©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Bcl-x Antagonizes the Protective Effects of Bcl-x(*)

(Received for publication, July 12, 1995; and in revised form, December 15, 1995)

Andy J. Minn (1) (2)(§) Lawrence H. Boise (1) (3)(¶) Craig B. Thompson (1) (2) (3) (4)(**)

From the  (1)Gwen Knapp Center for Lupus and Immunology Research, the (2)Committee on Immunology, the (3)Department of Medicine, and the (4)Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Bcl-x, a member of the Bcl-2 family, has two alternatively spliced forms, Bcl-x(L) and Bcl-x(S). Bcl-x(L), like Bcl-2, is able to protect cells from a wide variety of apoptotic stimuli. Bcl-x(S), as a result of alternative splicing, lacks 63 amino acids that comprise the region of greatest amino acid identity between Bcl-x(L) and Bcl-2. These amino acids contain the highly conserved BH1 and BH2 regions, which have been used to define the Bcl-2 family. We show that both Bcl-x(L) and Bcl-x(S) are able to regulate cell survival in a dose-dependent fashion. Bcl-x(L) is able to increase the cellular apoptotic threshold and is able to form stable complexes with Bax both in vitro and in vivo. In contrast, Bcl-x(S) can effectively inhibit the protective effects of Bcl-x(L) following growth factor withdrawal and chemotherapeutic drug treatment. However, compared with Bax, Bcl-x(S) binds to Bcl-x(L) weakly when assessed by in vitro binding assays. Coimmunoprecipitation from mammalian cells demonstrates that Bcl-x(S) does not show an observable ability to form heterodimers with other Bcl-2 family members. In addition, overexpression of Bcl-x(S) does not alter the ability of Bax to heterodimerize with Bcl-x(L)in vivo. Thus, Bcl-x(S) does not appear to function by competitively disrupting the formation of dimers composed of other Bcl-2 family members. This suggests that Bcl-x(S) can enhance cellular sensitivity to apoptosis via a mechanism of action distinct from other Bcl-2 family members that promote apoptosis.


INTRODUCTION

Apoptosis is an active cellular suicide process that is characterized by distinct biochemical and morphological changes such as DNA fragmentation, plasma membrane blebbing, and cell volume shrinkage (1, 2) . Apoptosis is recognized as an important physiological event involved in development, in organismal homeostasis, and possibly even in the prevention of neoplastic transformation(3, 4, 5, 6, 7, 8) . Recent data suggest that disruption or dysregulation of the genes that control apoptosis affect neuronal development, disturb lymphocyte homeostasis, initiate tumor progression, and enhance resistance to present cancer treatment modalities(9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) .

A number of genes have been described that function to regulate susceptibility to apoptosis. We have recently identified a gene, bcl-x, which is a member of the growing Bcl-2 family(20) . bcl-x has two alternatively spliced forms that encode two different protein products. Bcl-x(L) has the larger open reading frame of 233 amino acids and, like Bcl-2, functions to inhibit apoptosis induced by a variety of stimuli. As a result of alternative splicing, Bcl-x(S) lacks an internal 63 amino acids that comprise the region of greatest conservation between Bcl-x(L) and Bcl-2. Although Bcl-x(S) has been shown to inhibit the ability of Bcl-2 to protect from growth factor withdrawal, the ability of Bcl-x(S) to inhibit function or competitively bind to other family members has not been examined.

Within the internal 63-amino acid domain deleted in Bcl-x(S), there exists two homology regions that define members of the Bcl-2 family. These regions are referred to as BH1 and BH2 and have been suggested to be important in mediating protein interactions between Bcl-2 family members. Mutations in the BH1 or BH2 regions of Bcl-2 or Bcl-x(L) disrupt their ability to heterodimerize with Bax, a Bcl-2 family member that accelerates cell death when overexpressed in a growth factor-dependent cell line. Moreover, these mutations abrogate the ability of Bcl-2 and Bcl-x(L) to protect cells from apoptosis(21, 22, 23) . Another family member, Bad, preferentially heterodimerizes with Bcl-x(L) through BH1 and BH2 interactions and functions to antagonize the anti-apoptotic properties of Bcl-x(L) by disrupting Bax/Bcl-x(L) heterodimers(24) . The absence of BH1 and BH2 regions in Bcl-x(S) suggests that Bcl-x(S) may promote cell death by a mechanism distinct from Bax or Bad. However, analysis examining protein interactions using the yeast two-hybrid system has shown that Bcl-x(S) retains the ability to associate with Bcl-2 and Bcl-x(L) and argues that Bcl-x(S) may inhibit Bcl-2 function through a mechanism based on competitive dimerization(22, 25) . The demonstration that Bcl-2 family members are able to selectively interact with each other in both the yeast two-hybrid system and in mammalian cells suggests that protein interactions between these molecules may be an important mechanism to regulate the apoptotic threshold of a cell.

The present experiments were undertaken to further characterize the functional and biochemical properties of Bcl-x(S) in mammalian cells. Here we show that Bcl-x(S) is able to antagonize the protective effects of Bcl-x(L) in a dose-dependent manner. However, in comparison to Bcl-x(L), Bcl-x(S) has a substantially reduced ability to form immunoprecipitatable dimers with other Bcl-2 family members. These data suggest that Bcl-x(S) does not reduce resistance to apoptosis simply by acting as a competitive dimerization substrate.


