From The Burnham Institute, La Jolla, California 92037
Received for publication, December 11, 2000, and in revised form, February 20, 2001
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ABSTRACT |
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A novel human member of the Bcl-2 family
was identified, Bcl-B, which is closest in amino acid sequence homology
to the Boo (Diva) protein. The Bcl-B protein contains four Bcl-2
homology (BH) domains (BH1, BH2, BH3, BH4) and a predicted
carboxyl-terminal transmembrane (TM) domain. The BCL-B
mRNA is widely expressed in adult human tissues. The Bcl-B protein
binds Bcl-2, Bcl-XL, and Bax but not Bak. In transient
transfection assays, Bcl-B suppresses apoptosis induced by Bax but not
Bak. Deletion of the TM domain of Bcl-B impairs its association with
intracellular organelles and diminishes its anti-apoptotic function.
Bcl-B thus displays a unique pattern of selectivity for binding and
regulating the function of other members of the Bcl-2 family.
Bcl-2 family proteins play a central role in apoptosis regulation
in metazoan species. In humans, over 20 members of this family have
been identified to date, including proteins that suppress (Bcl-2,
Bcl-XL, Mcl-1, Bfl-1/A1, Bcl-W) and proteins that promote (Bax, Bak, Bok, Bad, Bid, Bik, Bim, Nip3, Nix) cell death (reviewed in
Refs. 1 and 2). Bcl-2 family proteins contain at least one of four
conserved regions, termed Bcl-2 homology
(BH)1 domains. Most members
of this family also contain a TM domain located near their carboxyl
terminus that anchors them in intracellular membranes of mitochondria
and other organelles (reviewed in Refs. 1 and 2).
Many Bcl-2 family proteins are capable of physically interacting,
forming homo- or heterodimers, and functioning as agonists or
antagonists of each other (1-3). Specificity for interaction partners
and tissue-specific patterns of expression combine to endow each
mammalian Bcl-2 family protein with a unique physiological role
in vivo, resulting for example in highly diverse phenotypes when members of this multigene family are individually knocked out in
mice (reviewed in Ref. 4). Thus, a need exists to identify comprehensively the members of the Bcl-2 family and to elucidate their
functional characteristics. In this report, we describe the molecular
cloning and initial characterization of a new human member of the Bcl-2
family, Bcl-B.
Cloning of BCL-B cDNAs--
TBLASTN searches of the human
expressed sequence tag (EST) data base using the amino acid sequence of
the mouse Boo/Diva as a query resulted in the identification of
homologous partial cDNAs. A human EST clone
(GenBankTM accession number AA098865) was obtained
(Research Genetics) and sequenced in its entirety, revealing an open
reading frame (ORF) encompassing the last 151 residues of a protein
with homology to Boo (Bcl-B) (submitted to GenBankTM with
accession number AF326964). The corresponding genomic sequence for this
cDNA was identified in the human genome data base (clone
CTD-2184D3), which was derived from human chromosome 15q21. Because the
EST clone lacked a candidate start codon, the corresponding 5'-end of
Bcl-B cDNAs was cloned by a reverse transcriptase polymerase chain
reaction (RT-PCR) approach, using the forward primer NKO118
(5'-CGGGCCAAGAAAACCAGCGAAGG-3'), which was designed to hybridize to a
region upstream of the Bcl-B ORF as predicted from the genomic data,
and the reverse primer NKO121 (5'-CACTCAAGGAAGAGCCATTTGCAT-3'), which
is complementary to a region downstream of the predicted Bcl-B ORF
corresponding to the 3'-untranslated region of the putative mRNA.
PCR amplification using human liver cDNA
(CLONTECH) as a template with the above primers
yielded a single ~0.9-kb product, which was cloned into pCR2.1-TOPO
(Invitrogen, following the manufacturer's instructions) to generate
TOPO-Bcl-B (pNK254) and sequenced.
RT-PCR Analysis--
Expression of BCL-B mRNA in
various tissues was examined by RT-PCR, using oligo(dT)-primed
first-strand cDNA derived from multiple adult human tissues
(CLONTECH) as templates. cDNAs were amplified
following the manufacturer's instructions using the forward primer
NKO120 (5'-GTGGTGACGCTCGTGACCTTCG-3') and NKO121 as the reverse primer.
Glyceraldehyde-3-phosphate dehydrogenase primers were used as a
positive control (5).
