCA C4742-95-cooled CCD camera operating with Simple PCI software (Compix, Inc.). Nearest neighbors deconvolution was performed using Simple PCI software. Confocal Microscopy was performed using a Zeiss LSM 510 confocal microscope. Images were captured with the Zeiss LSM510 v3.0 software and viewed using the Zeiss LSM 5 Image Browser. Images were processed using Adobe Photoshop 7.0.

Caspase 3 Activation Assay-- MDA-MB-468 cells were grown on 100-mm dishes and treated with 500 nM STS for 6 h. The media were collected, and the remaining cells were washed with PBS. The wash was collected. The remaining cells were trypsinized and combined with the media and wash. The cells were spun down, washed in PBS and resuspended in 4% paraformaldehyde (PF) and placed in 1.5-ml Eppendorf tubes. They were then incubated for 10 min at room temperature. The cells were then spun down, and the PF was removed. The cells were resuspended in ice-cold methanol and incubated for 20 min. The tubes were spun down, the methanol was removed, and the cells were washed in PBS. The cells were incubated in PBS with 5% goat serum for 30 min, followed by a 1-h incubation with a hamster monoclonal anti-human Bcl-2 antibody (6C8 clone, BD PharMingen) and a rabbit anti-active caspase 3 antibody (BD PharMingen). Cells were spun down, washed, and incubated with goat anti-hamster Alexa 647 and goat anti-rabbit Alexa 488. Cells were analyzed on a Becton-Dickinson LSR Flow Cytometer. Flow cytometry data were analyzed using Winlist (Verify Software House) and Microsoft Excel 2000.

Colocalization-- MDA-MB-468 cells stably transfected with FL-Bcl-2Cb5 or FL-Bcl-2 were grown in 2-chamber Lab-Tek II Chambered Coverglass Slides to 30% confluency. They were then fixed with 4% PF for 10 min. PF was removed, and cells were permeabilized with 0.5% Triton X-100 for 10 min. The cells were washed and incubated with PBS with 5% goat serum for 30 min. The cells were then incubated for 1 h with a 1:500 dilution of anti-Bcl-2 antibody (6C8 clone), followed by two washes in PBS with 5% goat serum. They were then incubated with goat anti-hamster Alexa 594 for 30 min. The cells were washed twice with PBS with 5% goat serum and then incubated with a monoclonal mouse anti-cytochrome c antibody (6H2.B4 clone, BD PharMingen) directly conjugated to Alexa 488. Conjugation was performed using the ZenonTM labeling kit (Molecular Probes). Cells were imaged using confocal microscopy as described previously.

MCF7 cells were transfected with MycBaxS184V or cotransfected with GFPBcl-2Cb5 and MycBaxS184V and incubated overnight. The cells were fixed with PF and stained with a monoclonal anti-Myc antibody (Clontech) at a 1:500 dilution as described previously. Cells were also stained with an antibody to cytochrome c oxidase IV (CoxIV) (Molecular Probes) as a mitochondrial marker. MycBaxS184V was visualized using a goat anti-mouse-Alexa 594 secondary antibody. CoxIV was visualized by directly binding the primary antibody with Alexa 488 using the ZenonTM labeling kit. Images of the cells were taken using epifluorescence microscopy as described earlier.

MCF7 cells were cotransfected with GFP-Cb5/HA-Bad3A, GFP-Cb5/pcDNA3, GFP-Bcl-2Cb5/HA-Bad3A, or GFP-Bcl-2Cb5/ pcDNA3 in 1:1 ratios. The cells were grown overnight (about 15 h) following the transfection. The cells were then fixed and permeabilized as described previously. They were then incubated with a mouse anti-HA antibody, conjugated to Alexa 594 at a 1:500 dilution. The cells were washed with PBS and imaged with epifluorescence microscopy as described previously.

