From the Institute of Biochemistry, University of
Lausanne, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland and
** MEMOREC Stoffel GmbH, Stoeckheimer Weg 1, D-50829 Koeln, Germany
Received for publication, November 21, 2000, and in revised form, March 20, 2001
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ABSTRACT |
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The Bcl-2 family of proteins plays a central
regulatory role in apoptosis. We have identified a novel, widely
expressed Bcl-2 member which we have named Bcl-rambo. Bcl-rambo shows
overall structural homology to the anti-apoptotic Bcl-2 members
containing conserved Bcl-2 homology (BH) motifs 1, 2, 3, and 4. Unlike
Bcl-2, however, the C-terminal membrane anchor region is preceded by a
unique 250 amino acid insertion containing two tandem repeats. No
interaction of Bcl-rambo with either anti-apoptotic (Bcl-2, Bcl-xL, Bcl-w, A1, MCL-1, E1B-19K, and BHRF1) or
pro-apoptotic (Bax, Bak, Bik, Bid, Bim, and Bad) members of the Bcl-2
family was observed. In mammalian cells, Bcl-rambo was localized to
mitochondria, and its overexpression induces apoptosis that is
specifically blocked by the caspase inhibitors, IAPs, whereas
inhibitors controlling upstream events of either the `death receptor'
(FLIP, FADD-DN) or the `mitochondrial' pro-apoptotic pathway
(Bcl-xL) had no effect. Surprisingly, the Bcl-rambo cell
death activity was induced by its membrane-anchored C-terminal domain
and not by the Bcl-2 homology region. Thus, Bcl-rambo constitutes a
novel type of pro-apoptotic Bcl-2 member that triggers cell death
independently of its BH motifs.
Apoptosis, the physiological process of cell death, is critical
for modeling tissues and maintaining homeostasis in multicellular organisms (1). Members of the Bcl-2 family of proteins are regulators
of apoptosis that can be grouped into three sub-families (2, 3). Bcl-2
and several of its close relatives including Bcl-x, Bcl-w, and
Caenorhabditis elegans CED-9 inhibit apoptosis, whereas
structurally similar relatives such as Bax, Bak, Bok, and distant
cousins such as Bik and Bim instead foster death. The Bcl-2 and Bax
subfamilies share three of the four conserved Bcl-2 homology
(BH)1 sequence motifs. In
contrast, mammalian Bad, Bik/Nbk, Blk, Hrk, Bid, Bim/Bod, Noxa, and
C. elegans EGL-1 share homology only within the short BH3
motif and are therefore called BH3-only proteins (2, 3). Most members
of the wider family possess a hydrophobic C-terminal segment, which
facilitates their interaction with the endoplasmic reticulum/nuclear
envelope and/or the outer mitochondrial membrane, where the prosurvival
members normally reside, and where most others assemble during apoptosis.
The ability of the prosurvival and antisurvival family members to form
heterodimers raises the possibility that each type might titrate the
other, potentially accounting for their opposing actions. The relative
concentrations of the opposing sub-family members would then determine
whether the cell lives or dies (4, 5). Mutagenesis of Bcl-2 initially
suggested that its ability to inhibit cell death required binding to a
pro-apoptotic family member (6), but Bcl-xL mutants have
been identified that do not bind Bax or Bak yet still block apoptosis
(7). Thus, it remains unclear whether the ability to associate with
other family members is central to regulating apoptosis.
To search for additional regulators of apoptosis, we have screened
public data bases with a profile defining Bcl-2 family members. This
search has yielded a novel BH4/BH3/BH1/BH2-containing protein, which we
have denoted Bcl-rambo. Here we report its initial characterization.
