From the Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Republic of Singapore
Received for publication, October 2, 2000, and in revised form, October 29, 2000
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ABSTRACT |
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A novel Bax-associating protein, named MAP-1
(Modulator of Apoptosis), has been identified
in a yeast two-hybrid screen. MAP-1 contains a BH3-like (BH:
Bcl-2 homology) motif and mediates
caspase-dependent apoptosis in mammalian cells when
overexpressed. MAP-1 homodimerizes and associates with the proapoptotic
Bax and the prosurvival Bcl-2 and Bcl-XL of the Bcl-2
family in vitro and in vivo in mammalian cells.
Mutagenesis analyses revealed that the BH3-like domain in MAP-1 is not
required for its association with Bcl-XL but is required
for association with Bax and for mediating apoptosis. Interestingly, in
contrast to other Bax-associating proteins such as Bcl-XL
and Bid, which require the BH3 and BH1 domains of Bax, respectively,
for binding, the binding of MAP-1 to Bax appears to require all three
BH domains (BH1, BH2, and BH3) of Bax, because point mutation of the
critical amino acid in any one of these domains is sufficient to
abolish its binding to MAP-1. These data suggest that MAP-1 mediates
apoptosis through a mechanism that involves binding to Bax.
Members of the Bcl-2 family of proteins are regulators of cell
death that can be grouped into subfamilies of prosurvival and proapoptotic molecules (1, 2). They are characterized by the presence
of several conserved motifs, known as the Bcl-2 homology (BH)1 domains, designated
BH1, BH2, BH3, and BH4 (1). Although the N-terminal BH4 domain is
restricted to some prosurvival members, BH1, BH2, and BH3 can generally
be found among members of both the prosurvival and proapoptotic
subfamilies (1, 2). Mutagenesis and structural studies revealed that
the BH1, BH2, and BH3 domains are important for dimerization function
(1, 2). Association of the prosurvival member Bcl-XL with
the proapoptotic member Bax requires the BH1, BH2, and BH3 domains of
the former but only the BH3 domain of the latter (3-5).
In addition to its role as a protein-protein interaction domain, the
BH3 domain of proapoptotic members appears to be important for
mediating their proapoptotic function (3, 4). This notion is further
supported by the recent discovery of a new group of cell death agonists
containing only the BH3 domain (6). Similar to other proapoptotic
members of the Bcl-2 family, the BH3 domain in these molecules plays an
important role in mediating proapoptotic function as well as
association with the prosurvival members of the Bcl-2 family.
The three-dimensional structure of Bcl-XL demonstrates that
the BH1, BH2, and BH3 regions form an elongated hydrophobic cleft to
which a BH3 amphipathic In our effort to gain further understanding of the functions of the BH
domains of Bax, we used the yeast two-hybrid screen to identify
Bax-associating proteins. A novel protein, termed MAP-1, was
identified. MAP-1 contains a putative BH3-like domain and induces
caspase-dependent apoptosis in mammalian cells when overexpressed. MAP-1 forms homodimers and associates with Bax, Bcl-2,
and Bcl-XL in vitro and in vivo in
mammalian cells. The BH3-like domain in MAP-1 appeared to be required
for mediating apoptosis and its binding to Bax, but not
Bcl-XL. Interestingly, in contrast to other Bax-associating
proteins, the binding of MAP-1 to Bax requires all three BH (BH1, BH2,
and BH3) domains of Bax. MAP-1 thus represents a novel Bax-associating
protein that may mediate apoptosis through binding to Bax.
Reagents and Cell Lines--
Mono- and polyclonal antibodies
against the Myc epitope (9E10, A14) and HA epitope (F7) were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against PARP
(C2-10) was from Dr. G. Poirier, Laval University Medical
Research Center, Québec Canada. MCF-7 (gift from Dr. V. Dixit),
293T, and SH-SY5Y cells were grown and maintained as described (8, 9).
HeLa cells were obtained from the American Type Culture Collection
(ATCC), and the cells were grown and maintained according to the
directions provided by the suppliers. The peptide protease inhibitors
YVAD-fmk, ZVAD-fmk, and DEVD-fmk were from Enzyme System Products.
