(Received for publication, November 13, 1995; and in revised form, February 5, 1996)
From the
Most members of the Bcl-2 protein family of apoptosis regulating
proteins contain two evolutionarily conserved domains, termed BH1 and
BH2. Both BH1 and BH2 in the Bcl-2 protein are required for its
function as an inhibitor of cell death and for heterodimerization with
the proapoptotic protein Bax. In this report, we mapped the region in
Bax required for heterodimerization with Bcl-2 and homodimerization
with Bax, using yeast two-hybrid and in vitro protein-protein
interaction assays. Neither the BH1 nor the BH2 domain of Bax was
required for binding to the wild-type Bcl-2 and Bax proteins. Moreover,
Bax (BH1) and Bax (
BH2) mutant proteins bound efficiently to
themselves and each other, further confirming the lack of requirement
for BH1 and BH2 for Bax/Bax homodimerization. Bax/Bax homodimerization
was not dependent on the inclusion of the NH
-terminal 58
amino acids of the Bax protein in each dimerization partner, unlike
Bcl-2/Bcl-2 homodimers which involve head-to-tail interactions between
the region of Bcl-2 where BH1 and BH2 resides, and an
NH
-terminal domain in Bcl-2 that contains another domain
BH4 which is conserved among antiapoptotic members of the Bcl-2 family.
Similarly, heterodimerization with Bcl-2 occurred without the
NH
-terminal domain of either Bax or Bcl-2, suggesting a
tail-to-tail interaction. The essential region in Bax required for both
homodimerization with Bax and heterodimerization with Bcl-2 was mapped
to residues 59-101. This region in Bax contains a stretch of 15
amino acids that is highly homologous in several members of the Bcl-2
protein family, suggesting the existence of a novel functional domain
which we have termed BH3. Deletion of this 15-amino acid region
abolished the ability of Bax to dimerize with itself and to
heterodimerize with Bcl-2. The findings suggest that the structural
features of Bax and Bcl-2 that allow them to participate in homo- and
heterodimerization phenomena are markedly different, despite their
amino-acid sequence similarity.
Programmed cell death and apoptosis are active forms of cell suicide which play a variety of important roles under normal physiological conditions and which, when dysregulated, can contribute to several diseases including cancer, autoimmunity, AIDS, and ischemia-associated tissue loss (reviewed in (1) and (2) ). The Bcl-2 family proteins regulate a distal step in an evolutionarily conserved pathway for programmed cell death(1, 3) . Several members of the Bcl-2 protein family can form physical interactions with each other in a complicated network of homo- and heterodimers(4, 5, 6) . Although many details remain unclear at present, in general, the ratio between antiapoptotic proteins such as Bcl-2 relative to pro-cell death proteins such as Bax determines the ultimate sensitivity of cells to various apoptotic stimuli(7) .
With the exception of some
relatively nonabundant isoforms that arise through alternative mRNA
splicing mechanisms, essentially all known members of the Bcl-2 protein
contain two conserved regions of amino acid similarity, which we have
previously termed Bcl-2 domains (BD) B and C but which are better known
as BH1 and BH2(4, 5, 8) . In addition, the
antiapoptotic proteins Bcl-2, Bcl-X, Mcl-1, A1, Nr-13, and
Ced-9 all contain an additional region of homology near their NH
termini, comprising a domain which we have termed BD-A but
hereinafter refer to as BH4 for Bcl-2 homology-4 domain. Mutagenesis
studies have shown that deletion of the BH1 or BH2 domain of Bcl-2 as
well as certain amino acid substitutions in these conserved domains
abolish Bcl-2 function as a suppressor of cell death and also abrogate
the ability of Bcl-2 to form heterodimers with
Bax(9, 10) . Similar mutations in the BH1 and BH2
domains of the antiapoptotic protein Bcl-X
have the same
effects on function and Bax binding(6) . These observations
suggest that for Bcl-2 and Bcl-X
to suppress apoptosis,
they must be able to heterodimerize with Bax. However, the situation is
likely to be more complicated because deletion of the BH4 domain of
Bcl-2 also destroys function but does not interfere with Bax
binding(10, 11) .
In contrast to Bcl-2, essentially nothing is known at present about structure-function relations in the Bax protein as pertains to homodimerization with itself and heterodimerization with Bcl-2. In this report, we map for the first time a dimerization domain in the Bax protein and show that this novel domain, which we have termed BH3, is distinct from BH1 and BH2.
