From the Department of Molecular Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
Received for publication, January 18, 2001, and in revised form, April 23, 2001
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ABSTRACT |
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2-5(A) synthetases are a family of
interferon-induced enzymes that polymerize ATP into 2'-5' linked
oligoadenylates that activate RNase L and cause mRNA degradation.
Because they all can synthesize 2-5(A), the reason for the existence of
so many synthetase isozymes is unclear. Here we report that the 9-2 isozyme of 2-5(A) synthetase has an additional activity: it promotes
apoptosis in mammalian cells. The proapoptotic activity of 9-2 was
isozyme-specific and enzyme activity-independent. The 9-2-expressing
cells exhibited many properties of cells undergoing apoptosis, such as
DNA fragmentation, caspase activation, and poly ADP-ribose
polymerase and lamin B cleavage. The isozyme-specific
carboxyl-terminal tail of the 9-2 protein was shown, by molecular
modeling, to contain a Bcl-2 homology 3 (BH3) domain, suggesting that
it may be able to interact with members of the Bcl-2 family that
contain BH1 and BH2 domains. Co-immunoprecipitate assays and confocal
microscopy showed that 9-2 can indeed interact with the anti-apoptotic
proteins Bcl-2 and BclxL in vivo and
in vitro. Mutations in the BH3 domain that eliminated the
9-2-Bcl-2 amd 9-2-BclxL interactions also eliminated the apoptotic activity of 9-2. Thus, we have identified an
interferon-induced dual function protein of the Bcl-2 family that can
synthesize 2-5(A) and promote cellular apoptosis independently.
Moreover, the cellular abundance of this protein is regulated by
alternative splicing; the other isozymes encoded by the same gene are
not proapoptotic.
Interferons are potent cytokines with a variety of effects on cell
physiology. Although their antiviral effects are the most well known,
IFNs affect cell growth and cell survival as well (1, 2). The cellular
effects of IFNs are mediated by the IFN-induced proteins, which number
in the hundreds (3). Among them are the family of enzymes called 2-5(A)
synthetases (4, 5). Three sets of IFN-induced genes encode three size
classes of these proteins. Within each size class, multiple members
arise as a result of alternative splicing of the primary transcript, giving rise to proteins with unique sequences at their carboxyl termini
(6, 7). Thus, the cellular abundance of individual isozymes is
regulated not only by IFN-induced transcription of the genes, but also
by alternative splicing. The enzymatic activity of 2-5(A) synthetases
requires a co-factor, double-stranded
(ds)1 RNA. In the presence of
dsRNA, these enzymes polymerize ATP into 2'-5'-linked oligoadenylates,
which in turn activate a latent ribonuclease, RNase L. Activated RNase
L can degrade cellular and viral RNAs and inhibit protein synthesis
(8).
We have been studying the structure-function relationships of 2-5(A)
synthetases and have identified their conserved catalytic domain, in
which three Asp residues form the catalytic triad (9). We have also
demonstrated that dimerization of the medium isozyme, P69, and
tetramerization of the small isozyme, 9-2, is necessary for their
enzyme activity (9, 10). It remains an enigma, however, why so many
isozymes exist. To address this issue, we have begun to examine the
cell growth regulatory and antiviral properties of individual isozymes.
The E16/3-9 small isozyme, when expressed by transfection, causes an
inhibition of replication of specific viruses, such as
encephalomyocarditis virus (11, 12). Similarly, ectopic expression of
the medium isozyme in permanently transfected P69 cells causes
inhibition of encephalomyocarditis virus replication and cell growth
(13). Similar attempts to establish cell lines that permanently express
the small isozyme 9-2 were unsuccessful and led to the unexpected
findings reported in this paper. The 9-2 protein caused cellular
apoptosis by specifically binding to the anti-apoptotic proteins of the
Bcl-2 family.
A large number of pro- and anti-apoptotic cellular proteins of the
Bcl-2 family regulate cellular apoptosis (14, 15). These proteins
contain one or more Bcl-2 homology (BH) domains, which are grouped into
four classes: BH1, BH2, BH3, and BH4. The BH3 domain interacts with a
pocket formed by the BH1 and BH2 domains, thus enabling many of these
proteins to homo-and heterodimerize and function as regulators of
apoptosis. Anti-apoptotic proteins, such as Bcl-2 and
BclxL, and proapoptotic proteins, such as Bax and Bak,
contain BH1, BH2, and BH3 domains, whereas other proapoptotic proteins,
such as Bad, Bid, and Bik, contain the BH3 domain only. The latter
proteins are thought to neutralize the anti-apoptotic proteins by
forming inactive heterodimers. Here, we report the identification of a
new member of the proapoptotic Bcl-2 family that contains only the BH3 domain.