Plasmids Construction--
Expression plasmids for hMAP-1,
hMAP-1 deletion mutants, mBax, hBcl-XL, hBcl-2, mBimL, and
mBid were generated by polymerase chain reaction (PCR) amplification of
cDNA clones using the Expand High Fidelity PCR system (Roche
Molecular Biochemicals, Germany), with primers incorporating
appropriate restriction sites, and subcloned into pXJ40HA or pXJ40Myc
mammalian expression vectors (10) driven by the CMV promoter.
Full-length MAP-1 cDNA was subsequently released from pXJHA-MAP-1
and cloned in-frame into GST fusion protein vector pGEX-TK4E (8). A
coding region of each construct was sequenced to ensure that no error
was introduced. All epitope tags were positioned at the N terminus.
Substitution mutants of MAP-1 and Bax were created using the
Transformer site-directed mutagenesis kit
(CLONTECH). Deletion mutants of MAP-1 were
generated by PCR with appropriate primers.
Yeast Two-hybrid Cloning--
Yeast two-hybrid screening was
done essentially as described previously (11). Briefly, mBax cDNA Library Screening--
cDNA fragment of B26 was
used to screen human brain (cerebellum) and mouse brain Northern Blot Analysis--
The human multiple tissue Northern
blot (CLONTECH) was hybridized with a
32P-labeled cDNA probe by using ExpressHyb
hybridization solution (CLONTECH) according to the
instructions of the manufacturer.
In Vitro Binding Assay--
Bacterial GST-MAP-1 fusion protein
was prepared as described previously (8, 12), and the recombinant
protein was immobilized onto glutathione-agarose beads. A GST binding
assay was carried out according to a previously established procedure
(8, 12). Briefly, 35S-labeled proteins were prepared by
in vitro transcription/translation of pXJ-Myc-cDNA
constructs using a TNT T7-coupled reticulocyte lysate system (Promega).
The integrity of the 35S-labeled proteins was verified by
SDS-polyacrylamide gel electrophoresis (PAGE). Equal amounts of
total 35S-labeled proteins (7 × 105 cpm
of trichloroacetic acid precipitable counts) were diluted into 0.2 ml
of GST binding buffer (20 mM Tris (pH 7.5), 1 mM EDTA, 150 mM NaCl, 0.2% Nonidet P-40) and
incubated for 1 h with GST-MAP-1 immobilized on the beads (~2
µg). Samples were subsequently washed six times with binding buffer
and boiled for 3 min in loading buffer before fractionation on 12%
SDS-PAGE. Bound proteins were visualized by autoradiography.
Coimmunoprecipitation--
293T cells seeded on a 100-mm plate
were transiently cotransfected with 10 µg each of expression plasmids
driven by the CMV promoter (pXJ40) (8, 12) encoding the indicated
N-terminal HA- and Myc-tagged proteins using the LipofectAMINE method
(Life Technologies, Inc.). 16 h after transfection, cells were
harvested for immunoprecipitation assay and analyzed as described (8, 12). Briefly, the cells were harvested and lysed in 1 ml of lysis
buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.2%
Nonidet P-40, 1 mM EDTA, 1 mM PMSF, 50 µg/ml
aprotinin, 10 µg/ml leupeptin). An aliquot (1%) of the cell lysate
was fractionated on SDS-PAGE for determining the expression of the
proteins by Western blot analysis using monoclonal anti-Myc or anti-HA
antibodies. The remaining lysates were subjected to immunoprecipitation
using 1 µg of polyclonal anti-Myc antibody for 1 h on ice and
then mixed with 20 µl of a 1:1 slurry of protein A-agarose and
incubated for another 1 h at 4 °C. The agarose beads were
washed once in 1 ml of lysis buffer, twice in 1 ml of lysis buffer
containing 500 mM NaCl, and once in 1 ml of lysis buffer
before fractionation on SDS-PAGE followed by Western blotting analysis.
Apoptosis Assay--
Apoptosis assays were carried out as
described previously (8). Briefly, cells seeded on a 35-mm plate at
70% confluency were transiently cotransfected with 1.5 µg each of
the expression plasmids or vector and 0.3 µg of
pCMV- Nuclear Staining of MAP-1-expressing Cells--
MCF-7 cells were
seeded onto glass coverslips at 70% confluency and cotransfected with
1.5 µg of expression plasmids of MAP-1 or vector and 0.3 µg of
pEGFP (CLONTECH) using LipofectAMINE. 24 h
after transfection, the cells were fixed, rinsed with
phosphate-buffered saline and then incubated for 2 min with Hoescht
33342 dye (Molecular Probes Inc.) to enable nuclear staining. The cells
were subsequently fixed and visualized using a Ziess Axioplan
microscope as described previously (9).