Colocalization images were taken with the Zeiss 63Xoil/1.4N.A. Plan Apochromat objective as 0.3-µm increment Z-stacks with as many as 10 images. The images were then run through a nearest neighbors deconvolution algorithm (Compix Inc.) to remove out of focus light.

Cytochrome c Release-- MDA-MB-468 cells transfected with Bcl-2Cb5, Bcl-2, or pCMV-tag2B were treated with 500 nM STS for 6 h. The cells were then fixed and stained as described for colocalization experiments. The cells were incubated with the anti-Bcl-2 antibody (6C8 clone) and anti-cytochrome c antibody (6H2.B4 clone), followed by incubation with goat anti-hamster Alexa 488 and goat anti-mouse Alexa 594. The cells were then imaged with epifluoresence illumination with a Zeiss 40Xoil/1.3N.A. Fluar objective.

Cross-linking-- MCF7 cells were grown to 50-60% confluency on a 100-mm dish and were cotransfected with Myc-BaxS184V/GFPCb5, Myc-BaxS184V/GFP-Bcl-2Cb5, pCMV-tag3A/GFPCb5, or pCMV-tag3A/GFP-Bcl-2Cb5 in a 3:2 ratio. After 12 h of transfection, they were incubated for 30 min with 1 mM BMH (Pierce Chemical) in media at 37 °C. The cells were trypsinized from the dish and washed in PBS. The cells were lysed (1% Triton, 0.1% SDS, 50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, phenylmethylsulfonyl fluoride, aprotinin) and then incubated on ice for 30 min. They were then spun down at 300 × g for 5 min at 4 °C to spin down nuclei and unbroken cells. Protein concentration was measured using the Bio-Rad protein assay. The lysate was boiled in sample buffer and run on a 10% SDS-PAGE gel. The gel was transferred onto a polyvinylidene difluoride membrane overnight. The membrane was blocked in 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBST). The membrane was then probed with a 1:1000 dilution of anti-Myc antibody (Clontech) in TBST with 5% nonfat milk. It was then washed three times in TBST, followed by a 30-min incubation with a 1:1000 dilution of goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences). The blot was then exposed on x-ray film (Fujifilm) and scanned into tiff format and processed using Adobe Photoshop 7.0.

Immunoprecipitation-- MCF7 cells were transfected and harvested as described in previous experiments with HA-Bad3A/FL-Bcl-2Cb5, pcDNA3/FL-Bcl-2Cb5, pcDNA3/pCMV-tag2B, HA-Bad3A/pCMV-tag2B, pcDNA3/FL-G145Ecb5, or HA-Bad3A/FL-G145Ecb5. Harvested cells were placed in lysis buffer and homogenized using a Tight Dounce homogenizer (Wheaton) with 25 strokes. The lysates were pre-incubated with 40 µl of protein G-agarose (Roche Molecular Biochemicals) for 3 h to remove nonspecific binding. The lysates were then incubated with anti-Bcl-2 antibody (6C8 clone) for 1 h. 40 µl of protein G-agarose was then added and incubated overnight. The protein G-agarose was spun down and washed once in Buffer 1 (Lysis Buffer) (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor mixture (Roche Molecular Biochemicals)). The samples were washed again in Buffer 2 (50 mM Tris-HCl, pH7.5, 500 mM NaCl, 0.1% Nonidet P-40, and 0.05% sodium deoxycholate) and then once more in Buffer 3 (10 mM Tris-HCl, pH 7.5, and 0.1% Nonidet P-40, and 0.05% sodium deoxycholate). The protein G-agarose was then boiled in sample buffer. The sample was run on an SDS-PAGE gel along with a cell lysate. Western blots were performed as described previously with a 1:2500 dilution of mouse anti-FLAG antibody (Stratagene) and 1:1000 dilution of anti-HA antibody (Covance, HA.11). An anti-mouse horseradish peroxidase secondary antibody was used.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ER-targeted Bcl-2 Localizes to the ER and Not the Mitochondria-- In order to study the role of Bcl-2 on the ER, the C-terminal insertion sequence of Bcl-2 was replaced with that of cytochrome b5, which was shown to restrict the subcellular localization of Bcl-2 to the ER (32). This finding was confirmed using confocal microscopy (Fig. 1). Fig. 1 shows MDA-MB-468 cells stably transfected with either wild type Bcl-2 or ER-targeted Bcl-2 (Bcl-2Cb5). Bcl-2 was imaged using a hamster monoclonal antibody and an Alexa 488-conjugated secondary antibody. In addition, these cells were stained with antibodies to calnexin, which is found in the ER lumen (Fig. 1, A and C) and cytochrome c, which is found in the intermembrane space of the mitochondria in healthy cells (Fig. 1, B and D). These images show that stably overexpressed Bcl-2 has strong colocalization with cytochrome c, but also localizes to the ER. Bcl-2Cb5 does not colocalize with cytochrome c but colocalizes strongly with calnexin, indicating that Bcl-2Cb5 localizes primarily to the ER and not the mitochondria.