Cell Culture and Transfection--
Human HT1080 cells were
cultured as described before (13). Where indicated, human IFN- Antibodies--
A monoclonal antibody recognizing all small
isozymes of synthetases has been described before (16). The
9-2-specific antipeptide antibody was raised in rabbit by Biosynthesis
Inc. The 17-mer peptide used as the antigen contained residues 355-371
of the protein (7). Rabbit anti-human Bcl-2 antibody was from
Pharmingen (San Diego, CA). Mouse monoclonal anti-human Bcl-2 antibody,
mouse monoclonal anti-human BclxL antibody, and rabbit
anti-Flag antibody were from Santa Cruz Biotechnology. Rabbit
anti-human Bclx antibody was from Transduction Laboratory and
mouse monoclonal anti-Flag antibody was from Sigma. Mouse monoclonal
anti-CD20 antibody was from Becton Dickinson. The horseradish
peroxidase-conjugated goat anti-rabbit, goat anti-mouse, donkey
anti-goat, and fluorescein isothiocyanate-conjugated goat anti-rabbit
antibody were from Life Technologies, whereas Texas Red-conjugated goat
anti-mouse antibody was from Molecular Probes.
Synthetase Isozymes and Their Mutants--
The isozymes 9-2, 3-9, L3, and P69 have been described before (7, 17, 18). They were all
expressed from pCDNA3 vector driven by cytomegalovirus early
promoters as Flag-tagged proteins (10). The enzymatically inactive 9-2 mutant, 9-2M, has two Asp residues in the catalytic domain substituted
by Ala (9). The 9-2 TM mutant does not form tetramers and is
enzymatically inactive (10). The 9-2 DM mutant had residues 345-414
deleted (7). 9-2 BH3M had the following mutations in the BH3 domain:
K376A, L378A, D383A, and F385A. The mutations were built into primers used for polymerase chain reaction cloning by the megaprimer method (10)
Fluorescence-activated Cell Sorter Selection of
CD20-expressing Cells--
Cells were co-transfected with a
cytomegalovirus early promoter-driven CD20 expression vector and
expression vectors for different synthetase isozymes at a ratio of
1:15. After 24 h, cells were trypsinized, washed with PBS twice,
incubated with fluorescein isothiocyanate-conjugated anti-CD20 antibody
for 30 min at 25 °C, and washed with PBS twice before separation of
the stained cells by fluorescent activated cell sorter (Becton Dickinson).
TUNEL Assay--
TUNEL assays were performed as described (19).
This assay detected apoptotic cells by DNA labeling, the transfected
protein by antibody staining, and cellular nuclei by DAPI staining of the nuclei. Apoptosis was quantitated by counting the number of protein-expressing cells that were TUNEL-positive. For this purpose, at
least 300 protein-expressing cells were scored by examining several
fields in a fluorescence microscope.
Caspase Activity Assays--
At a concentration of
108 cells/ml, cells were suspended in the lysis buffer
containing 25 mM HEPES, pH 7.5, 5 mM
MgCl2, 5 mM EDTA, 5 mM
dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, and 10 µg/ml leupeptin and disrupted by three
cycles of freeze-thawing. Supernatants were collected after a low speed
centrifugation. Caspase 3 and caspase 1 activities in the extracts were
measured by using the CaspACE assay system (Fluoremetric, Promega).
PARP Cleavage Assay--
Extracts of transiently transfected
cells were made in a buffer containing 62.5 mM Tris-Cl, pH
6.0, 6 M urea, 10% glycerol, 2% SDS, 5%
Lamin B Cleavage Assay--
Cells were extracted as for PARP
cleavage assay except that the buffer contained 10 mM
Tris-Cl, pH 8.0, 150 mM NaCl, 0.2% Nonidet P-40, 1% SDS,
10% glycerol, and 2 mM phenylmethylsulfonyl fluoride.
Proteins were analyzed by 10% polyacrylamide gel electrophoresis and a
polyclonal Lamin B antibody.
DNA Ladder Assay--
Cells at 72 h posttransfection were
scraped out and collected by centrifugation. The cell pellet was
incubated with 0.2 mg/ml proteinase K in 500 µl of buffer (100 mM Tris-Cl, pH 8.5, 5 mM EDTA, 200 mM NaCl, 0.2% SDS) at 37 °C. The DNA was precipitated with an equal volume of isopropanol. The precipitated DNA was treated
with 0.1 mg/ml RNase A at 37 °C, analyzed on a 2% agarose gel, and
detected by ethidium bromide staining.
Cytochrome C Release Assay--
For assaying release of
cytochrome C from mitochondria to cytosol, cells were washed with
chilled PBS, scraped off the plate in PBS, and harvested by
centrifugation. The cell pellet was washed once with PBS and then
suspended in 3 volumes of a buffer containing 20 mM HEPES,
pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, 10 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µM aprotinin, and 250 mM sucrose (29). After chilling on ice for 3 min, the cells
were disrupted by 25 strokes in a glass homogenizer. The extract was
centrifuged twice at 2500 × g to remove unbroken cells
and nuclei. Mitochondria were then pelleted from the supernatant by
centrifugation at 12,000 × g for 30 min. The
supernatant was removed and filtered successively through 0.2- and
0.1-µm Ultrafree MC filters (Millipore) to produce the cytosolic
fraction. Equal amounts of cytosolic protein were electrophoresed by
12% SDS-polyacrylamide gel electrophoresis and Western blotted with a
monoclonal antibody to cytochrome C (Pharmingen) at a 1:5000
dilution followed by a secondary goat anti-mouse horseradish
peroxidase-conjugated antibody at a 1:2000 dilution. The secondary
antibody was detected by enhanced chemiluminescence.
