From the Departments of Post-genomics & Diseases and
§ Surgery, Osaka University Graduate School of Medicine
and the ¶ Core Research for Evolutional Science and Technology of
the Japan Science and Technology Corporation, 2-2 Yamadaoka, Suita,
Osaka 565-0871, Japan
Received for publication, August 2, 2002, and in revised form, November 1, 2002
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ABSTRACT |
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The Bcl-2 family of proteins comprises well
characterized regulators of apoptosis, consisting of anti-apoptotic
members and pro-apoptotic members. Pro-apoptotic members possessing
BH1, BH2, and BH3 domains (such as Bax and Bak) act as a gateway for a
variety of apoptotic signals. Bax is normally localized to the
cytoplasm in an inactive form. In response to apoptotic stimuli, Bax
translocates to the mitochondria and undergoes oligomerization to
induce the release of apoptogenic factors such as cytochrome
c, but it is still largely unknown how the mitochondrial
translocation and pro-apoptotic activity of Bax is regulated. Here we
report that cytoplasmic protein 14-3-3 Regulation of programmed cell death, or apoptosis, is essential
for normal development and for the maintenance of homeostasis in most
metazoans. Various apoptotic signals eventually converge into a common
death mechanism, in which members of the cysteine protease family
(known as caspases) are activated and cleave various cellular proteins.
In mammals, the mitochondria play an essential role in apoptosis by
releasing apoptogenic factors including cytochrome c,
Smac/Diablo, and Omi/HtrA2 from the intermembrane space into the
cytoplasm (1-3). Once in the cytoplasm, cytochrome c binds to Apaf-1, a mammalian homologue of Ced-4, that recruits and activates initiator caspase-9, which subsequently activates effector
caspase-3/caspase -7 (4, 5), whereas Smac/Diablo and Omi/HtrA2
facilitate caspase activation by interacting with and inhibiting
IAPs, the endogenous caspase inhibitor family (3, 6, 7).
The Bcl-2 family of proteins includes the best characterized regulators
of apoptosis, comprising anti-apoptotic members, including Bcl-2 and
Bcl-xL, and pro-apoptotic members that include multi-domain Bax and Bak and various single-domain BH3-only proteins (1, 8).
Proteins of this family directly regulate the release of mitochondrial
apoptogenic factors. Many of the pro-apoptotic family members, such as
Bax, Bid, Bad, Bim, and Bmf, are localized in the cytoplasm, and
apoptotic stimulation results in their translocation to the
mitochondria and induction of the release of apoptogenic factors,
probably by inactivating anti-apoptotic members of the family and
activating multi-domain members like Bax and Bak (9-12). Translocation
of the BH3-only proteins appears to involve various post-translational
modifications. For instance, cytoplasmic Bid is cleaved by caspase-8
and then undergoes translocation to the mitochondria (13, 14).
Dephosphorylation by calcineurin frees Bad from cytosolic 14-3-3 and
allows it undergo translocation to the mitochondria (15). Although the
mechanism involved is still unknown, Bim and Bmf are freed from
microtubular dynein motor complexes and myosin V actin motor complexes,
respectively, during certain forms of apoptosis (16, 17). Bax has also
been shown to undergo translocation and integration into the
mitochondrial membrane during apoptosis (18-22), and the translocation
process has been suggested to involve a conformational change of the
Bax molecule, especially exposure of the C terminus (20, 21, 23). It
has also been reported that translocation of Bax to the mitochondria is
enhanced by caspases (20) or by intracellular alkalization (24) and is
negatively regulated by Bcl-2 through a still unidentified mechanism
(19, 21, 22).
The 14-3-3 proteins (seven isomers in mammals: In the present study, we showed that 14-3-3 Antibodies and Chemicals--
An anti-human Bax (N20) polyclonal
antibody that cross-reacted with mouse Bax was obtained from Santa Cruz
Biotechnology (Santa Cruz, CA), and an anti-human Bax (Ab-3) monoclonal
antibody (mAb)1 was obtained
from Oncogene (Darmstadt, Germany). Anti-sheep 14-3-3 Construction of Plasmid Vectors--
DNAs encoding human
14-3-3 Protein Purification--
Recombinant His-tagged human Bax was
expressed and purified as described previously (31). Recombinant human
14-3-3 Analysis of Protein-Protein Interaction--
For
immunoprecipitation experiments, HeLa and NIH3T3 cells were incubated
with 2 mM DTBP (a protein cross-linker) for 30 min. Then
the cells were lysed and sonicated in lysis buffer (50 mM Tris/HCl, pH 7.4, 142.5 mM KCl, 5 mM
MgCl2, 1 mM EGTA, and 0.2% Nonidet P-40)
containing proteinase inhibitors. To investigate the interaction with
exogenous Bax and Bad, 293T cells were transiently transfected with the
expression plasmids using LipofectAMINE in the presence of zVAD-fmk
(100 µM) to prevent Bax- or Bad-induced apoptosis. Then
the cells were lysed, and the lysates were subjected to
immunoprecipitation with the indicated antibodies, and the precipitates
were analyzed by Western blotting. To detect binding between purified
proteins, recombinant proteins were incubated for 8 h with either
GST-14-3-3 proteins or GST alone in 100 µl of the lysis buffer, and
then these proteins were incubated with glutathione-Sepharose for
3 h. After brief centrifugation, the beads were washed and
resuspended in the SDS-PAGE sample buffer, as described elsewhere (32).
The proteins were analyzed by Western blotting and autoradiography.
Surface Plasmon Resonance--
The affinity between Bax and
14-3-3 proteins was measured by surface plasmon resonance using a
Biacore2000 (Biacore). Equivalent molar amounts of GST-mock and
GST-14-3-3 proteins were immobilized on the sensor chip (CM5; Pharmacia
Corp.) by the amine-coupling method. Bax was added as the analite, and
the affinity was calculated from the difference between the resonance
units with GST-14-3-3 proteins and those with GST-mock.
Metabolic Labeling--
293T cells were transfected with
pUC-CAGGS-human Bax DNA or pUC-CAGGS-human FLAG-Bad DNA using
LipofectAMINE (Life Technologies, Inc.), according to the supplier's
protocol. The transfected cells were labeled for 24 h in
phosphate-free RPMI 1640 medium with [32P]orthophosphate
(4 µCi/106 cells).
Cell Fractionation--
Cell fractionation was performed using
digitonin, as described previously (22). Briefly, after washing twice
with phosphate-buffered saline, the cultured cells were collected and
treated with 10 µM digitonin for 5 min at 37 °C. The
cytosolic and organellar fractions were separated by centrifugation and
lysed with RIPA buffer. As a result, more than 92% of cytosolic
protein was recovered in the supernatant, and more than 95% of
mitochondrial protein was localized to the pellet. The heavy membrane
fraction enriched for mitochondria was prepared as follows. The cells
were washed twice in phosphate-buffered saline, resuspended in isotonic
buffer (20 mM potassium Hepes, pH 7.4, 1.5 mM
MgCl2, 10 mM KCl, and 250 mM
sucrose), and then homogenized with a Dounce homogenizer. After separation of nuclei and unbroken cells by centrifugation at 600 × g for 10 min, the post-crude nuclear supernatant was
centrifuged at 10,000 × g for 10 min to collect the
heavy membrane fraction.
