From The Burnham Institute, La Jolla, California 92037
Received for publication, July 5, 2000, and in revised form, October 10, 2000
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ABSTRACT |
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A new member of the Bcl-2 family was
identified, Bcl-G. The human BCL-G gene consists of
6 exons, resides on chromosome 12p12, and encodes two proteins
through alternative mRNA splicing, Bcl-GL (long) and
Bcl-GS (short) consisting of 327 and 252 amino acids in
length, respectively. Bcl-GL and Bcl-GS have
identical sequences for the first 226 amino acids but diverge
thereafter. Among the Bcl-2 homology (BH) domains previously recognized
in Bcl-2 family proteins, the BH3 domain is found in both
Bcl-GL and Bcl-GS, but only the longer
Bcl-GL protein possesses a BH2 domain. Bcl-GL mRNA is expressed widely in adult human tissues, whereas
Bcl-GS mRNA was found only in testis. Overexpression of
Bcl-GL or Bcl-GS in cells induced apoptosis
although Bcl-GS was far more potent than
Bcl-GL. Apoptosis induction by Bcl-GS depended
on the BH3 domain and was suppressed by coexpression of anti-apoptotic
Bcl-XL protein. Bcl-XL also
coimmunoprecipitated with Bcl-GS but not with mutants of
Bcl-GS in which the BH3 domain was deleted or mutated or
with Bcl-GL. Bcl-GS was predominantly localized
to cytosolic organelles, whereas Bcl-GL was diffusely
distributed throughout the cytosol. A mutant of Bcl-GL in
which the BH2 domain was deleted displayed increased apoptotic activity
and coimmunoprecipitated with Bcl-XL, suggesting that the
BH2 domain autorepresses Bcl-GL.
Bcl-2 family proteins are central regulators of apoptosis
(reviewed in Refs. 1-3). Bcl-2 family proteins are conserved
throughout the animal kingdom with homologs identified in both
vertebrates and invertebrates. These proteins contain up to four
conserved Bcl-2 homology
(BH)1 domains, BH1, BH2, BH3,
and BH4, which are recognized by their amino acid sequence similarity.
Both anti- and pro-apoptotic Bcl-2 family proteins have been
identified. These proteins control cell life/death decisions through
their effects on events such as mitochondrial release of proteins
involved in activation of caspase-family cell death proteases or by
binding sequestering caspase-activating proteins (reviewed in Refs.
2-5 and 7-8). Many Bcl-2 family proteins are capable of physically
interacting with each other, forming a complex network of homo- and
heterodimers, and these physical interactions sometimes play important
roles in the opposing effects of pro- and anti-apoptotic members of the family.
The pro-apoptotic members of the Bcl-2 family can be broadly classified
into two groups. One group, including Bax, Bak, and Bok in humans,
shares structural similarity with the pore-forming domains of certain
bacterial toxins and is capable of forming pores in synthetic membranes
in vitro (9-12). These proteins exhibit cytotoxic effects
independently of their ability to bind other Bcl-2 family proteins
including Bcl-2 and other cytoprotective members of the family such as
Bcl-XL, Bcl-W, Bfl-1, and Mcl-1. The second group of
pro-apoptotic Bcl-2 family proteins varies widely in their amino acid
sequences, often containing only a single region of similarity,
specifically, the BH3 domain. These "BH3-only" proteins appear to
possess no intrinsic or autonomous cytodestructive activity and instead
operate as trans-dominant inhibitors of the survival
proteins. Their antagonism of proteins such as Bcl-2 and
Bcl-XL depends on binding via their BH3 domains to a
hydrophobic pocket on target anti-apoptotic proteins (13).
Gene knockout studies in mice have demonstrated nonredundant roles for
various Bcl-2 family genes in regulating cell life and death in
specific tissues or under particular physiological or pathological
circumstances (14-18). Thus, it is important to identify all members
of the Bcl-2 family and to delineate the cellular contexts in which
they contribute to apoptosis regulation. In this report, we have
described the cloning and initial characterization of a new member of
the Bcl-2 family, Bcl-G.