Molecular Modeling of the BH3 Domains--
Amino acid sequences
of the BH3 domains of 9-2, BclxL, Bad, and other
proapoptotic family members were aligned using the homology module of
Insight II (Molecular Simulations, Inc.). The secondary structures were
determined using the PHD sec program at predict protein
server. Molecular modeling of the 9-2 and the Bad BH3 domains was done
using Insight II. For this modeling, the BH3 domain of
BclxL was as taken on the template to construct the backbone conformations of the two test BH3 domains.
In Vitro Protein-Protein Interaction Assays--
In
vitro coupled transcription-translation systems were used for
synthesizing radiolabeled Flag-tagged proteins 9-2, its mutants, Bcl-2,
and BclxL individually. Equal amounts of the interacting proteins were mixed in the binding buffer containing 10 mM
Tris-Cl, pH 8.0, 137 mM NaCl, 0.1% Nonidet P-40, 10%
glycerol, and 2 mM phenylmethylsulfonyl fluoride and
incubated at 4 °C for 1 h. 9-2 proteins were immunoprecipitated
by anti-Flag antibody bound to agarose (9). After washing twice with
the binding buffer containing 250 mM NaCl, the bound
proteins were analyzed by polyacrylamide gel electrophoresis and autoradiography.
Co-immunoprecipitation of Proteins Expressed in
Vivo--
Extracts of transfected or untransfected cells were made by
sonication of cells suspended in the binding buffer described above.
100 µg of protein was immunoprecipitated with anti-Flag, polyclonal
BclxL, or monoclonal Bcl2 antisera and protein A-Sepharose. Immunoprecipitated proteins or proteins in the cell extracts were analyzed by Western blotting with different antisera.
Confocal Microscopy for Protein Co-localization--
Flag-tagged
9-2 was transfected, and cells were fixed with 4% paraformaldehyde,
permeabilized in 0.1% Triton X-100, washed, and incubated in blocking
buffer and then with the primary antibodies. As primary antibodies,
rabbit anti-Flag antiserum at a 1:250 dilution and mouse monoclonal
anti-Bcl-2 antiserum at a 1:500 dilution were used. As secondary
antibodies, fluorescein isothiocyanate-conjugated anti-rabbit goat
antiserum and Texas Red-conjugated anti-mouse goat antiserum were used
at a 1:2000 dilution. The green and red colors were detected by
confocal fluorescent microscopy (13).
Expression of the 9-2 Isozyme in Human Cells--
We originally
cloned the 9-2 isozyme of 2-5(A) synthetase from a mouse cDNA
library along with another isozyme, 3-9 (7). The sequence of 3-9 was
almost identical to that of E16, a human isozyme, whereas all of 9-2 sequence was contained within that of E18, an alternatively spliced
sister of E16. Thus, we proposed that the mRNAs for E16/3-9, E18,
and 9-2 arise from the same primary transcript by alternative splicing
(Fig. 1A). As a result, all three proteins contain an identical 345 residues at their amino termini
and various numbers of unique residues at their carboxyl termini after
the alternative splice junction. Because we are interested in
determining specific cellular functions of these and other isozymes of
2-5(A) synthetases, we have been expressing them individually in human
cell lines. Because we have been using human cells for expression
analysis and the E16 and E18 isozymes were originally cloned from human
cells, it was important to establish that the 9-2 isozyme is also
expressed in human cells. Results presented in Fig. 1 demonstrate the
existence of the 9-2 isozyme in IFN-treated human HT1080 cells. To test
the existence of the 9-2 mRNA, RNA from IFN-treated cells was used
for reverse transcription-polymerase chain reaction using appropriate
primers. The sense primer was from the common region just upstream of
the splice junction, and the antisense primer was from the region
common to the E18 and 9-2 mRNAs placed right at the translation
termination point of the putative 9-2 protein (Fig. 1A). As
expected, two pieces of DNA were amplified from RNA of IFN-treated
cells (data not shown). Their sizes of 405 and 307 base pairs matched
exactly the sizes expected of E18 mRNA and 9-2 mRNA,
respectively. None of these DNAs were obtained without reverse
transcription. The two cDNAs were completely sequenced, confirming
the identities of the E18 and 9-2 mRNAs and the encoded proteins
(Fig. 1, B and C). This portion of the human 9-2 sequence was identical to that of the mouse 9-2. The E18 sequence had
two amino acid differences: a Thr to Ala and a Thr to Arg substitution
as compared with the published E18 sequence (6). Induction of 9-2 mRNA could be detected as early as 2 h after IFN treatment of
cells (data not shown). These data demonstrated that the alternatively
spliced 9-2 mRNA exists, albeit at a low level, in IFN-treated
human HT1080 cells.
