A Novel Splice Variant of the Cell Death-promoting Protein BAX*

Mei Zhou, Susan D. Demo, Thida N. McClure, Roberto Crea, and Catherine M. BitlerDagger

From the Department of Cell Biology, Neurex Corporation, Menlo Park, California 94025

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Cell death plays an important role in a number of physiological processes in all complex multicellular organisms. One of the molecules that regulates this process is BAX, an integral membrane protein, that promotes apoptosis. The function of BAX is countered by BCL-2 and BCL-XL. The differential expression of these proteins can influence the ability of the cell to die or survive. In this paper, we describe the cloning, biochemical, and functional characterization of a novel splice isoform of BAX, called BAX-omega . Transient overexpression of BAX-omega protein potentiates cell death at levels comparable to that of BAX-alpha overexpression.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

A family of BCL-2-related proteins has the effect of potentiating or attenuating apoptotic cell death (1-4). This family is specifically defined by four regions that share amino acid sequence homology designated BH1, BH2, BH3, and BH4. These domains are essential either for dimerization and/or the function of the different family members (5-7). BAX (8), the prototypical family member involved in potentiating apoptotic cell death, heterodimerizes with BCL-2 and BCL-XL and when overexpressed counters the protective effect of these two family members (8, 9). Many of the family members, including BCL-2 and BAX, have a putative transmembrane domain that anchors the proteins to intracellular membranes including mitochondrial, endoplasmic reticulum, and nuclear membranes (10-12). The BCL-2 gene encodes two proteins (26 and 22 kDa) that differ in their C termini as a result of alternative mRNA splicing mechanisms (13, 14). The smaller form, designated BCL-2-beta which lacks the C terminus, lacks the transmembrane domain and thus represents a soluble form of BCL-2 (14). Both the BCL-2-alpha and BCL-2-beta proteins enhance tumorigenicity of fibroblast NIH-3T3 cells (15), and both are able to malignantly transform rat embryo fibroblasts with the ras oncogene (16). In contrast, BCL-2-beta has been shown not to prolong cell survival nor suppress apoptosis (17). Although membrane attachment is not necessarily required for its protective effect (18), the smaller form of BCL-2 does not possess all of the characteristics of the longer form.

The BAX gene has also been shown to encode spliced variants (8). Four members of the BAX family have thus far been characterized including BAX-alpha that encodes for a 21-kDa protein; BAX-beta that encodes for a 24-kDa protein lacking the C terminus due to a termination codon within the coding region of intron 5; BAX-gamma , which is missing exon 2 resulting in a 4.5-kDa protein that prematurely terminates in exon 3 due to a translational frameshift; and BAX-delta (19) that is missing exon 3 but retains the same translational frame, the BH1 and BH2 domains, and the putative transmembrane domain. The function of these alternatively spliced variants is not yet known.

In this paper, we report the cloning, biochemical characterization, and functional analysis of a novel splice isoform that we have called BAX-omega . BAX-omega has a structure distinct from BAX-alpha including the absence of a putative transmembrane domain. BAX-omega is found in every tissue tested. Overexpression of BAX-omega increases basal levels of cell death but does not appear to potentiate death by other inducers. Interestingly, mouse fibroblast L929 cells stably transfected with BAX-omega exhibit properties distinct from both wild-type and vector-transfected cells when induced to undergo apoptosis in that the cells appear to be more resistant to other inducers of cell death. These data suggest that the putative function of this alternatively spliced form of BAX is to induce apoptosis, but under conditions of constitutive overexpression, BAX-omega directly or indirectly protects the cells from apoptotic cell death.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning BAX-omega -- Both human BAX-alpha and BAX-omega were cloned from human hippocampal cDNA (CLONTECH) by polymerase chain reaction (PCR)1 in a volume of 50 µl using 1 µM forward (5'-GGAATTCGCGGTGATGGACGGGTCCGG) and reverse (3'-TCTGGAAGAAGATGGGCTGAG) primers, 200 µM deoxynucleotide triphosphates, 1.5 mM MgCl2, 75 mM KCl, 10 mM Tris, pH 9.2 (Stratagene PCR Optimal Buffer 10) and 2.5 units of Taq polymerase (Boehringer Mannheim). The brain cDNA was amplified for 25 cycles as follows: denaturation at 94 °C for 1 min 20 s, annealing at 60 °C for 2 min, and extension at 72 °C for 1.5 min. Immediately following the last cycle, the mixture was heated to 94 °C for 1.5 min, 60 °C for 2 min, and 74 °C for 10 min. The resulting PCR product was end-filled with Klenow enzyme, blunt-ligated into the SmaI site of pBluescript (Stratagene, La Jolla, CA), and sequenced starting from the 5' end.