Preparation of Nuclear Extracts--
As described previously
(13), cells were washed twice with phosphate-buffered saline and
detached from plates by adding 1 ml of detachment buffer (150 mM NaCl, 1 mM EDTA, pH 8.0, 40 mM
Tris, pH 7.6). The cells were centrifuged at 300 × g,
and the supernatant was discarded. The pellet was resuspended in 400 µl of cold buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM
dithiothreitol, 0.5 mM PMSF, 2 mM benzamidine,
10 µg/ml aprotinin, and 10 µg/ml leupeptin). Nuclei were pelleted
by centrifuging at 2800 × g and resuspended in 50 µl
of buffer B (20 mM Hepes, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM
PMSF, 2 mM benzamidine, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). The nuclei were lysed by vigorous vortexing, and
the mixture was centrifuged at 15,000 × g for 5 min
and the supernatant was collected. Protein concentration was determined
by Bradford assay, and equal amounts of proteins were used for SDS-PAGE
followed by Western blotting analysis.
Reverse Transcriptase-linked PCR--
Total RNA was prepared
from various cell lines using TRIzol Reagent (Life Technologies, Inc.)
according to the manufacturer's protocol. 10 µg of total RNA from
various cell lines was annealed with oligo(dT)12-18 and
reversed-transcribed using Moloney murine leukemia virus reverse
transcriptase (Roche Molecular Biochemicals) for the synthesis of first
strand cDNA. 2 µl of the first strand cDNA was subjected to
the following PCR conditions: Initial incubation at 94 °C for 5 min,
then 25 cycles of amplification (94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min), ending with a final extension step of
72 °C for 10 min. For each experiment, PCR for glyceraldehyde-3-phosphate dehydrogenase was performed to ensure that
equal quality and quantity of cDNA were used. Negative controls containing water instead of cDNA were included to ensure that the
PCR product obtained was not due to cross-contamination of DNA. The
sequence of the primers used are as follows: hMAP-1(+) (5'-TAG AGG CTC
TTC AGC CTG CCC TGC-3'), hMAP-1( A yeast two-hybrid screen was performed to identify
Bax-associating proteins. Mouse cDNA encoding amino acids 1-171 of
Bax (Bax
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
helix can bind (5, 7). The other members of
the Bcl-2 family that have similar domains are predicted to have a
similar tertiary structure. Hence, proapoptotic members of the Bcl-2
family that contain multiple BH domains such as Bax are proposed to
exist in two different conformations: one that is similar to
Bcl-XL and the other with the BH3 domain rotated outside to
allow its insertion into the hydrophobic cleft of a prosurvival protein
(1). However, there is a lack of evidence to support the existence of a
Bcl-XL-like conformation among the proapoptotic molecules
that contain the BH1-3 domains.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-Zap human and mouse libraries were purchased from Stratagene (La
Jolla, CA).
C21 was
cloned in-frame with Gal-4 DNA binding domain in pAS2(HA) vector and
used to screen a yeast two-hybrid library derived from adult human
brain (CLONTECH). Screening and subsequent
protein-protein interaction studies were carried out in yeast Y190
reporter strain.
-ZAP
cDNA libraries (Stratagene) using standard techniques as described
(10).
-galactosidase. Vector plasmid was supplemented to bring the
total amount of plasmids for each transfection to 5 µg. Transfections
were carried out with LipofectAMINE for 6 h followed by change of
media, and caspase inhibitors were added to the fresh media at this
point where indicated. 18 h later, cells were fixed and incubated
in X-gal buffer to mark the
-galactosidase-expressing cells. Data
(mean ± S.D.) are plotted as the percentage of apoptotic cells
defined by the number of round blue cells over the total blue cells
counted (500-800 cells) from five randomly chosen fields.
) (5'-ACT CGT TGC CAT ATC CCT TCG
TGG-3'), glyceraldehyde-3-phosphate dehydrogenase(+) (5'-TGA AGG TCG
GTG TGA ACG GAT TTG-3'), and glyceraldehyde-3-phosphate dehydrogenase(
) (5'-GCC TAA ATG GCC TCC AAG GAG TAA-3').