In the next set of experiments, the presence of the 9-2 protein in
IFN-treated HT1080 cells was demonstrated. For this purpose, a
9-2-specific antibody was raised in rabbits using a peptide from its
specific carboxyl-terminal domain as an antigen. This peptide sequence
is underlined in Fig. 1C. For characterizing this
antibody, extracts of cells expressing high quantities of the 3-9/E16,
E18, or 9-2 isozyme were used. The 3-9 and E18 proteins were expressed
in transfected HT1080 cells, but because the 9-2 isozyme killed these
cells, we had to use a different source: insect cells infected with the
appropriate recombinant baculovirus (10). A high resolution Western
blot using a common antibody shows the presence of each isozyme in the
respective cell extracts (Fig. 1D, lanes 1-3). In contrast,
the newly raised 9-2-specific antibody recognized the 9-2 protein only
(Fig. 1D, lanes 4-6), thus establishing its specificity.
The 9-2-specific antibody was then used to analyze extracts of
IFN-treated and untreated cells. Because we anticipated that the level
of the 9-2 protein would be low, the 2-5(A) synthetases were enriched
from the cell extracts by binding to poly(I)·poly(C) agarose
before Western blot analysis using the 9-2-specific antibody. As shown
in Fig. 1D, the 9-2 protein was clearly present in the
extract of IFN-treated HT1080 cells (lane 7) but not in that
of untreated cells (lane 8). Immunofluorescence assays
showed that the 9-2 protein is primarily cytoplasmic (data not shown).
Proapoptotic Activity of 9-2--
Because ectopic expression of
9-2 by transfection of HT1080 cells caused cell death, we wanted to
investigate the underlying mechanism. Results shown in Fig.
2 demonstrated that it was due to
cellular apoptosis. 9-2-expressing cells were clearly
TUNEL-positive, an indication of the presence of multiple ends of DNA
produced by apoptosis-associated DNA fragmentation (Fig.
2A). In contrast, cells expressing the sister isozyme 3-9 were not TUNEL-positive. This was not due to a lower level of 3-9 expression; Western blotting of the cell extracts showed that 3-9 was
expressed better than 9-2 (Fig. 2B).
In the next series of experiments, the 9-2-mediated apoptotic process
was characterized further. For this purpose, 9-2 or 3-9 expression
vectors were co-transfected with an expression vector of the
cell-surface marker, CD20. CD20-expressing cells were sorted by
fluorescence-activated cell sorter, cultured for a day, and used for
biochemical analyses. As expected, DNA laddering assay showed DNA
fragmentation in cells expressing 9-2 but not in cells transfected with
3-9 (Fig. 2F). Cellular apoptosis is often associated with
the cleavage of the nuclear proteins PARP and Lamin B. Such cleavages
were detected by Western blotting appropriate cell extracts with
antibodies to the two proteins (Fig. 2, C and D).
As expected, the 115-kDa PARP was cleaved to an 85-kDa derivative in
cells expressing 9-2 but not in those expressing 3-9 (Fig.
2C). Lamin B (70 kDa) cleavage gives rise to a
characteristic 35-kDa fragment (Fig. 2D). Similar to PARP, Lamin B was also cleaved specifically in 9-2-expressing cells. DNA
fragmentation and protein cleavages in apoptotic cells are preceded by
the activation of specific caspases. Such caspase activation was
measured in the extracts of 9-2-expressing cells using caspase-specific
substrates and inhibitors. Caspase 3 was highly activated in
9-2-expressing cells (Fig. 2E) but not in 3-9-expressing
cells. In contrast, caspase 1 was not activated in these extracts,
demonstrating the specificity of caspase activation in 9-2-expressing
cells. Another hallmark of cells undergoing apoptosis is the release of
cytochrome C from mitochondria to cytoplasm. In 9-2-expressing cells,
cytosolic cytochrome C was readily detected (Fig. 2G). As
expected, no cytochrome C was present in the cytoplasm of
vector-transfected cells, but a large amount of it was present in the
cytoplasm of cells transfected with Bax, a known proapoptotic
protein (Fig. 2G). This series of experiments demonstrated
that 9-2 expression in cells causes cytosolic cytochrome C release and
caspase activation followed by PARP and Lamin B cleavage and DNA
fragmentation, a series of events known to occur in most cells
undergoing apoptosis.
Isozyme-specific and Enzyme Activity-independent Apoptosis Caused
by 9-2--
Once it was firmly established that 9-2 could cause
apoptosis, other 2-5(A) synthetase isozymes were tested for this
effect. These proteins were expressed by transfection, and individual cells were stained for protein expression and TUNEL positivity. Of 300 9-2-expressing cells, 225 (75%) were TUNEL-positive (Fig. 3A, bar 1). The corresponding
numbers for the two sister isozymes, E18 and 3-9, were 4 and 3, respectively (Fig. 3A, bars 2 and 3). No cells
expressing another small isozyme, L3, or the medium isozyme P69 were
TUNEL-positive (Fig. 3A, bars 4 and 5). Western
blotting showed that all isozymes were expressed at similar levels
(Fig. 3A, inset). Thus, the proapoptotic activity was an
exclusive property of the 9-2 isozyme.
