From the
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(
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).
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)
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.
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
In
vitro binding assays were performed by mixing 10-20 µg
of GST proteins immobilized on glutathione-Sepharose with 10 µl of
L-[
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-
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
Taken
together, these data are consistent with our previously proposed model
in which the NH
To further test some of
these ideas, experiments were performed using fusion proteins that
contained only the NH
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) (
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(
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
In addition to a requirement for
NH
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.
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
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
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.
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.
(
)
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.
(
)
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.
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.
-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.
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.
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).
-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.
-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 ().
-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).
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.
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.
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.
-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).
Table:
Two-hybrid analysis of Bcl-2/Bcl-2
homodimerization
-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
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.
-D-galactoside.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.