From the Drug Discovery Program, H. Lee Moffitt
Cancer Center and Research Institute, Department of Pharmacology and
Therapeutics, University of South Florida, Tampa, Florida 33612, ¶ Department of Genetics, National Children's Medical Research
Center, Tokyo 154, Japan, and
Imgenex Corporation, San
Diego, California 92121
Received for publication, February 19, 2001, and in revised form, March 12, 2001
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ABSTRACT |
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Bax is a proapoptotic member of the Bcl-2 protein
family that commits the cell to undergo programmed cell death in
response to apoptotic stimuli. To gain further insights into Bax
mechanisms, we have identified a novel Bax-binding protein, termed
Bif-1, by using a yeast two-hybrid cloning technique. Bif-1 is an
evolutionarily conserved cytoplasmic protein that contains a predicted
Src homology 3 (SH3) domain located near its C terminus but shares no
significant homology with members of the Bcl-2 family. A Northern blot
analysis indicates that Bif-1 is expressed in most tissues with
abundant expression in heart and skeletal muscle. Bif-1 is capable of
interacting with Bax as demonstrated by yeast two-hybrid,
coimmunoprecipitation, and immunofluorescence studies. Induction of
apoptosis in murine pre-B hematopoietic cells FL5.12 by interleukin-3
withdrawal results in increased association of Bax with Bif-1, which is
accompanied by a conformational change in the Bax protein.
Overexpression of Bif-1 promotes Bax conformational change, caspase
activation, and apoptotic cell death in FL5.12 cells following
interleukin-3 deprivation. Bif-1 thus represents a new type of
regulator of Bax-mediated signaling pathways for apoptosis.
Programmed cell death, or apoptosis, is defined as a physiological
process that plays a critical role in the normal development and
maintenance of tissue homeostasis by eliminating infected, mutated, or
damaged cells in essentially all multicellular organisms (1, 2).
Dysregulation of this physiological cell death process, resulting in
defects in normal cell turnover, is implicated in the pathogenesis of
many types of diseases, including cancer, autoimmune disease,
neurodegenerative disorders, and AIDS (3). Apoptosis is caused by the
activation within cells of a family of cysteine proteases, which
specifically cleave their substrates at aspartic acid residues. These
proteases are known as "caspases." The Bcl-2 family proteins appear
to control the "decision" step of apoptosis, determining whether
certain caspases will or will not become activated (4-6).
Antiapoptotic members of the Bcl-2 family such as Bcl-2 and
Bcl-xL tend to prevent activation of these terminal
effector proteases, whereas proapoptotic members Bax and BAK facilitate
caspase activation.
Bax is the first proapoptotic homologue of the Bcl-2 family, which was
identified by coimmunoprecipitation with the Bcl-2 protein (7).
Overexpression of Bax accelerates cell death induced by a wide range of
cytotoxic insults, whereas loss of Bax expression has been observed in
a wide variety of human cancers and also contributes to poor response
to chemotherapeutic drugs (8). Activation of this proapoptotic protein
appears to involve intracellular translocation and homodimerization
(9). Apoptotic stimuli induce a conformational change of the Bax
protein, resulting in exposure of its N and C termini that appears to
be required for the cytosolic Bax protein to move to the membranes of
mitochondria where it inserts as a homodimer (10-12).
Evidence has accumulated that mitochondria play an important role in
the control of apoptosis (13). Cytochrome c resides in the
intermembrane space of mitochondria of healthy cells. Once released
from mitochondria, cytochrome c binds to and activates Apaf-1, a human homologue of the Caenorhabditis elegans cell
death protein CED-4 (14). Activated Apaf-1 then forms complexes with pro-caspase 9, resulting in caspase activation and apoptosis induction. Overexpression of proapoptotic Bcl-2 family proteins Bax, BAK, and BID
induces cytochrome c release through a Bcl-2 suppressible mechanism (4). Thus, one possibility is that Bax may form selective channels for cytochrome c release from the
inter-membrane space of mitochondria into the cytosol, although exactly
how mitochondrial apoptogenic molecules escape during apoptosis
remains controversial.
To gain further insights into Bax action, we performed a yeast
two-hybrid screening to identify proteins that can bind to Bax. Here we
describe the molecular cloning and functional characterization of a
novel protein, termed Bif-1 for Bax-interacting factor-1, which
interacts physically with the Bax protein and influences cell life and death.