Because all isozymes could synthesize 2-5(A) and this was the only
known activity of this class of proteins, there was no obvious
explanation of the proapoptotic activity of the 9-2 protein. The first
indication that we may have uncovered an independent activity came from
testing the effect of 9-2 expression in RNase L A Putative BH3 Domain in 9-2--
Because the enzymatic activity
of 9-2 was not required for its apoptotic activity, we searched for an
alternative biochemical explanation for its apoptotic action. A
possible lead came from the observation that the 9-2 protein may
contain a putative Bcl-2 homology domain 3. This domain was present in
the isozyme-specific carboxyl-terminal tail of the protein between
residues 372 and 393 (Fig.
4A). When compared with the
sequences of the BH3 domains of several members of the Bcl-2 family,
all of them, including 9-2, contained predicted Interaction of 9-2 with Bcl-2 and BclxL--
Because
the 9-2 protein appears to have a BH3 domain, it may exert its
proapoptotic activity by binding to the anti-apoptotic proteins Bcl-2
and BclxL. These proteins contain the BH1 and BH2 domains
that form a pocket to which certain BH3 domains can bind. This
possibility was tested in the next series of experiments. In the first
experiment confocal microscopy was used to examine whether the
subcellular location of 9-2 partially overlaps with those of Bcl-2
(Fig. 5A). The distribution of
9-2 is shown in green and that of Bcl-2 in red. When the green image
was overlain with the red image, many yellow areas were observed,
indicating that subpopulations of 9-2 are located in the same
compartments where Bcl-2 resides in cells.
Physical interaction of 9-2 with Bcl-2 and BclxL was tested
by co-immunoprecipitation experiments. Endogenous 9-2 was induced in
cells by interferon treatment, and Bcl-2 or BclxL was
immunoprecipitated from cell extracts. 9-2 was co-precipitated with
both proteins but not with preimmune serum (Fig. 5B).
Importance of the BH3 Domain--
To further test our hypothesis
that the putative BH3 domain of 9-2 mediates its interaction with Bcl-2
and BclxL, specific mutations were introduced in this
region. In the mutant 9-2 BH3M, the two hydrophobic conserved residues,
Leu and Phe, and the two hydrophilic conserved residues, Lys and Asp,
in 9-2 (Fig. 4B) were replaced by Ala because similar
mutations are known to perturb the functions of the BH3 domain of
BclxL. A deletion mutant, 9-2 DM, missing all of the
9-2-specific carboxyl-terminal tail and the 9-2 BH3M mutant were tested
for their ability to bind to Bcl-2 and BclxL. When in
vitro translated proteins were mixed, both Bcl-2 (Fig.
6A, lanes 1-3) and
BclxL (Fig. 6A, lanes 4-6)
co-immunoprecipitated with WT 9-2 but not with either mutant.
Similarly, when the 9-2 proteins were expressed in cells by
transfection, only the WT 9-2 protein co-immunoprecipitated with Bcl-2
or BclxL (Fig. 6B, lanes 4-6), although similar
amounts of the mutant proteins were present in the cell extracts (Fig.
6B, lanes 1-3). The above experiments established that the
interaction of 9-2 with Bcl-2 and BclxL is mediated by its
BH3 domain. The contribution of that interaction to the apoptotic
activity of 9-2 is shown in Fig. 6C. Neither 9-2 DM nor 9-2 BH3M induced apoptosis. Quantitation of these results confirmed the
loss of ability of the 9-2 protein to cause apoptosis upon deletion or
point mutation of its BH3 domain (Fig. 3C).
We have reported here the identification of a new apoptotic
regulatory protein. 9-2 now joins the list of BH3-domain-only proapoptotic mammalian proteins of the Bcl-2 family, which includes Bad, Bid, Bim, Bik, Blk, and Hrk (20, 21). The authenticity of the 9-2 BH3 domain was first verified by molecular modeling exercises using the
known structure of the BclxL BH3 domain as the template
(22). In this experiment, we also modeled the BH3 domain of Bad, and
all three BH3 domains assumed an amphipathic Three features of the mode of synthesis of 9-2 and its function
are unique (Fig. 7). First, it is the
product of an IFN-inducible gene and the first IFN-induced protein of
the Bcl-2 family. It is worth pointing out that other 2-5(A) synthetase
isozymes have been shown to be induced by a variety of agents, such as
dsRNA, tumor necrosis factor, IFN-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was
used for treating these cells. All transfections were done using Fugene
6 reagent from Roche Molecular Biochemicals.