MATERIALS AND METHODS

Epitope Tagging of Bcl-x(L) and Bcl-x(S)

The HA epitope-tagged Bcl-x(L) was constructed by ligating a HindIII fragment from a HA-tagged Bcl-x(L) WGR to WAR mutant construct (kindly provided by Dr. Stanley Korsmeyer, Washington University, St. Louis, MO) into pBluescript-Bcl-x(L) plasmid cut with HindIII. Proper orientation was confirmed by restriction enzyme digest. The HA-Bcl-x(L) construct was then cut out of pBluescript with EcoRI and subcloned into pSFFV-Neo(20) . The FLAG epitope-tagged Bcl-x(S) was constructed by PCR (^1)using a 5` primer that incorporated an EcoRI site, a consensus translation initiation site, and the 8-amino acid FLAG epitope. The sequence for this primer is as follows: 5`-AGAGAATTCCCACCATGGACTACAAGGACGACGATGACAAGTCTCAGAGCAACCGGGAG-3`. The 3` primer used corresponded to sequence in the 3`-untranslated region of human Bcl-x(S) contained in a pBluescript-Bcl-x(S) plasmid and also incorporated an EcoRI site. The sequence for the 3` primer is as follows: 5`-GTGACTGGTAGGTGAGATCTTAAGCC-3`. PCR using pBluescript-Bcl-x(S) as the template DNA was performed through 30 cycles of PCR at 95 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 min. The PCR product was digested with EcoRI and cloned into pSFFV-Neo.

Cell Culture and Cell Transfections

The murine prolymphocytic IL-3-dependent cell line, FL5.12, was maintained as described previously(20) . Cotransfections with pSFFV-Neo-Bcl-x(L) and pSFFV-Neo-Bcl-x(S)(20) were performed using 10 µg of each plasmid, electroporated into 1 times 10^7 cells at 960 µF and 200 V. Neomycin-resistant cells were selected with 1 mg/ml of G418. Single cell clones from the bulk transfectants were derived by limiting dilution cloning. Clones were screened for Bcl-x(L) and Bcl-x(S) expression by immunoblotting with 2A1, a mouse monoclonal antibody to Bcl-x(26) . Cotransfections of FL5.12 cells with pSFFV-Neo-HA-Bcl-x(L) and pSFFV-Neo-FLAG-Bcl-x(S) (or pSFFV-Neo-Bcl-x(L)) were performed in a similar fashion, as was cotransfection of FL5.12 cells with pSFFV-Neo-Bcl-2 and pSFFV-Neo-FLAG-Bcl-x(S).

Cell Viability Assays

IL-3 deprivation assays were performed by washing cells three times with medium prepared without WEHI 3B supernatant (a source of IL-3) and resuspending the cells in this medium at a concentration of 5 times 10^5 cells/ml. Chemotherapeutic drug studies were performed by resuspending cells in medium without G418 at a concentration of 5 times 10^5 cells/ml and adding either 10 µg/ml of etoposide or 0.1 µg/ml of vincristine. Viability was determined over the course of 5 or 6 days by propidium iodide exclusion, as described previously(19) .

Laser Scanning Confocal Microscopy

1 times 10^5 FL5.12 cells expressing HA-Bcl-x(L) and FLAG-Bcl-x(S) were adhered to glass slides using a Cytospin 3 (Shandon) at 1200 rpm for 5 min. Cells were fixed with 2% paraformaldehyde for 10 min and permeabilized with PBS + 0.1% saponin for 15 min. Cells were then washed once with PBS + 0.03% saponin and blocked with 20% goat serum for 30 min. Labeling was done with either 0.01 µg/µl of 12CA5, an anti-HA antibody (Boehringer Mannheim), or M2, an anti-FLAG antibody (Kodak), in PBS + 3% bovine serum albumin for 30 min, after which time the slides were washed twice with PBS + 0.03% saponin for 5 min. Labeling with the secondary antibody (either fluorescein isothiocyanate-conjugated or Texas Red-conjugated) was also done for 30 min in PBS + 3% bovine serum albumin at a 1:100 dilution. The slides were washed twice as before and then mounted with Prolong (Molecular Probes) following the manufacturer's instructions. A Zeiss laser scanning confocal microscope was used to image the slides.

Metabolic Labeling and Immunoprecipitations

For each immunoprecipitation sample, 5 times 10^6 cells were resuspended in 5 ml of fresh medium and labeled overnight with 350 µCi of TransS-Label (ICN). Cells were lysed in 500 µl of NET-N buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris, 0.2% Nonidet P-40, pH 8.0) supplemented with 8 µg/ml aprotinin, 2 µg/ml leupeptin, and 170 µg/ml phenylmethylsulfonyl fluoride. Cellular debris was pelleted by centrifugation at 14,000 times g for 10 min at 4 °C. The supernatant was precleared with 25 µl of protein G-agarose (Life Technologies, Inc.) for 1 h at 4 °C on a rocking platform. 2 µl of 7B2 anti-Bcl-x mouse monoclonal antibody (production of 7B2 is the same as 2A1), 0.5 µg of 12CA5 anti-HA antibody, 5 µg of M2 anti-FLAG antibody, 2 µg of 6C8 anti-Bcl-2 antibody (PharMingen), or an isotype-matched control antibody (PharMingen) was added to the supernatant and rocked for 1 h at 4 °C. 20 µl of protein G-agarose was then added and rocked for another hour at 4 °C. The agarose beads were spun down and washed four times with NET-N buffer. The antigens were released and denatured by the addition of SDS sample buffer and heating at 95° for 5 min. Samples were analyzed using 15% SDS-PAGE and fluorography.