To obtain the 3' end of the BAX-omega gene it was necessary to use 3'-rapid amplification of cDNA ends technology. 3'-Rapid amplification of cDNA ends was performed according to the manufacturer's instructions (Life Technologies, Inc.) with the exception of the 3'-primer which was as follows: 5'-CGCAGATCTGATCGATGTCGTCATTTTTTTTTTTTT. Amplification of the cDNA was for 25 cycles as follows: denaturation at 94 °C for 1 min, annealing at 65 °C for 1 min, and extension at 72 °C for 40 s. The resulting product was subcloned into pCR3 using the TA-cloning kit (Invitrogen) and sequenced.

To determine whether the extra 49 bp contained within BAX-omega was encoded by intron 5, intron 5 was cloned by PCR amplification from human genomic DNA in a volume of 50 µl containing 1 µM of each of the primers, 200 µM deoxynucleotide triphosphates, 1.5 mM MgCl2, 75 mM KCl, 10 mM Tris, pH 9.2 (Stratagene PCR Optimal Buffer 10), and 2.5 units of Taq polymerase (Boehringer Mannheim). The 5'-primer was GAGCGGCTGTTGGGCTGGATCCAA, corresponding to sequence in exon 5, and the 3'-primer was the complement of TCTGGAAGAAGATGGGCTGAG, corresponding to sequence in exon 6. Amplification of the cDNA was for 30 cycles as follows: denaturation at 94 °C for 1 min, annealing at 65 °C for 1 min, and extension at 72 °C for 45 s. The resulting product was subcloned into pCR3 using the TA-cloning kit (Invitrogen), and the construct was sequenced.

For expression in monkey kidney E5 cells, BAX-omega was subcloned into the BamHI site of the plasmid, palpha 22beta galalpha GT (20).

Screening a cDNA Library-- A human frontal cortex-LambaZAP cDNA library (Stratagene) was screened (1 × 106 clones) with a probe corresponding to the first 452 nucleotides of BAX-alpha radiolabeled with 32P by random priming (Boehringer Mannheim). The screening used standard hybridization protocols (21) followed by washing in 0.1× SSC, 0.1% SDS at 55 °C.

RT/PCR Analysis-- Normal tissue was obtained from male Sprague-Dawley rats. Total RNA was isolated by the method of Chomczynski and Sacchi (22), and first strand cDNA synthesis was accomplished in a volume of 2.5 µl containing 150 ng of total RNA, Moloney murine leukemia virus reverse transcriptase (2 units; Boehringer Mannheim), and 1 µM complementary 3'-primer TCTGGAAGAAGATGGGCTGAGG (exon 6) at 42 °C for 30 min. Following first strand synthesis, 1 µM 5'-primer GAGCGGCTGTTGGGCTGGATCCAA (corresponding to sequence in exon 5), 200 µM dNTPs (dCTP was replaced with radiolabeled dCTP), and Taq DNA polymerase (10 units; Boehringer Mannheim) was added, and the reaction mix was heated to 95 °C for 3 min and amplified for 25 cycles with the following conditions: denaturation at 94 °C for 1 min, annealing at 63 °C for 1 min, and extension at 72 °C for 40 s. The products were electrophoresed on a 5% polyacrylamide/urea gel, and the gel bands were analyzed with a PhosphorImage Scanner (Molecular Dynamics, Sunnyvale, CA).

Ribonuclease Protection Analysis-- The RPA probe for BAX transcripts was prepared using the MAXIscript SP6 kit (Ambion, Inc., Austin, TX). The 400-bp BAX RNA probe was synthesized from a BamHI-linearized plasmid cDNA containing a 778-bp insert of human BAX cDNA in the vector, pcDNA3 (Invitrogen, Carlsbad, CA), and was radiolabeled with [alpha -32P]CTP that was included in the reaction mix. The control probe of 18 S rRNA was transcribed in vitro from an 18 S rRNA plasmid cDNA. The radiolabeled probes were purified on a 5% polyacrylamide urea gel. The full-length probes were cut from the gel and eluted overnight.

RPA quantitation of BAX was performed using the RPA 11 kit (Ambion Inc.), according to the manufacturer's protocol. Total human RNAs (20 µg) were hybridized with the radiolabeled BAX RNA and 18 S rRNA probes (as described above) and then digested with RNase A/RNase T1. The protected RNA fragments of BAX and 18 S rRNA were precipitated with ethanol and separated on a 5% polyacrylamide urea gel. Following gel electrophoresis, the protected fragments were visualized and quantitated using PhosphorImage scanning (Molecular Dynamics, Sunnyvale, CA).