Cloning and Sequences--
The expressed sequence tag
clone T48205 encodes a short splice form of human Bcl-rambo that shares
the N-terminal 1-200-amino acid (aa) of full-length Bcl-rambo. The
expressed sequence tag clone AA190545 contains C-terminal 255-485-aa
region. A cDNA library was used to amplify the fragment containing
the truncated part (aa 201-254) of Bcl-rambo using 5'-forward primer
JT1108 (5'-CCT TCA CCA GCA CAG GCT TTG ACC G-3') and 3'-backward primer JT1109 (5'-GGA CCA CCT CCT CCA CTT CAG TAG G-3'). The PCR product was
cloned into PCR-Blunt (Invitrogen) in the counterclockwise direction of
the vector. The NotI/BamHI fragment was inserted into the NotI and BamHI site of AA190545, which
yielded the 78-485-aa fragment of Bcl-rambo. Double PCR was carried
out to construct full-length Bcl-rambo. Two DNA fragments were
amplified from the plasmid carrying Bcl-rambo (78) using 5'-primer
JT1145 (5'-CAT ACC TCG AGG ACT ATT C-3') and 3'-primer flanked by
NotI site JT1144 (5'-TAG CGG CCG CCT ATT TCT TTC TCA G-3'),
and T48205 using 5'-primer flanked by EcoRI site JT1143
(5'-AAA GAA TTC ATG GCG TCC TCT TCT AC-3') and 3'-primer JT1146 (5'-GAA
TAG TCC TCG AGG TAT G-3'), respectively. Both DNA fragments were mixed
and amplified by PCR without primers followed by amplification with primers JT1143 and JT1144. The PCR product was cloned into PCR-Blunt, and the EcoRI/NotI fragment was subcloned into a
modified version of PCR3 (Invitrogen) in frame with a N-terminal FLAG
or HA peptide. Blast searches of GenBankTM revealed a
150,172-base pair human BAC clone (p1087l10) in the genomic data
base derived from chromosome 22q11 (GenBankTM AC006285),
which covers the complete Bcl-rambo gene.
Plasmid Construction--
The DNA fragment corresponding to
Bcl-rambo (1) was amplified by PCR from T48205 with 5'-forward
primer containing EcoRI site JT993 (5'-AAA GAA TTC ATG GCG
TCC TCT-3') and 3'-reverse primer containing NotI site JT994
(5'-TAG CGG CCG CTC ATA CCC AGC CAC C-3') and cloned into PCR-Blunt.
Other deletion mutants were constructed by PCR amplification using
full-length Bcl-rambo as a template with 5'-forward primer flanked by
EcoRI and 3'-reverse primer flanked by NotI as
follows: Bcl-rambo (1), 5'-primer JT1143 and 3'-primer JT1244
(5'-AT AGC GGC CGC CTA GTC ATT GCT ATC TTC GT-3'); Bcl-rambo (1),
5'-primer JT1143 and 3'-primer JT 1245 (5'-AT AGC GGC CGC CTA AGA CTT
GCC CTC AGA C-3'); Bcl-rambo (205), 5'-primer JT1251 (5'-AAA GAA
TTC AGT CTT GAG TCA GAG G-3') and 3'-primer JT1144; Bcl-rambo
(205), 5'-primer JT1251 and 3'-primer JT1245. The
EcoRI/NotI fragments were subcloned into a
modified version of PCR3 with N-terminal FLAG epitope: Bcl-rambo
( Cell Lines and Reagents--
The cell lines used here were grown
and maintained as previously described (8). zVAD-fmk was purchased from
Alexis. The antibody against Bcl-rambo (AL167) was generated in rabbits
against bacterial recombinant Bcl-rambo encompassing aa 1-223. The
antibody was affinity purified on immobilized antigen according to
standard procedures.
Northern Blots--
Northern blots
(CLONTECH, Palo Alto, CA) were hybridized using
random-primed radioactive probes in ExpressHyb buffer
(CLONTECH) for about 3 h and then washed at
room temperature for 40 min with several changes of 2× SSC/0.1% SDS
followed by 0.1× SSC/0.05% SDS for 40 min at 50 °C. The blots were
probed human Bcl-rambo or actin (data not included).