Cloning of Bcl-G cDNAs--
TBLAST searches of the public
databases using human Bcl-2 as a query sequence revealed a short EST
(GenBankTM/EBI no. AW000827) from colonic mucosa of 3 patients with Crohn's disease, which contains an open reading frame
(ORF) encoding sequences similar to the BH2 domain of Bcl-2 family
proteins. An oligonucleotide primer (5'-GTACTTGGTGCCAAAGCCCAGG-3') was
designed complementary to the EST sequence and used for 5'-RACE,
employing the SMARTTMRACE cDNA Amplification Kit
(CLONTECH) and human placental total RNA as
template. The 5'-RACE products were subcloned into pCR2.1-TOPO vector
using the TOPOTM TA Cloning kit (Invitrogen), and the DNA
sequence was determined revealing a complete ORF with start codon
within a favorable Kozak sequence context preceded by a 5'-UTR
containing stop codons in all three reading frames
(GenBankTM/EBI accession nos. AF281254 and
AF281255). Two additional EST clones, AI478889 and AI240211,
were identified by BLAST searches and correspond to overlapping partial
Bcl-G cDNAs that contain the 3'-UTR. BLAST searches of
GenBankTM/EBI also revealed a 190,858 base pair human BAC
clone (RPCI11-267J23) in the genomic database derived from chromosome
12p12 (GenBankTM/EBI no. AC007537), which contains the
BCL-G gene in its entirety.
Plasmids--
cDNAs containing the ORFs of
Bcl-GL and Bcl-Gs without additional flanking
sequences were generated by PCR using human placental cDNA as a
template and the following primers:
5'-GGCTCGAGCGATGTGTAGCACCAGTGGGTGTGACC-3', sense for both
Bcl-GL and Bcl-Gs;
5'-CCAAGCTTTAAGTCTACTTCTTCATGTGATATCCC-3', antisense for
Bcl-GL; 5'-CCAAGCTTTAAAATGCAGGCCATCAAACC-3',
antisense for Bl-GS. The resulting PCR products were
digested with restriction endonucleases and subcloned into the
XhoI and HindIII sites of pEGFP-C1
(CLONTECH). A mutant of Bcl-GS lacking
the BH3 domain was created by a two-step PCR method, using the
following primers: primer1,
5'-GGCTCGAGCGATGTGTAGCACCAGTGGGTGTGACC-3'; primer2,
5'-CCGGATCCGGCTAGTATTTGTTCTTCTTCATCTTTC-3'; primer3,
5'-CCGGATCCGACACTGCCTTCATCCCCATTCCC-3'; and primer4, 5'-CCAAGCTTTAAAATGCAGGCCATCAAACC-3'. The resulting PCR product was digested with Xhol/BamHI or with
BamHI/Hindlll respectively and ligated into
pEGFP-C1. Site-directed mutagenesis of Bcl-Gs was performed
to generate a L216E substitution mutation using the
QuikChangeTM Site-directed Mutagenesis kit (Stratagene)
following the manufacturer's protocol, with
pEGFP-C1/Bcl-Gs plasmid as DNA template and the mutagenic
primers: 5'-GCCAAAATTGTTGAGCTGGAGAAATATTCAGGAGATCAGTTGG-3' and
5'-CCAACTGATCTCCTGAATATTTCTCCAGCTCAACAATTTTGGC-3'. A mutant of
Bcl-GL lacking the BH2 domain was created by PCR using the same forward primer for Bcl-GL and
5'-GGAAGCTTCAGAAGTTCTCTTTCAGGTACTTGG as the reverse primer.
Measurements of Bcl-G mRNAs--
Bcl-G mRNAs were
detected by either Northern blotting or RT-PCR. For RT-PCR, we employed
multiple tissue cDNA panels (CLONTECH) containing first-strand cDNA generated from 16 different tissues. PCR was performed according to the manufacturer's protocol with the
following primers: (a) 5'-primer for both Bcl-Gs
and Bcl-GL, corresponding to exon 3, 5'-CTGAGGGTCTCTCCTTCCAGCTCCAAGG-3'; (b) 3'-primer for
Bcl-GL, corresponding to exon 5, 5'-GGCCGTGACGTCTATTACAAGGGCAGCC-3'; 3'-primer for Bcl-Gs,
corresponding to an alternatively spliced segment of exon 5, 5'-CAAGGGAATGGGGATGAAGGCAGTGTC-3'. Human
glyceraldehyde-3-phosphate dehydrogenase expression was examined by PCR
with the following primers: sense,
5'-TGAAGGTCGGAGTCAACGGATTTGGT-3'; antisense,
5'-CATGTGGGCCATGAGGTCCACCAC-3'.