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
C21), with the hydrophobic C-terminal transmembrane domain deleted to reduce toxicity in yeast (14), was cloned in-frame with the
GAL-4 DNA binding domain and used as "bait" to screen a yeast
two-hybrid human brain library (CLONTECH). From
approximately two million transformants, 12 clones from the primary
screen were identified, 2 of which, B26 and B100, were found to
interact specifically with Bax
C21 but not with other unrelated Gal-4
fusion proteins tested. Upon sequencing, these two clones were found to
contain two independent partial complementary DNA (cDNAs) derived
from the same gene. Using B26 cDNA fragment as probe, we screened a human brain (cerebellum) cDNA library and obtained several
cross-hybridizing cDNA clones. The longest clone, B9, contained a
2.2-kb cDNA insert with a 1056-nucleotide open reading frame
beginning with a translational initiation consensus sequence and ending
with an in-frame stop codon that encoded a protein of 351 amino acids
with a predicted molecular mass of 39 kDa (Fig.
1A). We named this protein
MAP-1 for modulator of apoptosis. A
corresponding murine cDNA clone was subsequently obtained by
screening a mouse brain cDNA library. The amino acid sequence
deduced from the murine cDNA clone is 77% identical to the human
clone (Fig. 1A). Data base searches revealed that the
predicted human MAP-1 protein shares extensive amino acid sequence
similarity with two other human proteins (Fig. 1A), Ma1
(58%) (15) and Ma2 (47%) (16), suggesting that MAP-1 is a member of a
gene family. Ma1 and Ma2 were both initially identified as antigens
recognized by autoantibodies present in the sera of some patients with
paraneoplastic neurological syndromes with underlying malignancy
(breast, colon, parotid for Ma1, and testicular cancer for Ma2) (15,
16). At present, no function has been described for the Ma1 and Ma2
proteins.
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Fig. 1.
Sequence, structure, and expression of
MAP-1. A, predicted amino acid sequences of human and
mouse MAP-1 are aligned with human Ma1 and Ma2. Amino acid
residues that are identical to human MAP-1 are boxed. The
two Bax-interacting clones isolated from the yeast two-hybrid screen
were fused to Gal-4 activation domain at the positions indicated by
B26 and B100. The putative BH3-like domains are
in boldface and underlined. B, MAP-1
contains a putative BH3-like motif. Sequence alignment of BH3-like
domain of MAP-1 with BH3-B from Bid and BH3 domains from selected
members of Bcl-2 family. Amino acids that are identical or functionally
conserved are boxed. C, Northern blot analysis of
MAP-1. A human tissue mRNA blot (CLONTECH) was
probed with random primed 32P-labeled cDNA insert from
clone B26. The blot was subsequently stripped and rehybridized with a
-actin probe.
Upon examination of the amino acid sequence of MAP-1, a region of eight amino acids (amino acids 120-127) was found to be highly similar to the BH3 domains present among members of the Bcl-2 family (Fig. 1B). The BH3-like motif identified appears to be most similar to the recently identified BH3-B domain (12) present in the N-terminal region of Bid (Fig. 1B). The amino acid sequences in Ma1 and Ma2, corresponding to the region that contains the BH3-like domain, appear to be less similar to BH-3 motif (Fig. 1A).
Northern blot analysis revealed a single 2.8-kb transcript of MAP-1 to be ubiquitously expressed but at a higher level in heart and brain (Fig. 1C). The messages of MAP-1 in total RNA isolated from MCF-7 cells could not be detected in Northern blot analysis. However, MAP-1 messages were readily detectable in MCF-7 and other human cell lines, including HeLa and SH-SY5Y with reverse transcription-linked PCR. Interestingly, Ma1 and Ma2 have a highly restricted expression patterns. Ma1 protein is detected only in brain and testis (15), whereas Ma2 protein is found exclusively in brain (16).
The ability of MAP-1 to associate with Bax was further demonstrated
in vivo in transient cotransfection experiments. HA-tagged MAP-1, Bax, Bid, Bcl-2, and Bcl-XL were transiently
coexpressed with Myc-tagged MAP-1 into 293T cells, and lysates were
prepared for immunoprecipitation. MAP-1 immunoprecipitated specifically with Bax, Bcl-2, and Bcl-XL but not Bid (Fig.