A murine bax cDNA (12) was employed for
mutagenesis experiments. Mutations were created using polymerase chain
reaction-assisted methods and specific primers, essentially as
described (5, 10, 13) (details available upon
request), and their DNA sequences were confirmed by routine dideoxy
sequencing methods. Bax mutants were expressed as fusion proteins
either with an NH-terminal LexA DNA binding domain in
pEG202 for yeast two-hybrid experiments or with an
NH
-terminal GST (
)domain in pGEX-4T-1 for
production of recombinant proteins in
bacteria(5, 10) . All versions of Bax and Bcl-2
employed here lacked the COOH-terminal transmembrane domains (TM)
(residues 172-191 in Bax; 219-239 in Bcl-2), thus avoiding
problems with targeting of proteins to the nucleus for two-hybrid
experiments and with solubility of bacteria-produced proteins.
The
procedures for transformation of yeast (EGY-191 strain), induction of
expression of Bax and Bcl-2 fusion proteins containing
NH-terminal transactivation (TA) domains using
galactose-containing media (repression on glucose), and performance of
two-hybrid reporter gene assays by auxotrophic growth on
leucine-deficient media as well as by
-galactosidase-based
colorimetric filter assays have been described in
detail(5, 10) . Expression of all LexA-Bax mutants was
confirmed by immunoblotting using a rabbit anti-LexA antiserum
(generous gift of Erica Golemis, Philadelphia, PA).
For in vitro protein-protein interaction assays, GST-fusion proteins were
produced in BL21-strain cells after induction with 1 mM isopropyl-1-thio--D-galactopyranoside and purified
on
glutathione-Sepharose(10, 14, 15, 16, 17) .
GST-fusion proteins (5 µg) were immobilized on
glutathione-Sepharose and incubated with 0.01 ml of in vitro translated, [
S]methionine-labeled Bcl-2,
Bax, Bax (
N), Bax (
BH1), or Bax (
BH2) proteins which
were prepared from Bluescript pSK-II templates using a coupled
transcription/translation system involving rabbit reticulocytes lysates
(Promega, Inc. TNT-lysates) and T7 RNA polymerase, as
described(10, 14, 15, 16, 17) .
Washing, SDS-polyacrylamide gel electrophoresis analysis, and
autoradiography were accomplished by previously published
methods(10, 14, 15, 16, 17) .
Preparation and characterization of all human Bcl-2 yeast two-hybrid and GST-bacterial expression plasmids and their encoded proteins, as well as a variety of negative control plasmids used for these studies, have been described(10, 14, 15, 16, 17) .
For initial explorations of the regions within the Bax
protein required for homodimerization with Bax and heterodimerization
with Bcl-2, a series of NH-terminal and COOH-terminal
truncation mutants were constructed in the two-hybrid plasmid pEG202
and expressed in yeast as fusion proteins with NH
-LexA DNA
binding domains (Fig. 1). These LexA-Bax proteins were then
tested by yeast two-hybrid assays for interactions with
``full-length'' Bax and Bcl-2 proteins (missing TM domains
only), which were expressed as fusions with a
NH
-transactivation domain (TA) under the control of a Gal-1
promoter using the plasmid pJG4-5(18) .
Figure 1:
Analysis of Bax protein regions
required for homodimerization with Bax and heterodimerization with
Bcl-2. The structure of the wild-type mouse Bax protein is depicted (top), showing the BH1, BH2, and TM (transmembrane) domains.
Also shown are the structures of the various Bax deletion mutants which
were expressed with NH-terminal LexA DNA binding domains in
pEG202 and tested by two-hybrid assays for specific interactions with
Bax and Bcl-2 TA-fusion proteins which were produced as full-length
proteins (with exception of removal of their TM domains), with TA
domains fused to their NH
termini in
pJG4-5(18) . Reactions were scored as positive (+)
if galactose-inducible growth on leucine-deficient plates occurred
within 5 days as well as conversion of X-gal substrate yielding an
unambiguous blue color within 1 h, and if no substantial growth on
leucine-deficient medium and no blue color development were observed
when tested against pJG4-5-Ras and pJG4-5 without an
insert. Some Bax mutants produced high background (HB) and
therefore could not be evaluated, i.e. growth on
leucine-deficient medium and conversion of X-gal substrate (blue color)
occurred when cells were plated on both galactose (activates Gal-1
promoter that drives expression of TA-Bax and TA-Bcl-2) and glucose
(represses Gal-1 promoter), and/or positive reactions were produced
with negative control plasmids, pJG4-5-Ras and
pJG4-5.