Transient Cell Transfection, Immunoprecipitation, and Western
Blotting--
For co-immunoprecipitation experiments, approximately 3 × 105 293T cells/10-cm culture dish were transfected with
the indicated expression plasmids by the calcium phosphate/HEPES
method (9). Cells were harvested 30 h after transfection and lysed
in complete Nonidet P-40 lysis buffer (1% Nonidet P-40 lysis buffer
with 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and
protease inhibitor mixture (Complete, Roche Molecular Biochemicals) on
ice. Postnuclear lysates were normalized for protein content,
precleared on Sepharose 6B (Sigma) for 2 h at 4 °C, and
subsequently subjected to immunoprecipitation with anti-FLAG-M2-agarose
beads (Sigma) for at least 2 h at 4 °C on a rotating wheel.
Immunoprecipitates were washed twice each with lysis buffer containing
1% or 0.05% Nonidet P-40, respectively, and washed beads were boiled
in reducing sample buffer before analysis by SDS-PAGE and Western blotting.
FLAG-, HA- and EE-tagged proteins were detected using anti-FLAG M2
(Sigma), the anti-HA (Babco) and anti-EE (Eurogentec) monoclonal antibodies, respectively. Cellular (endogenous) Bcl-rambo was detected
in cell lysate (50 µg) using an affinity-purified polyclonal rabbit
antibody (AL167) directed against a recombinant protein encompassing aa
1-223 of human Bcl-rambo. Blots were revealed with peroxidase-coupled
goat anti-mouse or anti-rabbit secondary antibodies (Jackson
ImmunoResearch Laboratories).
Apoptosis Assay--
293T cells (3 × 105
cells) were seeded on 60-mm culture plates the day before transfection
and transiently co-transfected with 2 µg of Bcl-rambo expression
plasmids together with 0.2 µg of EGFP expression vector
(pEGFP-C1, CLONTECH) or 0.5 µg of
pCMV- Subcellular Localization--
293T cells or HeLa cells (1 × 106 cells, 100-mm dish) were seeded the day before transfection.
293T cells were transfected with 0.5 µg of FLAG-tagged Bcl-rambo
constructs or FLAG-tagged Bcl-2 plus 9.5 µg of empty vectors for
12 h, whereas HeLa cells were transfected with 5 µg of
FLAG-tagged Bcl-rambo constructs plus 5 µg of empty vectors for
12 h. Cells were collected from four dishes and washed twice with
PBS. The cells were disrupted in 250 mM sucrose, 10 mM HEPES-OH (pH 7.4), 1 mM EDTA, 1 mM EGTA, and protease inhibitor mixture by dounce
homogenizer (20 strokes), and resultant cell lysates were centrifuged
(800 × g, 10 min) to remove unbroken cells and nuclei.
Postnuclear lysates were centrifuged (10,000 × g, 10 min) to yield heavy membrane fractions enriched with mitochondria. The
supernatants were then centrifuged (100,000 × g, 60 min) to separate cytosolic fraction (S100) and light membrane fractions
(P100). Proteins were separated by 10% SDS-PAGE and blotted with
anti-FLAG and anti-Bcl-2 antibodies.
Cytochrome c Release--
For cytochrome c release,
cells were incubated for 10 min on ice in 20 mM HEPES, pH
7.5, 10 mM KCl, 15 mM MgCl2, 1 mM EDTA, and 1 mM EGTA containing protease
inhibitors and then lysed with 15 strokes in a syringe (gauge G22). The
lysate was incubated for 10 min on ice and then centrifuged.
Supernatants and pellets were resuspended in reducing Laemmli's buffer
and probed for the presence of cytochrome c by Western
blotting (PharMingen).