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Fig. 1.   When stably transfected in MDA-MB-468 cells, FL-Bcl-2Cb5 goes to the ER and not the mitochondria. MDA-MB-468 cells transfected with FL-Bcl-2Cb5 (A and B) or FL-Bcl-2 (C and D) were fixed with PF and incubated with antibodies to calnexin (A and C), cytochrome c (B and D), and Bcl-2 (A-D). Cytochrome c and calnexin are shown in green and Bcl-2 is shown in red. Cytochrome c antibody was directly conjugated to Alexa 488, and Bcl-2 was stained with a secondary antibody conjugated to Alexa 594. Overlap is shown on the far right. Cells were examined using confocal microscopy. Scale bar is 10 microns.

ER-Bcl-2 Inhibits Caspase 3 Activation and Cytochrome c Release Induced by STS-- To study the effects of Bcl-2Cb5 on apoptosis, we assessed caspase-3 activation in cells undergoing apoptosis following treatment with staurosporine (STS). MDA-MB-468 cells, overexpressing Bcl-2 or Bcl-2Cb5 in a mixed population were treated with 500 nM STS for 6 h. These cells were fixed and stained with antibodies to Bcl-2 and active caspase 3. Transfected cells were identified by Bcl-2 antibody staining. Our findings show that expression of Bcl-2 or Bcl-2Cb5 protects cells against STS-induced cell death. Fig. 2 shows that cells transfected with Bcl-2Cb5 or Bcl-2 have low active caspase 3 levels (Fig. 2, A and B) and do not have apoptotic nuclei (Fig. 2A) following STS treatment in comparison to untransfected cells. This finding is consistent with previous results showing the protective effect of Bcl-2Cb5 (32).


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Fig. 2.   Bcl-2Cb5 overexpressing MDA-MB-468 cells are resistant to STS-induced caspase activation and apoptosis. MDA-MB-468 cells, stably transfected with pCMV-tag2B, FL-Bcl-2Cb5, or FL-Bcl-2 were treated with 500 nM STS or Me2SO for 6 h. They were then incubated with antibodies to Bcl-2 and active caspase 3 with Alexa 647 and 488 secondary antibodies. A, FL-Bcl-2Cb5-transfected cells were first analyzed using fluorescence microscopy. FL-Bcl-2Cb5 stained with Alexa 647 is shown in red (left) and active caspase 3 stained with Alexa 488 is shown in green (middle). DAPI is shown in white (right). B, cells were also analyzed by flow cytometry. Cells overexpressing Bcl-2 were gated using Bcl-2 antibody staining with Alexa 647 and analyzed for active caspase 3 expression. Experiments were done in triplicate. The error bars represent the S.E. of the mean.