The
essentially full-length Bax protein (amino-acids 1-171; i.e. missing only residues 172 COOH terminus to exclude TM
domain) produced strong two-hybrid interactions with both Bax and Bcl-2
when plated on galactose-containing medium (induces Gal-1 promoter in
pJG4-5), whereas little or no growth on leucine or positivity in
-galactosidase colorimetric assays occurred when cells where
plated on glucose-containing medium (represses Gal-1 promoter).
COOH-terminal truncation mutants of Bax containing only residues 1
159 or 1
117 retained the ability to interact strongly
with both Bax and Bcl-2, whereas a mutant that consisted only of amino
acids 1
68 did not produce two-hybrid interactions with either
full-length Bax or Bcl-2 (Fig. 1). Next, a series of
NH
-terminal truncation mutants were tested. Deletion of the
first 58 amino acids of Bax had no effect on its ability to form
two-hybrid interactions with either Bax or Bcl-2. In contrast, removal
of the first 99 or of the first 150 amino acids of Bax abolished all
reactivity with both Bax and Bcl-2 (Fig. 1).
Because this result suggested that the dimerization domain of Bax lies between residues 59 and 117, a series of Bax mutants which contained only the 59-117 region or various subfragments thereof were tested for interactions with Bax and Bcl-2. The Bax-(59-117) fragment produced strong two-hybrid interactions with both Bax and Bcl-2, which were comparable in strength to those seen with full-length Bax. In contrast, a smaller fragment of Bax containing only amino acids 69-117 completely lacked reactivity with both Bax and Bcl-2 (Fig. 1). Since the 59-117 region of Bax retains the well-conserved BH1 domain, a smaller fragment of Bax was produced which lacked this domain, Bax-(59-101). This protein, however, resulted in high amounts of background lacZ reporter gene transactivation, and thus could not be tested reliably by two-hybrid assay. A smaller fragment consisting of residues 69-101 was devoid of all reactivity with Bax or Bcl-2 in two-hybrid assays.
As
an alternative to the Bax-(59-101) construct, an internal
deletion mutant of Bax was produced which specifically lacked the
sequences encoding the BH1 domain, Bax (BH1). In addition, to
confirm the apparent lack of requirement for BH2, an internal deletion
mutant was also prepared which specifically lacked this domain, Bax
(
BH2). Both the Bax (
BH1) and the Bax (
BH2) mutants
produced strong two-hybrid interactions with full-length Bax and Bcl-2,
which were comparable in strength to the wild-type Bax protein. These
data argue that neither the BH1 nor the BH2 domain of Bax is required
for homodimerization with wild-type Bax or heterodimerization with
Bcl-2. With the exception of the Bax-(59-101) protein and a
Bax-(151-171) mutant, both of which suffered from background
problems, none of the Bax mutants described here resulted in
significant reporter gene activation with tested with
pJG4-5-Ha-Ras or the pJG4-5 parent plasmid lacking an
insert.
The mapping experiments described above suggested that the
Bax dimerization domain resided between residues 59 and 101, but we
were unable to test this through two-hybrid assays. For this reason, we
explored the binding properties of Bax-(59-101), as well as
several other Bax mutants through in vitro binding assays
where Bax mutants were expressed as GST fusions in bacteria and the
purified proteins were immobilized on glutathione-Sepharose and tested
for specific binding to in vitro translated,
[S]methionine-labeled Bax and Bcl-2.
Alternatively, in some experiments, Bax mutants were in vitro translated and tested for binding to wild-type Bax or Bcl-2
GST-fusion proteins. As shown in Fig. 2A, a
GST-Bax-(59-101) fusion protein bound to
S-Bax with
comparable efficiency to an essentially full-length GST-Bax fusion
protein (lacking only the TM domain). This 59-101 fragment of Bax
also bound effectively to in vitro translated Bcl-2 protein.