Immunostaining and Confocal Laser Scanning Microscopy--
The
day before transfection, HeLa cells were plated at 10-20% confluency
on 20-mm glass coverslips in 5.5-cm culture plates. Cells were
transfected the following day by the calcium phosphate/BES method and
harvested ~36 h after onset of transfection. Cells on coverslips were
washed twice with cold PBS, fixed for 12 min in 4% paraformaldehyde at
room temperature, washed twice with cold PBS, and permeabilized in PBS
with 0.1% saponin (PBSS) overnight at 4 °C. Coverslips were blocked
for 30 min at room temperature with 5% milk in PBSS (PBSSM), stained
with primary antibody anti-Bcl-rambo AL167 (1 µg/ml) for 1 h at
room temperature, washed three times with PBSS and stained with
Alexa-488-labeled (Molecular Probes) secondary anti-rabbit antibody
diluted 1/100 in PBSSM for 1 h in the dark. For mitochondrial
staining, cells were incubated for 1 h in the presence of
anti-Smac/Diablo antibodies (Alexis) and subsequently with the
anti-mouse Alexa-546 secondary antibody. Finally, coverslips were
washed three times with PBS and mounted with FluorSave reagent
(Calbiochem) on microscope slides. Confocal microscopy was performed on
a Zeiss Axiovert 100 microscope (Zeiss Laser Scanning Microscope 510).
Identification and Sequence of Bcl-rambo--
Using
the prf.Bcl-2
profile2 as a search tool, we
screened public data bases for novel Bcl-2 homologues and identified
Bcl-rambo. Subsequent DNA sequencing and identification of additional
expressed sequence tags allowed the isolation of the full-length human
Bcl-rambo cDNAs. The open reading frame of Bcl-rambo encodes a
protein of 485 amino acids (Fig.
1A). The methionine initiation
codon is preceded by a typical consensus Kozak sequence and
hydrophobicity analysis predicts the presence of a C-terminal
transmembrane domain. Comparison of protein sequences among different
Bcl-2 proteins indicated that Bcl-rambo contained all four BH (BH1, -2, -3 and -4) motifs that identify anti-apoptotic Bcl-2 members (Fig.
1A). Although in most other Bcl-2 members containing more
than one BH domain the Bcl-2 homology region is followed by the
C-terminal membrane anchor, these two regions are separated in
Bcl-rambo by a stretch of more than 250 aa that show no obvious
sequence homology with any other protein in the data base; we named
this domain BHNo domain because it contains no BH motif. Of note is the
presence of two distinct short tandem repeats (repeat A and repeat B,
Fig. 1B) within the BHNo domain. The presence of a zebrafish homologue containing the BHNo domain (data not shown) suggests that
Bcl-rambo is highly evolutionarily conserved. Using the cDNA of
Bcl-rambo, a human BAC clone from chromosome 22q11 was
identified in the human genomic data base covering the Bcl-rambo
sequence. Comparison with the cDNA shows that the coding region is
encompassed by six exons, displaying the typical genetic structure of
Bcl-2 family members (Fig. 1C). Interestingly, the
C-terminal part of the BH2 domain, the large BHNo insert, and the
membrane anchor domain are encoded by a single exon.
Expression and Subcellular Localization of Bcl-rambo--
The
expression of Bcl-rambo mRNA in diverse human tissues was examined.
As shown in Fig. 2A, a
Bcl-rambo transcript of 4.1 kilobases was detected in all tissues
studied. Highest mRNA levels were found in heart, pancreas, and
placenta. Two weaker bands of 2.1 and 1.2 kilobases may correspond to
alternatively spliced forms. Human cell lines of different origin
expressed Bcl-rambo mRNA at various levels (Fig. 2B) in
agreement with the observation that the Bcl-rambo protein was
detectable in all cell lines tested (Fig. 2C). Bcl-rambo has
a predicted molecular mass of 55 kDa. Hence, the apparent high
molecular mass of 85 kDa deduced from SDS-PAGE was surprising.
Expression of various fragments of Bcl-rambo indicated that the
unusual migration property of the protein was caused by the BHNo domain
that exhibited an apparent molecular mass of 52 kDa as compared with
its predicted molecular mass of 30 kDa (Fig. 2D).