Plasmids--
The human Bif-1 cDNA in pJG4-5 was obtained
from a yeast two-hybrid screening by using LexA-Bax ( Yeast Two-hybrid Assays--
Two-hybrid screens were performed
essentially as described (15) in Saccharomyces cerevisiae
EGY48 cells with plasmid pEG202 encoding LexA-Bax ( Northern Blot Analysis--
A human 12-lane multiple tissue
Northern blot (CLONTECH) was hybridized at 68 °C
overnight in Church buffer (0.5 M NaPO4, pH
7.1, 2 mM EDTA, 0.1% sodium pyrophosphate, 7% SDS)
containing 100 µg of single-stranded DNA and a
[32P]dCTP-labeled probe (1.8 × 109
cpm/ml) generated from an 0.8-kilobase N-terminal Bif-1 cDNA as
template and random primers (Life Technologies, Inc., Baltimore, MD).
Cell Transfections and Apoptosis Assays--
FL5.12 cells were
maintained in interleukin-3
(IL-3)1-containing medium and
transfected with 25 µg of pK-SFFV-Bif-1 or parental vector DNA (Neo)
by electroporation as described (20). 293 or 293T cells were
transfected by a calcium phosphate method. Stable transfectants were
selected by 1 mg/ml G418. The cell viability was determined by trypan
blue dye exclusion, 3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Thiazolyl
blue) assay, and flow cytometric analysis.
Coimmunoprecipitation Assay--
Cells were lysed in Nonidet
P-40 lysis buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 0.2% Nonidet P-40) or
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps)
lysis buffer (150 mM NaCl, 10 mM Hepes, pH7.4,
1% Chaps) containing 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1 µg/ml pepstatin A, and 5 µg/ml aprotinin.
Immunoprecipitation and immunoblot assays were performed as described
(18) with the indicated antibodies. The anti-Bif-1 polyclonal antiserum was generated in rabbits using glutathione
S-transferase-Bif-1 fusion protein as immunogen. The
glutathione S-transferase-Bif-1 protein was produced in
Escherichia coli DH5 Immunofluorescence Analysis--
Cells were cultured with 25 nM MitoTracker CMTMRos (Molecular Probes, Eugene, OR)
for 30 min prior to fixation in 4% paraformaldehyde and
permeabilization in 0.5% Triton X-100. Immunofluorescent staining was
performed as described (18) with anti-FLAG M2 monoclonal antibody
(Sigma, St. Louis, MO), anti-Bax N20 rabbit antiserum (Santa Cruz
Biotechnology, Santa Cruz, CA), or anti-Bif-1 monoclonal antibody (Imgenex Corp., San Diego, CA), which was detected by either
fluorescein isothiocyanate (FITC)-conjugated or rhodamine-conjugated secondary antibodies (Chemicon International, Inc., Temecula, CA).
Molecular Cloning of cDNAs for Bif-1--
To identify
cDNAs encoding proteins that can bind to Bax, we performed a yeast
two-hybrid screening using a LexA-Bax as the bait. A screen of 1.5 million independent transformants from a human brain Matchmaker
cDNA library yielded 68 clones positive for both
GAL1-LEU2 and GAL1-lacZ reporter gene expression.
Of these, 3 clones (numbers 3, 8, and 21) encoded the same protein, designated Bif-1, for Bax-interacting factor-1. The rest of the clones
were single clones, the sequences of which were not identifiable or
encoded artificial proteins. Surprisingly, no Bcl-2 family members were
found in this yeast two-hybrid screen, although they are no doubt
expressed in the human brain cDNA library used and previously have
been shown to interact with the Bax protein. All three Bif-1 cDNAs
contained an open reading frame encoding a predicted protein of 365 amino acids (Fig. 1).
Sequence alignment analysis revealed that Bif-1 contains a region
located in the C terminus of the protein with significant similarity to
the Src homology 3 (SH3) domain, which plays an important role in
signal transduction pathways and is involved in cell-cell communication
(21, 22). Bif-1, however, does not contain any of the conserved Bcl-2
homology (BH) domains of the Bcl-2 family proteins. We also performed
BLAST searches to identify additional previously undescribed homologues
of Bif-1 in mouse and possibly in the nematode C. elegans
and the fruit fly Drosophila melanogaster. As shown in Fig.
1, the mouse Bif-1 protein contains 365 amino acids and shares 96%
overall amino acid identity with human Bif-1, as deduced from expressed
sequence tag clones AA615579, AA517877, AA592742, and AI159401. The
C. elegans Bif-1 (AAB52640) is 366 amino acids in length and
shares 42% identity and 59% similarity with the human Bif-1 protein.
The D. melanogaster gene (AAF57578) encodes a protein of 426 amino acids that shares 39% identity and 57% similarity with human
Bif-1.