We next assessed cytochrome c release in cells treated with STS to demonstrate the effect of ER-Bcl-2 on mitochondrial events of apoptosis. MDA-MB-468 cells were treated with STS and the caspase inhibitor, z-VAD-FMK. z-VAD-FMK was used to maintain cell integrity during the experiment. Cytochrome c release was assessed using immunocytochemistry with paraformaldehyde fixation. Transfected cells were identified by costaining for Bcl-2. Cells overexpressing Bcl-2Cb5 or Bcl-2 did not release cytochrome c following STS treatment (Fig. 3A). These data indicate that mitochondrial events of apoptosis are inhibited by Bcl-2Cb5 and are consistent with the hypothesis that there may be some intermediary between the ER and mitochondria, as suggested by Hacki et al. (33).


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Fig. 3.   Bcl-2Cb5 inhibits Bax activation and cytochrome c release induced by STS. A, MDA-MB-468 cells transfected with Bcl-2Cb5 or Bcl-2 were treated with 500 nM STS for 6 h and incubated with antibodies to Bcl-2 (left panel) and cytochrome c (right panel) and secondary antibodies conjugated to Alexa 488 and Alexa 594. Cells were analyzed by fluorescence microscopy. Blue arrows indicate cells that have been not been transfected, and red arrows indicate cells that have been transfected. B, MDA-MB-468 cells, transfected with Bcl-2Cb5, Bcl-2, or empty vector were treated with 500 nM STS for 6 h and incubated with antibodies to Bcl-2 (shown in red) and a secondary antibody conjugated to Alexa 594. They were also incubated with the Bax6A7 antibody directly conjugated to Alexa 488 (shown in green). Treated and untreated vector-transfected cells are shown on top. Bcl-2- and Bcl-2Cb5-transfected cells are shown on the bottom. Blue arrows indicate cells that have not been transfected, and red arrows indicate cells that have been transfected.

Bcl-2Cb5 Inhibits the Activation and Oligomerization of Bax-- To address the mechanism of Bcl-2Cb5-mediated protection against apoptosis, we measured the activity of the pro-apoptotic Bcl-2 family member Bax using the 6A7 antibody, which preferentially recognizes the active conformation of Bax (36). MDA-MB-468 cells, stably transfected with Bcl-2Cb5 or Bcl-2 in mixed population were treated with 500 nM STS and z-VAD-FMK for 6 h. They were then stained with a Bcl-2 antibody and the 6A7 antibody. We found that vector control cells treated with STS and z-VAD-FMK stained positively for 6A7. Cells overexpressing Bcl-2Cb5 or Bcl-2 did not stain with the 6A7 antibody (Fig. 3B). These data suggest that Bcl-2Cb5 is inhibiting the activation of Bax, indicating that there is an intermediate between Bcl-2Cb5 and Bax, as the two proteins do not colocalize.

We next examined whether Bcl-2Cb5 could inhibit the activation of a constitutively mitochondrial form of Bax. This was done so that we could separate the proteins spatially, ensuring that they cannot interact with one another. For these experiments, we transiently transfected the S184V mutant of Bax into MCF7 cells. This form of Bax has a constitutive localization to the mitochondria (7). We first confirmed that Bcl-2Cb5 localized to the ER and not mitochondria following transient transfection in the MCF7 cells, by transfecting GFP-Bcl-2Cb5 and then staining for cytochrome c. GFP-Bcl-2Cb5 and GFP-Cb5 have ER patterns that are distinct from cytochrome c (Fig. 4A). Mitochondrial localization of BaxS184V was confirmed by transfecting Myc-tagged BaxS184V (Myc-BaxS184V) into MCF7 and costaining with an antibody to Myc and an antibody to the mitochondrial protein CoxIV (Fig. 4B). In addition, MCF7 cells were cotransfected with GFP-Bcl-2Cb5 and Myc-BaxS184V (Fig. 4B). Fig. 4B shows that Myc-BaxS184V localizes to the mitochondria and has a different subcellular localization from GFPBcl-2Cb5.