Assessment of the Integration of Bax into the Mitochondrial
Membrane--
Heavy membrane fractions enriched for mitochondria from
cells or isolated rat mitochondria were incubated in 0.1 M
Na2CO3 (pH 11.5), and then were centrifuged at
200,000 g for 45 min to separate the supernatant and pellet
as described elsewhere (21).
Analysis of Bax Translocation and Cytochrome c Release in
Vitro--
Mitochondria were prepared from the livers of male Donryu
rats in Mt-A buffer (0.3 M mannitol, 10 mM
potassium Hepes, pH 7.4, 0.1% fatty acid-free bovine serum albumin),
as described previously (33). Recombinant 14-3-3 In Vitro Assay of the Dissociation of Bax from
14-3-3
GST-14-3-3
14-3-3 In Vitro Assay of 14-3-3 Analysis of Cell Death--
293T cells were transiently
transfected with human Bax DNA (0.2 µg) with or without DNA
expressing human 14-3-3 Bax Interacts with 14-3-3--
Although translocation and
integration of cytoplasmic Bax into the mitochondrial membrane is a
critical step for its pro-apoptotic activity, the mechanism of action
is poorly understood. To improve our understanding of the regulation of
Bax, we searched for a molecule that interacted with Bax, modulated its
activity, and found that Bax was bound to protein 14-3-3 Both the N- and C-terminal Regions of Bax Are Required for
Interaction with 14-3-3 Interaction of Bax with 14-3-3 Bax Is Negatively Regulated by 14-3-3
The findings obtained using cell lysates were similar to those obtained
with living cells. As shown in Fig. 4d, lysates from VP16-treated cells were more efficient at causing Bax to dissociate from 14-3-3
The dissociation of Bax from 14-3-3 Caspases Directly Cleave 14-3-3
As shown in Fig. 4a, when 14-3-3 Bax Dissociates from 14-3-3 14-3-3
Next, we examined whether overexpression of 14-3-3 Bax is mainly found in cytoplasmic and/or peri-mitochondrial
locations in living cells, and apoptotic stimulation causes its stable
integration into the mitochondrial membrane, along with the induction
of cytochrome c release (18-22, 31, 40, 41). However, it is
still poorly understood how Bax remains inactive in healthy cells.
Although it has been suggested that Bax exists as a monomer in the
cytoplasm of healthy cells and forms dimers or oligomers on the
mitochondrial membrane during apoptosis (10, 21), the present study
clearly showed that a significant fraction of Bax interacts with
14-3-3 It has been suggested that Bax undergoes a conformational change during
apoptosis, on the basis of its increased susceptibility to proteolytic
cleavage (20) and binding with some antibodies (42). These changes can
be explained by our proposal that Bax dissociates from 14-3-3 during
apoptosis. Our finding that both N- and C-terminal regions of Bax were
required for its interaction with 14-3-3 The 14-3-3 proteins are highly conserved cytoplasmic molecules that
interact with various cellular proteins and that are thought to be
involved in the regulation of various cellular processes, including
apoptotic signal transduction (25). It has been reported that different
isoforms of 14-3-3 sequester different pro-apoptotic molecules through
a phosphorylated serine residue on the target molecule and thus inhibit
apoptosis (e.g. We showed that Bax dissociates from 14-3-3 In summary, we investigated the mechanisms by which translocation of
Bax into the mitochondrial membrane is regulated and found that 14-3-3 plays a crucial role in sequestering Bax to the cytoplasm, where
apoptotic stimulation causes it to release Bax in both a
caspase-independent and -dependent manner. Further studies
are required to identify the trigger that induces caspase-independent dissociation of Bax from 14-3-3 and the signals that enhance
translocation of Bax to the mitochondria.
binds to Bax and, upon
apoptotic stimulation, releases Bax by a caspase-independent mechanism,
as well as through direct cleavage of 14-3-3
by caspases. Unlike
Bad, the interaction with 14-3-3
is not dependent on the
phosphorylation of Bax. In isolated mitochondria, we found that
14-3-3
inhibited the integration of Bax and Bax-induced cytochrome
c release. Bax-induced apoptosis was inhibited by
overexpression of either 14-3-3
or its mutant (which lacked the
ability to bind to various phosphorylated targets but still bound to
Bax), whereas overexpression of 14-3-3
was unable to inhibit
apoptosis induced by a Bax mutant that did not bind to 14-3-3
. These
findings indicate that 14-3-3
plays a crucial role in negatively
regulating the activity of Bax.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
,
,
,
,
/
, and
) are highly conserved cytoplasmic molecules that form
homodimers and heterodimers and interact with various cellular proteins. These proteins seem to control various cellular processes by
sequestering regulatory molecules (25). The 14-3-3 proteins have also
been implicated in signaling for apoptosis through interaction with
apoptotic molecules such as Bad (26), ASK1 (27), and FKHRL1 (28).
Furthermore, 14-3-3
and
are known to act as mitochondrial import
stimulation factors (29) and appear to play a crucial role in
intracellular protein trafficking, although the precise mechanism by
which these isomers of 14-3-3 participate in protein translocation is
not yet understood.
protein was bound to Bax
in the cytoplasm of living cells and that Bax underwent dissociation
from this protein by caspase-independent and -dependent mechanisms during apoptosis to induce apoptotic changes of the mitochondria, indicating that 14-3-3
plays a crucial role in the
negative regulation of Bax activity in living cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(T16,
cross-reacting with human 14-3-3
), anti-rat 14-3-3
(C17, cross-reacting with human and mouse 14-3-3
), anti-human 14-3-3
(C16), and an anti-human 14-3-3
mAb (H8) that recognized the N-terminal region of various 14-3-3 family members were purchased from
Santa Cruz Biotechnology. An anti-mouse 14-3-3
/
mAb, an anti-Xpress mAb, and an anti-FLAG M2 mAb were purchased from Calbiochem (La Jolla, CA), Invitrogen, and Sigma, respectively. An anti-cytochrome c mAb (65981A) that cross-reacted with human cytochrome
c was obtained from PharMingen (San Diego, CA). Anti-Fas
antibody (CH-11) was purchased from MBL (Nagoya, Japan). The cleavable
protein cross-linker dimethyl-3,3'-dithiobispropionimidate-2HCl (DTBP) was purchased from Pierce, [32P]orthophosphate was
obtained from Amersham Biosciences, and zVAD-fmk was purchased from
Peptide Inc. (Minoh, Japan). Other chemicals were purchased from Wako
Co. (Tokyo, Japan).
,
,
, and various 14-3-3
mutants (K49E/V176D,
D239A,
1-6, and 1-239 with amino acid residues 1-161, and
1-239, respectively) were generated by the PCR using proofreading Pfu DNA polymerase (Stratagene, CA) and were
subcloned into a recombinant protein-producing vector (pGEx-1
T) and
two mammalian expression vectors, pUC-CAGGS (30) and pcDNA3.1
(Invitrogen), with an N-terminal His tag and an anti-Xpress epitope.