2A). Other members of the
Bcl-2 family, including BimL, Bak, Bad, and Bcl-w, also failed to
interact with MAP-1 in the immunoprecipitation assay under the same
experimental conditions (data not shown), suggesting that MAP-1 does
not bind universally to all members of the Bcl-2 family. In addition,
MAP-1 was able to homodimerize in vivo (Fig. 2A).
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It has been shown recently that the conformation of Bax can be affected by certain detergents such as Nonidet P-40 present in the lysis buffer, resulting in nonspecific interaction of it with some members of the Bcl-2 family (17). Because CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) was shown not to affect the N-terminal conformation of Bax (17), we performed the coimmunoprecipitation experiment as described in Fig. 2A in transient transfection analysis by lysing the 293T cells in buffer containing 0.2% CHAPS instead of Nonidet P-40. Similar to the results obtained with Nonidet P-40, MAP-1 self-associated and interacted with Bax, Bcl-2, and Bcl-XL, but not Bid under this condition (data not shown).
These interactions observed in the immunoprecipitation assays were further demonstrated in vitro by using the GST-pulldown assay. The in vitro translation of MAP-1 from the pXJ-Myc-MAP-1 plasmid in rabbit reticulocyte lysates generates MAP-1 products, which always appear as two bands when fractionated on SDS-PAGE. The upper band corresponds to the predicted molecular mass (39 kDa) of MAP-1 and appears to have the same mobility as the Myc-tagged MAP-1 expressed in 293T cells (data not shown). Furthermore, anti-Myc antibody was only able to immunoprecipitate efficiently the in vitro translation product of MAP-1 represented by the upper band (data not shown), suggesting that the protein represented by the lower band does not contain the Myc epitope, and it is likely to be generated from alternate usage of initiation codon. In agreement with the in vivo results, GST-MAP-1 was able to pulldown in vitro translated 35S-labeled MAP-1, Bax, Bcl-2, and Bcl-XL but not the 35S-labeled Bid (Fig. 2B).
We next examined the possible role of MAP-1 in modulating cell death by
transient transfection experiments. MCF-7 cells were cotransfected with
MAP-1 expression or control vector together with pCMV--galactosidase
or pEGFP as a marker for transfected cells. At 24 h
post-transfection, a significant percentage of total
-galactosidase-positive (blue) cells cotransfected with MAP-1 displayed apoptotic morphology as evidenced by the appearance of
shrunken and rounded cells (Fig.
3A). Nuclear condensation, another hallmark of apoptosis, was also observed in a significant percentage of total green fluorescence-positive (green)
cells cotransfected with MAP-1 (Fig. 3B). Proteolytic
cleavage of poly(ADP-ribose) polymerase (PARP), which serves as a
marker for the activation of caspases in cells undergoing apoptosis (8,
18), was also detected in cells transfected with MAP-1 (Fig.
3C). Furthermore, the cleavage of PARP to the signature
85-kDa apoptotic fragment in MCF-7 cells transfected with MAP-1 was
completely blocked by treatment with ZVAD-fmk (10 µM)
(Fig. 3C).
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A series of caspase inhibitors were tested for their ability to inhibit apoptosis triggered by MAP-1 overexpression. YVAD-fmk (50 µM), a specific inhibitor of caspase-1-like caspases, and DEVD-fmk (50 µM), an inhibitor of caspase-3-like caspases, conferred no and partial protection, respectively, against MAP-1-mediated killing (Fig. 3D). However, ZVAD-fmk (10 µM), a wide-spectrum caspase inhibitor, was able to abrogate the apoptotic activity of MAP-1 (Fig. 3D), suggesting that MAP-1 mediates apoptosis through a caspase-dependent pathway. Similar to Bax-mediated apoptosis, overexpression of Bcl-XL effectively blocked the apoptotic effect of MAP-1 (Fig. 3E). The effect of MAP-1 in mediating apoptosis is not restricted to MCF-7 cells, because overexpression of MAP-1 was able to induce apoptosis (judged by round cell and nuclear condensation assays) in both HeLa and SH-SY5Y cells (data not shown). Similar to human MAP-1, mouse MAP-1 induced apoptosis in MCF-7 cells and associated with Bax and Bcl-XL in immunoprecipitation assay in transient cotransfection experiments (data not shown).