In Vitro Translation and Immunoprecipitation

In vitro translation was done with the TNT Rabbit Reticulocyte Lysate System (Promega) according to the manufacturer's instructions. 0.5 µg of each cDNA-containing plasmid was used in the transcription/translation reaction. Immunoprecipitations were performed essentially the same as described above with the following exceptions. 20 µl of the in vitro translation reaction was added to 280 µl of NET-N (supplemented with protease inhibitors) for immunoprecipitation. 1 µl of 7B2, 4 µg of 12CA5, or 3 µg of M2 was used in the immunoprecipitations. After antibody addition, the sample was incubated overnight at 4 °C on a rocking platform. Samples were washed three times with NET-N and analyzed by 12% SDS-PAGE along with 2 µl of the original in vitro translation to represent the input prior to immunoprecipitation. Gels were processed for analysis either by fluorography or PhosphorImager analysis (Molecular Dynamics).

Immunoprecipitation and Immunoblotting

Immunoprecipitations from unlabeled cells were performed similarly to the immunoprecipitations from the metabolically labeled cells, except that lysis was done in 300 µl and that 10 µl of protein G-agarose was used in preclearing and complexing of antibody and antigen. For Bcl-x immunoblotting, samples were analyzed by 12 or 15% SDS-PAGE, transferred to a nylon membrane, and blocked with BLOTTO (5% milk + 0.2% Tween 20) for 1 h at room temperature. Membranes were then probed with a 1:10,000 dilution of 2A1 or a 1:1000 dilution of 13.4 (anti-Bcl-x rabbit polyclonal antisera) (26) in BLOTTO for 1 h at room temperature. The blot was washed 3 or 4 times for 5 min each in Tris-buffered saline + 0.2% Tween 20 and then probed with a horseradish peroxidase-conjugated secondary antibody for 30 min at room temperature. The blot was washed as before and developed using ECL (Amersham Corp.). For Bax immunoblotting, the protocol is the same except that 0.05% Tween 20 was used in the solutions. The anti-Bax antibody (sc 493, Santa Cruz) was used at 1 µg/ml. Films of developed immunoblots were quantitated using an Arcus Plus Scanner and NIH Image software (public domain program developed at the NIH and available from the Internet by FTP from zippy.nimh.nih.gov). The fraction of Bax not heterodimerized to Bcl-x(L) was determined by dividing the optical density of the band corresponding to the amount of Bax not heterodimerized by the band corresponding to the amount of total Bax.


RESULTS

Bcl-x(S) Antagonizes the Anti-apoptotic Properties of Bcl-x(L)

In order to determine if Bcl-x(S) could inhibit the protective properties of Bcl-x(L), we cotransfected the IL-3-dependent cell line FL5.12 with expression vectors for Bcl-x(S) and Bcl-x(L) and derived single cell clones by limiting dilution. Individual clones were screened for expression of both proteins by immunoblot. Because the monoclonal antibody used in the immunoblotting reacts with an identical epitope present in both Bcl-x(L) and Bcl-x(S), band intensities directly reflect the relative expression levels of each of the two proteins. Although we analyzed 64 individual clones isolated from three separate transfections, we have not been able to isolate a stable clone that substantially overexpressed Bcl-x(S) in comparison with Bcl-x(L). Most clones overexpress Bcl-x(L) relative to Bcl-x(S) or express Bcl-x(L) alone. No clones expressed high levels of Bcl-x(S) alone (data not shown).

Clones were categorized into two groups, intermediate Bcl-x(L) expressors and high Bcl-x(L) expressors. Clones in each of the two groups varied in their expression of Bcl-x(S) (Fig. 1). In the absence of Bcl-x(S) expression, Bcl-x(L) protected FL5.12 cells from apoptosis induced by IL-3 withdrawal in a dose-dependent fashion. Fig. 2A shows that clone 22, which expresses a high amount of Bcl-x(L), had a dramatically enhanced viability after growth factor removal when compared with the control transfected cells. Clone 7, in accord with its lower Bcl-x(L) expression levels, displayed a viability intermediate between the Neo control transfectants and clone 22.


Figure 1: Expression levels of Bcl-x(L) and Bcl-x(S). FL5.12 cells were cotransfected with equal molar amounts of pSFFV-Neo-Bcl-x(L) and pSFFV-Neo-Bcl-x(S). Single cell clones were derived by limiting dilution and screened by immunoblotting with 2A1, a mouse monoclonal antibody to Bcl-x. Bcl-x(L) migrates with an apparent molecular mass of 30 kDa, whereas Bcl-x(S) migrates with an apparent molecular mass of 21 kDa. Clones were grouped into intermediate Bcl-x(L) expressors (2, 14, 21, 7) and high Bcl-x(L) expressors(9, 19, 10, 22) . The lane labeled N represents a Neo control transfectant. Molecular masses are indicated in kilodaltons at the right of the gel.