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Fig. 4.   Bcl-2Cb5 inhibits oligomerization of the constitutively mitochondrial Bax mutant, BaxS184V. A, GFP-Bcl-2Cb5 and GFP-Cb5 go to the ER following transient transfection in MCF7 cells. Cells were transfected with GFP-Bcl-2Cb5 or GFPCb5. They were then fixed and permeabilized with 4% PF and 0.5% Triton. The cells were then stained with an antibody to cytochrome c. GFP (left) is shown in green. An Alexa 594 secondary antibody was used to visualize cytochrome c (middle), shown in red. Overlap is shown on the right. B, Myc-BaxS184V constitutively goes to the mitochondria in MCF7 cells and does not colocalize with Bcl-2Cb5. MCF7 cells were transiently transfected with Myc-BaxS184V or cotransfected with Myc-BaxS184V and GFP-Bcl-2Cb5. Cells transfected with MycS184V were stained with an anti-Myc antibody and a CoxIV antibody. Cells cotransfected with Myc-BaxS184V and GFP-Bcl-2Cb5 were stained with just the anti-Myc antibody. CoxIV (bottom) and GFP-Bcl-2Cb5 (top) are shown in green on the far left. Myc-BaxS184V is shown in red in the middle and the overlap is shown on the far right. C, GFP-Bcl-2Cb5 inhibits Myc-BaxS184V oligomerization. MCF7 cells were transiently cotransfected with Myc-BaxS184V and either GFP-Cb5 or GFP-Bcl-2Cb5. These cells were then treated with the homo-bifunctional sulfhydryl cross-linker bismaleimidohexane (BMH). The cells were then lysed, protein was collected, and run on SDS-PAGE. A Western blot was then analyzed using an antibody against the Myc epitope.

The homobifunctional sulfhydryl cross-linker, bismaleimidohexane (BMH) was used to assess Bax oligomerization in MCF7 cells following S184V transfection (Fig. 4C). MCF7 cells were cotransfected with GFP-Cb5 and pCMV-tag3A (lane 1), GFP-Bcl-2Cb5 and pCMV-tag3A (lane 2), GFP-Cb5 and Myc-BaxS184V (lane 3), or GFP-Bcl-2Cb5 and Myc-BaxS184V (lane 4) and cultured overnight. The cells were then treated with 1 mM BMH and subsequently lysed and run on SDS-PAGE. A Western blot was then performed using an anti-Myc antibody. As seen in Fig. 4C, there is a striking decrease in the amount of higher molecular weight Bax complexes in cells cotransfected with GFP-Bcl-2Cb5 than in cells transfected with GFP-Cb5. Based on these results, it appears that Bcl-2Cb5 can inhibit BaxS184V oligomerization without a direct interaction as the two proteins are spatially separated in the cell. Based on lanes 3 and 4 in the no BMH blot, it appears that there is an increased amount of MycBaxS184V with transfection of GFP-Bcl-2Cb5. This is most likely due to protection by GFP-Bcl-2Cb5 in cells over-expressing Myc-BaxS184V and only reaffirms the conclusion that GFP-Bcl-2Cb5 is inhibiting the action of Myc-BaxS184V.