Tissue Distribution of Bif-1--
Northern blot analysis was used
to assess the expression of Bif-1 mRNA in various human
tissues. Hybridization with a Bif-1 probe revealed expression of
Bif-1 in most tissues, with abundant expression in heart,
skeletal muscle, kidney, and placenta (Fig. 2). Interestingly, three different size
transcripts were detected for Bif-1, with major mRNAs of 1.5, 2, and 6 kilobases; it remains to be determined whether these different
size transcripts arise from alternative splicing mechanisms and whether
they encode different proteins.
Bif-1 Associates with Bax in Yeast and Mammalian Cells--
Yeast
two-hybrid analysis indicated that Bif-1, whether fused to a B42
transactivation domain (AD-Bif-1) or a LexA DNA-binding domain
(LexA-Bif-1), strongly interacted with Bax, as determined by assays of
To confirm that association of Bif-1 and Bax can occur in mammalian
cells, 293T cells were transiently transfected with expression plasmid
encoding FLAG-tagged Bif-1 or the same parental vector lacking Bif-1
cDNA. Immunoprecipitates were prepared using anti-FLAG antibody and
subjected to SDS-polyacrylamide gel electrophoresis (PAGE)/immunoblot
assays using anti-Bif-1 or anti-Bax antibodies, revealing that
endogenous Bax protein can coimmunoprecipitate with FLAG-Bif-1 (Fig.
3B). In addition, immunoblot analysis of the total lysates
indicated that overexpression of FLAG-Bif-1 has no effect on Bax
protein expression in 293T cells (Fig. 3B). More
importantly, endogenous Bax could be coimmunoprecipitated with
endogenous Bif-1 from the murine hematopoietic cell line FL5.12 (Fig.
3C), providing further evidence that the Bax-Bif-1 interaction occurs in the presence of physiological protein levels. Immunoprecipitations performed using preimmune serum (Fig.
3C) or empty vector (Fig. 3B) transfection
confirmed the specificity of these results.
Bif-1 Colocalizes with Bax in Cytosol--
To determine the
intracellular localization of Bif-1, we expressed Bif-1 as a green
fluorescent protein (GFP) fusion protein in 293 epithelial cells.
Fluorescence confocal microscopic analysis revealed an extranuclear
distribution of GFP-Bif-1 (Fig.
4A). Two-color analysis using
a mitochondrion-specific dye MitoTracker showed that a proportion of
the GFP-Bif-1 protein molecules was associated with mitochondria (Fig.
4Ac). Similar results were obtained for
endogenous Bif-1 protein in FL5.12 cells by immunofluorescence staining
with anti-Bif-1 specific monoclonal antibody (Fig. 4, B and
C). Upon induction of apoptosis by IL-3 withdrawal, Bif-1 was concentrated in punctate foci in the cytosol (Fig. 4,
Bd and Ce), suggestive of association with
mitochondria or other organelles. Double immunostaining with antibodies
specific for Bax and Bif-1 indicated that Bif-1 was partially
colocalized with Bax in the cytosol of FL5.12 cells (Fig.
4C).
Because certain apoptotic stimuli trigger Bax translocation from the
cytosol to the membranes of mitochondria, it was important to determine
whether Bif-1 affects the intracellular translocation of Bax in
response to apoptotic signals. For this purpose, 293 cells were
transiently cotransfected with GFP-Bax and FLAG-tagged Bif-1 and
treated without or with apoptosis-inducing agents, including staurosporine, vinblastine, and anti-Fas antibody. As shown in Fig.
5, GFP-Bax was located diffusely in
untreated cells but became concentrated in the cytosol following
apoptosis induction. This is consistent with previous studies (10, 23).
Cells labeled with anti-FLAG monoclonal antibody revealed a mostly
punctate distribution of FLAG-Bif-1 protein in the cytosol when
coexpressed with the proapoptotic protein Bax. Consistent with the
immunofluorescence data in FL5.12 cells, FLAG-Bif-1 was partially found
in the same cellular compartments with GFP-Bax in 293 cells.
Interestingly, the cytosolic redistribution of GFP-Bax in response to
apoptotic stimuli was not observed in some cells that failed to receive the FLAG-Bif-1 plasmid DNA (Fig. 5, D and G),
implying that Bif-1 may contribute to Bax translocation to mitochondria
during apoptosis.
IL-3 Deprivation Induces Bax Association with Bif-1--
To
determine whether apoptotic stimuli-induced changes in Bif-1
colocalization with Bax correlate with alterations in Bax heterodimerization with Bif-1, we performed coimmunoprecipitation experiments. As shown in Fig.
6A, increased association of
Bax with Bif-1 in FL5.12 cells was evident at 12 h after IL-3
deprivation and reached a maximum at 18 h. After 24 h of IL-3
withdrawal, the association between Bax and Bif-1 decreased (Fig.
