A Specific Isozyme of 2'-5' Oligoadenylate Synthetase Is a Dual Function Proapoptotic Protein of the Bcl-2 Family*

Arundhati Ghosh, Saumendra N. Sarkar, Theresa M. Rowe, and Ganes C. SenDagger

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Transfection-- Human HT1080 cells were cultured as described before (13). Where indicated, human IFN-beta was used for treating these cells. All transfections were done using Fugene 6 reagent from Roche Molecular Biochemicals.

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% beta -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.

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (30K):
[in this window]
[in a new window]
 
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-beta -treated (lane 7) and untreated (lane 8) HT1080 cells, after enriching them by poly(I)·poly(C) agarose chromatography.

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).


View larger version (22K):
[in this window]
[in a new window]
 
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.

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.


View larger version (24K):
[in this window]
[in a new window]
 
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.

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-/- 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.

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 alpha -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 alpha -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.


View larger version (25K):
[in this window]
[in a new window]
 
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 alpha -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.

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.


View larger version (37K):
[in this window]
[in a new window]
 
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-beta 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.

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).


View larger version (31K):
[in this window]
[in a new window]
 
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

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 alpha -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.

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-gamma , 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.


View larger version (16K):
[in this window]
[in a new window]
 
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).

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Vilcek, J., and Sen, G. C. (1996) in Fields Virology (Fields, B. N. , Knipe, D. M. , and Howley, P. M., eds) , pp. 375-399, Lippincott-Raven Publishers, Philadelphia
2. Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H., and Schreiber, R. D. (1998) Annu. Rev. Biochem. 67, 227-264[CrossRef][Medline] [Order article via Infotrieve]
3. Der, S. D., Zhou, A., Williams, B. R., and Silverman, R. H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15623-15628[Abstract/Free Full Text]
4. Rebouillat, D., and Hovanessian, A. G. (1999) J. Interferon Cytokine Res. 19, 1917-1924
5. Kerr, I. M. (1987) J. Interferon Res. 7, 505-510[Medline] [Order article via Infotrieve]
6. Benech, P., Mory, Y., Revel, M., and Chebath, J. (1985) EMBO J. 4, 2249-2256[Abstract]
7. Ghosh, S. K., Kusari, J., Bandyopadhyay, S. K., Samanta, H., Kumar, R., and Sen, G. C. (1991) J. Biol. Chem. 266, 15293-15299[Abstract/Free Full Text]
8. Silverman, R. H., and Cirino, N. M. (1997) in Gene Regulation (Morris, D. R. , and Hartford, J. B., eds) , pp. 295-309, John Wiley & Sons, New York
9. Sarkar, S. N., Ghosh, A., Wang, H. W., Sung, S. S., and Sen, G. C. (1999) J. Biol. Chem. 274, 25535-25542[Abstract/Free Full Text]
10. Ghosh, A., Sarkar, S. N., Guo, W., Bandyopadhyay, S., and Sen, G. C. (1997) J. Biol. Chem. 272, 33220-33226[Abstract/Free Full Text]
11. Chebath, J., Benech, P., Revel, M., and Vigneron, M. (1987) Nature 330, 587-588[CrossRef][Medline] [Order article via Infotrieve]
12. Rysiecki, G., Gewert, D. R., and Williams, B. R. (1989) J. Interferon Res. 9, 649-657[Medline] [Order article via Infotrieve]
13. Ghosh, A., Sarkar, S., and Sen, G. (2000) Virology 266, 319-328[CrossRef][Medline] [Order article via Infotrieve]
14. Chao, D. T., and Korsmeyer, S. J. (1998) Annu. Rev. Immunol. 16, 395-419[CrossRef][Medline] [Order article via Infotrieve]
15. Kelekar, A., and Thompson, C. B. (1998) Trends Cell Biol. 8, 324-330[CrossRef][Medline] [Order article via Infotrieve]
16. Ghosh, A., Desai, S. Y., Sarkar, S. N., Ramaraj, P., Ghosh, S. K., Bandyopadhyay, S., and Sen, G. C. (1997) J. Biol. Chem. 272, 15452-15458[Abstract/Free Full Text]
17. Ichii, Y., Fukunaga, R., Shiojiri, S., and Sokawa, Y. (1986) Nucleic Acids Res. 14, 10117[Medline] [Order article via Infotrieve]
18. Marie, I., and Hovanessian, A. G. (1992) J. Biol. Chem. 267, 9933-9939[Abstract/Free Full Text]
19. Ben Sasson, S. A., Sherman, Y., and Gavrieli, Y. (1995) Methods Cell Biol. 46, 29-39[Medline] [Order article via Infotrieve]
20. Adams, J. M., and Cory, S. (1998) Science 281, 1322-1326[Abstract/Free Full Text]
21. Boise, L. H., Gonzalez-Garcia, M., Postema, C. E., Ding, L., Lindsten, T., Turka, L. A., Mao, X., Nunez, G., and Thompson, C. B. (1993) Cell 74, 597-608[Medline] [Order article via Infotrieve]
22. Sattler, M., Liang, H., Nettesheim, D., Meadows, R. P., Harlan, J. E., Eberstadt, M., Yoon, H. S., Shuker, S. B., Chang, B. S., Minn, A. J., Thompson, C. B., and Fesik, S. W. (1997) Science 275, 983-986[Abstract/Free Full Text]
23. Gibson, L., Holmgreen, S. P., Huang, D. C., Bernard, O., Copeland, N. G., Jenkins, N. A., Sutherland, G. R., Baker, E., Adams, J. M., and Cory, S. (1996) Oncogene 13, 665-675[Medline] [Order article via Infotrieve]
24. Chiou, S. K., Tseng, C. C., Rao, L., and White, E. (1994) J. Virol. 68, 6553-6566[Abstract]
25. Otsuki, T., Yamada, O., Sakaguchi, H., Tomokuni, A., Wada, H., Yawata, Y., and Ueki, A. (1998) Br. J. Haematol. 103, 518-529[CrossRef][Medline] [Order article via Infotrieve]
26. Luchetti, F., Gregorini, A., Papa, S., Burattini, S., Canonico, B., Valentini, M., and Falcieri, E. (1998) Haematologica 83, 974-980[Medline] [Order article via Infotrieve]
27. Stark, G. R., Dower, W. J., Schimke, R. T., Brown, R. E., and Kerr, I. M. (1979) Nature 278, 471-473[Medline] [Order article via Infotrieve]
28. Etienne-Smekens, M., Vandenbussche, P., Content, J., and Dumont, J. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4609-4613[Abstract]
29. Pastorino, J. G., Chen, S. T., Tafani, M., Snyder, J. W., and Farber, J. L. (1998) J. Biol. Chem. 273, 7770-7775[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.