Cell Culture, Transfections, and Apoptosis Assay--
293T and
COS-7 cells were cultured in Dulbecco's modified Eagle's high glucose
medium (Irvine Scientific, Santa Ana, CA) containing 10% fetal bovine
serum. PC-3 cells were cultured with RPMI 1640 media containing 10%
fetal bovine serum. Transfection of cells was performed using SuperFect
(Qiagen, Chatsworth, CA). Both floating and adherent cells (after
trypsinization) were collected 24 h after transfection, fixed, and
stained using 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) for
assessing apoptosis based on nuclear fragmentation and chromatin
condensation (19, 20).
Coimmunoprecipitations and Immunoblotting--
Immunoblotting
was performed as described previously (19, 20). For
coimmunoprecipitations, cells were cultured in 50 mM benzocarbonyl-Val-Ala- Asp-fluoromethylketone (zVAD-fmk) to prevent apoptosis. Cells were suspended in lysis buffer (50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM EDTA, 50 mM NaF, 0.5% Nonidet P-40, 0.1 mM
Na3VO4, 20 µg/ml leupeptin, 20 µg/ml
aprotinin, 1 mM dithiothreitol, and 1 mM
phenylmethylsulfonyl fluoride). Lysates (0.2 ml diluted into 1-ml final
volume of lysis buffer) were cleared by incubation with 15 ml of
protein G-Sepharose 4B (Zymed Laboratories Inc.) and
then incubated with 15 µl of polyclonal anti-GFP antibody (Santa Cruz
Biotechnology) and 15 µl of protein G at 4 °C overnight. Beads
were then washed four times with 1.5 ml of lysis buffer before boiling
in Laemmli sample buffer and performing SDS-polyacrylamide gel
electrophoresis and immunoblotting.
Confocal Microscopy--
GFP-expressing cells were incubated
with 50 nM Mitotracker Red CMXRos (Molecular Probes) for 30 min at 37 °C in culture medium. The cells were then washed with
phosphate-buffered saline, fixed in 4% formaldehyde, and imaged by
confocal microscopy using a Bio-Rad MRC 1024 instrument (19, 21).
Identification and Sequence Analysis of the BCL-G gene and
cDNAs--
A short EST was identified during searches of the
public databases, which when conceptually translated revealed a
polypeptide sequence with similarity to the BH2 domain of Bcl-2 family
proteins. Full-length cDNAs were obtained revealing two potential
transcripts containing ORFs for proteins of 327 and 252 amino acids,
respectively, which we have termed Bcl-GL and
Bcl-GS (Fig. 1A).
The predicted Bcl-GL and Bcl-GS proteins are
identical for the first 226 amino acids and then diverge thereafter.
Comparison of the predicted amino acid sequences of Bcl-GL
and Bcl-GS with Bcl-2 family proteins revealed the presence
of a candidate BH3 domain in both Bcl-GL and
Bcl-GS (Fig. 1, A and B) and the
presence of a BH2 domain in Bcl-GL but not in
Bcl-GS (Fig. 1, A and C). Using the
Bcl-G cDNA sequences, the human genomic database was searched,
revealing a BAC clone from chromosome 12p12 containing the
BCL-G gene. Comparison with the cDNA sequences suggests
a 6-exon structure for the BCL-G gene. The
Bcl-GL and Bcl-GS cDNAs can be accounted
for by an alternative mRNA splicing mechanism in which different
splice acceptor sites associated with exon 5 are employed, resulting in
a change in the distal reading frame (Fig. 1D).