-mercaptoethanol, and 0.0125% bromphenol blue. 4 × 106 cells were suspended in 1 ml of buffer, sonicated at a
pulse of 15 s twice, and incubated at 65 °C for 15 min before
analyzing by 6% polyacrylamide gel electrophoresis. Western blotting
was done with a monoclonal anti-PARP antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of the 9-2 mRNA and protein in
human HT1080 cells. A, the alternative splicing pattern
that gives rise to the three mRNAs is schematically shown. The
thin lines denote the mRNAs, and the
rectangles show the proteins. The first 345 residues are
identical, and the rest are unique for each isozyme. The
arrows show the positions of the primers chosen to amplify
portions of the E18 9-2 and 9-2 mRNAs by reverse transcription
followed by polymerase chain reaction. B, partial sequences
of the two DNA fragments generated by polymerase chain reaction are
shown. Only the sequences at the ends and the splice junctions are
given. C, the amino acid sequences of the carboxyl-terminal
regions of the three isozymes after the alternative splice point are
shown. In the 9-2 sequence, the underlined peptide was used
to raise an isozyme-specific antibody. D, expression of the
9-2 protein in IFN-treated human HT1080 cells. Extracts of insect cells
expressing the 9-2 protein (lanes 1 and 4) and
HT1080 cells transfected with E18 (lanes 2 and 5)
and 3-9 (lanes 3 and 6) were analyzed by Western
blotting with a common antibody (lanes 1-3) or the new
9-2-specific antibody (lanes 4-6). The 9-2-specific
antibody was used to analyze extracts of 1000 units/ml IFN- -treated
(lane 7) and untreated (lane 8) HT1080 cells,
after enriching them by poly(I)·poly(C) agarose chromatography.
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Fig. 2.
Apoptosis by 9-2 expression.
A, TUNEL assay for measuring apoptosis. HT1080 cells
were transfected with expression vectors for Flag-tagged 9-2 or 3-9 proteins, and after 48 h, cells were assayed for DNA fragmentation
by TUNEL staining, for transfected protein expression by anti-Flag
antibody staining, and for nuclear localization by DAPI staining.
Several cells in each field are shown, some expressing the transfected
protein and the others not. B, levels of expression of 9-2 and 3-9 proteins in the extracts of cells shown in A as
measured by Western blotting with the common antibody. C-F,
cells transfected with 9-2 or 3-9 and CD20 were sorted for CD20
expression and extracts were prepared after 72 h. PARP cleavage
(C), Lamin B cleavage (D), caspase activation
(E), and DNA fragmentation (F) were assayed.
G, cells were transfected with expression vectors of Bax or
9-2 or nothing (Vec). Cell extracts were made after 72 h, and the cytosols were assayed for cytochrome C release by Western
blotting.
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Fig. 3.
Apoptotic properties of different synthetase
isozyme and 9-2 mutants. A, different isozymes. 300 protein-expressing cells were scored for TUNEL positivity. Bar
1, 9-2; bar 2, E-18; bar 3, 3-9; bar
4, L-3; bar 5, P69. Results are presented as percentage
of cells undergoing apoptosis. The inset shows the levels of
expression of the corresponding proteins in the cell extracts.
B, apoptosis of cells expressing two enzymatically inactive
mutants of 9-2: 9-2M, a mutant in the catalytic center (9) and 9-2 TM,
an oligomerization-defective mutant (10). Assay was done as in Fig.
2A. C, quantitation of apoptosis by different
mutants of 9-2: bar 1, WT 9-2; bar 2, 9-2M;
bar 3, 9-2 TM; bar 4, 9-2 DM, missing the
carboxyl-terminal tail; bar 5, 9-2 BH3M, a mutant in the BH3
domain. The inset shows the levels of protein
expression.
/
fibroblasts (a
gift of Robert Silverman). These cells were killed as efficiently as
the WT cells by 9-2, indicating that 2-5(A) activation of RNase L was
not required for the apoptotic effect (data not shown). This prompted
us to test whether the enzymatic activity of the 9-2 protein was
dispensable as well. We had previously generated an enzymatically
inactive mutant of 9-2, 9-2M, in which two Asp residues at its active
center were replaced by Ala residues (9). This mutant of 9-2 was active in causing apoptosis in the HT1080 cells as measured by TUNEL assay
(Fig. 3B). Another mutant of 9-2, 9-2 TM, is also
enzymatically inactive because it is a monomer (10). This mutant
retained the proapoptotic activity as well (Fig. 3B). The
two enzymatically inactive mutant proteins were expressed to the same
levels as the WT protein (Fig. 3C). When their apoptotic
activities were quantitated, 9-2M killed 67% of the cells and 9-2 TM
killed 61% of the cells as compared with 75% killing by the WT
protein (Fig. 3C, bars 1-3). These results clearly
established that the proapoptotic activity of 9-2 was independent of
its ability to synthesize 2-5(A) and must be mediated by a different pathway.
-helical structures
in this region. To probe their structures further, molecular modeling
studies were done using the known structure of the BH3 domain of
BclxL as the template (Fig. 4B). The BH3 domain
of BclxL is known to be an amphipathic
-helix.
The BH3 domain of Bad and the putative BH3 domain of 9-2 assumed very
similar structures when the BclxL residues were replaced by
the corresponding residues of the two other proteins. The hydrophilic
surface of the BclxL BH3 domain contains two charged
residues, Gln and Asp, and the corresponding hydrophobic surface
contains two residues, Leu and Phe, that are known to be required to
maintain the function of this domain. The Leu, Phe, and Asp residues
were conserved in similar locations in the BH3 domains of 9-2 and Bad
as well, whereas the Gln residue was replaced by Lys in 9-2 and by Arg
in Bad (Fig. 4B). These analyses strongly suggested that the
9-2 protein contains an authentic BH3 domain.