Figure 2: Bcl-x(L) protects cells from apoptosis in a dose-dependent fashion and is antagonized by Bcl-x(S). The clones shown in Fig. 1were washed 3 times in IL-3-free medium and resuspended at 5 times 10^5 cells/ml. Viability was determined over the course of 6 days by propidium iodide exclusion. A, comparison of viability between an intermediate Bcl-x(L) expressor (clone 7), a high Bcl-x(L) expressor (clone 9), and a Neo control transfectant. B, viability of intermediate Bcl-x(L) expressors in the presence of various levels of Bcl-x(S). C, viability of high Bcl-x(L) expressors in the presence of various levels of Bcl-x(S). These results are representative of at least three independent experiments.



Fig. 2B demonstrates that when Bcl-x(S) was expressed in conjunction with intermediate levels of Bcl-x(L), it took relatively little Bcl-x(S) to almost completely reverse the protective effects of Bcl-x(L) from IL-3 withdrawal. After exhibiting significant protection at 24 h, clones 2, 14, and 21 precipitously lost viability between 24 and 48 h and reached levels at or below the control transfectants (compare clones 2, 14, and 21 in Fig. 2B with the Neo clone from Fig. 2A). In the presence of high levels of Bcl-x(L) expression, it took correspondingly more Bcl-x(S) to see significant antagonism of Bcl-x(L) (Fig. 2C). The level of antagonism increased with increasing amounts of Bcl-x(S) (compare clones 10, 19, and 9).

To further confirm that Bcl-x(S) is able to antagonize the ability of Bcl-x(L) to protect FL5.12 cells from growth factor removal, we also supertransfected a Bcl-x(L) expressing clone with the Bcl-x(S) expression vector. Additionally, we employed a transient transfection assay to determine the effects of Bcl-x(S) on viability. In both cases, similar results to the cotransfectants were obtained (data not shown).

Bcl-x(S) Inhibits the Multidrug-resistant Phenotype Conferred by Bcl-x(L) Expression

We have previously demonstrated that expression of Bcl-x(L) can confer a multidrug-resistant phenotype to FL5.12 cells(19) . This mechanism of multidrug resistance may be clinically relevant because we and others have found Bcl-x(L) expressed at relatively high levels in both primary tumors and tumor cell lines. (^2)Therefore, we wished to determine if Bcl-x(S) could also antagonize the ability of Bcl-x(L) to protect FL5.12 cells from cell death induced by chemotherapeutic drugs. The clones described above were treated with either etoposide or vincristine and assayed for viability over the course of 5 days. In clones that expressed an intermediate level of Bcl-x(L), there was a good correlation between increasing Bcl-x(S) levels and enhanced sensitivity to both chemotherapeutic drugs (Fig. 1, 3A, and 3C). A comparison of clones 2 and 21 to clone 7 revealed that high Bcl-x(S) expression was quite effective at reversing the protective effects of Bcl-x(L), whereas low Bcl-x(S) expression (clone 14) was less effective.

In clones that expressed high amounts of Bcl-x(L), high levels of Bcl-x(S) were required to significantly antagonize Bcl-x(L) (Fig. 3, B and D). Only in clone 9 did we see significantly enhanced sensitivity to either etoposide or vincristine treatment when compared with clone 22.


Figure 3: Bcl-x(S) is able to antagonize the multidrug resistance conferred by Bcl-x(L). The clones from Fig. 1were resuspended in medium without G418 at a concentration of 5 times 10^5 cells/ml. Either 10 µg/ml of etoposide (A and B) or 0.1 µg/ml of vincristine (C and D) was added, and viability was determined over the course of 5 days by propidium iodide exclusion. These results are representative of at least three independent experiments.



Cellular Localization of Bcl-x(L) and Bcl-x(S)

Because Bcl-x(S) is an effective antagonist of Bcl-x(L), we wished to determine if these two proteins had a similar cellular localization. Because antibodies that can distinguish between Bcl-x(L) and Bcl-x(S) are not available, we epitope-tagged Bcl-x(L) with the hemagglutinin (HA) tag and epitope-tagged Bcl-x(S) with the FLAG tag. Expression vectors encoding these constructs were cotransfected into FL5.12 cells and tested for function. Both epitope-tagged proteins were found to be functional in assays identical to those shown in Fig. 2(data not shown). Next, cells that expressed both HA-Bcl-x(L) and FLAG-Bcl-x(S) were immunofluorescently stained with antibodies specific for each epitope and visualized by confocal microscopy. Both FLAG-Bcl-x(S) and HA-Bcl-x(L) exhibited cytoplasmic staining and faint perinuclear localization, and both proteins appeared to localize similarly when examined both separately and together (Fig. 4). The staining pattern was consistent with the mitochondria, endoplasmic reticulum, and perinuclear staining we have previously reported for Bcl-x(L)(27) . At the resolution utilized in this experiment, we could not detect any gross differences in cellular localization between Bcl-x(L) and Bcl-x(S).