The specificity of the binding was confirmed by use of control GST and
GST-CD40 cytosolic domain fusion proteins, as well as by failure of the
GST-Bax-(59-101) protein to interact with other irrelevant in
vitro translated proteins, including R-Ras, Raf-1, and baculovirus
p35 (Fig. 2A and data not shown).
Figure 2:
In vitro binding assays
demonstrate dependence on 59-101 region of Bax and independence
of BH1 and BH2 for dimerization of Bax mutants with wild-type Bax and
Bcl-2. GST-fusion proteins (5 µg (A, B, and C); 10 µM (D)) immobilized on
glutathione-Sepharose (10 µl) were tested for binding to
[S]methionine- labeled in vitro translated proteins (10 µl of reticulocyte lysates primed with
1 µg of plasmid DNA) as described (10, 17) .
Proteins that associated with GST fusions were analyzed by
SDS-polyacrylamide gel electrophoresis (12% gel) and radiofluorography.
Staining of gels with Coomassie Blue dye confirmed loading of similar
amounts of mostly intact GST-fusion proteins in all experiments (not
shown). As a control, 1 µl of reticulocyte lysates containing in vitro translated proteins was run directly in gels (IVT
Control). In A and D, full-length mouse Bax or
human Bcl-2 proteins were produced by in vitro translation,
whereas in B and C full-length Bax or Bax (
BH1),
Bax (
BH2), and Bax (
1-58) mutants were translated in vitro.
Because the
Bax-(59-101) fragment lacks the BH1 and BH2 domains, in vitro binding experiments were performed to confirm the lack of
dependence on the these conserved domains for dimerization with Bax and
Bcl-2. In vitro translated full-length Bax was compared with
Bax (BH1) and Bax (
BH2) for binding in vitro to
GST-Bax and GST-Bcl-2. Both the Bax (
BH1) and Bax (
BH2)
mutants bound to GST-Bax and GST-Bcl-2 with efficiencies comparable to
wild-type Bax, but did not bind to GST nonfusion, GST-CD40, or other
control GST-fusion proteins (Fig. 2B and data not
shown). In addition, the Bax (
BH2) and a Bax (
1-58)
mutant were also capable of binding in vitro to a
GST-Bax-(59-101) fusion protein, further indicating that the
59-101 region of Bax can bind to Bax independently of the BH2 and
NH
-terminal domains of Bax (Fig. 2C).
Interestingly, however, this 42-amino acid fragment of Bax (residues
59-101) was not capable of binding to Bax (
BH1) in
vitro. This result suggests that the BH1 domain, although perhaps
not directly required for Bax homodimerization, may be necessary to
facilitate the formation of an optimal binding site for the
59-101 Bax fragment (see below). In this regard, because the BH1
domain is located immediately adjacent to the 59-101 region, it
is possible that BH1 is necessary for proper folding or contextual
presentation of the 59-101 domain for homodimerization.
Finally, because the two-hybrid experiments above suggested an
important role for residues 59-69 of Bax for dimerization with
Bax and Bcl-2, a Bax-(59-117) fragment (which formed two-hybrid
interactions with Bax and Bcl-2 in two-hybrid experiments) was compared
with a Bax-(69-117) fragment (which did not react with Bax or
Bcl-2 in yeast). When expressed as GST-fusion proteins and tested for
binding in vitro to in vitro translated S-Bax and
S-Bcl-2, the GST-Bax-(59-117)
protein bound in vitro to Bax and Bcl-2 with efficiencies
comparable to the essentially full-length GST-Bax protein which was
missing only the TM domain (Fig. 2D and data not
shown). In contrast, the GST-Bax-(69-117) protein failed to bind
or, at best, bound very little of the
S-Bax or
S-Bcl-2 proteins in vitro, confirming the
two-hybrid results which suggested that residues 59-69 of Bax are
required for dimerization with Bax and Bcl-2.
An amino acid sequence
alignment was performed for the 59-69 region of Bax and several
other known members of the Bcl-2 family, including Bcl-2, Bcl-X, Mcl-1,
Ced-9, Bak, Bad, Bik, and Nr13. Significant homology was found in this
region of Bax with the Bak, Bcl-2, Bcl-X, and Mcl-1 proteins (Fig. 3A), suggesting the existence of a functionally
important, previously unrecognized conserved domain. We have termed
this homologous region the Bcl-2 homology domain-3 (BH3). Thus, four
evolutionarily conserved domains are envisioned in Bcl-2 family
proteins: the BH4 domain (previously termed the A-box (5, 8, 10) which is found in the
antiapoptotic members of the Bcl-2 family (Bcl-2, Bcl-X,
Mcl-1, Ced-9, A1, and Nr-13); the BH3 domain described here, and the
BH1 and BH2 domains (Fig. 3B).