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Fig. 4.
Molecular modeling of the BH3 domains.
A, on the top, the location of the putative BH3
domain in the 9-2 protein is shown by the black rectangle.
Below, the sequence of this domain is compared with the BH3
sequences of several proteins of the Bcl-2 family. The thick
arrows at the top of each sequence show predicted
-helix. The residues replaced by Ala in 9-2 BH3M are shown by
asterisks. B, the modeled structures of the BH3
domains of 9-2 and Bad using the known structure of BclxL
BH3 domain as the template.
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Fig. 5.
Interaction between 9-2 and Bcl-2.
A, immunofluorescence microscopy. Transfected Flag-tagged
9-2 distribution is shown in green, and the endogenous
(Bcl-2) is shown in red. When the two images were overlaid,
co-localization of the two proteins was indicated by the appearance of
yellow spots. B, co-immunoprecipitation of the
endogenous 9-2 protein with Bcl-2 and BclxL: cells were
treated with 1000 units/ml IFN- for 18 h (I) or left
untreated (C). Cell extracts were analyzed directly for 9-2 expression using 9-2-specific antibody for Western blotting, or they
were immunoprecipitated with preimmune (PI), anti-Bcl-2, or
anti-BclxL sera. The immunoprecipitates were analyzed as
indicated by Western blotting using 9-2, Bcl-2, or BclxL
antisera. Ten times more extracts were used for immunoprecipitation
than for straight Western blotting.
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Fig. 6.
Requirement of BH3 domain-mediated
interaction of 9-2 with Bcl-2 and BclxL for causing
apoptosis. A, in vitro interactions
between 9-2-Flag and Bcl-2 (lanes 1-3) or
BclxL (lanes 4-6) are shown. The proteins were
translated in vitro, mixed, immunoprecipitated with
anti-Flag antibody, and analyzed by gel electrophoresis. Lanes
1 and 4, WT 9-2; lanes 2 and 5, 9-2 BH3M; lanes 3 and 6, 9-2 DM. Lanes
1-3, Bcl-2; lanes 4-6, BclxL.
B, co-immunoprecipitations of 9-2 and its mutants with Bcl-2
and BclxL. Cells were transfected with WT 9-2-Flag
(lanes 1 and 4), 9-2 BH3M (lanes 2 and
5), or 9-2 DM (lanes 3 and 6). In the
left panel, cell extracts were Western blotted directly with
Flag, Bcl-2, or BclxL antisera. In the right
panel, extracts were first immunoprecipitated with anti-Flag
antisera and then Western blotted with preimmune (PI),
Bcl-2, and BclxL antisera as indicated. Three times more
extracts were used for immunoprecipitation than for straight Western
blotting. C, loss of the apoptotic activity of 9-2 BH3
mutants. Cells expressing 9-2 DM or 9-2 BH3M were analyzed for TUNEL
positivity as described in Fig. 2A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical
structure (Fig. 4). Although there was little sequence conservation
among the three BH3 domains, the putative structures were remarkably
similar. On the hydrophobic side of the helix, a Leu and a Phe residue
are conserved two helical turns apart in all of them. Similarly, on the
hydrophilic side, an acidic residue, Asp, in all three proteins is two
turns away from a basic residue that is different in the three
proteins: Lys in 9-2, Arg in Bad, and Gln in BclxL. The
functional importance of these residues of 9-2 was demonstrated by
their replacement with Ala, which caused a loss of its ability to
interact with Bcl-2 and BclxL and to promote apoptosis. The
contributions of other residues of the 9-2 BH3 domain in maintaining
its functional integrity remain to be evaluated. A detailed mutational
analysis of the BH3 domain of Bak has shown that many residues
influence its binding affinity for BclxL (22). Such
demanding requirements of specific residues are reflected in the fact
that all BH3 domains do not interact with all BH1-BH2 pockets. For
example, BclxL itself does not form an intermolecular
homodimer. It will therefore be necessary, in the future, to examine
the full repertoire of Bcl-2-family proteins with which 9-2 interacts.
, epithelial growth factor, nerve growth factor, and platelet-derived growth factor as well (see references in Ref. 7). If the same is true for 9-2, its cellular level
can be regulated by a variety of extracellular stimuli. This is in
sharp contrast with the other known BH3-only regulators of apoptosis;
they are all constitutively present in cells and their proapoptotic
functions are usually triggered by other events, not their synthesis.