Bcl-2Cb5 Inhibits Bax Activation Induced by Bad3A Overexpression-- The inhibition of BaxS184V oligomerization by Bcl-2Cb5 suggests that an intermediate is involved. Likely candidates for this intermediate are the members of the BH3 subfamily. For the following experiments, we used a constitutively active mutant of Bad, Bad3A (35). This mutant has serine to alanine mutations at three of its phosphorylation sites and therefore is not deactivated by phosphorylation. Initially, we assessed the ability of Bad3A to induce Bax activation, using the 6A7 antibody. MCF7 cells were transiently cotransfected with GFP-Cb5 or GFP-Bcl-2Cb5. The cells were then fixed and stained with the Bax 6A7 antibody. GFP expression was used as a marker of cotransfection. Cells were counted for GFP expression and 6A7 staining. Fig. 5A shows images of cells stained with 6A7 antibody following cotransfection with HABad3A and GFP-Cb5 or GFP-Bcl-2Cb5. When cotransfected with GFP-Cb5, HA-Bad3A induces Bax activation. When cotransfected with GFP-Bcl-2Cb5, there is significantly less Bax activation (Fig. 5, A and B). This result shows that GFP-Bcl-2Cb5 expression inhibits the activation of Bax by HA-Bad3A, indicating that Bcl-2 on the ER can intercede in the activation of Bax by Bad.


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Fig. 5.   Bad3A induces Bax activation in MCF7, which is inhibited by ER-Bcl-2. MCF7 cells were transiently cotransfected with the constitutively active Bad mutant, Bad3A and GFP-Cb5, or GFP-Bcl-2Cb5. They were then fixed with PF and stained with the 6A7 (anti-active Bax) antibody and an Alexa 594 secondary antibody (shown in red). A, Cells were then analyzed by fluorescence microscopy. GFP is shown in green (left) and 6A7 antibody stained with Alexa 594 is shown in red (right). B, approximately 100 cells were counted per dish. The data are representative of three separate experiments. Only GFP-positive cells were counted and assessed for 6A7 staining. Blinded, independent counting revealed similar results.

Bcl-2Cb5 Colocalizes and Can Interact with Bad3A-- Next, we examined the subcellular localization of HA-Bad3A during GFP-Bcl-2Cb5 cotransfection. Following cotransfection of GFP-Bcl-2Cb5 or GFP-Cb5 with HA-Bad3A, MCF7 cells were stained with anti-HA antibody fused to Alexa 594. These cells were visualized using epifluorescence microscopy. HA-Bad3A cotransfected with GFP-Cb5 has a mitochondrial pattern seen in Fig. 6A, distinct from the ER pattern associated with GFP-Cb5. This is in contrast to HA-Bad3A cotransfected with GFP-Bcl-2Cb5. When cotransfected with GFP-Bcl-2Cb5, HA-Bad3A has a reticular pattern and overlaps closely with GFP-Bcl-2Cb5 (Fig. 6, A and B). This indicates that these two proteins are colocalizing on the ER. HA-Bad3A also colocalized with stably expressed Bcl-2 following transient transfection in the 468 cells (data not shown). To assess whether this colocalization could be due to direct binding, we performed a coimmunoprecipitation between the HA-Bad3A and FL-Bcl-2Cb5 to determine if an interaction can occur between these two proteins. Fig. 6C shows that Fl-Bcl-2Cb5 can bind to HA-Bad3A. This is evident in lane 5 of the blot. When cotransfected, the two proteins co-immunoprecipitate. To demonstrate this further, a mutant of Bcl-2Cb5 was generated that would inhibit binding. The G145E is a mutation in the BH1 domain of Bcl-2 shown to disrupt dimerization with pro-apoptotic Bcl-2 family members (13). As shown in lane 6 of Fig. 6C Bad3A does not co-immunoprecipitate with Bcl-2G145E. In addition, Fig. 7A shows that HA-Bad3A maintains mitochondrial localization when cotransfected with Bcl-2Cb5G145E. The cells in Fig. 7A are stained with an antibody to CoxIV as a mitochondrial marker. Upon cotransfection of Fl-Bcl-2Cb5, HA-Bad3A appears to have a pattern distinct from CoxIV, while HA-Bad3A retains some co-localization with CoxIV following cotransfection with Fl-Bcl-2Cb5G145E. Fig. 7B shows that the G145E mutant is not effective in inhibiting Bax activation induced by Bad3A overexpression. HA-Bad3A was cotransfected into MCF7 with either pCMV-tag2B, Fl-Bcl-2Cb5, or Fl-Bcl-2Cb5G145E. Cells cotransfected with Fl-Bcl-2Cb5 were resistant to HA-Bad3A-induced Bax activation as measured by 6A7 antibody staining. Cells cotransfected with the G145E mutant were not resistant. These data suggest that an interaction between Bad3A and Bcl-2Cb5 is likely and demonstrates a likely mechanism for how Bcl-2Cb5 inhibits Bad3A-induced Bax activation.