6A), and more than half of the FL5.12 cells died (not
shown). Immunoblot analysis using whole cell lysates showed that the
protein levels of Bif-1 also slightly decreased following IL-3
withdrawal. Moreover, deprivation of IL-3 induced a conformational
change in Bax as demonstrated by immunoprecipitation with anti-Bax 6A7
monoclonal antibody that specifically recognizes the conformationally
changed Bax protein (24, 25). This correlated closely with the protein complex formation between Bif-1 and Bax in response to growth factor
withdrawal.
To determine whether Bax conformational change is required for its
binding to Bif-1, we compared the ability of Bax to heterodimerize with
Bif-1 under the presence of Nonidet P-40 versus Chaps. It has been reported that nonionic detergents such as Nonidet P-40 and
Triton X-100 can cause a conformational change in Bax, whereas the
zwitterionic detergent Chaps keeps Bax in its native conformation (24).
FL5.12 cells were cultured with or without IL-3 for 16 h, then
lysed with either 1% Chaps or 0.2% Nonidet P-40 and subjected to
immunoprecipitation with anti-Bax polyclonal antibody. When deprived of
IL-3, increased association of Bax with Bif-1 was observed in the
presence of Chaps but not Nonidet P-40 (Fig. 6B), suggesting
that once conformationally changed Bax no longer binds to Bif-1.
Bif-1 Promotes Apoptosis in IL-3-deprived FL5.12 Cells--
To
study the significance of Bax-Bif-1 interaction for regulation of
apoptosis, we stably transfected FL5.12 cells with expression plasmid
encoding human Bif-1 or the same parental vector lacking Bif-1 as a
control (Neo). FL5.12 cells are murine lymphoid progenitor cells that
die via apoptosis in the absence of IL-3, thus providing a model
commonly used to investigate the mechanisms of programmed cell death.
Immunoblot analysis of lysates prepared from the resulting polyclonal
bulk-transfected cell lines showed that levels of Bif-1 protein were
markedly elevated in Bif-1-transfected FL5.12 cells, compared with
FL5.12 cells that received the Neo control plasmid (Fig.
7B). These FL5.12 cells were
then cultured for various times in medium without IL-3, and cell
viability was assessed based on the ability to exclude trypan blue dye.
The kinetics of cell death was markedly accelerated in cultures of
IL-3-deprived Bif-1-expressing FL5.12 cells compared with FL5.12-Neo
control cells. As shown in Fig. 7A, for example, only
~40% of FL5.12 cells expressing Bif-1 remained viable at 12 h
after growth factor withdrawal, compared with nearly 75% of FL5.12-Neo
cells. These results were confirmed by 3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Thiazolyl blue
assay (Fig. 7C). Consistent with previous studies (20),
overexpression of Bcl-2 protected FL5.12 cells from apoptosis induced
by IL-3 withdrawal.
To further characterize the feature of cells undergoing apoptosis,
we performed flow cytometric analysis of the dead cells after staining
with annexin V, a Ca2+-dependent
phospholipid-binding protein that binds to apoptotic cells with exposed
plasma membrane phospholipid phosphatidylserine. As shown in Fig.
7D, 79% of Bif-1-transfected FL5.12 cells were annexin
V-positive, compared with 66% of FL5.12-Neo cells when cultured for
12 h in the absence of IL-3. Moreover, caspase activation was
examined by immunoblot analysis with antibodies specific for caspase-3
or caspase-cleaved D4-GDI fragment. When deprived of IL-3, the
protein levels of pro-caspase-3 dramatically decreased in FL5.12 cells
overexpressing Bif-1 compared with FL5.12-Neo control cells (Fig.
8A). Cleavage of D4-GDI
demonstrated further caspase activation in FL5.12-Bif-1 transfectants
following IL-3 withdrawal (Fig. 8A).
In addition, we also investigated Bax conformational change in FL5.12
cells expressing Bif-1 after IL-3 withdrawal by immunoprecipitation with 6A7 Bax monoclonal antibody. When cultured in the presence of
IL-3, no or very little conformationally changed Bax was detected in
the 6A7 immunoprecipitates from both FL5.12-Bif-1 and FL5.12-Neo transfectants (Fig. 8B, lanes 1 and
3). In contrast, 8 h after deprivation of IL-3, a large
proportion of Bax was immunoprecipitated with 6A7 antibody in
FL5.12-Bif-1 cells (Fig. 8B, lane 4) compared with FL5.12-Neo control cells (Fig. 8B, lane
2).
Using a yeast two-hybrid screening approach, we have identified
cDNAs encoding a novel Bax-binding protein, Bif-1, which is highly
conserved throughout evolution. All members of the Bcl-2 family of
proteins contain at least one of four evolutionarily conserved domains:
BH1, BH2, BH3, and BH4, which can be important for their function and
protein-protein interactions (26). Bif-1 interacts physically with Bax
but lacks identifiable similarity to all four of the conserved BH
domains, indicating that it is not a member of the Bcl-2 protein family.