We next proceeded to evaluate the requirement of the BH3-like domain in
MAP-1 for mediating protein-protein interaction and proapoptotic
functions. A series of Myc-tagged MAP-1 mutants was created (Fig.
4A). Deletion of the entire
BH3-like domain (M1) or substitution mutations of several conserved
amino acids (GHE/VLA) in the putative BH3-like domain (M2) of MAP-1
abrogated its interaction with Bax (Fig. 4B). The conserved
leucine residue in the BH3 domains of Bax and Bad was reported to be
critical for mediating interaction with Bcl-2 or Bcl-XL
(19, 20) as well as for the N-terminal BH3-B domain of Bid to interact
with its C terminus (12). Substitution of this conserved leucine of the
BH3 domain, but not the nonconserved histidine, to glutamic acid (M3,
M4), in MAP-1 was found to substantially weaken its interaction with
Bax (Fig. 4B). Interestingly, in contrast to most
proapoptotic members of the Bcl-2 family, the BH3-like domain in MAP-1
did not appear to be required for interaction with Bcl-XL
as deletion of the entire BH3-like domain (M1) of MAP-1 did not affect
its binding to Bcl-XL (Fig. 4C). In addition, deletion mutant containing the BH3-like domain (M6) failed to interact
with Bcl-XL, whereas the N-(M5) and C-(M7) terminal regions of MAP-1 were able to independently associate with Bcl-XL
(Fig. 4C).
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Similar to many BH3-domain containing molecules (1, 2), the BH3-like domain in MAP-1 appeared to be required for its cell death function as deletion of this domain (M1) or mutations of the highly conserved amino acids in this domain (M2, M3) resulted in nonapoptotic protein (Fig. 4D). The H126E (M4) mutant of MAP-1 that maintained binding to Bax also retained proapoptotic activity at a level similar to the wild-type MAP-1. Despite the presence of the BH3-like domain, the M6 mutant failed to induce apoptosis (Fig. 4D), suggesting that the BH3-like domain alone is not sufficient for mediating apoptosis. Although it has been reported that peptides encompassing essentially the BH3 domain are capable of inducing apoptosis (21, 22), the BH3 domain alone has also been shown to be insufficient for inducing apoptosis (23, 24). On the other hand, because no functional activity such as protein-protein interaction or apoptosis induction has been demonstrated with the M6 mutant, we cannot rule out the possibility that M6 mutant may not fold properly at all, which could result in a totally inactive protein. Additional experiments involving other BH3 domain-containing protein fragments or peptides of MAP-1 are clearly needed to resolve the issue whether the BH3-like region in MAP-1 is sufficient for mediating protein-protein interaction and/or inducing apoptosis. Taken together, these data suggest that the BH3-like domain in MAP-1 is required for interaction with Bax and mediating apoptosis. Although both the N- and C-terminal fragments of MAP-1 were able to associate with Bcl-XL, none of these mutants (M5 and M7) were found to be proapoptotic (Fig. 4D), suggesting that interaction with Bcl-XL is insufficient to render the molecule proapoptotic.
Several highly conserved amino acid residues of the BH1, BH2, and BH3
domains among Bcl-2 family members have been demonstrated to be
critical for heterodimerization functions (20, 25, 26). To evaluate the
requirement of the BH domains of Bax in mediating interaction with
MAP-1, substitution point mutations of these critical amino acid
residues in the BH1, BH2, and BH3 domains were made. In agreement with
previous data, mutation of the BH3 (L63E), but not the BH1 (G108A) or
BH2 (W151A) domains of Bax, abolished its binding to Bcl-XL
(Fig. 5A). However, none of
the point mutants were able to bind MAP-1 (Fig. 5B),
suggesting that the three BH domains (BH1, BH2, and BH3) of Bax are all
required for mediating protein-protein interaction with MAP-1. Yeast
two-hybrid experiments, performed with these Bax mutations in the
BaxC21 context to examine their interaction with the B26 clone,
yielded similar results (data not shown).
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In this report, we describe the cloning and characterization of a
Bax-associating protein, MAP-1. Several proapoptotic members of the
Bcl-2 family have been identified by using prosurvival members of the
Bcl-2 family as bait in the yeast two-hybrid system (27-29). However,
there has been no report in the literature for the identification of
protein partners of proapoptotic members of the Bcl-2 family such as
Bax using this system. This in part may be due to the toxicity
exhibited by some proapoptotic members of Bcl-2 family in yeast which
limits its utility in this system (14, 30). We took advantage of the
observation that, by eliminating the hydrophobic C-terminal tail of
Bax, the toxicity of Bax was substantially reduced in yeast, although
it was still capable of mediating apoptosis in mammalian cells (14).