Tissue-specific Expression of Bcl-GL and
Bcl-GS mRNAs--
Northern blotting demonstrated the
presence of Bcl-G transcripts of ~1.5-2.5 kilobase pairs in length
in several adult human tissues (Fig.
2A), but the highest levels of
Bcl-G mRNA by far were found in male gonad (testis), thus prompting
the moniker "Bcl-Gonad" (Bcl-G). Because Northern blotting failed
to resolve the mRNAs encoding Bcl-GL and
Bcl-GS, we designed RT-PCR assays using primers specific
for Bcl-GL and Bcl-GS sequences associated with
exon 5. The amplified bands corresponding to Bcl-GL and
Bcl-GS were excised and sequenced, confirming the validity
of the RT-PCR strategy (not shown). Bcl-GL mRNA was
clearly detected in lung, pancreas, prostate, and testis, with lower
levels present in some other tissues (Fig. 2B). In contrast,
Bcl-GS mRNA was uniquely expressed in testis. RT-PCR
amplification of a control mRNA, glyceraldehyde-3-phosphate dehydrogenase, demonstrated loading of nearly equivalent amounts of
mRNA from each tissue.
Induction of Apoptosis by Bcl-G Proteins--
To assess the
effects of Bcl-GL and Bcl-GS on apoptosis,
various cell lines, including COS-7, HEK293T, and PC3, were transiently transfected with plasmids encoding Bcl-GL or
Bcl-GS. For most experiments, Bcl-GL and
Bcl-GS were expressed as GFP fusion proteins so that
successfully transfected cells could be conveniently identified (Fig.
3A), but similar results were
obtained when FLAG-epitope tags were employed instead (not shown).
Overexpression of the shorter Bcl-GS protein reproducibly
induced striking increases in the percentage of cells undergoing
apoptosis, as determined by DAPI staining (Fig. 3A) and
other methods (not shown). In contrast, Bcl-GL was more
variable and less efficient at inducing apoptosis in these transient
transfection assays. Immunoblot analysis of lysates from transfected
cells demonstrated that the less potent effects of Bcl-GL
could not be accounted for by lower levels of protein production (Fig.
3A). Indeed, Bcl-GL protein accumulated to
levels ~10-fold higher in cells compared with Bcl-GS,
suggesting that Bcl-GS is a far more potent apoptosis
inducer. Analysis of the same blots with an anti-tubulin antibody
confirmed loading of essentially equivalent amounts of total protein
for each sample, thus validating the results. In additional
transfection experiments, Bcl-GL failed to demonstrate
cytoprotective activity in side by side comparisons with Bcl-2 and
Bcl-XL (data not presented). Also, when Bcl-GL
was coexpressed with Bcl-GS in cells, no synergy with or
nullification of Bcl-GS-induced apoptosis was observed.
The BH3 Domain of Bcl-GS Is Required for Its
Pro-apoptotic Activity--
The Bcl-GS protein contains a
BH3 domain but lacks other regions of homology with Bcl-2 family
proteins. Structural studies indicate that BH3 domains represent
amphipathic
Wild-type Bcl-GS potently induced apoptosis when
overexpressed in COS-7, PC3, HEK293T, and other cell lines, whereas
Bcl-GS- The BH2 Domain of Bcl-GL Negatively Regulates Its
Pro-apoptotic Activity--
Compared with Bcl-GS,
Bcl-GL only weakly induces apoptosis. Bcl-GL
contains a BH2 domain not found in Bcl-GS. A mutant of Bcl-GL lacking the BH2 domain was created.