DNAs encoding HA-tagged mouse Bax and its mutants (
N,
1,
BH3,
5/6, and
C, lacking amino acid residues 1-20, 22-37,
63-72, 106-153, and 172-193, respectively) were generated by PCR and
were subcloned into pBluescript SK (+/
) (Strategene, CA). DNA
encoding FLAG-tagged human Bad was subcloned into pUC-CAGGS.
,
, and
were expressed as GST fusion proteins in
Escherichia coli (strain DH5
) and were purified on a
glutathione-Sepharose column. 14-3-3
was released from GST by
cleavage with thrombin and purified to homogeneity by MonoQ
chromatography. Mock proteins were produced by the same method using
empty plasmids. In some experiments, GST-14-3-3 was used without
cleavage of GST. Recombinant caspase-3, -7, and -8 were expressed as
His-tagged proteins in E. coli (strain DH5
) and
purified on a Ni+-nitrilotriacetic acid column. One unit
was defined as the amount of enzyme that released 1 nmol of
7-amino-4-methylcoumarin in a buffer (50 mM
Tris/HCl, pH 7.4, 1 mM EDTA, and 1 mM EGTA)
containing 100 µM of substrate (Ac-DEVD-MCA, Ac-IETD-MCA,
or Ac-VEID-MCA) over 10 min at 30 °C. All of the proteins were
dissolved in a buffer composed of 20 mM Tris/HCl (pH 7.4),
2 mM MgCl2, and 1 mM
dithiothreitol. Mouse Bax and its mutants were produced using an
in vitro translation method. Briefly, DNAs were
transcribed/translated with a TNT T7 transcription/translation kit
(Promega, Japan) in the presence of [35S]methionine
according to the manufacturer's instructions.
(the indicated
amounts) and rBax (1 µg) were preincubated for 30 min at 25 °C and
then were added to the mitochondria (100 µg) and incubated for a
further 3 min at 25 °C in 100 µl of Mt-B buffer (Mt-A buffer plus
100 µM potassium phosphate and 4.3 mM
succinate). Next, the mixture was then centrifuged to collect the
mitochondria, and aliquots of mitochondria or supernatant were analyzed
by Western blotting using anti-Bax antibodies. To detect the release of
cytochrome c, mitochondria were centrifuged, and the
supernatant was analyzed by Western blotting with an anti-cytochrome c antibody.
--
GST-14-3-3
(2 µg) and rBax (5 µg) were
preincubated for 8 h at 4 °C in pH 7.5 buffer, and then the
mixture was incubated with glutathione-Sepharose for 3 h. After
brief centrifugation, the beads that bound GST-14-3-3
and
GST-14-3-3
-Bax complex were incubated with either cytosol (10 µg)
in pH 7.5 buffer for 12 h at 25 °C, with 200 µl of pH 6.5, pH
7.5, or pH 8.0 buffer (50 mM Tris/HCl, pH 6.5, 7.5 or 8.0, 0.2% Nonidet P-40) for 12 h at 4 °C, or with caspase-8 (600 units) for 4 h at 37 °C. After brief centrifugation, the beads
were washed and resuspended in the sample buffer and then analyzed by
Western blotting.
(2 µg) and rBax (5 µg) were preincubated for 30 min
at 4 °C in 10 µl of pH 7.5 or pH 8.0 buffer (50 mM
Tris/HCl, pH 7.5 or 8.0, 0.2% Nonidet P-40). The protein mixtures were
then incubated for 8 h at 4 °C in 200 µl of pH 7.5 or 8.0 buffer (50 mM Tris/HCl, pH 7.5 or 8.0, 0.2% Nonidet P-40)
and mixed with glutathione-Sepharose for 3 h. After brief
centrifugation, the beads were washed, and rBax bound to the beads was
analyzed by Western blotting.
(5 µg) was incubated with caspase-3 (600 units) or mock
protein for 4 h at 37 °C, rBax (2 µg) was added, and then incubation was done for 12 h at 4 °C. After addition of
Ni+ resin, 14-3-3
bound to rBax was collected and eluted
with 0.3 M imidazole (pH 6.8).
Cleavage by Caspases--
Cytosolic
fractions from healthy HeLa cells or recombinant 14-3-3
were
incubated for 5 h at 37 °C with or without caspases and in the
presence or absence of 200 µM zVAD-fmk. Then the cleavage of 14-3-3
was detected by Western blotting.
or its mutants (0.5 µg), plus 0.1 µg of
the green fluorescent protein (GFP) expression construct (pEGFP-N1;
Clontech). Transfected cells were incubated for
24 h at 37 °C and stained with 1 µM Hoechst
33342, after which the extent of apoptosis was calculated as the
percentage of GFP-positive cells showing nuclear fragmentation relative
to all GFP-positive cells.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in HeLa
cells (Fig. 1a). The same
interaction between Bax and 14-3-3
was also observed in NIH3T3 cells
(Fig. 1b). Because there are several isoforms of 14-3-3 (25), we next tested the interaction of Bax with other isoforms. As
shown in Fig. 1a, Bax was also bound to 14-3-3
and 14-3-3
in HeLa cells, whereas there was no interaction with
14-3-3
or 14-3-3
(data not shown). Furthermore, it has recently
been reported that Bax binds to 14-3-3
(34). Although the
interaction between Bax and 14-3-3 was initially detected in the
presence of a protein cross-linker (Fig. 1a, left
panel), a similar level of binding was observed in the absence of
the cross-linker (Fig. 1a, right panel).
Recombinant His-tagged Bax (rBax) showed binding to GST-fused
14-3-3
,
, and
but not to GST (Fig. 1c), indicating that Bax directly interacted with these 14-3-3 isoforms. Furthermore, surface plasmon resonance analysis revealed that 14-3-3
,
, and
all had a comparable affinity for rBax (Fig. 1d).
Estimation of the amount of each endogenous 14-3-3 isoform in HeLa
cells by comparison with recombinant isoforms on Western blots revealed that 10 µg of HeLa cell lysate contained ~35, 22, and 10 ng of 14-3-3
,
, and
, respectively (Fig. 1e). According
to these findings, although Bax interacted with 14-3-3
,
, and
, 14-3-3
was the major isoform in HeLa cells, so we studied its
role further. Although Bad is known to bind to 14-3-3
(26), we were
unable to detect any interaction between 14-3-3
and the other
pro-apoptotic Bcl-2 family members Bid or Bak, either by
immunoprecipitation or by the surface plasmon resonance method (data
not shown).
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Fig. 1.
Interaction of Bax with 14-3-3. a
and b, interaction of Bax with 14-3-3 in healthy cells. HeLa
(a, left panel) and NIH3T3 (b) cells
were preincubated with 2 mM DTBP (a cleavable protein
cross-linker), and the lysates were immunoprecipitated (IP)
with anti-Bax polyclonal antibody (N20) ( Bax) or normal rabbit IgG.