Western Blot Analysis-- Normal brain, liver, lung, kidney, and heart tissues were obtained from male Sprague-Dawley rats. Approximately 2 g of each tissue were homogenized in 500 µl of ice-cold RIPA containing aprotinin, phenylmethylsulfonyl fluoride, and leupeptin at 4 °C. Following homogenization, the tissue lysate was sonicated for 1 min and then incubated on ice for an additional 30 min. The protein lysate was centrifuged at 2500 rpm for 10 min at 4 °C, and the resultant supernatant was subjected to the Bradford method of protein analysis (Bio-Rad). Following the Bradford analysis, equal amounts of protein were denatured by boiling for 10 min in RIPA containing 100 µM dithiothreitol, and the proteins were electrophoresed on a 12% polyacrylamide gel (SDS-PAGE). Prestained molecular weight markers were run in parallel. The gel was electroblotted to polyvinylidene difluoride membrane (Bio-Rad) for 1 h at room temperature. For immunoblot analysis of proteins, the membrane was blocked in 5% non-fat dry milk (milk solution) for 30 min followed by incubation with the primary antibody, NXrBAX-1, for 1 h in milk solution, washing, incubating with the secondary goat anti-rabbit antibody coupled to peroxidase in milk solution for 1 h, washing and visualizing using ECL (Amersham Pharmacia Biotech), followed by autoradiography.

For Western analysis of cultured cells, L929 cells, which were either transfected with vector alone or with BAX-omega , were grown to subconfluent levels, removed from a 10-cm tissue culture plate with Versene, and centrifuged for 5 min at 1500 rpm. The cell pellet was resuspended in 500 µl of ice-cold RIPA containing aprotinin, phenylmethylsulfonyl fluoride, and leupeptin and was nutated at 4 °C for 1 h. The protein lysate was centrifuged at 2500 rpm for 10 min at 4 °C, and the resultant supernatant was subjected to the Bradford method of protein analysis (Bio-Rad). The samples were then processed essentially as above except for the use of P19 (Santa Cruz Biotechnology) as the primary antibody.

Transfection of Cells-- Mouse fibroblast L929 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (HyClone), referred to as complete medium, in an environment of 5% CO2 at 37 °C. Exponentially growing cells were seeded at 5 × 105 cells per 10-cm tissue culture plate (Falcon) in 10 ml of complete medium the day prior to transfection. The cells were electroporated in Dulbecco's phosphate-buffered saline containing 20 µg of BAX-omega -pcDNA3 (twice purified by CsCl2) with 250 volts and a capacitance of 250 microfarads, grown for 2 days in growth medium, and then selected in G-418 (Life Technologies, Inc.). Drug-resistant clones were transferred to 24-well dishes and grown to confluence with drug selection.

E5 cells were transfected with palpha 22beta galBAX-omega , palpha 22beta galBAX-alpha and palpha 22beta galBCL-2 using LipofectAMINE essentially as described by the manufacturer (Life Technologies, Inc.).

Measurement of Cell Death-- Exponentially growing L929 cells were seeded at 5-10 × 105 cells per 10-cm tissue culture plate (Falcon) in 10 ml of complete medium the day prior to the induction of cell death. Cell death was initiated by replacing the complete medium with Opti-MEM (Life Technologies, Inc.) containing 40 ng/ml TNF and 10 µg/ml cycloheximide. After various times, the cells were scraped from the plates, centrifuged at 1500 rpm for 5 min, and resuspended in Dulbecco's phosphate-buffered saline containing 0.2% trypan blue. The cells were incubated in the trypan blue solution for 5 min, transferred to a hemacytometer, and the number of viable (phase bright) and nonviable (blue) cells were recorded. For each sample, five fields were counted. We and others (23) found >95% death by 20 h.

In Vitro Protein Interactions-- Plasmids were constructed for coupled in vitro transcription/translation. Human BAX-alpha was subcloned into pcDNA3 (Invitrogen) from the PCR product obtained from the human hippocampal cDNA (as described above). Human BCL-2 cDNA was obtained from human hippocampal cDNA using the following primers: 5'-ggaattcgcggtgatggacgggtccgg-3' and 5'-ggaattctcagcccatcttcttccaga-3', and the resultant product was subcloned into pcDNA3. A partial BAX-omega clone, obtained from the library screen (see above), was ligated into PstI-digested pBluescriptSKII containing BAX-alpha , resulting in a complete clone. This full-length BAX-omega construct was then subcloned into EcoRI-digested pcDNA3. HA-truncated BAX-alpha (lacking the last 18 residues) and HA-BAX-omega were constructed into pcDNA3 by ligating a EcoRI/SalI or EcoRI, respectively, fragment containing the HA coding sequence in-frame with truncated BAX-alpha or full-length BAX-omega from the yeast expression vector pAS2-tBAX-alpha or pAS2-BAX-omega into EcoRI/XhoI or EcoRI-digested pcDNA3. Truncated BAX-alpha was constructed into pAS2 (CLONTECH) by standard PCR reactions. BAX-omega was constructed into pAS2 by ligating the BamHI/SalI fragment from a partial BAX-omega clone obtained from the library screen (see above) into BamHI/SalI-digested pAS2.

