©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure-Function Analysis of Bcl-2 Protein
IDENTIFICATION OF CONSERVED DOMAINS IMPORTANT FOR HOMODIMERIZATION WITH Bcl-2 AND HETERODIMERIZATION WITH Bax (*)

Motoi Hanada , Christine Aimé-Sempé (§) , Takaaki Sato (¶) , John C. Reed (**)

From the (1) La Jolla Cancer Research Foundation, Oncogene and Tumor Suppressor Gene Program, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Bcl-2 protein is a suppressor of programmed cell death that homodimerizes with itself and forms heterodimers with a homologous protein Bax, a promoter of cell death. Expression of Bax in Saccharomyces cerevisiae as a membrane-bound fusion protein results in a lethal phenotype that is suppressible by co-expression of Bcl-2. Functional analysis of deletion mutants of human Bcl-2 in yeast demonstrated the presence of at least three conserved domains that are required to suppress Bax-mediated cytotoxicity, termed domains A (amino acids 11-33), B (amino acids 138-154), and C (amino acids 188-196). In vitro binding experiments using GST-Bcl-2 fusion proteins demonstrated that Bcl-2(B)and Bcl-2(C) deletion mutants had a markedly impaired ability to heterodimerize with Bax but retained the ability to homodimerize with wild-type Bcl-2. In contrast, Bcl-2(A) and an NH-terminal deletion mutant Bcl-2(1-82) retained Bax binding activity in vitro but failed to suppress Bax-mediated cytotoxicity in yeast. Sequences downstream of domain C in the region 197-218 also were shown to be required for Bax-binding in vitro and anti-death function in yeast. Analysis of Bcl-2/Bcl-2 homodimerization using both in vitro binding assays as well as a yeast two-hybrid method provided evidence in support of a head-to-tail model for Bcl-2/Bcl-2 homodimerization and revealed that sequences within the NH-terminal A domain interact with a structure that requires the presence of both the carboxyl B and C domains in combination. In addition to further delineating structural features within Bcl-2 that are required for homo-dimerization, the findings reported here support the hypothesis that Bcl-2 promotes cell survival by binding directly to Bax but suggest that ability to bind Bax can be insufficient for anti-cell death function.


INTRODUCTION

Programmed cell death plays an important role in a wide variety of physiological processes, including for example removal of redundant cells during development, elimination of autoreactive lymphocytes, and eradication of older, differentiated cells in most adult tissues with self-renewal capacity (reviewed by Ellis(1991), Green et al.(1992), and Williams(1991)). Dysregulation of this physiological mechanism for cell death has been implicated in a variety of human diseases, ranging from cancer and autoimmunity, where insufficient cell death can figure prominently, to AIDS and neurodegenerative disorders, where excessive death of T-lymphocytes and neurons occurs (reviewed by Reed (1994a), Green et al. (1992), Gougeon and Montagnier(1993), and Bredesen(1994)). One of the major regulators of programmed cell death and its morphological equivalent ``apoptosis'' (Wyllie et al., 1980) is the bcl-2 gene. The bcl-2 gene was first discovered because of its involvement in t(14;18) chromosomal translocations found in the majority of non-Hodgkin's follicular B-cell lymphomas (Tsujimoto et al., 1985; Tsujimoto and Croce, 1986), where it contributes to neoplastic expansion of germinal center B-cells by prolonging cell survival rather than by accelerating the rate of cell proliferation (McDonnell et al., 1989; Katsumata et al., 1992). Studies of a homolog of bcl-2, termed ced-9, in the worm Caenorhabditis elegans have suggested that this gene plays a master switch role in deciding the life and death fates of cells during development (Hengartner and Horvitz 1994). Gene transfer studies in several types of mammalian cells have shown that elevations in Bcl-2 protein levels can protect cells from death induced by a wide variety of diverse insults and stimuli, suggesting that Bcl-2 controls a distal step in what may represent a final common pathway for apoptotic cell death (reviewed by Reed (1994a) and Vaux(1993)).

The protein encoded by the bcl-2 gene has a unique sequence that has failed to provide clues as to the biochemical mechanism by which this protein functions as a blocker of cell death. Of note is the presence of a stretch of hydrophobic amino acids in the carboxyl tail of the Bcl-2 protein that allows for its post-translational insertion into intracellular membranes, particularly the outer mitochondrial membrane, nuclear envelope, and portions of the endoplasmic reticulum (Tsujimoto et al., 1987; Chen-Levy and Cleary, 1990; Krajewski et al., 1993). This transmembrane domain, however, can be expendable for Bcl-2 function at least in some types of cells (Borner et al., 1994), although it may serve to optimize Bcl-2's ability to oppose cell death in some cases (Tanaka et al., 1993; Hockenbery et al., 1993; Nguyen et al., 1994). Several cellular and viral homologs of Bcl-2 have been identified recently (reviewed by Reed (1994a)). Some of these act similarly to Bcl-2 and block cell death (Bcl-X-L, Mcl-1, A1), whereas others oppose Bcl-2 and accelerate apoptosis (Bax, Bcl-X-S) (Oltvai et al., 1993; Boise et al., 1993).() Co-immunoprecipitation experiments have suggested that Bcl-2 can bind to Bax, presumably forming heterodimers or heteromultimers (Oltvai et al., 1993). In addition, based on yeast two-hybrid experiments, Bcl-2 also appears to be able to homodimerize with itself as well as to form heterodimers with Bcl-X-L, Bcl-X-S, and Mcl-1 (Sato et al., 1994a). These findings have suggested therefore that interactions among various members of the Bcl-2 protein family control the sensitivity or resistance of cells to apoptosis.