Figure 4: Bcl-x(L) and Bcl-x(S) exhibit similar immunofluorescent staining patterns. A polyclonal population of FL5.12 cells cotransfected with HA-Bcl-x(L) and FLAG-Bcl-x(S) were immunofluorescently stained with an anti-HA antibody and an anti-FLAG antibody. Specimens were analyzed by laser-scanning confocal microscopy. Top, Bcl-x(L) staining alone. Middle, the same field showing Bcl-x(S) staining. Bottom, composite image of Bcl-x(L) and Bcl-x(S) staining.



The cotransfected cells used in the immunofluorescent staining experiment were from a bulk transfection using equal molar amounts of each plasmid (Fig. 4). Because Bcl-x(S) increases sensitivity to cell death, cells that express high levels may show a survival disadvantage. Consistent with this prediction, the immunofluorescent staining of the polyclonal population of transfected cells showed that cells that expressed detectable Bcl-x(S) in the absence of Bcl-x(L) were rare. In contrast, cells that selectively express Bcl-x(L) (red) were easily detectable. For example, the bottom panel of Fig. 4shows several cells that expressed Bcl-x(L) only (red) but no cells that expressed Bcl-x(S) only (green). This pattern of Bcl-x(S) expression is in accord with the results we obtained from screening clones from various Bcl-x(L) and Bcl-x(S) cotransfections.

Bcl-x(S) Association with Bcl-x(L), Bcl-2, or Bax Is Not Detected in Vivo

It has recently been suggested that protein interactions among Bcl-2 family members may be an important property and a potential mechanism by which these proteins are functionally regulated(28) . Therefore, we wished to determine if Bcl-x(S) also associates with some of the known Bcl-2 family members in vivo, perhaps mechanistically explaining the apparent stoichiometric ability of Bcl-x(S) to antagonize Bcl-x(L).

Cells expressing both HA-Bcl-x(L) and FLAG-Bcl-x(S) were metabolically labeled, and coimmunoprecipitation studies were performed. Fig. 5A (first lane) shows that an anti-Bcl-x monoclonal antibody that recognizes both HA-Bcl-x(L) and FLAG-Bcl-x(S) immunoprecipitated both of these proteins, and furthermore, both were expressed at relatively high levels. As previously reported, Bax was coimmunoprecipitated with Bcl-x(L) and thus is also present in the first lane(24) . An additional band at approximately 26 kDa is also seen in the first lane. Based on its migration, this band appears to be endogenous Bcl-2. This was subsequently confirmed by Bcl-2 immunoblotting of Bcl-x immunoprecipitations (data not shown). Immunoprecipitation with the anti-HA monoclonal antibody precipitated HA-Bcl-x(L) and the associated proteins Bax and Bcl-2; however, FLAG-Bcl-x(S) was not coprecipitated (Fig. 5A, second lane). Conversely, the anti-FLAG antibody only immunoprecipitated FLAG-Bcl-x(S) (Fig. 5A, third lane). The failure of Bcl-x(S) to interact with Bcl-x(L) was also confirmed by immunoblotting with an anti-Bcl-x antibody after immunoprecipitating with an anti-HA antibody from FL5.12 cells cotransfected with HA-Bcl-x(L) and untagged Bcl-x(S) (data not shown).


Figure 5: Protein interactions between Bcl-2 family members in vivo. Cells were metabolically labeled overnight with [S]methionine and immunoprecipitated with antibodies against Bcl-x, the HA epitope, the FLAG epitope, or Bcl-2, as indicated at the top of each gel. A, immunoprecipitation from FL5.12 cells cotransfected with HA-Bcl-x(L) and FLAG-Bcl-x(S). B, immunoprecipitations from FL5.12 cells cotransfected with Bcl-2 and FLAG-Bcl-x(S) or cells cotransfected with HA-Bcl-x(L) and Bcl-x(L). The cell type used for the immunoprecipitation is indicated at the bottom of each gel, the positions of each Bcl-2 family member is indicated at the right of each gel, and the molecular masses are shown in kilodaltons at the left of each gel.



We also immunoprecipitated Bcl-2 from FL5.12 cells cotransfected with Bcl-2 and FLAG-Bcl-x(S). Fig. 5B (first lane) shows that the anti-Bcl-2 antibody coprecipitated Bax but not FLAG-Bcl-x(S). The anti-FLAG antibody only immunoprecipitated FLAG-Bcl-x(S) (Fig. 5B, second lane). Independent experiments using cells cotransfected with untagged Bcl-x(S) and Bcl-2 were used in immunoprecipitation/immunoblotting experiments to confirm the results seen in the first and second lanes of Fig. 5B. Neither immunoprecipitation of Bcl-2 followed by Bcl-x immunoblotting nor Bcl-x immunoprecipitation followed by Bcl-2 immunoblotting showed any evidence for Bcl-x(S) interacting with Bcl-2 (data not shown).

Finally, we immunoprecipitated with the anti-HA antibody from cells cotransfected with HA-Bcl-x(L) and untagged Bcl-x(L) to determine if Bcl-x(L) homodimerizes. As seen in Fig. 5B (third and fourth lanes), HA-Bcl-x(L) did not associate with untagged Bcl-x(L), suggesting that Bcl-x(L) does not preferentially homodimerize.