The immune complexes were analyzed by Western blotting using
anti-14-3-3 antibodies specific for the indicated isoform. The same
experiment was also performed without DTBP in HeLa cells (a,
right panel). lysate indicates the portion
(1/10) of the total lysate that was subjected to
immunoprecipitation. c and d, direct interaction
between Bax and several 14-3-3 isoforms (
,
, and
) with
comparable affinities. c, recombinant Bax (rBax, 2 µg) was
incubated with 2 µg of the indicated GST-14-3-3 proteins or the
equivalent amount of GST-mock protein for 8 h. Then GSH-Sepharose
was added for 3 h and collected by centrifugation, after which
bound rBax was analyzed by Western blotting. total indicates
the total amount of rBax used. d, the indicated amount (15 µg or 30 µg) of rBax or bovine serum albumin was run over a chip
containing immobilized GST-mock protein, GST-14-3-3
,
, or
,
and protein interactions were measured by surface plasmon resonance as
described under "Experimental Procedures." e, the amount
of each 14-3-3 isoform in HeLa cells. Lysates from healthy HeLa cells
(10 µg) and the indicated GST-14-3-3 isoforms (25 ng each) were
analyzed by Western blotting using antibodies specific for 14-3-3
(left panel), 14-3-3
(middle panel), and
14-3-3
(right panel). The amount of each of the 14-3-3 isoforms in 10 µg of lysate was estimated by comparison with the
GST-14-3-3 proteins using densitometric analysis and is shown below the
blots (in nanograms).
--
We then attempted to determine the
regions of Bax involved in binding to 14-3-3
by employing an
in vitro interaction assay using 35S-labeled,
HA-tagged mouse Bax mutants and GST-14-3-3
, because the level of
expression of the Bax mutants varied considerably in transfection
experiments. Wild-type Bax and three of its deletion mutants (
1
(lacking
-helix 1),
BH3 (lacking the BH3 region), and
5/6
(lacking the channel-forming
-helices 5 and 6)) showed binding to
GST-14-3-3
, whereas Bax
N and
C (lacking the N-terminal 20 amino acids and C-terminal 22 amino acids, respectively) did not bind
to GST-14-3-3
(Fig. 2), suggesting
that both the N- and C-terminal regions of Bax were involved in this
binding process. Although it was reported that some detergents, such as
Nonidet P-40, could enhance the conformational changes of Bax and
increase its homodimerization and heterodimerization with other Bcl-2
family members (35), the interaction between Bax and 14-3-3
was
decreased rather than enhanced by addition of Nonidet P-40 (data not
shown).
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Fig. 2.
Regions of Bax essential for interaction with
14-3-3 . The indicated mutants of mouse Bax were produced by
in vitro translation in the presence of
[35S]methionine and were incubated with GST-14-3-3
or
GST-mock protein. After GST-14-3-3 and GST-mock protein were
precipitated with GSH-Sepharose as described under "Experimental
Procedures," Bax bound to GST-14-3-3
or GST-mock was analyzed by
SDS-PAGE followed by autoradiography. A diagram of the Bax deletion
mutants is also shown. These deletion mutants retained the regions
shown by horizontal lines.
1-
7 indicate
the possible helices retained by Bax.
Is Independent of Bax
Phosphorylation--
The 14-3-3 proteins bind to various
phosphorylated proteins, such as Raf-1 and Bad, via phosphorylated
serine residues (26, 36), but these proteins are also known to bind to
several nonphosphorylated proteins (37). Therefore, we tested whether
phosphorylation of Bax was involved in its interaction with 14-3-3
.
As shown in Fig. 3a, although
Bax was bound to 14-3-3
, we could not detect any phosphorylation of
Bax in 293T cells when overexpressed Bax was labeled with
[32P]orthophosphate, a result consistent with previous
reports (38). Under the same experimental conditions, we readily
detected phosphorylation of Bad (Fig. 3a), which is known to
be phosphorylated before binding to 14-3-3
(26). Furthermore, the
immunoprecipitated Bax did not react with antibodies specific for
phosphoserine or phosphothreonine (data not shown). These results
indicated that phosphorylation of Bax was not necessary for interaction
with 14-3-3
. To further confirm that phosphorylation of Bax did not
play an essential role in the interaction with 14-3-3
, we examined
the binding of Bax to a mutant of 14-3-3
(K49E/V176D) that had
lost the ability to bind to various target phosphoproteins, including
Raf-1 and ASK1 (27). As shown in Fig. 3b, whereas wild-type
14-3-3
was co-immunoprecipitated with both Bax and FLAG-Bad,
14-3-3
K49E/V176D was co-immunoprecipitated with Bax but not with
FLAG-Bad, supporting our hypothesis that phosphorylation of Bax was not
necessary for interaction with 14-3-3
.
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Fig. 3.
Interaction of unphosphorylated Bax with
14-3-3 . a, lack of Bax phosphorylation. 293T cells were
transiently transfected with pUC-CAGGS-Bax (3 µg) or with
pUC-CAGGS-FLAG-Bad DNA (3 µg) and incubated in
phosphate-free medium containing [32P]orthophosphate (4 µCi/106 cells). After 24 h, the cells were lysed and
were immunoprecipitated (IP) with anti-Bax or anti-FLAG
antibodies or with normal rabbit IgG or mouse IgG. The immune complexes
were analyzed by SDS-PAGE followed by autoradiography (top
panel), as well as by Western blotting (WB) using
anti-FLAG (second panel), anti-Bax (third panel),
and anti-14-3-3
(bottom panel) antibodies. b,
interaction of 14-3-3
K49E/V176D with Bax, but not Bad. 293T cells
were transiently transfected with pUC-CAGGS-FLAG-Bad (left
panels) and pUC-CAGGS-Bax (right panels) DNA together
with pcDNA3.1- 14-3-3
or its K49E/V176D mutant in the
presence of zVAD-fmk (100 µM). After 24 h, the cells
were lysed and immunoprecipitated (IP) with anti-FLAG
antibody or NMI (left panels), as well as with anti-Bax
antibody or normal rabbit IgG (right panels). Then the
immune complexes were analyzed by Western blotting using anti-Xpress
(to detect 14-3-3
), anti-FLAG (left panels) and anti-Bax
(right panels) antibodies. lysate
indicates the portion (
and Dissociates during
Apoptosis in Both a Caspase-dependent and
Caspase-independent Manner--
To obtain some insight into the
biological significance of the interaction of Bax with 14-3-3
, we
next examined whether this interaction was altered during the
apoptotic process. As shown in Fig.
4a, treatment with VP16
(etoposide) caused the amount of 14-3-3
interacting with Bax to
decrease markedly, and a large fraction of Bax was translocated to the
mitochondria (Fig. 4b) with the release of cytochrome
c (Fig. 4a). To test whether Bax in the
mitochondrial fraction was stably integrated into the mitochondrial membrane, mitochondrial fractions were treated with an alkaline solution (pH 11.5) that only saved Bax, showing stable integration into
the membrane. Before incubation with VP16, a very small amount of Bax
was found in the heavy membrane fraction (Fig. 4b), half of
which was stably integrated into the mitochondrial membrane (Fig.