Amino acid sequence alignments of Bcl-2 with its various homologs have revealed three evolutionarily conserved domains, which we have previously termed Bcl-2 domains (BDs)() A, B, and C (Sato et al., 1994a, 1994b). Mutant forms of Bcl-2 that lack BD(B) or BD(C) or that contain particular amino acids substitutions in these conserved domains have been shown in mammalian cells to have impaired ability to co-immunoprecipitate with Bax and to block cell death, but remain capable of homodimerizing with endogenous wild-type Bcl-2 (Yin et al., 1994). Furthermore, during attempts to study Bcl-2/Bax interactions by use of yeast two-hybrid methods, we discovered that expression of Bax in yeast as fusion proteins with either an NH-terminal DNA-binding or trans-activation domain resulted in a lethal phenotype that could be specifically suppressed by co-expression of Bcl-2 (Sato et al., 1994a). Deletional analysis of Bcl-2 in this system revealed that removal of either the first 81 amino acids of Bcl-2 where BD(A) is located or deletion of amino acids 83-218 where BD(B) and BD(C) reside resulted in loss of suppression of Bax-mediated cytotoxicity without impairing ability to homodimerize with full-length Bcl-2 (Sato et al., 1994a). Taken together, these findings obtained in both mammalian cells and yeast suggest that, for Bcl-2 to function as a blocker of cell death, it must be able to heterodimerize with Bax. The importance of Bcl-2/Bcl-2 homodimers remains obscure at present.

To further delineate structure-function relations within the Bcl-2 protein, in this report we analyzed mutants of Bcl-2 lacking the conserved domains BD(A), BD(B), and BD(C) with regard to: 1) ability to neutralize Bax-mediated cytotoxicity in yeast and 2) binding to Bax and Bcl-2 in vitro; and 3) interactions with Bcl-2 deletion mutants in yeast two-hybrid assays.


MATERIALS AND METHODS

Plasmid Constructions

Yeast two-hybrid plasmids included pEG202 which utilizes a constitutive ADH promoter for production of fusion proteins containing a LexA DNA-binding domain at the NH terminus. The plasmid pJG4-5 contains a galactose-inducible Gal-1 promoter and was used for producing fusion proteins with an NH-terminal B42 trans-activation domain (Golemis et al., 1994; Zervos et al., 1993). The preparation of pEG202 and pJG4-5 plasmids encoding human Bcl-2 without its transmembrane domain (amino acids 1-218) or full-length mouse Bax (amino acids 1-191) in-frame with the upstream LexA and B42 sequences have been described, as well as the NH-terminal truncation mutants Bcl-2(72-218) and Bcl-2(83-218), the COOH-terminal truncation mutant Bcl-2(1-81), and plasmids that produce fusion proteins containing the Bcl-X-S protein without its transmembrane domain (Sato et al., 1994a). Using the pEG202-Bcl-2(1-218), pJG4-5-Bcl-2(1-218), and other previously described plasmids, nucleotides encoding amino acids 11-33 (A), 138-154 (B), and 188-196 (C) were deleted using the polymerase chain reaction overlap extension method (Ho et al., 1989) with specific primers and methods essentially as described (Tanaka et al., 1993). In addition, a Bcl-2 mutant was constructed that lacked amino acids 143-146 (NWGR), thus removing a well conserved sequence NWGR. Plasmids encoding the 205-amino acid human Bcl-2 protein were generated by first subcloning a BamHI-AvaII fragment encompassing the splice site and downstream intron sequences from the genomic clone p18-21H (Tsujimoto and Croce, 1986) into pSKII-bcl-2- (Tanaka et al., 1993) which had been prepared by digestion of the vector with SpeI, blunting with Klenow fragment and T4 DNA polymerases, followed by cleavage within the bcl-2 cDNA with BamHI to liberate a 0.2-kilobase pair fragment and gel purification of the larger plasmid band. A 0.2-kilobase pair BamHI/NotI fragment from the resulting pSKII-bcl-2 plasmid was then subcloned into pEG202-Bcl-2(1-218) (Sato et al., 1994a) which had been digested with BamHI and NotI, thus replacing the sequences beginning at the BamHI site in bcl-2 with bcl-2. A COOH-terminal deletion mutant of Bcl-2 lacking all sequences located downstream of residue 196 was also created by annealing 200 pmol each of the oligonucleotides 5`-GATCCAGGATAACGGAGGCTGGGATTGAC-3` and 5`-TCGAGTCAATCCCAGCCTCCGTTATCCTG-3`, and then subcloning into EcoRI/XhoI-digested pEG-202, thus introducing a stop codon after residue 196. The cDNAs encoding Bcl-2 and Bcl-2(1-196) were removed from their pEG202 vectors by digestion with EcoRI and XhoI and subcloned into the corresponding sites in pJG4-5. The expression of all LexA and B42-fusion proteins in yeast was confirmed by immunoblotting using an antiserum specific for LexA (kindly provided by E. Golemis) or a monoclonal antibody specific of the hemagglutinin-epitope tag (Boeringer Mannheim) incorporated into the B42 fusion proteins, essentially as described previously (Sato et al., 1994a). For expression as GST fusion proteins in Escherichia coli, the cDNA inserts encoding wild-type and mutant Bcl-2 proteins were excised from their pEG202 or pJG4-5 plasmids using EcoRI and XhoI and subcloned into pGEX-4T1 (Pharmacia Biotech Inc.). Proper construction of all plasmids was confirmed by DNA sequencing.

Plasmid Transformations in Yeast and Two-hybrid Assays

Yeast strain EGY191 (MAT, trp1, ura3, his3, LEU2::pLexAop1-LEU2) containing the lacZ reporter gene plasmid pSH 18-34 (contains URA3 marker) was co-transformed with 5 µg each of pEG202-Bax(1-191) and pJG4-5 plasmids containing Bcl-2(1-218) or various Bcl-2 deletion mutants using a LiOAc method as described previously (Sato et al., 1994a). Half of the cells were then plated on galactose-containing and half on glucose-containing Burkholder's minimal media lacking tryptophan, histidine, and uracil but containing leucine for selection of the pEG202 (HIS3) and pJG4-5 (TRP1) plasmids, respectively. Cells were grown at 30 °C for 4-7 days, and growth was scored as positive or negative based on the number of colonies that formed having a diameter of 1 mm.