The predicted amino acid sequence of Bif-1 contains an SH3-like domain
at residues 308-364 and shares significant similarity to several
SH3-containing proteins, including endophilin and GRB2-like proteins.
The SH3 domain, which contains ~60 amino acids, binds to proline-rich
sequences in many intracellular proteins. These protein-protein
interactions regulate the cellular localization of protein-tyrosine
kinases and their substrates such as those involved in signaling at the
cell surface or regulating the cytoskeleton (22). Recently, these
interactions also are implicated in the regulation of apoptosis (27,
28). The SH3 domain-containing protein SETA binds to AIP1/Alix,
apoptosis-linked gene 2 (ALG-2)-interacting protein 1 or
ALG-2-interacting protein X, and sensitizes astrocytes to
UV-induced apoptosis (27). Similarly, overexpression of dynamin-2, an
SH3-interacting GTPase, triggers apoptosis in a
p53-dependent manner (28). Moreover, the deletion mutant of
dynamin-2 lacking the proline/arginine-rich domain triggers apoptosis
more potently than the wild-type, suggesting that the SH3-binding
domain mediates negative regulation of an apoptotic activity in the
dynamin-2 protein (28).
We have identified a novel SH3 domain-containing protein, Bif-1, which
also participates in regulating apoptosis possibly through activating
the Bax-mediated cell death pathway. Bax resides largely in the cytosol
of healthy cells, despite the presence of a typical TM domain near its
C terminus (10, 29). Apoptotic stimuli cause a conformational change in
Bax, inducing its translocation and integration into the membranes of
mitochondria and promoting apoptosis (10, 11, 30). Our studies indicate
that Bif-1 binding to Bax may contribute to induction of Bax
conformational change in response to apoptotic signals. In murine
hematopoietic cells, IL-3 withdrawal induced Bif-1 association with
mitochondria and colocalization with Bax (Fig. 4). In addition,
overexpression of Bif-1 in 293 cells seems to promote the translocation
of Bax to mitochondria following apoptosis induction (Fig. 5).
Moreover, deprivation of IL-3 induced increased association of Bax with Bif-1 in FL5.12 cells, which was accompanied by induction of Bax conformational change (Fig. 6). Interestingly, the nonionic detergent Nonidet P-40, which can induce a conformational change in Bax, dramatically reduced Bax heterodimerization with Bif-1 (Fig.
6B).
Based on these results, we propose a "hit-and-run" model for
Bax-Bif-1 interaction that Bif-1 binds to the "inactive" form of
Bax in the cytosol and induces a conformational change in this protein.
Once conformationally changed or integrated into intracellular membranes, Bax no longer requires interaction with Bif-1. Indeed, overexpression of Bif-1 in FL5.12 cells promoted Bax conformational change, caspase activation, and apoptotic cell death following growth
factor withdrawal. However, how apoptotic stimuli trigger the
interaction of Bif-1 with Bax is unclear. One possibility is that the
ability of Bif-1 to induce Bax conformational change is controlled by
mechanisms of post-translational modifications. For example, the
ability of Bad, a proapoptotic member of the Bcl-2 family, to
heterodimerize with Bcl-xL to induce apoptosis is mediated
by mechanisms controlling the state of phosphorylation of Bad (31, 32).
In addition, cleavage of Bid, another proapoptotic member of the Bcl-2
family, by caspase-8 or granzyme B generates a truncated Bid fragment
that binds to and induces Bax conformational change during apoptosis
(33-36). Although additional work is clearly required to further study
the molecular mechanism of Bax-Bif-1 interaction, the data shown here
argue that Bif-1 may promote apoptosis by inducing a conformational
change of Bax leading to its mitochondrial targeting.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TM) as bait.
After digestion with EcoRI and XhoI, the Bif-1
cDNA was subcloned into the yeast two-hybrid vector pEG202 (15) or
the mammalian expression vector pK-SFFV that was generated by replacing
the CMV promoter of pcDNA3.1 (Invitrogen, Carlsbad, CA) with the
SFFV-LTR promoter from the SFFV-neo plasmid (16). The Bif-1 open
reading frame was cloned in-frame into the
EcoRI/XhoI-digested vector pEGFP-C2
(CLONTECH Lab, Inc., Palo Alto, CA), pGEX4T-1
(Amersham Pharmacia Biotech, Inc., Piscataway, NJ), or the
EcoRI/SalI-digested pFLAG-CMV2 vector (17). All
other plasmids have been described (18).