4c). Note that a much larger amount of
VP16 sample
was analyzed than that of +VP16 in Fig. 4c. On the other
hand, the majority of Bax was found in the heavy membrane fraction
after VP16 treatment (Fig. 4b), most of which was stably
integrated into the membrane (Fig. 4c). In the presence of
zVAD-fmk, which completely inhibited caspase activation, dissociation
of Bax from 14-3-3
was only partly inhibited (Fig. 4a),
indicating that dissociation occurred in both a caspase-independent and
caspase-dependent manner. As shown in Fig. 4c,
integration of Bax into the mitochondrial membrane was also partly
inhibited by zVAD-fmk, whereas translocation of Bax to the mitochondria
was not affected by this caspase inhibitor (Fig. 4b). Note
that the amount of caspase-independent dissociation of Bax-14-3-3
was well correlated with the caspase-independent mitochondrial
integration of Bax (Fig. 4, a and c).
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Fig. 4.
Dissociation of Bax from 14-3-3 and
integration into the mitochondria during apoptosis. a,
dissociation of Bax from 14-3-3
during apoptosis. HeLa cells were
incubated for the indicated time with 200 µM VP16 in the
absence or presence of 200 µM zVAD-fmk. Equivalent
aliquots of the cells were lysed and immunoprecipitated with anti-Bax
antibody, and the immune complexes were subjected to Western blotting
using anti-14-3-3
(top panel) or anti-Bax antibodies
(middle panel). Aliquots of the cell lysates were also
subjected to Western blotting with anti-14-3-3
antibody (lower
panel). The extent of apoptosis is also shown in the figure. The
remaining cells were fractionated into organellar and cytosolic
fractions using 10 µM digitonin, and each fraction (5 µg of protein) was analyzed by Western blotting with anti-cytochrome
c antibody. b, translocation of Bax from the
cytosol to the mitochondria was not inhibited by zVAD-fmk during
VP16-induced apoptosis. HeLa cells were incubated for the indicated
time with 200 µM VP16 in the absence or presence of 200 µM zVAD-fmk. Heavy membrane (HM) and cytosolic
fractions were prepared by differential centrifugation without alkaline
treatment. Then the fractions were analyzed by Western blotting using
anti-Bax antibody. c, integration of Bax, but not 14-3-3
,
into the mitochondrial membrane. HeLa cells were incubated for 24 h with 200 µM VP16 in the absence or presence of 200 µM zVAD-fmk. Heavy membrane (HM) fractions
were prepared by differential centrifugation, incubated in pH 7.5 buffer (
) or in 0.1 M Na2CO3 (pH
11.5) (+) on ice for 30 min, and then centrifuged at 200,000 g for 45 min to yield a supernatant (S) and a
pellet containing heavy membranes (P). The fractions were
analyzed by Western blotting using anti-Bax and anti-14-3-3
antibodies. Equivalent amounts of the HM fraction from +VP16 and +VP16
+zVAD
fmk cultures were analyzed, but a much larger amount of the
fraction from
VP16 cultures was assessed. d, dissociation
of Bax from 14-3-3
induced by the cytosol of apoptotic cells.
GST-14-3-3
(2 µg) and rBax (5 µg) were preincubated for 8 h
at 4 °C, and then the mixture was incubated with
glutathione-Sepharose for 3 h. After brief centrifugation, the
beads retaining 14-3-3
-Bax complex were incubated with the indicated
cytosol (10 µg) for 12 h at 25 °C. After brief
centrifugation, the beads were washed. Then the combined
supernatants (sup) and the beads resuspended in sample
buffer were analyzed by Western blotting using anti-Bax and
anti-14-3-3
antibodies. total indicates the amount
of rBax in the pellet after incubation with
glutathione-Sepharose.
than lysates from normal cells. Interestingly, this dissociation was partially inhibited in the presence of the caspase inhibitor zVAD-fmk, indicating that dissociation of Bax from 14-3-3
occurred via both caspase-dependent and -independent
mechanisms in the cell lysates (Fig. 4d), as it did in
living cells (Fig. 4a). All of these results suggested that
14-3-3
had a role in the sequesteration of Bax.
during apoptosis suggested that
14-3-3
was a negative regulator of Bax. To test this possibility, we
examined whether 14-3-3
affected the mitochondrial translocation of
Bax using isolated mitochondria. As shown in Fig.
5, the addition of recombinant 14-3-3
(or
) protein inhibited the integration of Bax into the
mitochondrial membrane (Fig. 5a) as well as Bax-induced
release of cytochrome c (Fig. 5b).
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Fig. 5.
Inhibition of the mitochondrial integration
of Bax by 14-3-3. GST-14-3-3 , GST-14-3-3
(50 µg), and the
equivalent amount of GST-mock were incubated with or without rBax (25 µg) for 3 h and then were incubated with the mitochondria (1 mg)
for 20 min at 25 °C. Supernatants and pellets were obtained by
centrifugation. The pellets were incubated in 0.1 M
Na2CO3 (pH 11.5) on ice for 30 min, followed by
centrifugation, and then each pellet was analyzed by Western blotting
using anti-Bax antibody (a). Cytochrome c in the
supernatant was also analyzed by Western blotting using an
anti-cytochrome c antibody (b). total
represents the total amount of rBax used (a) or an
equivalent aliquot of mitochondria (b).
to Release Bax--
To
investigate the mechanism of the caspase-dependent
dissociation of Bax from 14-3-3
, we examined whether caspases were able to cleave 14-3-3
. As shown in Fig.
6a, 14-3-3
and 14-3-3
, but not 14-3-3
, were cleaved during apoptosis, and their cleavage was completely inhibited by the caspase inhibitor zVAD-fmk. When recombinant caspase-3 was added to normal cell lysates, 14-3-3
was
cleaved in a z-VAD-fmk-sensitive manner (Fig. 6b).
Furthermore, recombinant caspase-3 also cleaved recombinant 14-3-3
(Fig. 6c), indicating that 14-3-3
is a direct target of
caspase-3. As shown in Fig. 6c, 14-3-3
was also cleaved
by caspase-7 and caspase-8. In contrast, 14-3-3
was cleaved by
caspase-3 in the presence of cell lysate but not in its absence (Fig.
6, b and c), indicating that 14-3-3
is not a
direct target of caspases, unlike 14-3-3
. To identify the caspase
cleavage site in 14-3-3
, various Asp to Ala mutants were produced.
Among them, only the D239A mutant was not cleaved during apoptosis,
indicating that Asp239 (not present in 14-3-3
and
14-3-3
) is the caspase cleavage site.
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Fig. 6.
Caspase-dependent dissociation of
Bax from 14-3-3 . a, cleavage of 14-3-3
and
during
apoptosis. HeLa cells were incubated for 24 h with or without 200 µM VP16 in the absence or presence of 200 µM zVAD-fmk. Equivalent aliquots of the cells were lysed
and analyzed by Western blotting using anti-14-3-3
,
, and
antibodies. The arrows indicate cleaved 14-3-3. b
and c, direct cleavage of 14-3-3
, but not 14-3-3
, by
caspases. b, the cytosolic fraction of HeLa cells was
incubated with (+) or without (
) recombinant caspase-3 (600 units) in
the presence (+) or absence (
) of 200 µM zVAD-fmk for
5 h at 37 °C. The lysates were analyzed by Western blotting
using anti-14-3-3
and anti-14-3-3
antibodies. The
arrows indicate cleaved 14-3-3. c, recombinant
14-3-3
, 14-3-3
, and Bid were incubated with 600 units of
caspases-3, -7, and -8 in the presence (+) or absence (
) of 200 µM zVAD-fmk for 5 h at 37 °C. Each protein was
analyzed by Western blotting using specific antibodies. Cleavage of Bid
was performed to confirm caspase activity. The arrow
indicates cleaved 14-3-3
. d, determination of the site of
cleavage of 14-3-3
by caspases. Top panel, structure of
14-3-3
. The
-helix is indicated at the top, and the
C-terminal amino acid sequence is indicated below. An
asterisk indicates the site of cleavage by caspases.