Bcl-GL-( Bcl-GS Associates with Bcl-XL in a
BH3-dependent Manner--
The pro-apoptotic activity of
BH3-only members of the Bcl-2 family depends on their ability to
dimerize with and suppress the activity of survival proteins such as
Bcl-XL (reviewed in Ref. 13). We, therefore, explored
whether Bcl-GL and Bcl-GS are capable of
associating with other Bcl-2 family proteins by coimmunoprecipitation
assays. Bcl-GS association with the survival proteins
Bcl-XL and Bcl-2 was readily detected by
coimmunoprecipitation using lysates from transiently transfected cells,
whereas no association with pro-apoptotic proteins Bax, Bak, Bid, or
Bad was observed (Fig. 4A and
not shown). Interaction of Bcl-GS with Bcl-2 and Bcl-XL, but not with Bax or Bak, was also confirmed by
yeast two-hybrid assays (not shown). Yeast two-hybrid assays also
suggested that no homo- or hetero-dimerization occurred among the
Bcl-GS and Bcl-GL proteins. In contrast,
association of the longer Bcl-GL protein with Bcl-2 or
Bcl-XL was not easily detected by coimmunoprecipitation assays (Fig. 4A). With much longer x-ray film exposure
times, however, small amounts of Bcl-XL were observed in
association with Bcl-GL immunocomplexes, suggesting either
low affinity binding of Bcl-GL to Bcl-XL or
implying that only a small portion of total Bcl-GL proteins
are competent to bind Bcl-XL (not shown). The interaction
of Bcl-GS with Bcl-XL was
BH3-dependent, as determined by comparisons of wild-type
Bcl-GS with the Bcl-GS- Bcl-GS Is Associated with Cytosolic
Organelles--
Many Bcl-2 family proteins, such as Bcl-2,
Bcl-XL, and Bak, contain a hydrophobic stretch of amino
acids near their carboxyl terminus that anchors them in intracellular
membranes of mitochondria, endoplasmic reticulum, or nuclear envelope
(reviewed in Refs. 1-3). However, some pro-apoptotic Bcl-2 family
proteins such as Bax, Bid, and Bim are found in the cytosol and must be
induced to translocate to mitochondria and other organelles where the Bcl-2-family proteins to which they dimerize reside (25-28). We explored the intracellular locations of the Bcl-GL and
Bcl-GS protein by two-color confocal microscopy analysis of
cells expressing GFP-tagged proteins. GFP·Bcl-GL protein
was located diffusely throughout cells, similar to GFP control protein
(Fig. 5). In contrast, Bcl-GS
was found in a punctate cytosolic pattern (Fig. 5), and partially
colocalized with a mitochondria-specific dye (Mitotracker).
Surprisingly, deletion of the BH3 domain from Bcl-GS did
not disrupt the punctate distribution (Fig. 5), indicating that other
regions of the Bcl-GS protein are sufficient for
subcellular targeting. Subcellular fractionation experiments confirmed
these observations, demonstrating association of Bcl-GS and
Bcl-GS- We describe here a new member of the BCL-2 gene family
in humans, BCL-G. The BCL-G gene potentially
encodes two protein products, Bcl-GL and
Bcl-GS. Bcl-2 family proteins contain up to four conserved BH domains. The shorter Bcl-GS protein contains only the
BH3 domains, similar to several other pro-apoptotic Bcl-2 family
proteins, including Bad, Hrk, Bik, Bim, Blk, Noxa(APR), and Egl1
(reviewed in Refs. 13, 29, 30). In contrast, the longer
Bcl-GL protein contains BH2 and BH3 domains. No other
examples of Bcl-2 family proteins are known that combine BH2 and BH3
domains in the absence of BH1. Though the Bad protein was originally
suggested to contain a BH2 domain (31) and has been shown to possess
the BH3 domain, inspection of the BH2 region reveals very little
similarity of amino acid sequence with other BH2 domains (32). In
contrast, the BH2 of Bcl-GL contains a stretch of 8 of 8 residues showing identity or conservative amino acid substitutions with
the BH2 domains of other family members. By comparison, the Bad
sequence reveals only 3 of 8 identical or similar amino acids in the
same region. Thus, Bcl-GL defines a novel structural
variant within the Bcl-2 family of apoptosis-regulating proteins.