TM), fusion
protein (19), and human fetal brain cDNA library cloned into
pJG4-5 (CLONTECH). Candidate clones were isolated
from yeast colonies formed on leucine-deficient agar plates with
detectable
-galactosidase activity and retested by cotransformation
with the bait expressing plasmids.
cells and purified by
glutathione-Sepharose according to manufacturer's recommendations (Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Bif-1 is an evolutionarily conserved SH3
domain-containing protein. The predicted amino
acid sequence of human Bif-1 protein and the homologous mouse, C. elegans, and D. melanogaster proteins are aligned, with
identical residues in boxes. The predicted SH3 domain is
indicated in bracket.
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Fig. 2.
Tissue distribution of human Bif-1. A
human 12-lane multiple tissue Northern blot was hybridized with a human
Bif-1-specific radiolabeled riboprobe. PBL, peripheral blood
leukocyte.
-galactosidase activity (Fig.
3A). The only region in the
Bif-1 protein that shares significant amino acid homology to other
known proteins is an SH3-like domain located between residues 308 and
364 of the human Bif-1 protein. To explore whether binding of Bif-1 to
Bax requires this domain, a C-terminal deletion mutant of Bif-1
(residues 1-284) that lacks the SH3-like domain was expressed as a
LexA-fusion protein and tested for its ability to interact with AD-Bax
in budding yeast. As shown in Fig. 3A, LexA-Bif-1 (1)
interacted with AD-Bax to a degree comparable with the interactions
between full-length LexA-Bif-1 and AD-Bax. In contrast, AD-Bax failed
to form two-hybrid interactions with LexA-Bif-1 (285), a deletion
mutant of Bif-1 protein that essentially contained only the SH3-like
region, indicating that the SH3-like domain of Bif-1 is not required
for its interaction with Bax.
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Fig. 3.
Specific interaction of Bax with Bif-1.
A, interactions of pairs of fusion proteins containing
either an N-terminal LexA DNA-binding domain (BD) or B42
transactivation domain (AD) were determined by yeast
two-hybrid assay using lacZ reporter genes under the control
of lexA operators. Five independent transformants were
tested for their ability to produce blue color on
X-5-bromo-4-chloro-3-indolyl -D-galactopyranoside
(X-gal) plates containing either galactose (Gal) or glucose
(Glu), to either induce or repress, respectively, the gal-1
promoters in the two-hybrid plasmids. B, 293T cells were
transiently transfected with pFLAG-CMV2-Bif-1 or parental pFLAG-CMV2
vector DNA. Immunoprecipitations were performed 2 days later using
anti-FLAG M2 antibody, followed by SDS-PAGE and immunoblot analysis of
the resulting immune complexes with either anti-Bif-1 or anti-Bax
polyclonal antibodies. In addition to immune complexes, the total
lysates (25 µg) were directly analyzed. C, FL5.12 cells
were lysed in Chaps lysis buffer containing protease inhibitors, and
immunoprecipitation was performed using anti-Bif-1 polyclonal antiserum
or preimmune serum (NRS) as a negative control, followed by Western
blot analysis using anti-Bax 6A7 (PharMingen, San Diego, CA) or
anti-Bif-1 (Imgenex, San Diego, CA) mouse monoclonal
antibodies.
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Fig. 4.
Subcellular localization of Bif-1.
A, 293 cells were transiently transfected with expression
plasmids encoding GFP-Bif-1. One day after transfection, cells were
incubated with MitoTracker Red dye and analyzed by fluorescence
confocal microscopy using appropriate filters for visualization of
green (a), red (b), or combined (c)
fluorescence resulting from the GFP and MitoTracker molecules.
B, FL5.12 cells were cultured in the presence
(a-c) or absence (d-f) of
IL-3 for 14 h. After a 30-min incubation with MitoTracker Red dye,
cells were fixed and stained with anti-Bif-1 monoclonal antibody,
followed by FITC-conjugated goat anti-mouse IgG secondary antibody.
Stained cells were visualized by fluorescence confocal microscopy using
filters appropriate for visualization of FITC (a,
d), MitoTracker (b, e), or both
(c, f). C, FL5.12 cells were cultured
with (a-c) or without
(d--f) IL-3 for 14 h prior to fixation and
double staining with anti-Bax rabbit antiserum and anti-Bif-1
monoclonal antibody, followed by application of FITC-conjugated goat
anti-rabbit IgG and rhodamine-conjugated goat anti-mouse IgG secondary
antibodies. Cells were analyzed by fluorescence confocal microscopy
using appropriate filters for visualization of green
(a, d), red (b,
e), or combined (c, f) fluorescence
resulting from the FITC and rhodamine molecules.
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Fig. 5.