Based on confocal microscopic analysis of HeLa cells transfected with a
Bcl-rambo expression plasmid, Bcl-rambo protein was found to be
cytosolic, localizing to intracellular organelles in a pattern typical
of Bcl-2 family proteins (Fig.
3A). Identical results were
obtained when 293T cells were analyzed (data not shown). The majority
of Bcl-rambo co-localized with the IAP inhibitor Smac/DIABLO
(11, 12) to mitochondria. When the C-terminal membrane anchor was
removed, the punctate cytoplasmic staining disappeared and was replaced
by a diffuse nuclear staining (Fig. 3A). Thus, in agreement
with the crucial role of this region in the subcellular distribution of
other Bcl-2 family proteins (reviewed in Refs. 2 and 3), the apparent
mitochondrial localization of Bcl-rambo is dictated by its hydrophobic
C terminus.
The subcellular localization studies were confirmed by cellular
fractionation studies (Fig. 3B). Full-length Bcl-rambo and Bcl-rambo lacking the BHNo domain ( Absence of Interaction with Other Bcl-2 Members--
Prosurvival
and antisurvival Bcl-2 family members are known to frequently form
heterodimers (reviewed in Ref. 13). We thus investigated interactions
between Bcl-rambo and different anti- and pro-apoptotic Bcl-2 family
proteins using the 293T overexpression system. As shown in Fig.
4A, there was no interaction
found with the pro-apoptotic members Bax, Bak, Bik, Bid, Bim, Bad, and
Bcl-rambo, whereas the interaction of these BH3-only members with
Bcl-xL (Fig. 4C) was readily demonstrable. Of
interest, the co-expression of some of these pro-apoptotic Bcl-2
members with Bcl-rambo significantly decreased their expression (Fig.
4A). To demonstrate that the lack of interactions between
Bcl-rambo and pro-apoptotic Bcl-2 members was not due to increased cell
death of transfected cells, the experiments were repeated in the
presence of the Pan-caspase inhibitor z-VAD-fmk; the
outcome, however, was identical. We further tested interactions between
Bcl-rambo and several anti-apoptotic Bcl-2 members (Bcl-2,
Bcl-xL, Bcl-w, A1, MCL-1, E1B-19K, and BHRF1) (Fig.
4B); but again no interaction was observed. Bcl-rambo also did not interact with Bcl-2 family members only when the Bcl-2 homology
region of Bcl-rambo (aa 1-223) was expressed, and moreover, no
Bcl-rambo homodimerization was detected (data not shown).
Induction of Apoptosis by Bcl-rambo--
The ability of Bcl-rambo
to regulate apoptosis in mammalian cells was investigated. In 293T
cells, transfection with expression vectors encoding Bcl-rambo resulted
in significant caspase-3 activation and cell death (Fig.
5A). The pro-apoptotic effect
of Bcl-rambo was specific because transfection of the empty plasmid did
not affect cell survival. Overexpression of the N-terminal Bcl-2
homology region (aa 1-223 or 1-203) had little effect on cell
viability, whereas the expression of the C-terminal BHNo domain (aa
205-485) induced slightly higher caspase-3 activation than that
observed with the full-length protein despite identical protein
expression (Fig. 5A). The pro-apoptotic activity of
Bcl-rambo and its BHNo domain was completely dependent on the
C-terminal membrane anchor (MA) as its removal resulted in the complete
loss of cell death activity. We therefore investigated whether the
pro-apoptotic activity of the C-terminal region of Bcl-rambo was solely
due to its MA region or whether the presence of the BHNo domain was also required. A mutant protein was generated where the MA region was
directly fused to the BH domain, thus lacking the BHNo region (
Bax and Bak induce apoptosis by causing cytochrome c
release from mitochondria which then binds to APAF-1 resulting in
caspase-9 activation and apoptosis. Similar to Bax and Bak,
overexpression of Bcl-rambo resulted in the appearance of cytochrome
c in the cytosol (Fig. 5B) indicating that
mitochondrial damage occurs during Bcl-rambo-induced cell death.