Figure 3:
Sequence and domain comparisons of Bcl-2
family proteins. In A, amino acid sequence alignments are
presented for the BH3 domains of mouse Bax, human Bak, human Bik, human
Bcl-2, human Bcl-X, and human Mcl-1. Residues that are
identical or similar in at least 4 of the 5 proteins are boxed. In B, the domain structures of the known
cellular members of the Bcl-2 family are presented, showing the
locations of the BH1, BH2, BH3, BH4, and TM
domains.
To confirm the
importance of the BH3 domain for dimerization of Bax with itself and
with Bcl-2, a Bax (BH3) deletion mutant was tested for binding to
Bax, Bax (
BH3), and Bcl-2 by two-hybrid assays. Although
immunoblot analysis confirmed that the Bax (
BH3) protein was
produced at levels comparable to the wild-type Bax protein (both when
fused with a LexA-DNA binding domain or a B42 transactivation domain)
(not shown), the Bax (
BH3) mutant failed to react with wild-type
Bax, itself, or with Bcl-2 in two-hybrid assays (Fig. 4A). Thus, the BH3 domain is required for Bax
homodimerization and for heterodimerization with Bcl-2. Interestingly,
an isoform of Bax (Bax-
) has recently been described which arises
because of alternative splicing(19) . The predicted Bax-
protein lacks residues 30-77 where the BH3 domain resides and
thus should be incapable of dimerizing with either Bcl-2 or Bax, based
on the findings shown here.
Figure 4:
Testing of Bax mutants for binding to
Bcl-2 and Bax mutants. In A, examples of two-hybrid results
are presented based on -galactosidase filter assays. Three
independent transformants were plated on either galactose (Gal)- or glucose (Glu)-containing medium. Although
not shown here, all LexA-Bax mutants were also tested for reactivity
with TA-Bcl-2, TA-Ras, and TA revealing that all mutants which reacted
with TA-Bax also were positive with TA-Bcl-2 but did not react with
TA-Ras or TA. In addition, all TA-Bax mutants were also tested for
reactivity with LexA-Fas and LexA, revealing no two-hybrid interactions
with these control proteins. In B, in vitro binding
studies were performed using GST-fusion proteins and a
[
S]methionine-labeled, in vitro translated mutant of Bax containing residues
59-191.
Our previous analysis of the domains
required for Bcl-2/Bcl-2 homodimerization suggested an anti-parallel,
head-to-tail model wherein the NH-terminal domain of Bcl-2
where the BH4 domain resides (residues 1-81) binds to structures
contained in a downstream region of Bcl-2 where the BH1, BH2, and BH3
domains are located (amino acids 83-218)(5) .
Furthermore, those studies showed that physical interactions of the
NH
-terminal Bcl-2 domain (residues 1-81) and
downstream region in Bcl-2 (amino acids 83-218) require the
simultaneous presence of both the BH1 and BH2 domain in the downstream
region of Bcl-2 and the BH4 domain in the NH
-terminal
segment of Bcl-2(10) . However, if one dimerization partner was
missing BH1 and BH2 (but retained BH4) and the other was lacking BH4
(but still had BH1 and BH2), then interactions could
occur(10) . This antiparallel fashion in which Bcl-2
homodimerizes promoted us to further explore the nature of Bax/Bax
homodimers by testing binding of additional Bax mutants to each other
by two-hybrid assays. As shown in Fig. 4A, a LexA DNA
binding domain-Bax (
N) fusion protein containing Bax residues
59-171 formed strong interactions with a TA domain
Bax-(59-171) fusion protein. Thus, the NH
-terminal 58
amino acids of Bax are not required for homodimerization of Bax
(
N) to Bax (
N) mutants, implying that Bax/Bax
homodimerization occurs via a tail-to-tail interaction. Fig. 4A shows in addition that the Bax (
BH1) and
Bax (
BH2) internal deletion mutants were able to form strong
two-hybrid interactions with themselves and each other, further
confirming the data presented in Fig. 2C which
suggested an absence of a requirement for BH1 or BH2 for
homodimerization of Bax (Fig. 4A). The ability of the
Bax (
BH1) mutant in particular to homodimerize with itself
supports the idea that if BH1 is involved in Bax homodimerization, it
plays only an indirect or facilatory role compared to BH3. These same
mutants of Bax (Bax (
N), Bax (
BH1), Bax (
BH2)) also
formed two-hybrid interactions with Bcl-2 with strengths comparable to
the wild-type Bax protein (not shown). The specificity of these
interactions was confirmed by use of various irrelevant proteins (Fig. 4A; see legend).