Apoptotic stimuli induce Bax
translocation. 293 cells were transiently
cotransfected with pEGFP-Bax and pFLAG-CMV2 plasmids encoding
FLAG-Bif-1. One day after transfection, cells were treated without
(A-C) or with 50 nM staurosporine
(D-F), 0.25 µg/ml vinblastine
(G-I), or 5 ng/ml anti-Fas antibody CH11 plus 5 µg/ml cycloheximide (J-L) for 16 h. Cells
were fixed, stained with anti-FLAG monoclonal antibody, and detected as
described in Fig. 4.
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Fig. 6.
IL-3 withdrawal induces Bax
heterodimerization with Bif-1. A, FL5.12 cells were
cultured in the absence of IL-3 for various times prior to preparation
of cell lysates in Chaps lysis buffer containing protease inhibitors.
Immunoprecipitation (IP) was performed using anti-Bax
polyclonal antiserum or anti-Bax monoclonal antibody 6A7, followed by
SDS-PAGE/immunoblot analysis with antibodies specific for Bax or Bif-1.
B, FL5.12 cells were cultured with or without IL-3 for
16 h and lysed in lysis buffer containing either 1% Chaps or
0.2% Nonidet P-40. Immunoprecipitation was performed with anti-Bax
polyclonal antiserum or preimmune serum as control, followed by
immunoblot analysis of the resulting immune complexes with antibodies
specific for Bax or Bif-1. In addition to immune complexes, the lysates
(30 µg of protein) were analyzed directly.
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Fig. 7.
Bif-1 promotes apoptosis in IL-3-deprived
FL5.12 cells. FL5.12 cells were stably transfected with control
vector (Neo), pK-SFFV-Bif-1, or Bcl-2 expression plasmids as
indicated. B, stably transfected FL5.12 cells were analyzed
by immunoblotting (25 µg of protein) with anti-Bif-1 monoclonal
antibody. Cells were cultured without IL-3 for various times, and the
percentage of viable cells was determined by exclusion of trypan blue
dye (A), or
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Thiazolyl
blue assay (C). In D, transfected FL5.12 cells
were cultured at 105 cells/ml in medium with or without
IL-3 for 12 h. Apoptotic cells were stained with annexin V-FITC
(PharMingen) according to the manufacturer's recommendation and
analyzed by flow cytometry.
View larger version (26K):
[in a new window]
Fig. 8.
Bif-1 promotes caspase activation and Bax
conformational change. A, stably transfected FL5.12
cells were cultured in the presence or absence of IL-3 for 10 h
and lysed in Nonidet P-40 lysis buffer containing protease inhibitors.
Cell lysates were normalized for protein content and subjected to
SDS-PAGE/immunoblotting assay (30 µg of protein per lane) with
monoclonal antibodies specific for caspase-3 (Imgenex), cleaved D4-GDI
(Imgenex), or tubulin (Sigma) control protein. B, FL5.12
transfectants were cultured with or without IL-3 for 8 h, and cell
lysates were prepared with Chaps lysis buffer containing protease
inhibitors. The conformationally changed Bax protein was
immunoprecipitated by anti-Bax 6A7 antibody and detected by
immunoblotting with anti-Bax rabbit antiserum. Also, the total lysates
were applied directly to SDS-PAGE/immunoblot analysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank the Molecular Biology, Flow Cytometry, and Molecular Imaging core facilities at the H. Lee Moffitt Cancer Center and Research Institute for support.
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FOOTNOTES |
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* This work was supported in part by American Cancer Society grant IRG-032.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.
§ These authors contributed equally to this work.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF350371.
** To whom correspondence should be addressed: Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612. Phone: (813) 979-6764; Fax: (813) 979-6748; E-mail: wanghg@moffitt.usf.edu.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M101527200
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ABBREVIATIONS |
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The abbreviations used are: IL-3, interleukin-3; SH3, Src homology 3; BH, Bcl-2 homology; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; ALG-2, apoptosis-linked gene 2.
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REFERENCES |
---|
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---|
1. | Raff, M. C. (1992) Nature 356, 397-400[CrossRef][Medline] [Order article via Infotrieve] |
2. | Vaux, D. L., and Korsmeyer, S. J. (1999) Cell 96, 245-254[Medline] [Order article via Infotrieve] |
3. | Thompson, C. B. (1995) Science 267, 1456-1462[Medline] [Order article via Infotrieve] |
4. | Reed, J. C. (1998) Oncogene 17, 3225-3236[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Adams, J. M.,
and Cory, S.