Plasmid Construction--
The ORF encoding Bcl-B was
PCR-amplified from TOPO-Bcl-B (pNK254) using the forward primer NKO101
(5'-GGAATTCATGGTTGACCAGTTGCGGGAG-3') and reverse primer NKO103
(5'-CCGCTCGAGTCATAATAATCGTGTCCAGAG-3'). The PCR products were digested
with EcoRI and XhoI and cloned into the
EcoRI and XhoI sites of pcDNA3-Myc
(Stratagene), and the EcoRI and SalI sites of
pcI-Neo-FLAG (Invitrogen) and pEGFP-C2 (CLONTECH). A plasmid encoding Bcl-B lacking its
COOH-terminal transmembrane domain (Bcl-B Cell Culture, Transfection, and Apoptosis Assays--
HEK293,
COS7, HT1080, and PPC1 cells were maintained in Dulbecco's modified
Eagle's medium (Irvine Scientific) supplemented with 10% fetal bovine
serum, 1 mM L-glutamine, and antibiotics. For
transient-transfection apoptosis assays, cells (5 × 105) in six-well dishes were co-transfected using Superfect
(Qiagen) with 0.5 µg of pcDNA3-Bax plus 0.5 µg of green
fluorescence protein (GFP) marker plasmid pEGFP
(CLONTECH) or 0.5 µg of pEGFP-Bak, and 1 µg of
pcDNA3, pcDNA3-Myc-Bcl-B, pcDNA3-Myc-Bcl-B
For stable transfections, HeLa cells in 100-mm dish were transfected
with pcDNA3 (control), pcDNA3-Myc-Bcl-B, or pRC-CMV-Bcl-2 plasmids using LipofectAMINE plus (Life Technologies, Inc.). Two days
later, complete medium containing G418 (800 µg/ml) (Omega Scientific
Inc.) was used to select stably transfected cells. Several of the
resulting G418-resistant clones were recovered using cloning cylinders
and individually expanded. G418-resistant clones were screened for the
expression of desired genes by immunoblotting with antibodies. For
apoptosis assays, stably transfected clones (5 × 105
cells) in six-well dishes (30 mm diameter) were cultured in medium containing various concentrations of staurosporine (Calbiochem) (0.2-1
µM) or of recombinant TRAIL (Biomol) (10-100
ng/ml) for 8-10 h. Both floating and adherent cells were collected,
fixed, and subjected to DAPI staining, enumerating the percentage
apoptosis cells by UV microscopy.
Immunofluorescence and Subcellular
Fractionation--
The intracellular location of Bcl-B was examined
using fluorescence confocal microscopy and subcellular fractionation
methods, essentially as described (6, 7).
Co-immunoprecipitation and Immunoblotting Assays--
293T cells
(5 × 105) cultured with 50 µM
benzoyl-Val-Ala-Asp-fluoromethylketone (Bachem) were co-transfected
with 1.5 µg of pcDNA3-Myc-Bcl-B, pcI-Neo-FLAG-Bcl-B, pcDNA3-human
calcyclin-binding protein (used as a control), or
pcDNA3-FLAG-Bcl-XL, together with 1.5 µg of pEGFP,
pEGFP-Bcl-B, pcDNA3-HA-BAG1, pcDNA3-HA-Bax,
pcDNA3-FLAG-Bcl-XL, pRC-CMV-Bcl-2, or pEGFP-Bak. At 24-h
post-transfection, cells were collected and resuspended in lysis buffer
(142.4 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.4), 0.5 mM EGTA, 0.2% Nonidet
P-40) containing 12.5 mM During TBLASTN searches of the publicly available EST data bases
using the amino acid sequence of the mouse Boo/Diva as a query, we
discovered an EST clone (GenBankTM accession number
AA098865) encoding a predicted polypeptide harboring a BH1 domain. PCR
methods were used to obtain cDNAs containing the complete ORF
corresponding to a 204-amino acid protein (Fig.
1A). The predicted ORF was
initiated by an AUG start codon within a favorable Kozak context. The
predicted protein contains regions resembling the BH1, BH2, BH3, and
BH4 domains typical of anti-apoptotic members of the Bcl-2 family, as
well as a putative carboxyl-terminal TM domain (Fig. 1B).
Comparisons of the sequence of this predicted protein with all known
Bcl-2 family members by BLAST search indicated that it is most similar to the murine Bcl-2 family protein Boo (also known as Diva) (9, 10),
sharing 47% amino acid sequence identity, and thus prompting the
moniker "Bcl-2 family protein resembling Boo" (Bcl-B). The BCL-B gene is located on chromosome 15 (map 15q21), as
determined by in silico screening of the human genome data base at
NCBI. Comparison of the BCL-B cDNA sequence with genomic
data indicates a two-exon structure, in which the region encoding
residues Trp163 and Asp164 (within the BH2
domain) of the Bcl-B protein are interrupted by an ~2.3-kb intron.