The coupled in vitro transcription and translation was performed with a commercially available kit (Promega). The 35S-labeled proteins were incubated overnight at 4 °C with anti-HA antibody (Boehringer Mannheim) in immunoprecipitation buffer (50 mM Tris (pH 7.5), 150 mM NaCl, and 0.2% Nonidet P-40) bound to protein A/G agarose beads (Santa Cruz Biotechnology) and washed three times with the same buffer containing 0.01% Nonidet P-40. The immunoprecipitated products were analyzed on 14% SDS gels. Gels were dried and exposed autoradiographically.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Cloning of a Novel BAX Splice Variant from Human Brain-- Human hippocampal cDNA was used to screen for brain-specific BAX homologues using the technique of reverse transcriptase/polymerase chain reaction (RT/PCR). By using synthetic primers, we obtained three different splice variants of BAX, including the previously described (8) BAX-alpha and BAX-gamma . Additionally, we obtained a novel splice variant that we have called BAX-omega . As shown in Fig. 1, BAX-omega is identical to BAX-alpha through the region of the protein encoded by exon 5; at this point, BAX-omega is generated as a result of the splice donor site on exon 5 joined to a site 49 base pairs 5' to the BAX-alpha acceptor site on exon 6 (Fig. 2). We have confirmed that the extra 49-bp insert found in BAX-omega is derived from the 3' end of intron 5 by sequencing a genomic clone of BAX (data not shown). The addition of 49 bp results in a translational frameshift and a protein distinct from BAX-alpha . In order to obtain the complete coding sequence for BAX-omega , it was necessary to use 3'-rapid amplification of cDNA ends as described under "Experimental Procedures." The complete coding sequence for BAX-omega is shown in Fig. 2. While BAX-alpha encodes a 21-kDa protein, the novel transcript predicts a larger 24-kDa protein. An interesting characteristic of this novel protein is that it contains no putative transmembrane domain, suggesting a cytosolic rather than membrane localization. BH1, BH2, and BH3 domains are almost entirely conserved, however.


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Fig. 1.   BAX-omega is generated by alternative RNA splicing. The alignment of these three proteins was accomplished using the Higgins-Sharp algorithm. Identical residues are indicated with black background. The areas of greatest homology are the three domains previously identified as BH1, BH2, and BH3. BAX-omega diverges from BAX-alpha at the end of the BH2 domain as a result of a translational frameshift.


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Fig. 2.   Alternative splicing generates multiple forms of BAX. The black boxes in the upper portion of this figure represent the different exons that comprise BAX-alpha and BAX-omega that are translated into protein. The smaller hatched box is that part of exon 6 that is translated into protein for BAX-omega only as a result of the translational frameshift. The hatched larger boxes represent untranslated mRNA. The thin dark lines represent the introns of the gene, and the heavy dark line represents the portion of intron 5 retained by BAX-omega . The actual amino acids contained within each of the two proteins beginning with the BH2 domain are shown at the bottom of the figure.

To confirm that this new form of BAX is not a cloning artifact, a human frontal cortex library was screened with a BAX-omega probe. Seven independent clones were obtained and sequenced. Sequence analysis of the clones showed that three of the clones encode nucleotides 71-800 of BAX-omega , whereas the other four encode BAX-alpha (data not shown). The discovery of this novel isoform of BAX in human brain suggested that it may represent a brain-specific form of BAX. To address this question, we evaluated the expression of BAX-omega and BAX-alpha (and other possible splice variants) in a number of different tissues.

BAX-omega Is Widely Distributed-- Ribonuclease protection analysis was performed to evaluate the tissue distribution of BAX-omega versus BAX-alpha . RNA from human brain, liver, heart, lung, and kidney were hybridized with an RNA probe synthesized from a cDNA clone containing a portion of the BAX-omega gene corresponding to nucleotides 300-600, which includes the unique 49-bp insert of BAX-omega . The resultant RNA probe was designed to recognize all of the known splice variants of BAX. As shown in Fig. 3, we observed three main transcripts that correspond to the predicted sizes for BAX-alpha , BAX-beta , and BAX-omega . Bgl II was used to digest the resultant bands to confirm their identity (data not shown). While BAX-alpha is the predominant transcript in human lung and kidney, both BAX-omega and BAX-beta are expressed at levels comparable to BAX-alpha in human brain, liver, and heart.


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Fig. 3.   BAX-omega , BAX-alpha , and BAX-beta mRNA are differentially expressed. Total RNA was isolated and RPA was performed as described under "Experimental Procedures." The RPA products were electrophoresed on an SDS-PAGE, and autoradiography was used to visualize the separated products. The tissues are as follows: lane 2, brain; lane 3, liver; lane 4, heart; lane 5, lung; lane 6, kidney. Lane 1 shows molecular mass markers.

We also evaluated the levels of protein of the two forms of BAX, BAX-alpha and BAX-omega , in rat tissues. As shown in Fig. 4, BAX-alpha and BAX-omega are expressed in all of the tissues examined including rat brain, heart, kidney, liver, and lung. Interestingly, the pattern of expression of the two proteins is quite distinct from one another. For example, BAX-omega is highly expressed in brain (upper panel, lane 1). BAX-alpha is highly expressed in lung (lower panel, lane 4), moderately expressed in brain and liver (lower panel, lanes 1 and 2), and barely detectable in heart and kidney (lower panel, lanes 3 and 5). These results are consistent with those of Krajewski et al. (42). In brain and liver we see a doublet for BAX-omega . When this antibody is preadsorbed with peptide, the lower band disappears suggesting that the upper band represents either a post-translationally modified form of BAX-omega , possibly a phosphorylated form, or that the upper band represents a nonspecific cross-reacting antigen. If the latter is true, then BAX-omega protein is undetectable in heart, lung, and kidney by this method.