In Vitro Analysis of Bcl-x(S) Protein Interactions

It previously has been reported that Bcl-x(S) interacts with Bcl-x(L) and Bcl-2 in the yeast two-hybrid system(22, 25) . Therefore, we sought another method to investigate the ability of Bcl-x(S) to interact with Bcl-x(L). For this purpose, we in vitro translated HA-Bcl-x(L) together with Bax and/or FLAG-Bcl-x(S) and analyzed for associations by coimmunoprecipitation. When HA-Bcl-x(L), Bax, and FLAG-Bcl-x(S) were translated together in vitro and immunoprecipitated using the anti-HA antibody, HA-Bcl-x(L) preferentially interacted with Bax in comparison with Bcl-x(S), despite the presence of near equal molar quantities of all three proteins prior to immunoprecipitation (Fig. 6A). Quantitation using a PhosphorImager showed that after normalization for methionine content, Bax coprecipitated with HA-Bcl-x(L) 3.5-fold better than Bcl-x(S). The stronger association between Bcl-x(L) and Bax compared to Bcl-x(L) and Bcl-x(S) was confirmed when HA-Bcl-x(L) was cotranslated with Bax and FLAG-Bcl-x(S) separately (Fig. 6B). Finally, when the ability of FLAG-Bcl-x(S) to associate with Bcl-2 or Bax were investigated in vitro, the inability of FLAG-Bcl-x(S) to interact with either molecule was confirmed (data not shown). Thus, as determined by both in vivo and in vitro analysis, the ability of Bcl-x(S) to interact with Bcl-x(L) is at best a weak interaction, and Bcl-x(S) does not interact with either Bcl-2 or Bax.


Figure 6: Bcl-x(L) preferentially interacts with Bax over Bcl-x(S)in vitro. cDNA-containing plasmids for HA-Bcl-x(L), FLAG-Bcl-x(S), and/or Bax were in vitro transcribed/translated and immunoprecipitated as described under ``Materials and Methods.'' A, HA-Bcl-x(L), FLAG-Bcl-x(S), and Bax were translated together and immunoprecipitated with an anti-HA antibody. Lane 1 represents one-tenth of the input volume used for the immunoprecipitation. Lane 2 represents the immunoprecipitation. Quantitation of the FLAG-Bcl-x(S) and the Bax signal intensities using a PhosphorImager (Molecular Dynamics) showed that after normalization, Bax coprecipitates with HA-Bcl-x(L) 3.5-fold better than FLAG-Bcl-x(S). B, HA-Bcl-x(L) was cotranslated with either FLAG-Bcl-x(S) (lanes 1, 3, and 5) or Bax (lanes 2, 4, and 6) and immunoprecipitated with an anti-HA antibody. Lanes 1 and 2 represent one-tenth of the input volume used for the immunoprecipitations. Lanes 3 and 4 are the immunoprecipitations, and lanes 4 and 5 are a 4-fold longer exposure of lanes 3 and 4.



In vitro binding studies also revealed that Bax and Bcl-x(S) have different requirements for binding to Bcl-x(L). Bcl-x(S) and Bcl-x(L) have a conserved region contained within 25 amino acids located at the NH(2) terminus of both proteins that is absent in Bax. As seen in Fig. 7, when these 25 amino acids were deleted from the NH(2) terminus of Bcl-x(L), the truncated protein had an enhanced ability to bind to FLAG-Bcl-x(S). In contrast, the NH(2)-terminal truncation of Bcl-x(L) abolished binding to Bax. When the conserved NH(2)-terminal region was removed from Bcl-x(S), the binding to HA-Bcl-x(L) was also abolished (data not shown). Thus, in order for Bax to bind to Bcl-x(L), the NH(2)-terminal region must be present in Bcl-x(L). In contrast, in order for Bcl-x(S) to bind to Bcl-x(L), the NH(2)-terminal region need only be present in Bcl-x(S). Removal of the NH(2)-terminal region from Bcl-x(L) actually enhances binding to Bcl-x(S).


Figure 7: In vitro binding to Bcl-x(L) by Bax but not Bcl-x(S) requires a 25-amino acid region within the NH(2) terminus of Bcl-x(L). A deletion mutant of Bcl-x(L) was constructed that lacked the first 25 NH(2)-terminal amino acids (DeltaN Bcl-x(L)). cDNA-containing plasmids of DeltaN Bcl-x(L) and FLAG-Bcl-x(S) (lanes 1 and 2) or DeltaN Bcl-x(L) and Bax (lanes 3 and 4) were in vitro transcribed/translated and immunoprecipitated with an anti-FLAG antibody (lane 2) or an anti-Bcl-x antibody (lane 4). Lanes 1 and 3 represent one-tenth of the input volume used for immunoprecipitation. The band that migrates slightly below Bax (*) in lanes 1-4 is a premature termination product of the NH(2)-terminal deleted Bcl-x(L), as it is observed when DeltaN Bcl-x(L) is translated by itself and after immunoprecipitation by the anti-Bcl-x antibody.