PCR analysis suggested that the BCL-B mRNA is widely
expressed in adult human tissues (Fig. 1C).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
TM) was constructed by
PCR-based mutagenesis using primers NKO101 and NKO131
(5'-CCGCTCGAGTCATGTTTTCTCCAAAAAGCCAGTG-3'). The resulting PCR product
was digested with EcoRI and XhoI and cloned into
pcDNA3-Myc.
TM, or pcDNA3-FLAG-Bcl-XL. The total amount of DNA was normalized
to 3 µg per transfection using pcDNA3. At 24 h
post-transfection, both adherent and floating cells were collected,
fixed, and stained with 0.1 µg/ml 4',6-diamidino-2-phenylindole
(DAPI). The percentages of apoptotic cells were determined by counting
the GFP-positive cells having nuclear fragmentation and/or chromatin
condensation (mean ± S.D.; n = 3).
-glycerophosphate, 2 mM NaF, 1 mM Na3VO4, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, and a protease inhibitor mixture (Roche
Molecular Biochemicals). Soluble lysates were incubated with 10 µl of
anti-Myc (Santa Cruz) or anti-FLAG (Sigma) antibody-conjugated
Sepharose beads overnight at 4 °C. Beads were then washed four times
in 1.5 ml of lysis buffer and boiled in Laemmli gel-loading solution
before performing SDS-PAGE/immunoblotting using the following
polyclonal or monoclonal antibodies: polyclonal rabbit anti-GFP (Roche
Molecular Biochemicals), monoclonal rat anti-HA (Roche Molecular
Biochemicals), monoclonal mouse anti-FLAG (Sigma), monoclonal mouse
anti-Myc (Santa Cruz), rabbit anti-huBcl-2, rabbit anti-hu
Bcl-XL, rabbit anti-hu Bax, or rabbit anti-hu Bak (8).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence analysis and the expression of Bcl-B
cDNA. A, the predicted Bcl-B amino acid sequence is
presented with the TM underlined (GenBankTM
accession number AP326964). B, alignments of BH1, BH2, BH3,
and BH4 domains of Bcl-B and other Bcl-2 family proteins are shown.
Numbers on the left indicate the position of the
amino acid in each protein based on GenBankTM accession
numbers AAD08703 (murine Boo), Q90343 (chicken Nr13), AAA35591 (hu
Bcl-2), CAA80661 (hu Bcl-XL), and P41958 (C. elegans CED9).
Identical and similar residues are indicated by black and
gray boxes, respectively. Asterisks under the BH2
alignment indicate the intron junction for hu BCL-B,
BCL-2, and BCL-X genes. C, expression
of BCL-B in adult human tissues. First-strand cDNAs made
from RNA samples from various adult human tissues were PCR-amplified
using BCL-B-specific primers. The reverse primer was
downstream of the intron, thus avoiding amplification of contaminating
genomic DNA. PCR products were size-fractionated by electrophoresis in
1% agarose gels, stained with ethidium bromide, and photographed under
UV illumination. Primers specific for glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) were also used for PCR as a positive
control.
The Bcl-B protein was tested for interactions with other Bcl-2 family
proteins by co-immunoprecipitation experiments, wherein Bcl-B was
expressed in HEK293T or HT1080 cells with various
NH2-terminal epitope tags. These studies indicated that
Bcl-B is capable of associating with itself, Bax, Bcl-2, and
Bcl-XL, but not with Bak (Fig.
2).
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The function of the Bcl-B protein was explored by transient
transfection in a variety of cell lines, including HEK293T, COS7, HT1080, and PPC1. Overexpression of Bcl-B did not induce apoptosis, nor
did it negate suppression of apoptosis caused by overexpression of
Bcl-2 or Bcl-XL (not shown), suggesting that Bcl-B is not a pro-apoptotic protein. We therefore tested the possibility that Bcl-B
is a cytoprotective protein by ascertaining its effects on apoptosis
induced by the pro-apoptotic proteins Bax and Bak. Co-expressing Bcl-B
markedly suppressed apoptosis induced by Bax but not Bak (Fig.
3), thus correlating with protein binding
data demonstrating that Bcl-B associates with Bax but not Bak (Fig. 2).