The production of different protein isoforms by alternative mRNA
splicing is a common feature of BCL-2 family genes,
including BCL-2, Bcl-X, MCL-1, BAX, and BIM
(33-37). Unlike BCL-X, which encodes a long and short
protein, Bcl-XL and Bcl-XS, possessing anti-apoptotic and pro-apoptotic functions, respectively, the longer
isoform of Bcl-G did not display anti-apoptotic activity. When
overexpressed, Bcl-GL induced modest and variable increases in apoptosis, whereas the shorter Bcl-GS protein
consistently exhibited potent cytotoxic activity. This behavior is
reminiscent of the proteins encoded by the BIM gene, which
include Bim-short (BimS), Bim-long (BimL), and
Bim-Extra-Long (BimEL) (34). The longer proteins,
BimL and BimEL, are sequestered in complexes with dynein light-chain (DLC) in association with microtubules, thus
preventing them from interacting with target proteins such as
Bcl-XL on the surface of mitochondria and other organelles (26). In contrast, because the shortest isoform, BimS, does not associate with DLC, it is free to interact with Bcl-XL,
Bcl-2, and other survival proteins and hence displays far more potent apoptotic activity when overexpressed in cells. By analogy, the longer
Bcl-GL protein could be sequestered in an inactive complex with an unidentified protein.
Besides interactions with sequestering proteins, the activity of
pro-apoptotic Bcl-2 family proteins can be suppressed by other
mechanisms, including post-translational modifications. For example,
the Bad protein is inactivated by phosphorylation. This protein can be
directly or indirectly phosphorylated by several protein kinases,
including PKA, PKB (Akt), Raf1, and Pak1, thus preventing it from
dimerizing with target proteins such as Bcl-2 and Bcl-XL
(reviewed in Refs. 30, 38). The intracellular location of Bad varies,
depending on its phosphorylation state, with phosphorylated Bad
residing in the cytosol and unphosphorylated Bad associated with
mitochondria and other intracellular organelles where Bcl-2 and
Bcl-XL are located. In this regard, the Bcl-GL
protein contains candidate phosphorylation sites for PKA and PKC,
including some not found in Bcl-GS. However, we have been
unable to demonstrate in vivo phosphorylation of
Bcl-GL in pilot experiments (unpublished observations).
Another post-translational modification shown previously to active
latent pro-apoptotic Bcl-2 family proteins is proteolysis. Specifically, the Bid protein contains a N-terminal domain of ~58
amino acids that masks its BH3 domain, reducing its ability to dimerize
with other Bcl-2 family proteins. Upon cleavage by caspases, however,
removal of the N-terminal domain exposes the BH3 domain and is
associated with translocation of Bid from the cytosol to mitochondria,
where it induces cytochrome c release and apoptosis (27,
28). Whereas Bcl-GL contains candidate caspase recognition
sites, we have been unable to demonstrate significant cleavage of
Bcl-GL in vitro using purified active caspases
or in cells during apoptosis (unpublished observations). We cannot
exclude the possibility, however, that a specific caspase not yet
tested is capable of cleaving and activating Bcl-GL.
Though possessing no hydrophobic region that might anchor it in
membranes, the Bcl-GS protein was constitutively associated with intracellular organelles. Interestingly, removal of the BH3 domain
did not interfere with organellar-targeting of Bcl-GS but did abolish dimerization with Bcl-XL. Thus, the BH3 domain
apparently is not responsible for association of Bcl-GS
with intracellular organelles. This BH3-independent targeting of
Bcl-GS differs from some other BH3-only Bcl-2 family
proteins such as Bad, where it has been observed that removal of the
BH3 domain abrogates binding to anti-apoptotic Bcl-2 family proteins as
well as association with mitochondria (39)
The BCL-G gene resides on chromosome 12p12, a region deleted
in ~50% of prostate cancers, ~30% of ovarian cancers, and ~30% of childhood acute lymphocytic leukemias (ALLs) (40-42). Given that at
least one of the protein products of the BCL-G gene exhibits pro-apoptotic function, it is possible that BCL-G represents
a tumor suppressor gene. However, thus far, we have detected neither somatic mutations in the exons of BCL-G nor evidence of
deletion of both BCL-G alleles in tumor cell lines or
primary tumor specimens (data not shown). Further studies are required
therefore to determine whether loss of BCL-G expression
occurs in tumors by means other than somatic alterations in gene
structure and DNA sequence such as changes in gene methylation or
aberrant transcriptional or post-transcriptional regulation.