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Fig. 4.   BAX-omega protein is detectable in rat tissues. Protein lysates were isolated from normal rat tissues as described under "Experimental Procedures." The protein lysates were electrophoresed on an SDS-PAGE and then probed with either NXrBAX-1 (top panel) or N20 (bottom panel; Santa Cruz Biotechnology). Antigen was detected using ECL (Amersham Pharmacia Biotech). Peptide antigen blocked antibody binding (data not shown). Lane 1, brain; lane 2, liver; lane 3, heart; lane 4, lung; lane 5, kidney.

This differential expression of BAX splice variants in different tissues suggested to us that the function of BAX-omega may have different relative importance from tissue to tissue depending on the expression of other BCL-2 family members in those tissues. Co-immunoprecipitation analysis was done to determine whether the translational product of BAX-omega interacts with other known members of the BCL-2 family of proteins.

Interaction of BAX-omega with Other BCL-2 Family Members-- Full-length BAX-omega , BAX-alpha , BCL-2, and a hemagglutinin protein of human influenza virus (HA)-tagged form of full-length BAX-omega (HA-BAX-omega ) and a HA-tagged form of truncated BAX-alpha (lacking its transmembrane domain, HA-tBAX-alpha ) were prepared by in vitro transcription and co-translation and assayed for their ability to co-immunoprecipitate. Translation of HA-BAX-omega produces a major product that migrates slightly higher than the untagged protein (Fig. 5, lower panel, compare 1st and 4th lanes). Lower molecular weight bands are also apparent, probably resulting from internal initiations at the first BAX-omega methionine and an internal methionine at amino acid residue 20. A similar pattern of one major product and several internal initiations is seen with the translation of HA-tBAX-alpha (Fig. 5, upper panel, 1st lane).


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Fig. 5.   BAX-omega interacts with BAX-alpha and BCL-2 by co-immunoprecipitation analysis. BAX-omega , BAX-alpha , and BCL-2 were translated in vitro alone or in combination with HA-tagged BAX-omega or HA-tagged truncated BAX-alpha . The translation products were either electrophoresed as lysates or were first immunoprecipitated with HA-specific antibodies and then electrophoresed on a 14% SDS-PAGE and visualized by autoradiography. Lysates of BAX-alpha (2nd lane, both panels), BAX-omega (4th lane, both panels), BCL-2 (6th lane, both panels), and SNAP25 (8th lane, both panels). Top panel, immunoprecipitates with HA-specific antibody of HA-tBAX-alpha  + BAX-alpha (1st lane), HA-tBAX-alpha  + BAX-omega (3rd lane), HA-tBAX-alpha +BCL-2 (5th lane), HA-tBAX-alpha  + SNAP25 (7th lane). Lower panel, immunoprecipitates with HA-specific antibodies of co-translated HA-BAX-omega  + BAX-alpha (1st lane), HA-BAX-omega  + BAX-omega (3rd lane), HA-BAX-omega  + BCL-2 (5th lane), or HA-BAX-omega  + SNAP25 (7th lane).

Different combinations of the cDNAs were co-transcribed and co-translated using [35S]Met-labeled proteins and were incubated with an anti-HA antibody. The anti-HA antibody immunoprecipitated HA-BAX-omega and HA-tBAX-alpha (Fig. 5, 1st lane, both panels) but not BAX-omega , BAX-alpha , or BCL-2 (data not shown). When HA-tBAX-alpha or HA-BAX-omega was co-translated with BCL-2, the anti-HA antibody co-precipitated BCL-2 indicating the interaction of both HA-tBAX-alpha and HA-BAX-alpha with BCL-2 (Fig. 5, upper panel, 5th lane; lower panel, 5th lane). When HA-tBAX-alpha was co-translated with BAX-omega or when HA-BAX-omega was co-translated with BAX-alpha , the anti-HA antibody co-precipitated the BAX-omega in the case of HA-tBAX-alpha (Fig. 5, upper panel, 3rd lane) and BAX-alpha in the case of HA-BAX-omega (Fig. 5, lower panel, 1st lane) indicating an interaction of full-length and truncated BAX-alpha with BAX-omega .

Dimerization of truncated BAX-alpha with full-length BAX-alpha was seen when HA-tBAX-alpha was co-translated with BAX-alpha and immunoprecipitated with the anti-HA antibody (Fig. 5, upper, lane 1). Dimerization of BAX-omega with itself was more difficult to establish. The lower molecular weight bands that result from translation of the HA-tagged proteins appear to co-precipitate with the tagged proteins (Fig. 5, lower panel, 3rd lane). This could result from the cross-reactivity of the antibody with these proteins or, alternatively, from the interaction of these proteins with HA-tBAX-alpha and HA-BAX-omega . We favor the latter explanation since Western blotting analysis showed that the lower bands are not directly recognized by the anti-HA antibody (data not shown). Co-precipitation of these lower bands with the HA-tBAX-alpha and HA-BAX-omega suggests an interaction of HA-tBAX-alpha with tBAX-alpha and HA-BAX-omega with BAX-omega .