Thus, 9-2 is the first example of a bona fide cytokine-induced
proapoptotic protein of the Bcl-2 family. The second important feature
of 9-2 is that its synthesis is regulated by alternative splicing. It
is only one of the possible three products of the gene. This feature
adds a second layer of regulation of 9-2 synthesis, although little is
known about what regulates alternative splicing and how the relative
proportions of the products can be altered in a cell. Alternative
splicing affecting the properties of apoptotic proteins is well
documented in the case of Bclx protein. BclxL and
BclxS are encoded by two alternatively spliced mRNAs; BclxL is anti-apoptotic, and BclxS is
proapoptotic (21). Similarly, alternative splicing is also involved in
the synthesis of Bcl-w (23). The third unique feature of the 9-2 protein is its dual function: it has an enzymatic activity and a
proapoptotic activity. Mutational studies firmly established that the
two activities are independent of one another. 9-2 DM and 9-2 BH3M were
as active in synthesizing 2-5(A) as the WT protein, although they did
not cause apoptosis; conversely, 9-2M and 9-2 TM were enzymatically inert but proapoptotic. The enzymatic activity of 2-5(A) synthetases requires dsRNA as a cofactor. Although potential up- or down-regulation of the apoptotic activity of 9-2 by dsRNA cannot be ruled out, it
definitely does not require dsRNA. Again, this dual functional nature
of the 9-2 protein is unique among the known members of the Bcl-2
family.
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Fig. 7.
A model for 9-2 synthesis and its dual
functions. IFN induces enhanced transcription of the gene, and one
of three alternatively spliced mRNA encodes the 9-2 protein. The
isozyme-specific carboxyl-terminal sequence of E18 is different from
that of 9-2. The active center and the oligomerization domains of the
protein required for its ability to synthesize 2-5(A) in the presence
of dsRNA have been identified. A distinct region of the protein
containing the BH3 domain mediates its interaction with the Bcl-2 and
BclxL proteins, causing apoptosis.
To demonstrate the apoptotic effects of 9-2, we had to express it
by transfection of the cDNA because the only other known way to
induce it is by IFN treatment of cells, which induces hundreds of other
proteins as well. Because transfection may cause overexpression of a
protein, one has to be cautious about interpreting the results. For
this reason, we took the trouble of expressing other isozymes of 2-5(A)
synthetase and various mutants of the 9-2 at the same level as the wild
type 9-2 protein (see Fig. 3). Although expressed equally well, other
isozymes and some mutants of 9-2 did not cause apoptosis, indicating
that the observed apoptotic effect of the 9-2 protein is
physiologically relevant. Clearly, in the cell lines tested, 9-2 is
only one product of alternative splicing. It appears that both in mouse
Ehrlich ascites tumor cells, from which the murine 9-2 was first
cloned, and in human HT1080 cells, used in the current study, only a
low quantity of the 9-2 mRNA and the protein are synthesized. This
quantity of 9-2 is presumably not enough to cause apoptosis because IFN
treatment of HT1080 cells does not kill these cells. Alternatively,
other IFN-induced proteins may protect cells from the apoptotic effects
of 9-2. It is quite possible, however, that its expression primes the cells, by partially titering out the anti-apoptotic proteins, to be
prone to apoptosis in response to other signals. One can imagine that
such signals are generated by virus infection, so that 9-2-expressing
cells are killed faster upon virus infection, thus aborting the virus
replication cycle and promoting the antiviral activity of IFN.
Alternatively, 9-2 may interact with viral anti-apoptotic proteins,
such as the adenoviral E1B 19-kDa protein (24), and block their action.
Again, the consequence will be a promotion of apoptosis of IFN-treated
virus-infected cells and an inhibition of spreading of the virus
infection, a major physiological function of the IFN system. Another
possible function of 9-2 could be manifested in selected cell types.
IFNs are known to have differential anti-growth activities in different
tumor cell lines. Daudi cells, for example, are exquisitely sensitive
to IFNs. In some cases, such effects have been connected to cellular
apoptosis (25, 26). It is possible that such apoptosis is mediated by
the induction of a high level of the 9-2 protein, a scenario that can
be experimentally tested. What is unique about the 9-2-mediated
apoptosis as compared with the two other proapoptotic IFN-induced
systems, protein kinase, RNA-dependent and RNase L (2), is that
no activator, such as dsRNA, is required for triggering it. Thus, it is
conceivable that the 9-2 protein is used for tissue remodeling in
interferon-unrelated physiological regulations. It is pertinent in this
context to note that very high levels of 2-5(A) synthetase are
expressed in resorbing chick oviducts (27) and partially hepatectomized livers (28).
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ACKNOWLEDGEMENTS |
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We thank Alex Almasan and Robert Silverman for valuable reagents and helpful discussion. Thanks are also due to Guan Chen, Yoshihiro Sokawa, Amy Raber, Judy Drazba, Jim Lang, and Shen-Shu Sung.
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FOOTNOTES |
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* This research was supported by National Institutes of Health Grants CA68782 and CA62220.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.
To whom correspondence should be addressed: Dept. of Molecular
Biology/NC20, The Lerner Research Institute, The Cleveland Clinic
Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-0636;
Fax: 216-444-0513; E-mail: seng@ccf.org.
Published, JBC Papers in Press, April 25, 2001, DOI 10.1074/jbc.M100496200
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ABBREVIATIONS |
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The abbreviations used are: dsRNA, double-stranded RNA; BH, Bcl-2 homology; PARP, poly ADP-ribose polymerase; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; WT, wild type.
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