Yeast two-hybrid experiments were performed exactly as described previously (Sato et al., 1994a). Briefly, after transformation with various pEG202 and pJG4-5 plasmids and plating on galactose-containing minimal media lacking tryptophan, histidine, and uracil, at least six independent colonies were transferred to plates containing similar medium, with the following modifications: (a) galactose+, leucine-; (b) glucose+, leucine-; (c) galactose+, X-gal+; (d) glucose+, X-gal+. Galactose-dependent growth on leucine-deficient media was then assessed 4-7 days later, and galactose-dependent production of -galactosidase was assayed 1-6 h later, by plate and filter assays as described by Sato et al. (1994a).

Preparation of GST Fusion Proteins and in Vitro Binding Assays

The pGEX-4T1 plasmids containing various wild-type or mutant bcl-2 cDNA inserts were transformed into XL-1 Blue strain E. coli (Stratagene, Inc.). Bacteria were grown at 37 °C in 0.5 liter of LB medium containing 50 µg/ml ampicillin to an OD of 0.6, then cells were transferred to 30 °C and 1 mM isopropyl-1-thio--D-galactopyranoside was added to the medium. After incubation for 4-6 h, bacteria were recovered by centrifugation, and the resulting pellet was frozen overnight at -80 °C. After resuspension in 10 ml of 50 mM Hepes (pH 7.4), 150 mM NaCl, 2 mM EDTA, 5 mM 2-mercaptoethanol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and lysozyme (10 mg) were added, and the samples were incubated for 0.5 h on ice prior to sonication on ice twice for 60 s using a 2-mm diameter probe at setting 3.5 (Heat Systems model CL4; Farmingdale, NY). After centrifugation at 5,000 g (Sorvall SS-34 rotor at 8,000 rpm) for 40 min at 4 °C, 1.5-ml aliquots of the resulting supernatants were mixed with 0.15 ml of glutathione-Sepharose (Pharmacia) for 2 h at 4 °C. The beads were then washed four times in HKM solution (10 mM Hepes (pH 7.2), 142.5 mM KCl, 5 mM MgCl, 1 mM EGTA, 0.2% Nonidet P-40) and resuspended in 0.15 ml of the same solution. A 5-µl aliquot of the suspension was then subjected to SDS-PAGE, and the gels were stained with Coomassie Blue to estimate the amount of GST fusion protein bound per µl of beads based on comparisons with a standard curve run in the same gel consisting of from 2.5-20 µg of bovine serum albumin per lane.

In vitro binding assays were performed by mixing 10-20 µg of GST proteins immobilized on glutathione-Sepharose with 10 µl of L-[S]methionine-labeled in vitro translated Bcl-2 or mouse Bax proteins in 0.15-0.2 ml of HMK solution for 2 h at 4 °C. Beads were washed four times in HMK before boiling in Laemmli buffer. Eluted proteins were analyzed by SDS-PAGE (12% gels), and the resulting gels were fixed in 25:65:10 isopropanol/water/acetic acid and stained with Coomassie Blue to verify loading of approximately equal amounts of mostly intact GST fusion proteins, prior to impregnation of gels with fluorographic reagent (Amplify; Amersham Corp.), drying, and exposure to x-ray film (Kodak XAR) with intensifying screens at -80 °C. The in vitro translated proteins were prepared using rabbit reticulocytes lysates (TNT-lysate; Promega) and the plasmids pSKII-bcl-2- or pSKII-bax, essentially as described in detail elsewhere (Tanaka et al., 1993; Miyashita et al., 1994; Krajewski et al., 1994).

RESULTS

Effects of Deletion of BD(A), BD(B), or BD(C) on Bcl-2/Bcl-2 Homodimerization

Bcl-2 deletion mutants lacking either BD(A), BD(B), or BD(C) were tested for ability to homodimerize using a yeast two-hybrid method. For these experiments, Bcl-2 proteins were expressed as fusion proteins with NH-terminal extensions representing either a LexA DNA-binding domain or B42 trans-activation domain. The COOH-terminal 21 amino acids that encode the transmembrane domain of Bcl-2 were omitted by introduction of a stop codon after position 218, so as to avoid problems with targeting of proteins to the nucleus. Protein-protein interactions were detected in yeast co-transformed with pairs of LexA- and B42-expression plasmids, resulting in trans-activation of LEU2 and lac-Z reporter genes under the control of lexA operators.

As summarized in , co-transformation of cells with a LexA expression plasmid that encodes ``full-length'' Bcl-2 protein (amino acids 1-218) with B42 expression plasmids encoding mutant versions of Bcl-2 with deletions of BD(A), BD(B), or BD(C) resulted in activation of both the LEU2 reporter gene (as determined by ability to form colonies on leucine deficient semisolid medium) and the lacZ reporter gene (based on strong blue color of cells grown on medium containing X-gal). In addition, a deletion mutant lacking only the well conserved NWGR motif within BD(B) at residues 143-146 also mediated interactions with the LexA-Bcl-2(1-218) protein (). Similar results where obtained when the BD(A), BD(B), BD(C), and NWGR deletion mutants were expressed as LexA fusion proteins and the full-length Bcl-2(1-218) protein was expressed as a B42 fusion protein. In addition, fusion proteins incorporating Bcl-2(1-218) also interacted with fusion proteins containing the 205-amino acid Bcl-2- protein, a form of Bcl-2 that arises through alternative splicing and that diverges in its sequence precisely after the BD(C) domain (Tsujimoto and Croce, 1986). These results thus indicate that BD(A), BD(B), BD(C) when deleted individually, as well as sequences located between BD(C) and the transmembrane domain(199-218) when substituted with the completely nonhomologous sequences in Bcl-2-, are not essential for homodimerization with wild-type Bcl-2 protein. At least qualitatively, there was no gross difference in the strength of the interactions of these deletion mutants of Bcl-2 with wild-type Bcl-2, compared to wild-type Bcl-2 with itself (not shown).