The specificity of these interactions was confirmed by co-translation of HA-tBAX-alpha or HA-BAX-omega with SNAP-25 and immunoprecipitation with the anti-HA antibody. In these experiments, no interaction was observed between HA-tBAX-alpha or HA-BAX-omega with SNAP-25 (Fig. 5, upper panel, 7th lane; lower panel, 7th lane). As shown in Fig. 5 the lysates alone were run alongside the immunoprecipitations to validate the size of the protein being co-immunoprecipitated (upper and lower panels, 2nd, 4th, 6th, and 8th lanes).

Expression of BAX-omega protein in the rabbit reticulocyte lysate system and its subsequent interaction with BAX-alpha and BCL-2 proteins showed that the BAX-omega mRNA encodes for a functional protein. To evaluate its physiological function in vivo, we transfected BAX-omega into the mouse fibroblast cell line, L929, selected clones, and evaluated the expression level of the protein in the various clonal cell lines using Western blot analysis.

Expression of BAX-omega in Mammalian Cells-- An expression plasmid derived from pcDNA3 (Invitrogen) was constructed placing BAX-omega under the control of the CMV promoter. The resultant pcDNA3-BAX-omega plasmid was stably transfected into the mouse fibroblast cell line, L929, by electroporation. Twenty four clonal cell lines were obtained, and 12 were evaluated by Western analysis for the expression levels of BAX-omega protein using the BAX-alpha antibody, BAX (P-19) (Santa Cruz Biotechnology). The translational product of BAX-omega migrated at approximately 28 kDa in SDS-polyacrylamide gel electrophoresis as shown in Fig. 6. Two other bands are detected with this antibody that migrate at approximately 42 and 60 kDa. Although we do not know the nature of these bands, we can say that the 42-kDa band appears infrequently and randomly and, thus, appears to be nonspecific. The 60-kDa band is frequently seen but with a number of different antibodies, as well as with preimmune serum, suggesting that it too is a nonspecific band. Several of the transfected cell lines express BAX-omega protein (Fig. 6). Although all of the cells lines have detectable BAX-omega mRNA levels as shown by RT/PCR analysis (data not shown), some of the cell lines express no detectable levels of the protein using this antibody. One possibility for this difference is that the BAX-omega protein expressing cell lines express a more stable form of the protein. Another possibility is that the cellular milieu of some of the transfected cell lines is more amenable to the expression of or stability of BAX-omega protein (i.e. depending on where the gene inserts). To evaluate the function of BAX-omega , we evaluated its role in a model of apoptotic cell death, namely TNF-induced cell death.


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Fig. 6.   Transfected L929 cells express BAX-omega protein by Western analysis. Mouse fibroblast L929 cells were electroporated with pcDNA3-BAX-omega , and clonal cells lines were isolated. Proteins were prepared from some of the clonal cell lines and electrophoresed on SDS-PAGE. The proteins were transferred to polyvinylidene difluoride membranes and probed with antibody BAX(P19). Products were visualized on XAR-5 film by chemiluminescence (ECL; Amersham Pharmacia Biotech). Lane 1, vector-transfected L929; lane 2, clone 1; lane 3, clone 3; lane 4, clone 2; lane 5, clone 4; lane 6, clone 5; lane 7, clone 7; lane 8, clone 8; and lane 9, clone 11.

BAX-omega Expression Decreases TNF-induced Cell Death-- The BAX-omega -transfected L929 cells were used to determine whether BAX-omega has an activity similar to or distinct from BAX-alpha . Vector-transfected L929 or BAX-omega -transfected cells were treated with TNF (40 ng/ml) and cycloheximide (10 µg/ml) for 12 h. Cell death was measured using the technique of trypan blue exclusion. As shown in Fig. 7, of the three cell lines tested, pcDNA3BAX-omega 1, pcDNA3BAX-omega 3, and pcDNA3BAX-omega 8 all showed increased viability relative to the vector-transfected control. These data indicate that BAX-omega has a function opposite to that of BAX-alpha . To determine how long after treatment with 40 ng/ml TNF BAX-omega remained protective, a time course of treatment was done. As shown in Fig. 8, BAX-omega was protective up to 12 h following treatment. After 20 h, a majority of the cells died, probably because cycloheximide prevented the ongoing translation of all proteins. These data suggest that constitutive overexpression of BAX-omega protects L929 cells from TNF-induced cell death.