Using BaxC21 as bait, we managed to screen for protein partners of
Bax. Interestingly, we failed to identify any prosurvival members of
the Bcl-2 family, including Bcl-XL in our screen. Two
possible reasons may account for this observation. First, cDNAs
encoding prosurvival members of the Bcl-2 family may be in extremely
low abundance in the library. Second, Bax may associate only weakly
with the prosurvival members of the Bcl-2 family in this system.
Indeed, we noted that interaction of Bax with Bcl-XL was
relatively weak in the yeast two-hybrid system (data not shown).
MAP-1 contains a putative BH3 domain and associates with Bax, Bcl-XL, and Bcl-2 in vitro and in vivo in mammalian cells. It mediates caspase-dependent apoptosis when overexpressed. The BH3-like domain in MAP-1 is required for binding to Bax, but not Bcl-XL, and for mediating apoptosis. Interestingly, in contrast to other known Bax-associating proteins, the binding of Bax to MAP-1 requires all of the BH (BH1, BH2, and BH3) domains of Bax, because point mutations affecting any one of the BH domains abolished its binding to MAP-1. MAP-1 thus represents the first protein partner of Bax identified that requires all the BH domains of Bax for association.
Why do some proapoptotic members of the Bcl-2 family contain BH1, BH2, and BH3 domains resembling those of the prosurvival members of Bcl-2 family? The crystal structure of Bcl-XL suggested that it shares similarity to the pore-forming domains of bacterial toxins such as colicins A1 and E1 and diphtheria toxin (7). It has been reported that regions encompassing part of the BH1 and BH2 domains may have pore-forming function (2). In addition, Bax was shown to form an ion channel in vitro (2). However, it still remains to be determined whether the Bcl-2 family of proteins actually forms channels in vivo and whether these proteins regulate apoptosis via the creation of ion channels (31).
Instead of the BH1, BH2, and BH3 domains forming a receptor structure,
as in the case of Bcl-XL (6), the BH domains in Bax
have been suggested to serve independent functions. The BH3 domain of
Bax has been proposed to be involved in binding the permeability
transition pore and inducing permeability transition and cytochrome
c release from the mitochondria (31, 32). In addition, it
has been proposed that the BH3 domain of Bax mediates its proapoptotic
effect by binding to the prosurvival molecule Bcl-XL and,
hence, displacing the binding of Apaf-1 from Bcl-XL leading
to caspase-9 activation (33, 34). However, the validity of this model
was recently challenged, because a physiologically relevant level of
stable interaction between Apaf-1 and Bcl-XL cannot be
demonstrated (35, 36). Recently, the BH3-domain-only molecule Bid was
found to associate with Bax through its BH1 domain (26). Thus, Bid is
the only proapoptotic member of the Bcl-2 family known to associate
with Bax prior to MAP-1. This association appears to alter the
conformation of Bax and lead to the release of cytochrome c
from the mitochondria (37). Hence, multiple mechanisms are likely to be
responsible for the apoptotic effects mediated by Bax. Identification
of MAP-1 as a novel Bax-associating protein may thus provide further
opportunities for investigating the complex mechanisms operated by Bax.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Suzanne Cory and David Huang for providing us with many of the cDNA clones employed in this study.
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FOOTNOTES |
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* This work was supported by grants from the National Science and Technology Board of Singapore.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF305550 (human MAP-1) and AF305551 (mouse MAP-1).
To whom correspondence should be addressed: Tel.: 65-874-3740 and
65-874-6862; Fax: 65-779-1117; E-mail: mcbyuck@imcb.nus.edu.sg.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M008955200
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ABBREVIATIONS |
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The abbreviations used are:
BH, Bcl-2 homology;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel
electrophoresis;
PCR, polymerase chain reaction;
X-gal, 5-bromo-4-chloro-3-indolyl -D-galactopyranoside;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PARP, poly(ADP-ribose) polymerase;
GFP, green fluorescence protein;
HA, hemagglutinin;
CMV, cytomegalovirus;
PMSF, phenylmethylsulfonyl
fluoride;
kb, kilobase(s).
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