Our previous studies of Bcl-2/Bcl-2 homodimerization suggested that this protein-protein interaction involves a head-to-tail arrangement where a structure or structures present in the NH-terminal domain of Bcl-2 (amino acids 1-81) interacts with elements located in the COOH-terminal portion of Bcl-2(83-218) (Sato et al., 1994a). We therefore tested the ability of various deletion mutants of Bcl-2 to interact with other Bcl-2 deletion mutants. As indicated in , when BD(A), BD(B), BD(C), or NWGR was deleted in both two-hybrid partners, no interaction was detected. Further analysis of various pairs of deletion mutants revealed that some mutations can complement others where Bcl-2/Bcl-2 homodimerization is concerned (). For example, deletion mutants of Bcl-2 lacking BD(A) interacted with deletion mutants lacking BD(B) or BD(C) but not with mutants lacking BD(A). In addition, deletion mutants lacking BD(B) interacted with mutants lacking BD(A) but not BD(B) or BD(C). The NWGR deletion mutant exhibited interaction properties identical to the Bcl-2(B) mutant. Similarly, a mutant lacking BD(C) was capable of interacting with the deletion mutants lacking BD(A) but not BD(B) or BD(C) (). Studies of the interactions of the Bcl-X-S protein with various Bcl-2 deletion mutants yielded results comparable to those obtained for the Bcl-2(NWGR), Bcl-2(B), and Bcl-2(C) mutants. Bcl-X-S mediated interactions with Bcl-2(1-218) and Bcl-2(A), but not with the Bcl-2(NWGR), Bcl-2(B), and Bcl-2(C) mutants, consistent with the structural features of this version of the Bcl-X protein which lacks BD(B), BD(C), and the sequences located between these domains because of an alternative splicing event (Boise et al., 1993). Finally, the Bcl-2 protein also behaved similar to Bcl-2(NWGR), Bcl-2(B) and Bcl-2(C), interacting with Bcl-2(1-218) and Bcl-2(A) but not with the Bcl-2(NWGR), Bcl-2(B), or Bcl-2(C). Thus, sequences located downstream of BD(C), where the Bcl-2- sequence diverges from Bcl-2-, evidently are also important for Bcl-2/Bcl-2 homodimerization.

Taken together, these data are consistent with our previously proposed model in which the NH-terminal portion of Bcl-2 where BD(A) is located interacts with the COOH-terminal part of Bcl-2 where BD(B) and BD(C) reside. The findings extend those previous observations by showing that sequences within the conserved BD(A), BD(B), and BD(C) domains are required for these interactions. Furthermore, these experiments indicate that for the formation of Bcl-2/Bcl-2 homodimers, one of the two partners must have an intact BD(A) domain and the other must contain both the BD(B) and BD(C) regions simultaneously. As shown in , if either BD(B) or BD(C) is deleted in one partner, then interactions can no longer occur with versions of Bcl-2 that retain a BD(A) domain. In addition to the simultaneous presence of intact B and C domains, sequences located downstream of BD(C), between BD(C) and the transmembrane domain of Bcl-2 (i.e. residues 197-218), also appear to be required for binding to the NH-terminal region of Bcl-2, based on the results obtained with the Bcl-2 protein ().

To further test some of these ideas, experiments were performed using fusion proteins that contained only the NH-terminal first 81 amino acids of Bcl-2 (residues 1-81) where BD(A) resides or the COOH-terminal domain from position 83 to the transmembrane domain (i.e. residues 83-218) where BD(B) and BD(C) are located. As summarized in , the Bcl-2(1-81) fragment interacted only with the wild-type Bcl-2 protein(1-218) and the Bcl-2(A) mutant, but not with deletion mutants that lacked either BD(B), the NWGR motif within BD(B), or BD(C). Thus, for the Bcl-2(1-81) region to interact with Bcl-2, both the BD(B) and BD(C) domains must be present. Similarly, experiments performed with the fusion proteins containing the Bcl-2(83-218) fragment revealed interactions with wild-type Bcl-2(1-218), Bcl-2(B), Bcl-2(NWGR), and Bcl-2(C), but not with the Bcl-2(A) mutant which is lacking BD(A). These results thus indicate that, for the Bcl-2(83-218) region to interact with Bcl-2, the BD(A) domain must be present but there is no requirement for BD(B) or BD(C).

Finally, to further confirm these results, a fragment of the Bcl-2 protein containing only residues 1-81 and a mutant version of this fragment lacking BD(A) (11-33) were expressed as fusion proteins in yeast with an NH-terminal B42-trans-activation domain. The ability of the Bcl-2(1-81) and Bcl-2(1-81/A) proteins to interact with a fusion protein consisting of a LexA DNA binding domain linked to a fragment of the Bcl-2 protein containing only residues 83-218 was then tested by two-hybrid assays. As summarized in , the Bcl-2(1-81) protein interacted with Bcl-2(83-218), whereas Bcl-2(1-81/A) did not. Thus, the BD(A) region which encompassed residues 11-33 is required for the 1-81 fragment of Bcl-2 to interact with the 83-218 region of Bcl-2. In addition, deletion mutants of the 83-218 fragment were created that lacked either BD(B) (residues 138-154) or BD(C) (residues 188-196). Neither the Bcl-2(83-218/B) nor the Bcl-2 (83-218/C) proteins mediated interactions with Bcl-2(1-81). Moreover, deletion of only the four residues (NWGR) located within the BD(B) domain at 143-146 was sufficient to prevent the 83-218 portion of Bcl-2 from interacting with Bcl-2(1-81). These findings thus are again consistent with the idea that the NH-terminal region of Bcl-2 from residues 1-81 interacts in a BD(A)-dependent fashion with the COOH-terminal portion of Bcl-2 from 83-218 in a manner that requires the simultaneous presence of intact BD(B) and BD(C) domains.