Finally, the structural
features of Bax/Bcl-2 heterodimers were explored by testing the binding
of Bcl-2/mutants to Bax mutants. As shown in Fig. 4B,
an in vitro translated N-truncation mutant of Bax missing the
first 58 amino acids bound with comparable efficiencies in vitro to an essentially full-length GST-Bcl-2 protein (missing only TM
domain; i.e. has residues 1-218) and a GST-Bcl-2
N-truncation mutant missing the first 82 amino acids of the Bcl-2
protein (83-218) but failed to bind to a C-truncation mutant of
Bcl-2 comprised only of Bcl-2 residues 1-81 fused to GST. In
contrast, in vitro translated S-Bcl-2 bound to
both the GST-Bcl-2-(1-81) and GST-Bcl-2-(83-218) proteins
employed for this experiment (not shown), confirming the integrity of
the GST-Bcl-2-(1-81) protein despite its failure to bind to Bax.
The specificity of the in vitro interactions of the Bax
(
N) protein (residues 59
COOH terminus (191)) with
GST-Bcl-2 and GST-Bax was confirmed by use of GST, GST-CD40, and
GST-R-Ras control proteins. Unlike Bcl-2/Bcl-2 homodimerization,
therefore, the heterodimerization of Bcl-2 with Bax appears to occur in
a parallel, tail-to-tail fashion in which the NH
-terminal
domains of neither Bcl-2 nor Bax are required.
Taken together, these
observations suggest that dimerization of Bax with itself and with
Bcl-2 occurs independently of the well-conserved BH1 and BH2 domains.
Thus, the structural features of Bax that allow it to physically
interact with other members of the Bcl-2 family are strikingly
different from Bcl-2, which does require BH1 and BH2 for
heterodimerization with Bax, as well as for homodimerization with
itself, at least when testing for binding of mutant Bcl-2 to mutant
Bcl-2(9, 10) . Moreover, the data presented here
indicate that another region in the Bax protein, where the BH3 domain
resides, is both necessary and sufficient for binding to the wild-type
Bax and Bcl-2 proteins and thus defines a novel dimerization domain for
this family of apoptosis-regulating proteins. The finding that this
region of Bax shares strong amino acid sequence homology with some
other members of the Bcl-2 protein family, including proapoptotic (Bak,
Bik) and antiapoptotic (Bcl-2, Bcl-X, Mcl-1) proteins, also
raises the possibility that the BH3 domain may be required for
interactions among other Bcl-2 family proteins. In this regard, while
this paper was under review, a report appeared showing that the region
of Bak that contains BH3 is sufficient both for heterodimerizing with
Bcl-X
and for promoting apoptosis in mammalian
cells(20) . It remains to be determined however whether the BH3
domain of Bak promotes apoptosis by directly engaging the cell death
pathway (i.e. effector domain) versus by acting as a
decoy (i.e. regulatory domain) that ties up antiapoptotic
protein such as Bcl-X
, thereby preventing them from forming
effective interactions with the full-length endogenous Bak or Bax
proteins. In addition, a new member of the Bcl-2 protein family was
described Bik that: (a) promotes apoptosis, (b)
contains a region with strong homology to the BH3 domain but lacks the
BH1 and BH2 domains (Fig. 3, A and B), and (c) which binds to Bcl-2 in a BH3-dependent manner. When taken
together with the data presented here, therefore, these findings
confirm the functional importance of the BH3 domain. It will be
interesting in future investigations to determine whether the
BH3-mediated homodimerization of Bax with itself is necessary for
promotion of apoptosis by the Bax protein.