Investigation of the tissue distribution of Bcl-GL and
Bcl-GS mRNAs by RT-PCR revealed that Bcl-GL
mRNA is found in several normal adult tissues, whereas
Bcl-GS was detected only in testis. This finding indicates
tissue-specific regulation of Bcl-GS mRNA splicing.
Tissue-specific splicing of other Bcl-2 family mRNAs has been
observed previously. For example, Bcl-X mRNA splicing events, which
generate the pro-apoptotic Bcl-XS protein occur in the
thymus during T-cell ontogeny and in the mammary gland during
postlactation involution, in association with extensive apoptosis
induction (35, 6). Future studies therefore should include analysis of
the differential mRNA splicing patterns of Bcl-G transcripts during
fetal development and following various scenarios in the adult where
apoptosis occurs as part of a normal physiological response or an
abnormal pathological reaction to environmental insults.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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REFERENCES
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ABSTRACT
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DISCUSSION
REFERENCES
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Fig. 1.
Sequence analysis of Bcl-G cDNAs.
A, the predicted amino acid sequences of the
Bcl-GL and Bcl-Gs proteins are presented with
the BH2 and BH3 domains underlined and residue numbers
indicated. The predicted proteins are identical from residues 1-226.
The unique C-terminal region of Bcl-GS is indicated in
italics. B, an alignment is presented of the
BH3 domains of Bcl-GL and several other Bcl-2 family
proteins. Identical and similar residues are shown in black
and gray blocks, respectively. C, an alignment is
presented of the BH2 domains in Bcl-G and several other Bcl-2 family
proteins, as above. D, the exon-intron organization of the
BCL-G gene is presented. The human BCL-G gene
contains 6 exons, spanning a ~30 kilobase region of chromosome 12. Alternative splicing at the 5'-end of exon 5 accounts for the
production of the Bcl-GL and Bcl-GS proteins,
where splice-acceptor sites at nucleotide positions 63,870 versus 63,797 in BAC clone RPCI 11-267J23
(GenBankTM/EBI no. AC007537) are utilized for
Bcl-GL and Bcl-GS, respectively. The positions
of the start and termination codons are indicated, with coding regions
in gray blocks and noncoding 5'- and 3'-UTR sequence in
open blocks. The BH3 domain is located in exon 4 of both
Bcl-GL and Bcl-GS, whereas the BH2 domain
resides in exon 5 of Bcl-GL.
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Fig. 2.
Expression of Bcl-Gs
and Bcl-GL in human tissues. The
expression of transcripts encoding Bcl-GL or
Bcl-GS was examined by Northern blotting (A) and
RT-PCR (B). For Northern blotting,
poly(A)+-selected RNA samples from various adult tissues
were analyzed by hybridizing the blot with a 32P-labeled
Bcl-GL cDNA probe. The blot was stripped and reprobed
with a -actin cDNA (bottom). Molecular mass markers
are indicated in kilobase pairs. For RT-PCR, first-strand cDNA
prepared using RNA samples from various adult human tissues was PCR
amplified using primers specific for Bcl-GL and
Bcl-GS based on differences in splice-acceptor utilization
in exon 5. The primers flank an intron in both cases, thus excluding
amplification because of contaminating genomic DNA. PCR products were
size-fractionated in 2% agarose gels, stained with ethidium bromide,
and then photographed under UV-illumination.
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Fig. 3.
Induction of apoptosis by Bcl-G.
A, plasmids encoding GFP, GFP·Bcl-Gs, or
GFP·Bcl-GL were transfected into COS-7 cells alone or in
combination with a plasmid encoding Bcl-XL. Apoptosis was
examined by DAPI staining at 24 h post-transfection (mean ± S.D., n = 3, top). Levels of GFP and
GFP·Bcl-G fusion proteins were examined by immunoblotting lysates
from transfected COS-7 cells (20 µg per lane) and anti-GFP antibody
with ECL-based detection (middle). Equal loading was
confirmed by reprobing the same membrane with anti-tubulin antibody
(bottom). B, plasmids encoding GFP,
GFP·Bcl-GS, or the mutant proteins,
Bcl-GS- BH3 and GFP·Bcl-Gs-L216E were
transfected into COS-7 cells. The percentage of apoptotic cells was
examined 1 day later as above (top). Protein expression was
assessed by immunoblotting as above, using anti-GFP (middle)
or anti-tubulin (bottom) antibodies. C, plasmids
encoding GFP, GFP·Bcl-GL, or Bcl-GL-
BH2
were transfected into COS-7 cells. The percentage of apoptotic cells
was examined 1 day later as above (top). Protein expression
was assessed by immunoblotting as above, using anti-GFP
(middle) or anti-tubulin (bottom)
antibodies.