Bcl-rambo-induced cell death was caspase-dependent
and was completely blocked by the Pan-caspase-inhibitor
zVAD-fmk (data not shown). In contrast, co-expression of c-FLIP (14), a
dominant negative form (DN) of FADD (15), CrmA (16) (Fig.
6), suggests that Bcl-rambo induces cell
death independent of the death receptor pathway. Moreover,
Bcl-xL as well as DN-caspase-9 (17) only marginally
prevented Bcl-rambo-induced cell killing indicating that cell death
could still proceed in the absence of an active cytochrome
c-Apaf1-caspase-9 pathway. Only IAPs (18) displayed a strong inhibitory
effect, in particular X-IAP and to a lesser extend c-IAP1 and c-IAP2.
These IAP members have been shown to bind and inactivate caspase-3, -6, and to a lesser degree caspase-9, but not caspase-1, -6, -8, or -10 (18).
In this report we describe the cloning and the initial
characterization of a novel Bcl-2 family member, Bcl-rambo. Bcl-rambo contains all four conserved BH motifs and a C-terminal membrane anchor and is therefore predicted to be a member of the anti-apoptotic Bcl-2 subfamily. Unlike other members of this subfamily, such as Bcl-2
or Bcl-xL, the BH-containing domain is separated from the
membrane anchor by a unique 250-aa-long region. This C-terminal BHNo
domain, which shares no significant sequence homology with other
proteins in the data base, reveals very little information about a
possible domain structure and function. The only notable feature is the
presence of two short tandem repeats that both are rich in serines. One
is located at a position where Bcl-2 members usually contain the
membrane anchor domain, whereas the longer second repeat unit is found
further downstream immediately preceding the transmembrane domain of
Bcl-rambo (see Fig. 1).
Surprisingly, it is this BHNo domain and not the Bcl-2 homology
region that more efficiently induces cell death upon overexpression. It
is currently unclear how this segment causes apoptosis. Bcl-rambo is
localized to mitochondria, and because removal of the C-terminal membrane anchor results not only in the loss of mitochondrial localization but also of its cell death activity, it is likely that
Bcl-rambo initiates the cell death pathway by inflicting damage to
mitochondria. Indeed, cytochrome c release was detected upon
Bcl-rambo overexpression. For the pro-apoptotic Bcl-2 members such as
Bax, it was proposed that they cause mitochondrial outer membrane
damage by forming channels in agreement with their structural homology
with bacterial toxins (19). Bcl-rambo appears to induce apoptosis, at
least in part, independently of known mitochondrial signaling pathways.
Overexpression of Bcl-xL (Fig. 6) and Bcl-2 (data not
shown), which are known to efficiently block apoptosis induced by
pro-apoptotic Bcl-2 members by inhibiting cytochrome c
release, had little effect on Bcl-rambo-mediated cell death. Moreover,
a dominant-negative form of caspase-9, which blocks apoptosis induced
by Apaf-1, showed only a moderate inhibitory effect suggesting that
Bcl-rambo death activity is not dependent on the classical
mitochondrial pathway. This conclusion is supported by the
absence of interaction with more than 10 different Bcl-2 pro- and
anti-apoptotic family members tested. Similarly, FLIP and a
dominant-negative form of FADD, both potent inhibitors of the death
receptor signaling pathway, do not block the Bcl-rambo-induced caspase-3 activation. Only inhibitors that interfere with the activity
of the downstream caspase-3, such as members of the IAP family, are
effective at blocking Bcl-rambo death activity. The p53-induced protein
p53AIP1 is another example of a protein that is localized within
mitochondria and that triggers apoptosis with an unknown mechanism
(10).