The BD(A), BD(B), and BD(C) Regions Are Important for Neutralization of Bax-mediated Cytotoxicity in Yeast

To explore the effects of various deletion mutants on the ability of Bcl-2 to abrogate Bax-mediated cytotoxicity in yeast, B42-expression plasmids encoding the same Bcl-2 mutants described above were co-transformed with a LexA-expression plasmid that produces a fusion protein consisting of the full-length Bax protein with an appended NH-terminal LexA DNA-binding domain (Sato et al., 1994a), and colony formation was assessed after plating cells on appropriate selective medium containing either galactose to induce the Gal-1 promoter that controls the expression of B42-Bcl-2 fusion proteins or on glucose-containing medium which represses this promoter. summarizes the results for all mutants tested, and Fig. 1B shows the results from a representative experiment. In contrast to full-length Bcl-2(1-218) protein, internal deletion mutants of Bcl-2 that lacked BD(A), BD(B), or BD(C) were ineffective at blocking Bax-mediated cytotoxicity (). In addition, a Bcl-2 deletion mutant lacking only four residues (NWGR at 143-146) was also unable to neutralize Bax-induced cytotoxicity in yeast. Bcl-2- also failed to neutralize Bax, implying that the sequences downstream of BD(C) in Bcl-2- ineffectively substitute for the usual Bcl-2 sequences. Consistent with these findings obtained for Bcl-2-, a COOH-terminal deletion mutant of Bcl-2 that retained only residues 1-196 of Bcl-2 and thus was lacking all sequences downstream of BD(C) where Bcl-2- and Bcl-2- diverge was also unable to neutralize Bax-mediated cytotoxicity in yeast (). As reported previously, NH-terminal truncation mutants of Bcl-2 (72-218 and 83-218) that contain BD(B) and BD(C) but not BD(A), as well as a COOH-terminal truncation mutant (1-81) that contains BD(A) but not BD(B) or BD(C) were also unable to neutralize Bax-mediated cytotoxicity. Immunoblot assays verified that all of these Bcl-2 deletion mutants were produced at levels equivalent to or greater than the Bcl-2(1-218) full-length protein in yeast (Sato et al., 1994a) (data not shown), excluding protein instability as a trivial explanation for the findings.


Figure 1: Bax inhibits growth of yeast through a Bcl-2 suppressible mechanism. In A, the structure of the human Bcl-2 protein is depicted, showing the locations of BD(A), BD(B), and BD(C), and the transmembrane (TM) domain. In B, the results of an experiment are presented where EGY191 cells were co-transformed with 5 µg of pEG202-Bax and 5 µg of various pJG4-5 expression plasmids as indicated. Equal aliquots of the transformed cells were then plated on His-, Tryp-, Ura- medium containing leucine and either galactose () or glucose () to activate or repress, respectively, the Gal1 promoter in pJG4-5. The number of colonies with diameter 1 mm was counted 7 days later. In C, an example of the Bax colony assay is shown. EGY191 cells were transformed with 5 µg of pEG202-Bax and 5 µg of pJG4-5 plasmids encoding B42 fusion proteins with Bcl-2(1-218) or Bcl-2(72-218), as indicated. Transformed cells were plated on His-, Tryp-, Ura- medium containing leucine and either galactose or glucose. Plates were photographed at 7 days. As a control, EGY191 cells were also transformed with a pEG202 plasmid in which the bax cDNA was subcloned in reverse (antisense) orientation.



Fig. 1C shows an example of the results of this Bax suppression assay. When plated on galactose-containing medium, cells transformed with the LexA-Bax plasmid and a plasmid that produces a B42/Bcl-2(1-218) protein formed a significant number of colonies. In contrast, when an aliquot of these same transformants were plated on glucose-containing medium which represses the Gal-1 promoter that controls production of the B42/Bcl-2(1-218) protein, very few or no colonies appeared. Deletion mutants of Bcl-2(1-218) such as an NH-terminal truncation mutant of Bcl-2 missing the first 71 amino acids were ineffective at suppressing Bax-mediated inhibition of colony formation. When a control LexA expression plasmid was used in which the bax cDNA had been subcloned in antisense orientation into pEG202, large numbers of colonies formed regardless of the presence or absence of Bcl-2, indicating that the bax cDNA sequence is not nonspecifically toxic when introduced into yeast. The difference in the numbers of colonies formed when pEG202-Bax and pEG202-Bax-antisense plasmids were transformed, however, suggests that Bcl-2(1-218) only partially suppresses cytotoxicity mediated by the Bax protein in yeast, at least under the conditions of these functional assays.

Analysis of Binding of Bcl-2 Mutants to Wild-type Bax and Bcl-2 Proteins in Vitro

Next, the findings from the yeast two-hybrid assays and the Bax cytotoxicity studies in yeast described above were compared with the physical binding characteristics of mutant Bcl-2 proteins using in vitro protein interaction assays. For these experiments, the various deletion mutants of Bcl-2 described above were expressed in E. coli as GST fusion proteins and affinity-purified. These GST fusion proteins, immobilized on glutathione-Sepharose, were then incubated with either S-labeled Bcl-2 or S-labeled Bax that had been prepared by in vitro translation using rabbit reticulocyte lysates. After extensive washing, specific binding was detected by SDS-PAGE followed by fluorography.