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Fig. 7.   BAX-omega promotes cell survival. Vector-transfected L929 cells and three BAX-omega overexpressing clonal cell lines were exposed to 40 ng/ml TNF with 10 µg/ml cycloheximide for 12 h in Opti-MEM (Life Technologies, Inc.). Following incubation, the cells were removed from the plates, stained with trypan blue, and counted. The percent viability represents the number of live cells divided by the total number of cells. Each data point represents the average of triplicate sample. Statistical analysis using the Peritz' F test (41) shows a significant difference between the various treatment groups (p < .03). Error bars are S.D. for three experiments.


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Fig. 8.   Time course of TNF-induced cell death in L929 and BAX-omega -transfected L929 cells. Vector-transfected L929 cells and three BAX-omega overexpressing clonal cell lines were exposed to 40 ng/ml TNF with 10 µg/ml cycloheximide for 4.5, 6.5, 12, and 20 h in Opti-MEM (Life Technologies, Inc.). Following incubation, the cells were removed from the plates, stained with trypan blue, and counted. The percent viability represents the number of live cells divided by the total number of cells. Each data point represents the average of triplicate samples. bullet  is vector-transfected L929 cells (control); open circle  is BAX-omega -transfected clone 1; triangle  is BAX-omega -transfected clone 3; diamond  is BAX-omega -transfected clone 8. Statistical analysis using the Peritz' F test (41) shows a significant difference between the various treatment groups (p < 0.05). Error bars are S.D. for three experiments.

Transient Overexpression of BAX-omega Increases Basal Levels of Cell Death in Monkey E5 Cells-- The small number of transfectants obtained from the BAX-omega -transfected L929 cells, relative to BCL-2-transfected cells, suggested to us that BAX-omega may have been deleterious to the cells. To address this question, we evaluated cell survival in cells transiently expressing BAX-omega .

BAX-omega was subcloned into plasmid, palpha 22beta galalpha 4GT, a plasmid previously used to transfect cells subsequently superinfected with herpes simplex virus deletion mutant, d120 (20). High transfection frequencies were obtained when this plasmid was used to transfect E5 cells or primary mouse cortical neurons.2 The palpha 22beta galalpha 4GT plasmid encodes for lacZ under the control of the alpha 22 promoter and gives rise to the expression of lacZ in E5 cells which is used as a measure of cells expressing our gene of interest. As shown in Fig. 9, fewer cells express BAX-omega than any of the other proteins, suggesting that transient overexpression of BAX-omega actually increases cell death. It can also be seen that neither BAX-omega nor BAX-alpha potentiates cell death induced by TNF or staurosporine (Fig. 10); however, BCL-2 transfected cells are significantly protected from cell death induced by these two effectors. Similar results were obtained with PC12, COS 7 cells, and primary cortical neural transfectants (data not shown).


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Fig. 9.   Transient overexpression of BAX-omega decreases cell survival in monkey kidney E5 cells. Monkey kidney E5 cells were transfected with palpha 22beta gal-BAX-omega , palpha 22beta gal-BAX-alpha , palpha 22beta gal-BCL-2 or vector only and the number of lacZ (+) cells were counted 48 h later. Error bars are the S.E. for 6 fields of triplicate samples (total = 18 fields).


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Fig. 10.   BAX-omega transient overexpression does not potentiate or attenuate TNF or staurosporine-induced cell death. Monkey kidney E5 cells were transfected with palpha 22beta gal-BAX-omega , palpha 22beta gal-BAX-alpha , palpha 22beta gal-BCL-2 or vector only, treated with TNF (10 ng/ml; upper panel) or staurosporine (lower panel) 24 h following transfection, and the number of lacZ (+) cells were counted an additional 24 h later. The results are presented as percent cell survival relative to untreated cultures. Error bars are the S.E. for 6 fields of triplicate samples (total = 18 fields).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Cell death is important to protect cells from uncontrolled cell growth, as a mechanism to protect surrounding cells from stress or trauma, and is a normal feature of embryonic or postnatal development of major tissues. The prototypical mammalian programmed cell death pathway gene is BCL-2 (2). BCL-2 functions as a repressor of cell death (24-26) and is the best understood member of this gene family.

The BCL-2 family members interact with each other as shown by the yeast two-hybrid (27) or co-immunoprecipitation analysis. The BH1, BH2, BH3, and BH4 (6) domains have all been shown to be critical for these protein-protein interactions (5, 28). BAX protein homodimerizes or heterodimerizes with BCL-2 or BCL-XL to affect cell death (5, 29). Overexpression of BAX-alpha counteracts the cell death repressing activity of BCL-2 and BCL-XL (5, 9). It has been proposed that the ratio of BCL-2 to BAX determines survival or death following an apoptotic signal (30). Site-directed mutagenesis studies (5) have shown that mutations that disrupt the heterodimerization interaction of BCL-2 with BAX but that still maintain the ability of BCL-2 to homodimerize, completely abrogate the death repressor action of BCL-2, suggesting that BAX alone or as a homodimer is sufficient to trigger death. Recently it has been demonstrated that the BH3 domain is critical for both homo- and heterodimerization between BCL-2, BCL-XL, and BAX (31, 32).

