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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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.
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RESULTS |
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.
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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.
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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.
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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.
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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.
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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.
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DISCUSSION |
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.