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Fig. 6.   Bcl-2Cb5 binds Bad3A, preventing it from localizing to the mitochondria. A, MCF7 cells were transiently cotransfected with the constitutively active Bad mutant, Bad3A, and GFP-Cb5 or GFP-Bcl-2Cb5. Bad3A was HA tagged. Cells were then fixed with PF and stained with an antibody to HA with an Alexa 594 secondary antibody. Cells were then analyzed by epifluorescence microscopy. Scale bar is 10 microns. B, magnification of ER strands. GFP-Bcl-2Cb5 is shown in green and HA staining is shown in red. Scale bar is 10 microns. C, HA-Bad3A binds to ER-Bcl-2. MCF7 cells were cotransfected with HA-Bad3A and Fl-Bcl-2Cb5 or a mutant deficient in hetrodimerization, Fl-Bcl-2Cb5G145E. The cells were lysed and preincubated with an antibody to Bcl-2. The lysate was then incubated with protein G-agarose, and the Bcl-2 complex was immunoprecipitated. The precipitate was analyzed by Western blotting.


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Fig. 7.   Interaction between Bcl-2Cb5 and Bad3A is required to inhibit Bax activation. A, HA-Bad3A and Fl-Bcl-2Cb5 or Fl-Bcl-2Cb5G145E were cotransfected into MCF7 cells and fixed with PF. They were stained with an antibody to CoxIV with an Alexa 594 secondary antibody (shown in red) and an antibody to HA, directly conjugated to Alexa 488 (shown in green). B, HA-Bad3A and pCMV-tag2B, Fl-Bcl-2Cb5, or FL-Bcl-2Cb5G145E were cotransfected into MCF7 cells and fixed with PF. The cells were then stained with an antibody to HA, directly conjugated to Alexa 488 and with the active Bax antibody, 6A7 with a 594 secondary antibody. Transfected cells were identified by HA staining. Bax activation was assessed by 6A7 staining. Approximately 100 cells were counted per dish. The data shown are averaged from three separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous Bcl-2 targeting studies have provided valuable information about the role of subcellular localization in the anti-apoptotic activity of Bcl-2. The first localization requirement for Bcl-2 was discovered from studies of a C-terminal truncation mutant that localized to the cytoplasm. It was found that the activity of Bcl-2 was dependent on insertion into an intracellular membrane, but there was some residual function of the cytoplasmic form (37, 38). Later studies suggested that Bcl-2 could inhibit apoptosis induced by a variety of stimuli when targeted to either the ER or mitochondrial membranes (32). These stimuli include ceramide, Myc overexpression, Bax overexpression, and staurosporine. Bcl-2Cb5 was shown to inhibit caspase 3 activation, mitochondrial membrane depolarization, and cytochrome c release (32, 39, 40). The finding that Bcl-2Cb5 can inhibit apoptosis suggests that Bcl-2 can protect mitochondrial membranes by an indirect mechanism.

The data presented in this paper are consistent with the growing evidence that Bcl-2 blocks apoptosis by inhibiting the pro-apoptotic activity of BH3-only proteins. These data show that Bcl-2 can inhibit the action of a BH3-only member at a location distinct from the mitochondria and Bax. Several studies have shown that mutants of Bcl-2 and Bcl-xL that are deficient in Bax binding are still functional (11, 12). Recent work by Cheng et al. (13) showed that mutants of Bcl-xL that bind BH3 proteins but not Bax still retained anti-apoptotic activity, while mutants that could not bind BH3 proteins were not active.