Taken together, Bcl-2-rambo, via its BHNo and MA domain, is likely to
trigger death via an as yet to be discovered signaling pathway that
joins other pro-apoptotic pathways at the level of caspase-3. It will
be interesting to determine whether the Bcl-2 homology region of
Bcl-rambo, carrying all four BH domains, also has an anti-apoptotic
effect for example by inhibiting the pro-apoptotic activity of the BHNo
domain. Identification of Bcl-rambo may therefore provide further
insight into the complex mechanism of cell death regulation by the
Bcl-2 family members.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
207-456); the BHNo region was deleted according to standard procedures.
-galactosidase expression vector. Postnuclear lysates were used
to measure fluorescence intensity of EGFP,
-galactosidase
activity, and caspase-3 activity. Caspase-3 activity was measured by
mixing 10 µl of cell lysate (30-40 µg of protein) with 100 µl of
reaction buffer (10 mM Tris-HCl, pH 7.4, 0.1% CHAPS, 2 mM MgCl2, 1 mM dithiothreitol, 5 mM EGTA, and 150 mM NaCl) containing 50 µM of a fluorogenic caspase-3 substrate (Ac-DEVD-AMC,
Alexis). The mixture was incubated for 60 min in an enzyme-linked
immunosorbent assay titer plate and fluorescence was measured in a
Fluoroskan enzyme-linked immunosorbent assay reader (excitation 355 nm,
emission 460 nm). The caspase-3 activity (fold induction) was
normalized with the fluorescence intensity of EGFP or
-galactosidase activity, respectively.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence of Bcl-rambo and comparison to
anti-apoptotic members of the Bcl-2 family. A, the
predicted amino acid sequences of human Bcl-rambo with the predicted
BH1, BH2, BH3, BH4, and membrane anchor (MA) region boxed
are aligned to the sequences of Bcl-2, Bcl-xL, and Bcl-w.
The locations of the two tandem repeats are indicated. Identical and
similar amino acids are shown in black and gray
blocks, respectively. B, alignment of the tandem
repeats. C, exon-intron organization of the human Bcl-rambo
gene. The positions of the start and stop codons as well as the BH
domains are indicated. The non-coding region is shown in
gray.
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Fig. 2.
Expression of Bcl-rambo. Northern blots
of various human tissues (A) and tumor cell lines
(B) (CLONTECH) were probed with a
random-primed 32P-labeled cDNA fragment of the
N-terminal part of Bcl-rambo. B, expression of Bcl-rambo
protein in various human and mouse (mu) cell lines. Western
blot analysis was performed with an affinity-purified antibody (AL167)
generated against the Bcl-2 homology region of Bcl-rambo (aa 1-223,);
Jurkat and EL-4 (T cell leukemias); A20, Raji, Ramos, Bjab, and Raw (B
cell lymphomas); THP-1, U937, and K562 (monocytic cell lines); HeLa
(cervical carcinoma); HEK293 and 293T (embryonic kidney cell line).
D, various FLAG-tagged Bcl-rambo expression plasmids (aa
1-485, 1-223, 205-485) were transfected into 293T cells, and cell
extracts were assayed using the anti-FLAG antibody M2 (left
panel) or the polyclonal antibody against Bcl-rambo (right
panel).
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Fig. 3.
Subcellular localization of
Bcl-rambo. A, HeLa cells were transfected with plasmids
encoding full-length Bcl-rambo (1) or Bcl-rambo lacking the
C-terminal membrane anchor domain ( MA, 1-459), and their
subcellular localization was determined 48 h after transfection by
confocal laser microscopy using the anti-Bcl-rambo antibody AL167 or an
antibody detecting Smac/DIABLO. The overlay of Bcl-rambo fluorescence
(green) and Smac/DIABLO (red) is shown in
yellow. Bar, 10 µm. B, 293T cells
and HeLa cells were transfected with the various Bcl-rambo constructs
(see Fig. 5), and subcellular localization was analyzed by separating
heavy membranes (HM, including mitochondria), light
membranes (LM), and cytosol. As marker protein, VSV-tagged
Bcl-2 was co-transfected in 293T cells and detected using an anti-FLAG
antibody. In both cell lines, endogenous (end) Bcl-2 protein
was detected with an anti-Bcl-2 antibody.