This suppression was not due to reduced levels of Bax protein, as
determined by immunoblotting. In contrast to Bcl-B, co-expression of
Bcl-XL suppressed apoptosis induced by either Bax or Bak
(Fig. 3).
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To further explore the effects of Bcl-B on apoptosis, HeLa cells were
stably transfected with a plasmid encoding Myc-tagged Bcl-B,
versus control (empty) plasmid. Several stably transfected clones were tested for Bcl-B expression by immunoblotting, and their
responses to apoptosis induced by staurosporine (STS) or TRAIL were
compared. Comparisons were also made to HeLa cells stably transfected
with a Bcl-2-encoding plasmid. Fig. 3, CE, show
representative results, where control transfected (vector) cells were
compared with two Bcl-B-transfected clones. The Bcl-B-expressing clones
shown here (clones 9 and 16) produced different relative amounts of
Myc-Bcl-B protein, as determined by immunoblotting, with clone
16 containing ~5 times higher levels of Bcl-B than clone 9. HeLa cell
clones such as clone 16, which contained higher amounts of
Myc-Bcl-B, displayed resistance to apoptosis induced by STS and
TRAIL, compared with control (vector)-transfected cells. In contrast,
HeLa cell clones such as clone 9, which contained lower levels of
Myc-Bcl-B, demonstrated only slight resistance to these
apoptotic stimuli (Fig. 3, D and E). These data
thus demonstrate that Bcl-B can suppress apoptosis induced by exogenous stimuli if expressed at sufficient levels. However, even HeLa cell
clones with higher levels of Bcl-B did not manifest the profound resistance to apoptosis seen in Bcl-2-overexpressing cells (Figs. 3,
D and E).
Many Bcl-2 family proteins associate with mitochondria in cells
(reviewed in Refs. 1 and 2). Expression of GFP-tagged Bcl-B in cells
revealed a punctate cytosolic pattern and partial co-localization with
a mitochondria-specific dye (MitoTracker), as determined by
two-color confocal microscopy (Fig.
4A). Crude subcellular
fractionation analysis revealed that Myc-tagged Bcl-B protein resides
predominantly in the mitochondria-containing HM fraction, similar to
Bcl-2, as determined by immunoblot analysis of the cellular fractions
(Fig. 4, B and C). In contrast to full-length Bcl-B, a truncation mutant of Bcl-B lacking the carboxyl-terminal TM
domain (Bcl-BTM) targeted less efficiently to the HM fraction (Fig.
4D). The Bcl-B
TM protein also was ineffective at blocking Bax-induced apoptosis (Fig. 4E), even though this protein
was produced at comparable levels with the full-length Bcl-B protein. Thus, efficient organellar targeting appears to be required for optimal
function of Bcl-B.
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DISCUSSION |
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We describe a new member of the human Bcl-2 family protein, Bcl-B. This protein is most similar in amino acid sequence to the murine Boo (Diva) protein and the most similar among all human Bcl-2 family proteins to the CED9 protein of Caenorhabditis elegans. The Boo (Diva) protein interacts selectively with some Bcl-2 family proteins but not others, although controversy exists as to the details (9, 10). Interestingly, one report has suggested that the Boo protein can bind Bak but not Bax, and accordingly provided evidence that Boo suppresses apoptosis induced by overexpression of Bak but not Bax (10). Conversely, we observed that Bcl-B selectively binds and suppresses apoptosis induction by Bax, but fails to interact with or negate apoptosis triggered by Bak overexpression.
The murine Boo (Diva) protein has been variably reported to either suppress or promote apoptosis (9, 10). In transient transfection assays performed in four different human tumor cell lines, we consistently observed an anti-apoptotic action of Bcl-B. Stable overexpression of Bcl-B in HeLa cells also resulted in increased resistance to diverse apoptotic stimuli. However, because Bcl-B is capable of associating with either the anti-apoptotic proteins Bcl-2 and Bcl-XL or with the pro-apoptotic protein Bax, it is possible that Bcl-B could display different phenotypes under some circumstances depending on cellular context. A similar phenomenon has been reported for some other Bcl-2 family proteins. For example, Bcl-2 can reportedly promote apoptosis in photoreceptor cells of the retina, while Bax can suppress cell death in some types of neurons (11, 12).