Bcl-x(S) Does Not Affect Bax Heterodimerization with Bcl-x(L) in Vivo

Disrupting Bcl-x(L) heterodimerization with Bax to increase the amount of Bax homodimers has been suggested to be one way cells can be sensitized to apoptosis (22, 23, 24) . Because we could only detect a weak in vitro association between Bcl-x(S) and Bcl-x(L) and no association between Bcl-x(S) and Bax, this argued that Bcl-x(S) did not effect the ability of Bax to bind to Bcl-x(L). Additionally, the requirement for an amino-terminal region in Bcl-x(L) for in vitro binding to Bax but not Bcl-x(S) also suggested that Bcl-x(S) and Bax did not compete for the same binding site within Bcl-x(L). In order to determine if the presence of Bcl-x(S) could affect the amount of Bax heterodimerized with Bcl-x(L)in vivo, we lysed cells that either expressed both Bcl-x(L) and Bcl-x(S) or expressed Bcl-x(L) alone and performed two sequential Bcl-x immunoprecipitations from two-thirds of the cleared lysate. Half of this immunoprecipitate and half of the supernatant left following Bcl-x immunoprecipitation along with the one-third of the original lysate were analyzed for Bax by immunoblotting (Fig. 8, top). Thus, the amount of Bax from one-third of the lysate is equal to the amount of Bax present in the Bcl-x immunoprecipitate and supernatant combined. The amount of Bax in the Bcl-x immunoprecipitate represents the amount of Bax heterodimerized with Bcl-x(L) (Fig. 8, H). The amount of Bax left in the supernatant represents the amount of Bax not heterodimerized with Bcl-x(L) (Fig. 8, U). Finally, the amount of Bax from one-third of the original lysate is representative of the relative amount of total Bax (Fig. 8, T). The other half of the immunoprecipitate and the supernatant were analyzed by Bcl-x immunoblotting to confirm the efficiency of the Bcl-x immunoprecipitation (Fig. 8, bottom).


Figure 8: Bcl-x(S) does not alter the amount of Bax not heterodimerized with Bcl-x(L). Clones 2, 7, 9, and 22 were lysed in 0.2% Nonidet P-40. Two-thirds of the lysate was immunoprecipitated twice with 7B2, a mouse monoclonal antibody to Bcl-x. The one-third left from the original lysate and the supernatant left from the immunoprecipitation were saved. Top, one-third of the original lysate, one-half of the 7B2 immunoprecipitation, and one-half of the supernatant left from the immunoprecipitation were analyzed by 12% SDS-PAGE and immunoblotting with an anti-Bax antibody. The amount of Bax in the lysate represents the relative amount of total Bax (labeled T). The amount of Bax in the 7B2 immunoprecipitation represents the relative amount of Bax heterodimerized to Bcl-x (labeled H). The amount of Bax left in the supernatant represents the relative amount of Bax not heterodimerized with Bcl-x (labeled U). Also shown is the control antibody immunoprecipitation and its supernatant (last two lanes). The 28-kDa band that is present in the H lanes probably represents light chain from the Bcl-x antibody. Bottom, the remaining one-half of the 7B2 immunoprecipitation and the remaining one-half of the supernatant were analyzed by 12% SDS-PAGE and immunoblotting with 13.4, a rabbit anti-Bcl-x polyclonal antisera. The lanes labeled I represent the 7B2 immunoprecipitation, and the lanes labeled S represent the supernatants from these immunoprecipitations. The last two lanes represent a control antibody immunoprecipitation and its supernatant.



The top panel of Fig. 8shows that there was little difference between the amount of Bax not heterodimerized in clone 2, which expresses Bcl-x(L) and Bcl-x(S), compared with clone 7, which expresses Bcl-x(L) alone (compare first and third lanes to fourth and sixth lanes). Densitometry scanning of three independent experiments showed that the fraction of Bax not heterodimerized to Bcl-x in clone 2 was 0.37 ± 0.07, and in clone 7 the value was 0.31 ± 0.15. The failure to see a substantial increase in the amount of Bax not heterodimerized was also evident when comparing clone 9 and clone 22 (compare seventh and ninth lanes to tenth and twelfth lanes). In two independent experiments, the amount of Bax not heterodimerized to Bcl-x was less than 0.10 for both clones.

The bottom panel of Fig. 8demonstrates that the immunoprecipitations with the anti-Bcl-x antibody effectively removed nearly all of the Bcl-x. It should be noted that this immunoblot was done with 13.4, a rabbit Bcl-x-specific polyclonal antisera, to eliminate reaction with Ig light chain present in the immunoprecipitation. This antibody seems to preferentially recognize Bcl-x(L), which is why the ratios between Bcl-x(L) and Bcl-x(S) observed in this immunoblot differ from the immunoblot presented in Fig. 1.


DISCUSSION

In this study we demonstrate that Bcl-x(S) can inhibit the ability of Bcl-x(L) to protect cells from apoptosis induced by growth factor withdrawal or chemotherapeutic drugs. A previous study demonstrated that Bcl-x(S) can also block the protective effects of Bcl-2. The inhibitory property of Bcl-x(S) does not appear to involve observable in vivo heterodimerization with Bcl-x(L), Bcl-2, or Bax. Bcl-x(S) can weakly interact with Bcl-x(L)in vitro, but this interaction seems to be fundamentally different from the Bcl-x(L)/Bax association in both strength and structural requirements. Consistent with these binding properties, Bcl-x(S) does not significantly decrease the amount of Bax heterodimerized with Bcl-x(L)in vivo.