-helices in which the hydrophobic surface of the
-helices of apoptosis-inducing BH3 peptides bind to a pocket on
survival proteins such as Bcl-XL (22). We, therefore,
compared the apoptosis-inducing activity of the wild-type
Bcl-GS protein with mutants lacking the BH3 domain (
BH3)
or in which leucine 216 within the BH3 domain of Bcl-GS was
chosen for mutation to the charged glutamic acid, based on comparisons
with previously described BH3 mutagenesis experiments demonstrating a
critical requirement for the analogous leucine in other pro-apoptotic
Bcl-2 family proteins (23, 24).
BH3 and Bcl-GS-L216E did not (Fig.
3B and data not shown). Immunoblot analysis confirmed
production of the Bcl-GS-
BH3 and
Bcl-GS-L216E proteins at levels exceeding the amounts of
wild-type Bcl-GS protein. We conclude, therefore, that the
BH3 domain of Bcl-GS is critical for its pro-apoptotic activity.
BH2) induced apoptosis in COS-7 cells as
potently as Bcl-GS (Fig. 3C). Thus the BH2
domain negatively regulates the pro-apoptotic activity of
Bcl-GL.
BH3 and
Bcl-GS-L216E proteins (Fig. 4, B and
C). Thus, the pro-apoptotic activity of Bcl-GS
correlates with it ability to bind Bcl-XL. When the BH2 domain of Bcl-GL was deleted, this mutant
Bcl-GL associated with Bcl-XL (Fig.
4D), thus providing further evidence that the BH2 domain
autorepresses this protein.
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Fig. 4.
Interactions of Bcl-Gs and
Bcl-GL with Bcl-xL. 293T cells were
transiently cotransfected with plasmids encoding Bcl-XL and
GFP, GFP·Bcl-GL, GFP·Bcl-Gs (A),
GFP·Bcl-GS- BH3 (B),
GFP·Bcl-GS-L218E (C), or
Bcl-GL-
BH2 (D). Cells were lysed 1 day later,
and immunoprecipitations were performed using anti-GFP antibody.
Immunocomplexes (prepared from 0.5 mg of lysate, top) and
lysates (20 µg of protein, bottom) were subjected to
SDS-polyacrylamide gel electrophoresis/immunoblot analysis using
anti-Bcl-XL (top) and anti-GFP
(bottom) antibodies, respectively.
BH3 predominantly with organelle-containing heavy
membrane fractions, with scant amounts in the soluble cytosolic
compartment (not shown).
View larger version (39K):
[in a new window]
Fig. 5.
Microscopic evaluation of intracellular
distributions of Bcl-GL and Bcl-GS.
Plasmids encoding GFP, GFP·Bcl-GL,
GFP·Bcl-GS, and GFP·Bcl-Gs- BH3 were
transfected into COS-7 cells. Cells were incubated with 50 nM Mitotracker Red for 30 min, fixed in 4% formaldehyde,
and examined by two-color confocal microscopy.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by CaP-CURE and National Institutes of Health Grant GM60554.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) AF281254, AF281255.
Recipient of a Postdoctoral Traineeship Award from the United
States Army MRMC Breast Cancer Research Program.
§ To whom correspondence should be addressed: The Burnham Inst., 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3140; Fax: 858-646-3194; E-mail: jreed@burnham-inst.org.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M005889200
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ABBREVIATIONS |
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The abbreviations used are: BH, Bcl-2 homology domain; EST, expressed sequence tag; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; GFP, green fluorescent protein; PKA, cAMP-dependent kinase; PKC, protein kinase C; UTR, untranslated region.
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