Dimerization of molecules is a mechanism that cells utilize to control their environment and destiny. For example, how does one regulate molecules that are themselves regulators? One way to do this is to change the phosphorylation state of the molecule, which in turn changes the partnering of the molecule. Partnering between BCL-2 family proteins may be dependent on the phosphorylation state of the different members since it has been demonstrated that phosphorylation of BCL-2 interferes with its ability to inhibit apoptotic cell death (33). It has been suggested that activation of Raf 1 kinase may be responsible for its phosphorylation, based on inhibitor studies (34).

BAX-alpha , BCL-2-alpha , and BCL-XL-alpha all possess a C-terminal hydrophobic domain that is thought to target the proteins to internal membranes. Interestingly, BRAG-1 (35), BFL-1 (36), or A1 (37), BCL-2 family members as well as BCL-XL-beta (38), a naturally occurring splice variant of BCL-XL, and BCL-2 Delta C22 (6), an artificially constructed form of BCL-2, all lack their hydrophobic domains and thus do not contain within their primary structure the conventional transmembrane hydrophobic domain and yet are still capable of repressing cell death.

Recently, the predicted three-dimensional structure of BCL-XL was solved (39). An arrangement of alpha -helices in BCL-XL that mimic the membrane translocation domain of bacterial toxins, such as diphtheria toxin, was demonstrated. The predicted membrane pore-forming domain is quite N-terminal to the transmembrane domain, suggesting that proteins lacking a conventional transmembrane domain, nevertheless, may be localized to membranes. Zha et al. (7) have shown in fact that a construct of BAX lacking its transmembrane domain (BAX Delta TM) retained its cell death function, dimerized with wild type BAX-alpha and localized to mitochondria. Thus, a conventional transmembrane domain is not essential for either organellar localization nor for function. We now describe another BCL-2 family member that is missing a hydrophobic domain and acts to potentiate cell death. BAX-omega , which lacks a transmembrane domain, retains the ability to bind to both BCL-2 and BAX-alpha proteins (and possible other death-effecting proteins) and potentiates basal cell death in BAX-omega -transfected cells. Overexpression of BAX-omega in the transient-transfected cells show that BAX-omega increases cell death comparable to BAX-alpha but that it does not potentiate cell death effected by other inducers such as TNF or staurosporine. BCL-2 transfectants were protected from TNF and staurosporine-induced cell death. Together these data suggest that BCL-2 and the two BAX isoforms converge with the death pathways of TNF and staurosporine.

Our results with the stably transfected cell lines, on the other hand, suggest that constitutive BAX-omega overexpression can protect a cell from further insult, possibly through the constitutive up-regulation of a protective protein. The absence of such a protein in a particular cell may have deleterious effects. For example, experiments with a BAX knock-out mouse (40) suggest a role for BAX (either BAX-alpha , BAX-omega , or both) in cell survival. In these mice, BAX-/BAX- male mice were infertile and showed increased apoptosis in the testes with clusters of apoptotic germ cells, whereas thymocytes and B cells displayed hyperplasia. This study suggested that, depending on the cell type, the action of BAX may be more anti-apoptotic than pro-apoptotic. It may be that BAX-omega is the predominant or, at least, an essential splice transcript in the testes, so that elimination of BAX eliminates not only BAX-alpha but BAX-omega . Thus, BAX deficiency is manifested as an increase or decrease in cell death depending on the cellular context.

The present set of investigations demonstrates that another form of BAX exists in most cell types. It will be interesting to determine under what conditions the ratio of the two forms of BAX change and how those changes affect the state of the cells. This work and the work of other labs suggest that RNA splicing may be an important form of control that the cell utilizes to express proteins with different functions (not necessarily opposing functions). Further investigations will be necessary to distinguish between these two forms of BAX and to determine under what conditions each of these molecules contributes to cell death and how they affect cell death within a cell. It will also be interesting to determine whether cells undergoing degeneration modify their expression of BAX-alpha and BAX-omega proteins by differential splicing, and what molecules are involved in that control. This has important implications for cancer, male fertility (as described above), as well as stroke, Alzheimer's disease, and other neurodegenerative processes.

    ACKNOWLEDGEMENTS

We thank Dr. Robert Sapolsky (Stanford University) for the expression vector, palpha 22beta gal-BCL-2, and Dr. Dora Ho (Stanford University) for help and advice. We thank Mr. Frank Chang for DNA sequencing and analysis. We thank Dr. Richard Scheller (Stanford University) and Dr. William Hopkins (Neurex Corp.) for critically reading the manuscript and for thoughtful comments.

    FOOTNOTES

* 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.

1 The abbreviations used are: PCR, polymerase chain reaction; RT/PCR, reverse transcriptase/polymerase chain reaction; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; TNF, tumor necrosis factor; RPA, ribonuclease protection analysis.

2 C. M. Bitler, M. Dimant, and M. Zhou, unpublished observations

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Results
Discussion
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