In vitro translated wild-type Bcl-2 appeared to bind with roughly comparable efficiency to all GST-fusion proteins tested, including Bcl-2(1-218) (full-length), Bcl-2(1-196), Bcl-2(A), Bcl-2(B), Bcl-2(C), Bcl-2-, Bcl-2(1-81), Bcl-2(72-218), and Bcl-2(83-218) ( Fig. 2and data not shown). In general, approximately 5-10% of the total in vitro translated Bcl-2 protein was recovered in association with the GST-Bcl-2 fusion proteins. The specificity of these results were confirmed by experiments performed using various control GST fusion proteins, including CD40, TNF-R1, TNF-R2, BAP-1, and Fas, as well as GST nonfusion protein ( Fig. 2and data not shown). In addition, in vitro translated S-labeled Lyn kinase did not bind to any of the GST-Bcl-2 fusion proteins, providing further evidence that these protein-protein interactions are specific (data not presented).


Figure 2: Analysis of binding of Bcl-2 deletion mutants to Bcl-2 and Bax by in vitro binding assay. GST fusion proteins (10-20 µg) were immobilized on glutathione-Sepharose and incubated with 10 µl of reticulocyte lysates containing in vitro translated S-labeled Bcl-2 (top) or Bax (bottom). After extensive washing, beads were boiled in Laemmli buffer and eluted proteins were analyzed by SDS-PAGE (12% gels) and detected by fluorography. In some lanes, 1 µl of in vitro translated (IVT) proteins were run directly in the gel as a control. GST fusion proteins encoding portions of the tumor necrosis factor type II-receptor (TNF-R2), the 14-3-3 protein BAP-1, or CD40 were used as additional negative controls. The lanes marked GST represent GST nonfusion proteins. All GST-Bcl-2 fusion proteins lacked the transmembrane domain and thus terminated at residue 218 because of an introduced stop codon. Results from two independent experiments are presented; (A) and (B).



In contrast to Bcl-2, in vitro translated wild-type Bax protein physically interacted only with the full-length GST-Bcl-2(1-218), Bcl-2(A), and Bcl-2(83-218) proteins (Fig. 2). None of the other internal or end deletions of Bcl-2 retained the ability to bind to Bax in vitro at appreciable levels under these conditions. Again, the specificity of these results was confirmed by use of control GST nonfusion and fusion proteins, to which Bax failed to bind ( Fig. 2and not shown).

DISCUSSION

Bcl-2 represents the first member of a family of homologous proteins that regulate programmed cell death and apoptosis. This protein has been shown to both homodimerize with itself, as well as to form heterodimers with other members of the Bcl-2 protein family (Oltvai et al., 1993; Sato et al., 1994a; Yin et al., 1994). The Bcl-2 protein is produced at high levels in many types of cancer, including about 90% of colorectal, 30-60% of prostate, 70% of breast, 20% of non-small cell lung cancers, and 65% of lymphomas (reviewed by Reed (1994b). In vitro, the levels of Bcl-2 protein have been demonstrated to be an important regulator of the relative response of tumor cells to induction of apoptosis by chemotherapeutic drugs and radiation, with gene transfer-mediated elevations in Bcl-2 associated with marked resistance to anticancer agents and antisense-mediated decreases correlated with increased sensitivity (Miyashita and Reed, 1992, 1993; Kitada, et al., 1994; Campos et al., 1994). In vivo, expression of Bcl-2 has been associated with poor responses to therapy in at least some subgroups of cancer patients, including some patients the lymphomas, acute leukemia, and prostate cancer (Yunis et al., 1989; Offit et al., 1989; Campos et al., 1993; McDonnell et al., 1992). Improved understanding of the structural details of how Bcl-2 participates in homo- and heterotypic interactions with itself and other members of the Bcl-2 protein family thus may create opportunities for eventually pharmacologically modulating the activity of this oncoprotein for the improved treatment of cancer.

Bcl-2/Bcl-2 Homodimerization

The data presented here suggest that a region located in the NH-terminal portion of Bcl-2 that includes BD(A) is required for binding to a more carboxyl region in partner Bcl-2 molecules during homodimerization. Necessary structures within this more distal region within Bcl-2 appear to include both the BD(B) and BD(C) domains, since deletion mutants lacking either of these segments were unable to homodimerize with Bcl-2(1-81) containing an intact BD(A) domain, in yeast two-hybrid assays. Sequences located downstream of BD(C), between residues 196 and 219, also appear to be important, based on the findings obtained with the Bcl-2 protein which diverges from the usual Bcl-2 protein beyond position 196. Thus, presumably the BD(B) and BD(C) domains together with some addition downstream sequences cooperate to form a structure that is recognized by sequences in the NH-terminal portion of Bcl-2 that include or depend on the BD(A) domain. It remains to be determined however whether these represent actual contact sites in the Bcl-2 protein that participate directly in homodimerization, versus segments that play an indirect role in helping the molecule to assume an active conformation or that are required for proper spacing of other domains. The strong conservation of the amino acid sequences of the the BD(B) and BD(C) domains across broad evolutionary distances, however, tends to support the former possibility. In contrast to BD(B) and BD(C), the region between BD(C) and the transmembrane domain is not well conserved among Bcl-2 homologs (Sato et al., 1994b), raising the possibility that it contributes more indirectly in facilitating Bcl-2/Bcl-2 homodimerization.