Bottom panel, cleavage of 14-3-3
, but not 14-3-3
D239A,
during apoptosis. HeLa cells were transiently transfected with
pUC-CAGGS-His-14-3-3
(wild) and
pUC-CAGGS-His-14-3-3
D239A (D239A) for 24 h and
treated with (+) or without (
)
-Fas antibody (CH-11, 0.25 µg/ml)
for 18 h. Then the lysates were analyzed by Western blotting using
anti-Xpress (to detect 14-3-3
) and anti-Bid (as a positive control)
antibodies. e, interaction of Bax with 14-3-3
but not
with 14-3-3
after cleavage by caspases. Recombinant 14-3-3
(5 µg) was treated with caspase-3 (600 units) or the mock protein for
4 h at 37 °C (input), and then rHis-Bax (2 µg) was added at
4 °C for 12 h. By addition of Ni+ resin, rBax and
interacting 14-3-3
were collected. The proteins were eluted from the
Ni+ resin with 0.3 M imidazole (pH 6.8) and
were analyzed by Western blotting (bound) with anti-14-3-3
antibody
and anti-Bax antibody. f, dissociation of Bax from 14-3-3
by caspase treatment. GST14-3-3
(5 µg) and rBax (2 µg) were
incubated at 4 °C for 12 h. After collecting the Bax-14-3-3
complex with GSH-Sepharose, caspase-8 (600 units) was added, and
incubation was done at 37 °C for 4 h. Bax released from and
bound to GST-14-3-3
was detected by Western blotting using anti-Bax
antibody.
was cleaved during
apoptosis, cleaved 14-3-3
was not co-immunoprecipitated with Bax,
suggesting that cleaved 14-3-3
has a decreased affinity for Bax. In
fact, full-length r14-3-3
interacted with rBax, whereas this
interaction was greatly diminished when r14-3-3
was cleaved by
caspase-3 (Fig. 6e). Furthermore, when the complex formed by
GST-14-3-3
and rBax was treated with caspase-8, rBax was released,
and this release was completely inhibited by a caspase inhibitor (Fig. 6f). These results indicated that
caspase-dependent dissociation of Bax from 14-3-3 could be
ascribed to cleavage of 14-3-3
by caspases.
under Basic and Acidic
Conditions--
It has been demonstrated that in the early phase of
apoptosis induced by a variety of stimuli, including cytokine
deprivation, cytoplasmic alkalization occurs and induces a
conformational change of Bax that results in its integration into the
mitochondria (24). Therefore, we examined whether alkalization had an
influence on the interaction of Bax with 14-3-3
. As shown in Fig.
7a, the interaction of Bax
with 14-3-3 was weaker at pH 8.0 than at pH 7.5. Treatment of Bax, but
not 14-3-3
, with an alkaline solution (pH 8.0) decreased their
affinity (Fig. 7a), supporting the earlier finding that
alkalization induced a conformational change of Bax (24). Dissociation
of Bax from 14-3-3
at an alkaline pH (Fig. 7b) implies
that cytoplasmic alkalization during apoptosis may be one of the
initial caspase-independent mechanisms promoting dissociation of the
complex between Bax and 14-3-3
. Dissociation of Bax from 14-3-3
was also observed at an acidic pH of 6.5 (Fig. 7c). It has
been reported that cells show cytoplasmic acidification in the early
phase of apoptosis induced by staurosporine and anti-Fas antibodies
(39), so acidification may also be a trigger for the dissociation of
Bax from 14-3-3
.
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Fig. 7.
Caspase-independent dissociation of Bax from
14-3-3 . a, decrease of Bax binding to 14-3-3
by
treatment at pH 8.0. Upper panel, the interaction between
rBax and GST-14-3-3
at pH 7.5 or 8.0 was analyzed using surface
plasmon resonance as in Fig. 1d. Lower panel,
GST-14-3-3
(2 µg) and rBax (5 µg) were preincubated for 30 min
at 4 °C at the indicated initial pH and then were mixed for 8 h
at 4 °C in buffer at the indicated terminal pH. The mixture was then
incubated with glutathione-Sepharose for 3 h. After a brief
centrifugation, the beads were washed, and the amount of rBax and
14-3-3
on the beads was analyzed by Western blotting.
total indicates the total amount of rBax used. b
and c, the pH-dependent dissociation of Bax from
14-3-3
. GST-14-3-3
(2 µg) and rBax (5 µg) were preincubated
at 4 °C for 8 h, and then the mixture was incubated with
glutathione-Sepharose for 3 h. After brief centrifugation, the
beads were treated in buffer (pH 7.5, pH 8.0 (b), or pH 6.5 (c)) at 4 °C for 12 h. The amount of rHis-Bax and
14-3-3
on the beads was then analyzed by Western blotting.
Inhibits Bax- and Fas-induced Apoptosis--
Finally, we
examined the physiological role of the interaction between 14-3-3
and Bax in the regulation of apoptosis. If 14-3-3
negatively
regulates Bax, overexpression of 14-3-3
could be expected to inhibit
Bax-induced apoptosis. As shown in Fig. 8, (a, left panel,
and b), apoptosis induced by transfection of Bax DNA was significantly
reduced by co-transfection of 14-3-3
DNA, and integration of Bax
into the mitochondrial membrane was also inhibited. Importantly, a
mutant form of 14-3-3
(K49E/V176D) that bound to Bax but not to
phosphorylated targets, including Bad (Fig. 3b), also
inhibited Bax-induced apoptosis (Fig. 8a, left
panel), suggesting that the inhibition was due to direct association with Bax and not to the influence of various other 14-3-3-binding proteins, including Bad, Raf-1, and forkhead protein. Furthermore, a caspase-cleaved mutant of 14-3-3
(1-239) with a weak
affinity for Bax (Fig. 6f) caused less inhibition of
Bax-induced apoptosis, whereas another mutant (14-3-3
-
1-6) that
did not bind to Bax (data not shown) could not inhibit such apoptosis (Fig. 8a, middle panel). Incomplete suppression
of Bax-induced apoptosis by overexpression of 14-3-3
was probably
due to the abundance of endogenous 14-3-3
. Moreover, as shown in
Fig. 8a (right panel), 14-3-3
did not inhibit
apoptosis induced by Bax
N, to which it did not bind (Fig. 2).
Consistent with the inability of 14-3-3
to sequester Bax
N, we
found that Bax
N showed efficient translocation to the mitochondria
(Fig. 8c) and induced more apoptosis than wild-type Bax
(Fig. 8a). These results suggested that 14-3-3
inhibits
Bax-induced apoptosis in an interaction-dependent
manner.
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Fig. 8.