207-459;
BHNo in
Fig. 5) were both recovered in heavy membrane fractions containing
mitochondria, whereas the full-length Bcl-rambo lacking the membrane
anchor domain was present mostly in the fraction containing soluble
proteins. An identical subcellular fractionation pattern was found with Bcl-2.
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Fig. 4.
No association of Bcl-rambo with various
Bcl-2 family members. 293 T cells were transiently transfected
with FLAG- (A) or HA-tagged (B) Bcl-rambo
together with FLAG-tagged anti-apoptotic Bcl-2 members or EE-tagged
pro-apoptotic Bcl-2 members in the presence of z-VAD-fmk. The
postnuclear lysates were immunoprecipitated with anti-FLAG M2-agarose
and blotted with anti-EE or anti-HA antibody, respectively. C, control
experiment: 293T cells were transfected with FLAG-tagged
Bcl-xL together with EE-tagged Bax, Bak, Bik, Bid, Bim, or
Bad for 22 h in the presence of z-VAD-fmk. The postnuclear lysates
were immunoprecipitated with anti-FLAG M2-agarose and blotted with an
anti-EE antibody.
BHNo). Bcl-rambo
BHNo induced apoptosis when overexpressed
although with reduced efficiency (Fig. 5A). We conclude that
the MA as well as the BHNo domain are important for optimal
pro-apoptotic activity.
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Fig. 5.
Bcl-rambo induces apoptosis via its
C-terminal domain. A, 293T cells were transfected with
2 µg of various Bcl-rambo constructs (right panel)
together with 0.2 µg of the EGFP expression vector (pEGFP-C1) for
42 h. The activity of caspase-3 and the fluorescence intensity of
EGFP in duplicate determinations was measured. The caspase-3 activity
(fold increase) was normalized with the fluorescence
intensity of transfected EGFP. Protein expression was assessed in
cell extracts by Western blot analysis using the anti-FLAG antibody M2.
B, release of cytochrome c from mitochondria was
determined in 293T cells transfected with Bcl-rambo and for control
purposes with Bax and Bak.
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Fig. 6.
Bcl-rambo-induced cell death is blocked by
IAPs. A, 293 T cells were transfected with 2 µg of
Bcl-rambo plasmid and 0.5 µg of expression vectors encoding various
inhibitors of apoptosis together with 0.5 µg of the -galactosidase
expression vector for 27 h. B, as in A but 293 T cells
were transfected with 2 µg of Bcl-rambo encoding aa 205-485 (BHNo
domain and membrane anchor domain). The activity of caspase-3 and
-galactosidase in duplicate determinations was measured. The
caspase-3 activity (fold increase) was normalized
based on the
-galactosidase activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. David Huang and Andreas Strasser for providing us with many of the cDNA clones employed in this study. We thank Drs. Chris Benedict, Margot Thome, and Kim Burns for helpful comments.
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FOOTNOTES |
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* This work was supported in part by a grant of the Swiss National Science Foundation.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) AF325209.
§ Present address: Dept. of Bioengineering, Tokyo Inst. of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
¶ These authors contributed equally to this work.
Supported by a grant of the Alliance des Recherches sur
le Cancer.
To whom correspondence should be addressed. Tel.:
41-21-692-5738; Fax: 41-21-692-5705; E-mail:
Jurg.tschopp@ib.unil.ch.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M010520200
2 F. Martinon, K. Hofmann, and J. Tschopp, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: BH, Bcl-2 homology; aa, amino acid(s); PCR, polymerase chain reaction; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered solution; MA, membrane anchor; VSV, vesicular stomatitis virus.
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