Although stably transfected clones of HeLa cells, which contained higher levels of Bcl-B, exhibited resistance to exogenous apoptotic stimuli, the resistance afforded by Bcl-B was not as profound as that observed for Bcl-2 overexpression. This difference in potency of Bcl-B could be due to variations in the relative amounts of Bcl-B and Bcl-2 produced in transfected cells, or it could reflect a fundamental difference in the mechanisms of these proteins. In this regard, because Bcl-2 blocks cell death induced by both Bax and Bak, whereas Bcl-B inhibits apoptosis induced only by Bax but not Bak, it seems likely that Bcl-B may be less efficacious under circumstances where both Bax and Bak contribute to apoptosis induction. Bcl-B therefore may provide a mechanism for selectively inhibiting Bax-dependent apoptotic processes in vivo, while allowing Bak-dependent cell death to proceed normally.
The mouse Boo (Diva) protein was reported to associate with the
caspase-activating Apaf1 protein (a homologue of C. elegans CED-4) (9, 10). Although we have observed weak interactions of Bcl-B
with Apaf1 in co-immunoprecipitation assays, functional analysis has
failed to reveal an effect of Bcl-B on Apaf1-induced apoptosis (not
shown). Since Apaf1 is a soluble cytosolic protein (13), the inability
of Bcl-BTM to suppress Bax-induced apoptosis also suggests that
Bcl-B does not play a significant role in suppressing Apaf1. Moreover,
the observation that Bcl-B suppresses apoptosis induced by Bax but not
Bak also argues against a role for Bcl-B as an Apaf1 suppresser, given
that both Bax and Bak induce mitochondrial release of the Apaf1
activator, cytochrome c (14, 15).
The correlation between membrane targeting and function is reminiscent
of some other Bcl-2 family proteins and suggests that the site of
action of Bcl-B is close to the intracellular organelles, including
mitochondria, with which it associates. Although roughly half of the
Bcl-BTM protein was associated with the HM membrane fraction in
cells, this may be due to its dimerization with other resident Bcl-2
family proteins. A membrane site of action for Bcl-B would be
consistent with evidence that several Bcl-2 family proteins are capable
of forming ion channels or pores in membranes (reviewed in Ref. 16).
Indeed, molecular modeling of Bcl-B on the structure of
Bcl-XL suggests that it possesses a similar overall fold
and that it contains amphipathic
-helices similar to the putative
pore-forming
5 and
6 of Bcl-XL (not shown).
The differences observed in the functions and protein interaction
partners of murine Boo and human Bcl-B proteins suggest that Bcl-B does
not represent the human orthologue of mouse Boo/Diva. Also consistent
with this interpretation is the difference in the patterns of
expression of Bcl-B and Boo. Whereas Boo (Diva) is expressed
predominantly in ovary, testis, and epididymis in adult mice (9, 10),
RT-PCR analysis suggests that the BCL-B mRNA is widely
expressed in adult human tissues. Comparisons of the sequence of
BCL-B cDNAs with human genome sequence data indicate that the BCL-B gene is comprised of two exons interrupted by
a ~2.3-kb intron. Interestingly, the location of this intron
corresponds precisely to the intronic interruption in the coding region
of the anti-apoptotic BCL-2 and BCL-X genes
(corresponding to the motif GGW^D or GGW/D in BH2 (see
Fig. 1B). (The genomic sequence of murine boo/diva is unfortunately unavailable for comparison.) In
contrast to BCL-B, the pro-apoptotic genes BAX
and BAK have more complicated exon-intron organizations, in
which the coding regions of the gene are spread over 5 (Bak) or 6 (Bax)
exons. The similar genomic organization of the BCL-2,
BCL-XL, and BCL-B genes thus suggests they
evolved from a common ancestor and indirectly implies a similar mechanism of action for their encoded proteins.
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Addendum |
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While this manuscript was in preparation, the cDNA sequence of Bcl-B was deposited into GenBankTM (accession number AF285092) by L. H. H. Zhang (unpublished data).
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM-60554, by CaP-CURE, and by United States Army Medical Research and Materiel Command Grant DAMD17-99-1-9511).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) AF326964.
To whom correspondence should be addresses: The Burnham Inst.,
10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3140; Fax:
858-646-3194; E-mail: jreed@burnham.org.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.C000871200
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ABBREVIATIONS |
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The abbreviations used are: BH, Bcl-2 homology; TM, transmembrane; EST, expressed sequence tag; ORF, open reading frame; RT-PCR, reverse transcriptase polymerase chain reaction; kb, kilobase pair(s); GFP, green fluorescence protein; DAPI, 4',6-diamidino-2-phenylindole; HM, heavy membrane; LM, light membrane; PAGE, polyacrylamide gel electrophoresis; STS, staurosporine; hu, human; HA, hemagglutinin; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.
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