Involvement of 14-3-3 in the regulation of
apoptosis. a, inhibition of Bax-induced apoptosis by
co-transfection of 14-3-3
. 293T cells were transfected with
pUC-CAGGS-human 14-3-3
or its mutant (0.5 µg), with or without
pUC-CAGGS-HA-tagged Bax (0.2 µg) or a Bax mutant (
N),
together with the GFP expression construct (0.1 µg) (to identify
DNA-transfected cells). After 24 h, apoptosis was assessed from
the nuclear morphology by Hoechst 33342 staining of GFP-positive cells.
The data are expressed as the means ± S.D. (n = 4). b, prevention of Bax translocation to the mitochondria
by expression of 14-3-3
. A similar experiment to that described in
a was conducted. DNA-transfected cells were fractionated,
and the extent of Bax translocation to the mitochondria was assessed by
Western blotting using anti-Bax antibody. c, efficient
translocation of Bax
N to the mitochondria. 293T cells were
transfected with pUC-CAGGS-HA-tagged Bax (0.2 µg) or a Bax mutant
(
N) and then were fractionated. The extent of Bax
translocation to the mitochondria was assessed by Western blotting
using anti-HA antibody. d, inhibition of Fas-mediated
apoptosis by a caspase-noncleavable mutant of 14-3-3
. HeLa cells
were transfected with pUC-CAGGS-human 14-3-3
or its mutant (0.4 µg) together with the GFP expression construct (0.1 µg) (to
identify DNA-transfected cells). After 24 h, the cells were
treated with 0.25 µg/ml of anti-Fas antibody (CH-11), and apoptosis
was assessed after another 12 h from the nuclear morphology by
Hoechst 33342 staining of GFP-positive cells. The data are expressed as
the means ± S.D. (n = 4).
could inhibit
apoptosis induced by an anti-Fas antibody. Because we found that
caspase-8 cleaved 14-3-3
to release Bax, we also tested a
caspase-resistant (noncleavable) mutant of 14-3-3
(14-3-3
D239A). As shown in Fig. 8d, overexpression of 14-3-3
D239A
significantly inhibited apoptosis induced by anti-Fas antibody,
suggesting that the cleavage of 14-3-3
by caspases could facilitate
Fas-induced apoptosis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in living cells and that this interaction negatively
regulates Bax by sequestering it to the cytoplasm. Among seven
isoforms, 14-3-3
, 14-3-3
(this study), and 14-3-3
(34) also
bind to Bax and probably play a redundant role.
is also consistent with
previous observations that translocation of Bax to the mitochondria is
stimulated by N-terminal deletion (Fig. 8c and Ref. 20) as
well as by mutation of charged residues in the N- and C-terminal
regions (23, 24). We also showed that Bax dissociates from 14-3-3
under both alkaline and acidic conditions, which is consistent with the
previous observation that translocation of Bax to the mitochondria is
enhanced at an alkaline pH (24). Taken together, it seems likely that a
significant fraction of Bax is sequestered by cytoplasmic 14-3-3 in
healthy cells, whereas apoptotic stimuli cause the dissociation of Bax from 14-3-3 and translocation to the mitochondria.
for ASK1 (27),
for Bad (26), and
for FKHRL1 (28)). The different 14-3-3 proteins therefore appear to
protect cells from apoptosis at various steps of the death signaling
pathway by sequestering different pro-apoptotic proteins (37). Most of
the 14-3-3-binding proteins interact with 14-3-3 via phosphorylated
serine or threonine residue (25, 36). In contrast, we showed that the
interaction of Bax with 14-3-3 occurs in a phosphorylation-independent
manner, based on the observations that phosphorylation of Bax was
undetectable (Fig. 3a) and that Bax still bound to a mutant
form of 14-3-3
(K49E/V176D) lacking the ability to bind to
various phosphoprotein targets (Fig. 3b). It has been
reported that 14-3-3 also interacts with nonphosphorylated proteins
such as ADP-ribosyltransferase Exoenzyme S (ExoS) from
Pseudomonas aeruginosa (43). Our preliminary study suggested
that a region from
-helix 7 to the C terminus of 14-3-3
, the
three-dimensional structure of which could not be identified (possibly
because of its high flexibility) (44, 45), was crucial for the
interaction with Bax (data not shown).
-Helix 7 and the more
C-terminal
-helix 8 comprise the box-1 region, where phosphorylated
target proteins mainly bind by hydrophobic interaction (36, 46, 47),
and this region probably undergoes a conformational change upon binding
of a phosphoprotein to 14-3-3. It is therefore conceivable that Bax
binds to 14-3-3 in healthy cells, and unidentified phosphoprotein(s)
may interact with 14-3-3 to release Bax after an apoptotic stimulus is delivered.
by caspase-independent
and -dependent mechanisms. For the caspase-independent process, one possible trigger is alteration of cytosolic pH
(acidification or alkalization), which has been shown to occur in the
early phase of apoptosis (24, 39), and indeed we found that this
induced the dissociation of Bax from 14-3-3
. Bax also underwent
dissociation after the direct cleavage of 14-3-3
by caspases. The
caspase-dependent dissociation of Bax from 14-3-3
and
subsequent integration of Bax into the mitochondrial membrane probably
represents a positive feedback loop for death signal transduction. In
death receptor-mediated apoptotic signaling, however, casapase-8 (which
cleaves 14-3-3
) is activated upstream of the mitochondria, so the
caspase-dependent dissociation of Bax from 14-3-3
acts
as an initial trigger for apoptotic mitochondrial changes. When HeLa
cells were treated with anti-Fas antibody, cleavage of 14-3-3
,
dissociation of Bax from 14-3-3
, and integration of Bax into the
mitochondria were observed simultaneously (data not shown).
Furthermore, overexpression of caspase-uncleavable 14-3-3 mutant
(D239A) conferred stronger resistance to Fas-mediated apoptosis than
overexpression of wild-type 14-3-3
(Fig. 8d), suggesting
that cleavage of 14-3-3
by caspase-8 is one of the crucial steps in
Fas/TNF-mediated apoptosis. An essential role of Bax in Fas-mediated
apoptosis has been shown by gene-targeting studies, because hepatocytes
of Bax/Bak-deficient mice are resistant to Fas-mediated apoptosis,
whereas hepatocytes from Bax-deficient, Bak-deficient, and wild-type
mice are all equally sensitive to Fas-mediated apoptosis (11).
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FOOTNOTES |
---|
* This work was supported in part by a Scientific Research on Priority Areas grant, a Center of Excellence Research grant, a grant from the Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, and Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of Japan.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.
Both authors contributed equally to this work.
** To whom correspondence should be addressed. E-mail: tsujimot@gene.med.osaka-u.ac.jp.
Published, JBC Papers in Press, November 7, 2002, DOI 10.1074/jbc.M207880200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: mAb, monoclonal antibody; DTBP, dimethyl-3,3'-dithiobispropionimidate-2HCl; HA, hemagglutinin; GST, glutathione S-transferase; GFP, green fluorescent protein.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Tsujimoto, Y., and Shimizu, S. (2000) FEBS Lett. 466, 6-10[CrossRef][Medline] [Order article via Infotrieve] |
2. | Martinou, J.-C., and Green, D. R. (2001) Nat. Rev. 2, 63-67[CrossRef] |
3. | Wolf, B. B., and Green, D. R. (2002) Curr. Biol. 12, R177-R179[CrossRef][Medline] [Order article via Infotrieve] |
4. | Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479-489[Medline] [Order article via Infotrieve] |
5. | Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413[Medline] [Order article via Infotrieve] |
6. | Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000) Cell 102, 33-42[Medline] [Order article via Infotrieve] |
7. | Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, G. E., Moritz, R. L., Simpson, R. J., and Vaux, D. L. (2000) Cell 102, 43-53[Medline] [Order article via Infotrieve] |
8. |
Adams, J. M.,
and Cory, S.