At present the functional significance of Bcl-2/Bcl-2 homodimers remains unknown. In studies in which mutant forms of Bcl-2 were expressed in mammalian cells that contain endogenous Bax and Bcl-2, it was shown that Bcl-2 mutants that failed to bind to Bax had lost function in terms of blocking cell death, and yet still retained the ability to bind to endogenous wild-type Bcl-2 protein (Yin et al., 1994). Although these experiments have been interpreted as evidence against a functionally significant role for Bcl-2/Bcl-2 homodimers in regulating cell death, it remains possible that Bcl-2/Bcl-2 homodimers act as cell death blockers and that these Bcl-2 mutants act in a dominant-negative fashion to prevent homodimerization of wild-type Bcl-2 proteins and thus fail to rescue cells from apoptosis. Alternatively, if Bcl-2/Bcl-2 homodimers are not operative in an anti-cell death pathway and instead Bcl-2 binding to Bax is critical for Bcl-2's function as an anti-apoptotic protein, then Bcl-2/Bcl-2 homodimerization conceivably could contribute indirectly to cell death by sequestering Bcl-2 molecules in a form that prevents them from simultaneously forming heterodimers with Bax. In this scenario, mutations that prevent Bcl-2/Bcl-2 homodimerization but that do not interfere with Bax/Bcl-2 heterodimerization might be more potent as cell death blockers than wild-type Bcl-2. Although additional mutagenesis studies are underway, to date we have yet to identify a mutant of Bcl-2 that retains the ability to heterodimerize with Bax but that has lost the capacity to bind to wild-type Bcl-2. Consequently, it has not been possible to test this hypothesis that such mutants would have a gain of function.

In either model for explaining the relative significance of Bcl-2/Bcl-2 and Bcl-2/Bax dimers, mutants of Bcl-2 that retain the ability to homodimerize with wild-type Bcl-2 but that fail to bind to Bax could potentially function as dominant inhibitors of wild-type Bcl-2. This is presumably how the Bcl-X-S protein, for example, promotes cell death and antagonizes Bcl-2, since it binds to Bcl-2 but not Bax (Sato et al., 1994a). Similarly, the Bcl-2 protein conceivably could function as an inhibitor of Bcl-2, since it was capable of binding to Bcl-2 based on yeast two-hybrid experiments and in vitro binding assays but failed to interact significantly with Bax protein in vitro and was considerably less active than Bcl-2 in abrogating the lethal effects of Bax in yeast. In this regard, we have shown previously that, when expressed in interleukin-3-dependent 32D.3 cells, Bcl-2 failed to provide protection against apoptosis induced by lymphokine withdrawal and, in fact, slightly accelerated the rate of cell death (Tanaka et al., 1993). Conversely, both Bcl-2 and Bcl-2 increased the rate of tumor formation by NIH-3T3 fibroblasts in nude mice (Reed et al., 1988). Tumorigenicity, however, is a complex phenotype with many factors and selection pressures contributing to the final outcome, and thus it is difficult to assess the relevance of this observation to cell death regulation.

Bcl-2/Bax Heterodimerization

Analysis of Bcl-2 deletion mutants demonstrated that not all mutants which retain ability to heterodimerize with Bax in vitro can neutralize Bax-mediated cytotoxicity in yeast. Specifically, deletion of the first 82 amino acids of Bcl-2 or of residues 11-33 (A) abrogated function in yeast but had no discernible effects on binding to Bax in vitro. In contrast, the Bcl-2(B), Bcl-2(NWGR), Bcl-2(C), Bcl-2(1-81), and Bcl-2(1-196) deletion mutants, as well as the Bcl-2- protein, failed to both nullify Bax lethality in yeast and to bind to Bax in vitro. The failure of all the Bcl-2 deletion mutants described here as well as Bcl-2 to inhibit Bax-mediated cytotoxicity in yeast was not due to instability of these proteins in Saccharomyces cerevisiae, based on immunoblot comparisons of the relative levels of wild-type and mutant Bcl-2 proteins (Sato et al., 1994a) (data not shown). Furthermore, the structures of these Bcl-2 deletion mutants presumably were not grossly distorted, given that they were still capable of binding to wild-type Bcl-2 both in vitro and in yeast two-hybrid assays. These data thus argue that while all three of the conserved domains [BD(A), BD(B), BD(C)] in Bcl-2 are important for anti-Bax function in yeast, the NH-terminal 82 amino acids of Bcl-2 are expendable for binding to the Bax protein in vitro. This finding suggests that the NH-terminal domain of Bcl-2 participates in cell death regulation through a mechanism that is independent of heterodimerization with Bax. In this regard, although many potential explanations can be advanced that are consistent with the data presented here, some possibilities are that: (a) the NH-terminal domain of Bcl-2 is needed for binding to some other third protein or for masking a binding site on Bax for an additional protein and (b) the NH-terminal domain of Bcl-2 may indirectly regulate post-translational modifications of Bax, such as phosphorylation.

In addition to a requirement for NH-terminal sequences within Bcl-2 for negating Bax function in yeast, a role for COOH-terminal sequences located downstream of BD(C) was also found. In this regard, both a Bcl-2(1-196) COOH-terminal truncation mutant that is missing all residues downstream of BD(C) and the Bcl-2 protein failed to bind to Bax in vitro and were relatively ineffective at suppressing Bax-lethality in yeast. The 22-kDa Bcl-2 protein diverges from the usual 26-kDa Bcl-2 protein precisely after BD(C), because of an alternative splicing event (Tsujimoto and Croce, 1986). The sequences found downstream of the splice site in Bcl-2 share essentially no homology with the corresponding region in p26-Bcl-2. Thus, in addition to the previously documented important role for BD(C) [also known as BH2] (Yin et al., 1994), our findings lend additional support to other data derived from analysis of Bcl-2 deletion mutants in mammalians cells which suggested that sequences located downstream of BD(C), between 196 and 203 play a role either directly or indirectly in suppression of cell death (Borner et al., 1994). In that report, however, binding to Bax was not tested and thus the molecular explanation for the findings was unclear. Although sequences downstream of BD(C) appear to be required in some way for binding to Bax in vitro and suppression of Bax lethality in yeast, the lack of sequence homology in this region of Bcl-2 proteins isolated from various species including human, mouse, rat, and chicken (Sato et al., 1994b), as well as the dearth of similarity in this region in Bcl-2 and other Bcl-2 family proteins such as Bcl-X-L and Mcl-1 that can bind to and functionally neutralize Bax (Sato et al., 1994a), suggests that the amino acid sequence criteria required in this region are relatively nonspecific. Consistent with this idea, we previously showed that a chimeric protein in which the first 195 amino acids of Bcl-2 were fused with a portion of the IL-2 receptor- chain was functional at prolonging cell survival in a hemopoietic cell line (Tanaka et al., 1993), suggesting that even some heterologous sequences can functionally and structurally substitute for the usual sequences found downstream of BD(C).