(1998)
Science
281,
1322-1326 |
6. | Korsmeyer, S. J. (1999) Cancer Res. 59, 1693s-1700s[Medline] [Order article via Infotrieve] |
7. | Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J. (1993) Cell 74, 609-619[Medline] [Order article via Infotrieve] |
8. | Reed, J. C. (1996) in Behring Institute Mitteilungen (Krammer , and Nagata, eds), Vol. 97 , pp. 72-100, Behring Institute Mitteilungen, Marburg, Germany |
9. |
Gross, A.,
McDonnell, J. M.,
and Korsmeyer, S. J.
(1999)
Genes Dev.
13,
1899-1911 |
10. |
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 |
11. |
Gross, A.,
Jockel, J.,
Wei, M. C.,
and Korsmeyer, S. J.
(1998)
EMBO J.
17,
3878-3885 |
12. |
Nechushtan, A.,
Smith, C. L.,
Hsu, Y. T.,
and Youle, R. J.
(1999)
EMBO J.
18,
2330-2341 |
13. |
Green, D. R.,
and Reed, J. C.
(1998)
Science
281,
1309-1312 |
14. | 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] |
15. | Golemis, E. A., Gyuris, J., and Brent, R. (1994) in Current Protocols in Molecular Biology (Ausubel, F. M. , Brent, R. , Kingston, R. E. , Moore, D. D. , Seidman, J. G. , Smith, J. A. , and Struhl, K., eds) , pp. 13.14.1-13.14.17, J. Wiley & Sons, Inc., New York |
16. | Fuhlbrigge, R. C., Fine, S. M., Unanue, E. R., and Chaplin, D. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5649-5653[Abstract] |
17. | Wang, H.-G., Rapp, U. R., and Reed, J. C. (1996) Cell 87, 629-638[Medline] [Order article via Infotrieve] |
18. | Komatsu, K., Miyashita, T., Hang, H., Hopkins, K. M., Zheng, W., Cuddeback, S., Yamada, M., Lieberman, H. B., and Wang, H. G. (2000) Nat. Cell Biol. 2, 1-6[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Sato, T.,
Hanada, M.,
Bodrug, S.,
Irie, S.,
Iwama, N.,
Boise, L. H.,
Thompson, C. B.,
Golemis, E.,
Fong, L.,
Wang, H.-G.,
and Reed, J. C.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
9238-9242 |
20. | Wang, H.-G., Millan, J. A., Cox, A. D., Der, C. J., Rapp, U. R., Beck, T., Zha, H., and Reed, J. C. (1995) J. Cell Biol. 129, 1103-1114[Abstract] |
21. | Morton, C. J., and Campbell, I. D. (1994) Curr. Biol. 4, 615-617[Medline] [Order article via Infotrieve] |
22. | Pawson, T. (1995) Nature 373, 573-580[CrossRef][Medline] [Order article via Infotrieve] |
23. | Nishita, M., Inoue, S., Tsuda, M., Tateda, C., and Miyashita, T. (1998) Exp. Cell Res. 244, 357-366[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Hsu, Y. T.,
and Youle, R. J.
(1997)
J. Biol. Chem.
272,
13829-13834 |
25. |
Hsu, Y. T.,
and Youle, R. J.
(1998)
J. Biol. Chem.
273,
10777-10783 |
26. |
Zha, H.,
Aime-Sempe, C.,
Sato, T.,
and Reed, J. C.
(1996)
J. Biol. Chem.
271,
7440-7444 |
27. |
Chen, B.,
Borinstein, S. C.,
Gillis, J.,
Sykes, V. W.,
and Bogler, O.
(2000)
J. Biol. Chem.
275,
19275-19281 |
28. |
Fish, K. N.,
Schmid, S. L.,
and Damke, H.
(2000)
J. Cell Biol.
150,
145-154 |
29. |
Hsu, Y.-T.,
Wolter, K. G.,
and Youle, R. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3668-3672 |
30. |
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 |
31. | Zha, J., Harada, H., Yang, E., Jockel, J., and Korsmeyer, S. J. (1996) Cell 87, 619-628[Medline] [Order article via Infotrieve] |
32. |
Wang, H.-G.,
Pathan, N.,
Ethell, I. M.,
Krajewski, S.,
Yamaguchi, Y.,
Shibasaki, F.,
McKeon, F.,
Bobo, T.,
Franke, T.,
and Reed, J. C.
(1999)
Science
284,
339-343 |
33. | Li, H., Zhu, H., Xu, C. J., and Yuan, J. (1998) Cell 94, 491-501[Medline] [Order article via Infotrieve] |
34. | Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. (1998) Cell 94, 481-490[Medline] [Order article via Infotrieve] |
35. |
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 |
36. |
Heibein, J. A.,
Goping, I. S.,
Barry, M.,
Pinkoski, M. J.,
Shore, G. C.,
Green, D. R.,
and Bleackley, R. C.
(2000)
J. Exp. Med.
192,
1391-1402 |