(1998)
Science
281,
1322-1326 |
9. | Antonsson, B., Montessuit, S., Lauper, S., Eskes, R., and Martinou, J. C. (2000) Biochem. J. 345, 271-278[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Antonsson, B.,
Montessuit, S.,
Sanchez, B,
and Martinou, J. C.
(2001)
J. Biol. Chem.
276,
11615-11623 |
11. |
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 |
12. | Puthalakath, H., and Strasser, A. (2002) Cell Death Differ. 9, 505-512[CrossRef][Medline] [Order article via Infotrieve] |
13. | Li, H., Zhu, H., Xu, C., and Yuan, J. (1998) Cell 94, 491-501[Medline] [Order article via Infotrieve] |
14. | Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. (1998) Cell 94, 481-490[Medline] [Order article via Infotrieve] |
15. |
Wang, H. G.,
Pathan, N.,
Ethell, I. M.,
Krajewski, S.,
Yamaguchi, Y.,
Shibasaki, F.,
McKeon, F.,
Bobo, T.,
Franke, T. F.,
and Reed, J. C.
(1999)
Science
284,
339-343 |
16. | Puthalakath, H., Huang, D. C., O'Reilly, L. A., King, S. M., and Strasser, A. (1999) Mol. Cell 3, 287-296[Medline] [Order article via Infotrieve] |
17. |
Puthalakath, H.,
Villunger, A.,
O'Reilly, L. A.,
Beaumont, J. G.,
Coultas, L.,
Cheney, R. E.,
Huang, D. C.,
and Strasser, A.
(2001)
Science
293,
1829-1832 |
18. |
Hsu, Y. T.,
Wolter, K. G.,
and Youle, R. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3668-3672 |
19. |
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 |
20. |
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 |
21. |
Gross, A.,
Jockel, J.,
Wei, M. C.,
and Korsmeyer, S. J.
(1998)
EMBO J.
17,
3878-3885 |
22. |
Nomura, M.,
Shimizu, S.,
Ito, T.,
Narita, M.,
Matsuda, H.,
and Tsujimoto, Y.
(1999)
Cancer Res.
59,
5542-5548 |
23. |
Nechushtan, A.,
Smith, C. L.,
Hsu, Y. T.,
and Youle, R. J.
(1999)
EMBO J.
18,
2330-2341 |
24. |
Khaled, A. R.,
Kim, K.,
Hofmeister, R.,
Muegge, K.,
and Durum, S. K.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14476-14481 |
25. | Aitken, A. (1996) Trends Cell Biol. 6, 341-347[CrossRef] |
26. | Zha, J., Harada, H., Yang, E., Jockel, J., and Korsmeyer, S. J. (1996) Cell 87, 619-628[Medline] [Order article via Infotrieve] |
27. |
Zhang, L.,
Chen, J.,
and Fu, H.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
8511-8515 |
28. | Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, M. J., Arden, K. C., Blenis, J., and Greenberg, M. E. (1999) Cell 96, 857-868[Medline] [Order article via Infotrieve] |
29. | Alam, R., Hachiya, N., Sakaguchi, M., Kawabata, S., Iwanaga, S., Kitajima, M., Mihara, K., and Omura, T. (1994) J. Biochem. (Tokyo) 116, 416-425[Abstract] |
30. | Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene (Amst.) 108, 193-199[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Narita, M.,
Shimizu, S.,
Ito, T.,
Chittenden, T.,
Lutz, R. J.,
Matsuda, H.,
and Tsujimoto, Y.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14681-14686 |
32. | Shimizu, S., Narita, M., and Tsujimoto, Y. (1999) Nature 399, 483-487[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Shimizu, S.,
Eguchi, Y.,
Kamiike, W.,
Funahashi, Y.,
Mignon, A.,
Lacronique, V.,
Matsuda, H.,
and Tsujimoto, Y.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
1455-1459 |
34. |
Samuel, T.,
Weber, H. O.,
Rauch, P.,
Verdoodt, B.,
Eppel, J.-T.,
McShea, A.,
Hermeking, H.,
and Funk, J. O.
(2001)
J. Biol. Chem.
276,
45201-45206 |
35. |
Hsu, Y. T.,
and Youle, R. J.
(1997)
J. Biol. Chem.
272,
13829-13834 |
36. | Muslin, A. J., Tanner, J. W., Allen, P. M., and Shaw, A. S. (1996) Cell 84, 889-897[Medline] [Order article via Infotrieve] |
37. |
Masters, S. C., Fu, H.
(2001)
J. Biol. Chem.
276,
45193-45200 |
38. |
Gilmore, A. P.,
Metcalfe, A. D.,
Romer, L. H.,
and Streuli, C. H.
(2000)
J. Cell Biol.
149,
431-445 |
39. | Matsuyama, S., Llopis, J., Deveraux, Q. L., Tsien, R. Y., and Reed, J. C. (2000) Nat. Cell Biol. 2, 318-325[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Jurgensmeier, J. M.,
Xie, Z.,
Deveraux, Q.,
Ellerby, L.,
Bredesen, D.,
and Reed, J. C.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
4997-5002 |
41. |
Marzo, I.,
Brenner, C.,
Zamzami, N.,
Jurgensmeier, J. M.,
Susin, S. A.,
Vieira, H. L. A.,
Prevost, M. C.,
Xie, Z.,
Matsuyama, S.,
Reed, J. C.,
and Kroemer, G.
(1998)
Science
281,
2027-2031 |
42. |
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 |
43. | Masters, S. C., Pederson, K. J., Zhang, L., Barbieri, J. T., and Fu, H. (1999) Biochemistry 38, 5216-5221[CrossRef][Medline] [Order article via Infotrieve] |
44. | Liu, D., Bienkowska, J., Petosa, C., Collier, R. J., Fu, H., and Liddington, R. (1995) Nature 376, 191-194[CrossRef][Medline] [Order article via Infotrieve] |
45. | Xiao, B., Smerdon, S. J., Jones, D. H., Dodson, G. G, Soneji, Y, Aitken, A., and Gamblin, SJ. (1995) Nature 376, 188-194[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Ichimura, T.,
Uchiyama, J.,
Kunihiro, O.,
Ito, M.,
Horigome, T.,
Omata, S.,
Shinkai, F.,
Kaji, H.,
and Isobe, T.
(1995)
J. Biol. Chem.
270,
28515-28518 |
47. | Ichimura, T., Ito, M., Itagaki, C., Takahashi, M., Horigome, T., Omata, S., Ohno, S., and Isobe, T. (1997) FEBS Lett. 413, 273-276[CrossRef][Medline] [Order article via Infotrieve] |