By in vitro analysis, we found that removing 25 amino acids from the NH(2) terminus of Bcl-x(L) enhanced the ability of Bcl-x(S) to interact with Bcl-x(L). In contrast, the removal of these amino acids from Bcl-x(L) eliminates the ability of Bcl-x(L) to heterodimerize with Bax. The amino terminus of Bcl-x(L) most likely interacts with its own BH1/BH2 region as suggested by the yeast two-hybrid studies (25) and our unpublished data. (^3)Thus, it is likely that removal of the amino terminus of Bcl-x(L) allows the identical amino-terminal region in Bcl-x(S) to more readily interact with the BH1/BH2 region of Bcl-x(L). Consistent with this explanation, truncation of the amino terminus from Bcl-x(S) prevents it from interacting with Bcl-x(L). Because intramolecular interactions are more favorable than intermolecular interactions, this would also explain why the Bcl-x(S)/Bcl-x(L) association is weak. The failure to see Bcl-x(L) homodimerize in vivo and in vitro (data not shown) can also be explained by this intramolecular versus intermolecular competition. Finally, a potential explanation for why studies using the yeast two-hybrid system showed strong associations between Bcl-x(S) and either Bcl-x(L) or Bcl-2 is because the yeast two-hybrid system involves the use of fusion proteins that may disrupt the intramolecular association between the amino terminus and the BH1/BH2 region. If the amino terminus of Bcl-x(L) competes with the amino terminus of Bcl-x(S) for binding to the BH1/BH2 region of Bcl-x(L), disrupting these intramolecular interactions within Bcl-x(L) would facilitate Bcl-x(S) binding.

We cannot be sure whether the weak association between Bcl-x(S) and Bcl-x(L) observed in vitro is a physiologically relevant interaction or an in vitro artifact. One possibility is that Bcl-x(S)/Bcl-x(L) interaction occurs but simply cannot be detected using the in vivo methods employed due to the weakness of the interaction. Overexpression of Bcl-x(S) relative to Bcl-x(L) may facilitate seeing association in vivo; however, we found it difficult to obtain such clones. Another possibility is that if the Bcl-x(S) association with Bcl-x(L) is mediated by an intermolecular interaction involving the amino terminus of Bcl-x(S), the in vitro associations observed may be an artifact of a misfolded Bcl-x(L) aggregating with Bcl-x(S). In vivo, chaperones may prevent such products from forming. Finally, even if the interaction between Bcl-x(S) and Bcl-x(L) is physiologically relevant, it is unlikely to be the mechanism by which Bcl-x(S) promotes cell death not only because the interaction is weak but also because Bcl-x(S) fails to interact with Bcl-2 either in vivo or in vitro, despite being a potent antagonist of Bcl-2 function.

Regardless of the significance of the Bcl-x(S)/Bcl-x(L) association, Bcl-x(S) does not appear to antagonize Bcl-x(L) by directly or indirectly liberating Bax. Increasing the amount of Bax homodimers has been postulated to be a general mechanism for enhancing sensitivity to cell death(28) . Bad is a recently cloned antagonist of Bcl-x(L) that may work by effectively competing with Bax for Bcl-x(L) binding, resulting in an increase in free or homodimerized Bax(24) . However, we did not observe any significant changes in the amount of Bax not heterodimerized to Bcl-x(L) when Bcl-x(S) was present. Our results suggest that increasing the sensitivity to apoptosis does not absolutely require an increase in free or homodimerized Bax.

If Bcl-x(S) does not liberate Bax or antagonize Bcl-x(L)/Bcl-2 by direct association, how might Bcl-x(S) work? Bcl-x(S) could promote cell death by binding to a yet unidentified factor(s) that is regulated by Bcl-x(L) and Bcl-2 to promote survival. The binding of this factor by Bcl-x(S) sequesters the factor into a complex that now accelerates cell death. Another possibility is that Bcl-x(S) does not act as a competitor in protein-protein interactions, rather Bcl-x(L) and Bcl-2 are active effectors of cell survival, and Bcl-x(S) counters these molecules by performing the opposite effector function. Irrespective of how these molecules are working to influence cell survival, it is becoming clear that at least two independent domains, the NH(2)-terminal 25 amino acids and the internal BH1/BH2 domain found in both Bcl-x(L) and Bcl-2, are required to produce a protein that increases the apoptotic threshold of a cell. Production of a protein containing only one of these domains such as Bcl-x(S), which lacks the BH1/BH2 domain, or Bax, which lacks the NH(2)-terminal homology domain, results in a transdominant inhibitor.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI35294. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Medical Scientist Training Program.

Fellow of the Leukemia Society of America.

**
To whom correspondence should be addressed: The University of Chicago, 924 E. 57th St., Rm. R413A, Chicago, IL 60637-5420. Tel.: 312-702-4360; Fax: 312-702-1576.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; IL-3, interleukin 3; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.

(^2)
A. J. Minn, L. H. Boise, and C. B. Thompson, unpublished data.

(^3)
S. W. Muchmore, C. B. Thompson, and S. W. Fesik, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Stanley Korsmeyer and Brian Chang for providing reagents and Therese Conway for assisting in the preparation of the manuscript.


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