Also located downstream of BD(C) is the hydrophobic stretch of amino acids that normally allows the Bcl-2 protein to insert into membranes. Versions of the human Bcl-2 protein that lack the COOH-terminal transmembrane domain (residues 219-237) have been shown to retain cell death blocking activity in mammalian cells, although in some circumstances such membrane anchor-deficient Bcl-2 proteins have reduced function compared to the wild-type Bcl-2 protein (Borner et al., 1994; Tanaka et al., 1993; Hockenbery et al., 1993). In the experiments described here, Bax fusion proteins contained the COOH-terminal transmembrane domain of Bax, whereas the Bcl-2 fusion proteins were lacking a membrane anchore. The transmembrane domain of Bcl-2 therefore is not absolutely required for Bcl-2 protein function in yeast, although we cannot exclude the possibility that inclusion of membrane-anchoring sequences would improve the efficiency of Bcl-2/Bax interactions and thus possibly enhance the ability of Bcl-2 to functionally neutralize Bax.

Although the BD(A), BD(B), and BD(C) deletion mutants of Bcl-2 described here were not functionally analyzed in mammalian cells, recent reports have shown that BD(B) and BD(C) (also known as BH1 and BH2) are essential for co-immunoprecipitation of Bcl-2 with Bax and for prolongation of cell survival in the setting of lymphokine withdrawal from a factor-dependent murine hemopoietic cell line (Yin et al., 1994). In addition to BD(B) and BD(C), a deletion mutant of Bcl-2 lacking amino acids 4-29 (which encompasses most of the BD(A) region examined in this report (residues 11-33)) has been shown to be impaired in its ability to block tumor necrosis factor-induced cytotoxicity in L929 fibroblasts and to prolong survival of nerve growth factor-deprived rat sympathetic neurons (Borner et al., 1994). Moreover, a COOH-terminal truncation mutant of Bcl-2 lacking all sequences downstream of residue 196 has been shown to have markedly impaired cell death blocking activity in mammalian cells (Borner et al., 1994), consistent with our results in yeast and with our in vitro binding data demonstrating markedly impaired Bax binding by a COOH-terminal Bcl-2 truncation mutant, Bcl-2(1-196). The findings presented here therefore where the function of Bcl-2 mutants was assessed in yeast are in excellent agreement with analogous studies performed using mammalian cells, suggesting that elements of the Bax/Bcl-2 pathway are conserved from the simplest unicellular eukaryotic organisms to the most complex multicellular species. As such, these observations suggest that S. cerevisiae can be employed reliably as a rapid readout for assessing the function of other Bcl-2 mutants, as well as for structure-function studies of Bax. They also raise the possibility that classical yeast genetic approaches could be taken for mapping portions of the Bcl-2/Bax pathway for cell death regulation, using either inhibition or accentuation of Bax-induced lethality in yeast as a screening assay.

  
Table: Two-hybrid analysis of Bcl-2/Bcl-2 homodimerization

Two-hybrid assays were performed using LEU2 and lacZ reporter genes under the control of lexA operators for detection of protein-protein interactions between plasmid-produced fusion proteins containing either a LexA DNA-binding domain (pEG202) or a B42 trans-activation domain (pJG4-5). In all cases, a positive signal was not produced when cells were plated on glucose-containing media which represses the Gal-1 promoter in pJG4-5 (not shown). Data are representative of at least two independent experiments. At least six independent transformants were tested for each experiment. Plasmids encoding portions of Fas or Ras were employed as negative controls, and have been described previously (Sato et al., 1994a). At least two additional negative controls were also employed for each plasmid, further confirming the specificity of the interactions detected here (not shown). Data were scored as positive (+) or negative (-) for interaction. In all (+) cases, unambiguous growth on leucine deficient medium and production of an intense blue color in -galactosidase filter assays was obtained. Negative (-) interactions yielded either no or very little growth (leucine-deficient medium) and blue color production (-Gal).


  
Table: Neutralization of Bax-mediated cytotoxicity by Bcl-2 in yeast

EGY191 cells were transformed with plasmid DNAs (5 µg each) and plated on His-, Tryp-, Ura- medium containing leucine and either 2% galactose (Gal) or glucose (Glu) to induce or repress, respectively, the Gal-1 promoter in pJG4-5. A positive (+) score indicates the presence of >100 colonies of 1-mm diameter. Negative (-) scores indicate that the number of colonies was 15% of that obtained when cells containing pEG202-Bax and pJG4-5-Bcl-2(1-218) were plated on galactose-containing medium. In pEG202-Bax (antisense), the bax cDNA was subcloned in reverse orientation as a negative control. Data represent results from two to four experiments.



FOOTNOTES

*
This work was supported in part by a grant from CaP CURE. 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.

§
Fellow of l'Association pour la Recherche sur le Cancer.

Fellow of the United States Army Research Development Command/Breast Cancer Research Project.

**
Scholar of the Leukemia Society of America. To whom correspondence should be addressed: La Jolla Cancer Research Foundation, Oncogene and Tumor Suppressor Gene Program, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-455-6480; Fax: 619-455-0181.

M. Hanada, C. Aimé-Sempé, T. Sato, and J. C. Reed, unpublished data.

The abbreviations used are: BD, Bcl-2 domain; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; X-gal, 5-bromo-4-chloro-3-indoyl -D-galactoside.


ACKNOWLEDGEMENTS

We thank Erica Golemis for the two-hybrid system, C. Thompson for the Bcl-X-S cDNA, and A. M. Pendergast for GST-BAP-1.


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