These data point to BH3 proteins as intermediates between Bcl-2 and Bax. We propose a model, whereby Bcl-2 protects the mitochondrial membrane by binding the active form of BH3 proteins. As an example, Bad is normally sequestered in the cytoplasm by 14-3-3 until it becomes dephosphorylated and dissociates during interleukin-3 withdrawal (15) or disruption of calcium homeostasis (16). Bcl-2 most likely sequesters Bad following dephosphorylation, thus preventing unbound Bad from accumulating on the mitochondria and inducing apoptosis. Bcl-2 and Bcl-xL would therefore function as a checkpoint of BH3 protein-mediated apoptosis following initial activation.

These data also provide mechanisms for divergent pathways of apoptosis suggested by Lee et al. (39), who demonstrated that Bcl-2Cb5 could not inhibit apoptosis by etoposide, while wild-type or mitochondrial-targeted Bcl-2 could. It is likely that this is due to the number of different BH3 proteins with different properties and subcellular localizations. It is possible that some BH3 proteins require Bcl-2 to be on the mitochondria for their activity. For instance, Bbc3/Puma is constitutively on the mitochondria (41, 42), which might make it impossible for Bcl-2Cb5 to intercede in its ability to activate Bax or Bak, as there would be little access of Bcl-2Cb5 to sequester this BH3 proteins.

How BH3 domain only proteins induce Bax activation is still unclear. It is possible that BH3 proteins directly bind to Bax, although there are only a few examples of such interactions occurring. Another possibility is that BH3 proteins affect mitochondria or mitochondrial membranes in such a manner as to cause Bax activation. There is evidence that the BH3 protein, Bim induces apoptosis by binding to the VDAC channel, inducing loss of mitochondrial membrane potential (43). This is supported by data suggesting that Bax oligomerization is preceded by disruption of mitochondrial membrane potential (9).

This study provides a unique insight into Bcl-2, as it shows that Bcl-2 on the ER can inhibit mitochondrial events of apoptosis by inhibiting the oligomerization of Bax on the mitochondria. The ability of Bcl-2Cb5 to inhibit Bax oligomerization calls into question the notion that Bcl-2 inhibits Bax by a direct biochemical effect on Bax or on mitochondrial membranes. The more likely scenario is that Bcl-2 prevents the pro-apoptotic activity of BH3 proteins, suggesting a model where BH3 proteins activate Bax by either a direct interaction or by affecting mitochondria. More generally, this study begins to address the question of what Bcl-2 is doing on the ER membrane.

    ACKNOWLEDGEMENTS

We thank Dr. Minh Lam and the Confocal Microscopy Core Facility for assistance with confocal microscopy (P30 CA43703-12). We thank Michael Sramkowski, Megan Gottlieb, and the Flow Cytometry Core Facility (NCI CA43703) for assistance with flow cytometry. We are grateful to Dr. Tom Chittenden and Dr. Xiao-Mai Zhou (ImmunoGen, Inc.) for the use of their HA-Bad3A vector. We also thank Dr. Alex Almasan, Dr. John Mieyal, Dr. Yu-Chung Yang, Michael Malone, and Karen McColl for technical and intellectual assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants RO1 CA85804 (to C. W. D.) and T32 CA73515 (to M. J. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Division of Hematology/Oncology, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4937. Tel.: 216-368-1175; Fax: 216-368-1166; E-mail: cwd@po.cwru.edu.

Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M208878200

    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; PBS, phosphate-buffered saline; STS, staurosporine; CoxIV, cytochrome c oxidase IV; HA, hemagglutinin; PF, paraformaldehyde; FL, FLAG; GFP, green fluorescent protein; Cb5, cytochrome b5; z-VAD-FMK, z-Val-AlaDL-Asp-fluoromethylketone.

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