(Received for publication, March 28, 1997)
From IDUN Pharmaceuticals, Inc., La Jolla, California 92037
Interactions among proteins in the Bcl-2 family regulate the onset of programmed cell death. Previous work has shown that the death-inhibiting family members Bcl-2 and Bcl-xL form heterodimers with the death-promoting homologue Bax and that certain site-directed mutants of Bcl-2 and Bcl-xL lose both biological activity and the ability to bind Bax. To better understand the structural basis of heterodimer formation, we have used a yeast two-hybrid assay to screen for mutants of Bax that regain the ability to bind to these inactive Bcl-2(G145A) and Bcl-xL(G138A) mutants. This screen identified a series of C-terminally truncated Bax molecules that contain complete BH3 (Bcl-2 homology domain 3) domains but that have lost BH1 and BH2 sequences. These results indicate that while the Bcl-2 and Bcl-xL mutants fail to bind full-length Bax, they still retain a binding site for the critical BH3 domain. This suggests that conformational constraints in full-length Bax regulate its ability to bind to other Bcl-2 family members. Furthermore, we demonstrate that the normally inert Bcl-2(G145A) mutant effectively blocks apoptosis induced by a C-terminally truncated Bax molecule, but does not block apoptosis induced by wild-type Bax. This demonstrates that cell protection can be effected by directly binding pro-apoptotic members of the Bcl-2 family.
The bcl-2 gene family encodes proteins that regulate programmed cell death (1, 2). For example, in the nematode Caenorhabditis elegans, the bcl-2 homologue ced-9 acts to inhibit developmental cell deaths. This is evidenced by the elevated levels of cell death that characterize the embryos of loss-of-function ced-9 mutants (3, 4). Similarly, in cultured mammalian cells, apoptosis is inhibited by the expression of several family members, including Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 (5-8). Furthermore, gene ablation experiments in mice have further confirmed the anti-apoptotic effects of bcl-2 and bcl-x (9, 10). In contrast to the anti-apoptotic members of the Bcl-2 family, mammalian cells also express family members that promote apoptosis. For example, Bax and Bak have been shown to kill when overexpressed in mammalian cells and can antagonize the cell-protective functions of Bcl-2 and Bcl-xL (11-14).
While the mechanisms by which Bcl-2 family members modulate cell death remain unclear, a variety of studies indicate that many of the family members can interact with each other and that these interactions are functionally important. Immunoprecipitation studies using genetically engineered cells first demonstrated that members of the Bcl-2 family can form protein-protein dimers with each other. These studies demonstrated that the Bcl-2 molecule can form a heterodimer with Bax and can also form a homodimer with another Bcl-2 molecule (11, 15). These conclusions were subsequently confirmed using several systems of yeast two-hybrid analysis (16-19). Similarly, Bcl-xL and Bax can form heterodimers, although there has been controversy concerning the ability of Bcl-xL to homodimerize (18, 20, 21).
Sequence alignments of Bcl-2 family proteins have identified several conserved domains, denoted BH1, BH2, and BH3, that are common to all homologues and a fourth domain, BH4, present in at least some of the family members (15, 22, 23). Site-directed mutagenesis has been used to assess the functional importance of these domains. Mutations in the BH1 and BH2 domains have been shown to abrogate the death-inhibiting functions of Bcl-2 and Bcl-xL. For example, mutation of the highly conserved glycine 145 to alanine in Bcl-2 (15), or the homologous G138A mutation in Bcl-xL (17), renders the respective proteins functionally inactive. These mutations also abrogate the ability of Bcl-2 and Bcl-xL to dimerize with Bax (15, 17). Based on these results and the results obtained with additional BH1 and BH2 mutants (15), it was suggested that the death-inhibiting function of Bcl-2 and Bcl-xL is dependent on their ability to form Bax heterodimers. Further mutagenesis work, however, has identified Bcl-xL mutants that do not bind Bax, but that still block apoptosis, suggesting that inhibition of cell death does not require heterodimerization (24). Nevertheless, the observation that the G145A and G138A mutations in Bcl-2 and Bcl-xL, respectively, are inactive suggests that the structure of this region of these molecules is important for biological function.
Mutagenesis studies on the death-promoting Bcl-2 family members Bak and Bax indicate that the BH3 domains of these molecules are critical both for induction of apoptosis and for binding to Bcl-xL and Bcl-2 (22, 23). The three-dimensional structure of Bcl-xL, derived by x-ray diffraction and NMR techniques, demonstrates the presence of a hydrophobic groove on the Bcl-xL surface (25) that has been shown to bind the BH3 domain of Bak (26). Glycine 138 forms part of this groove, and it was therefore further suggested that the bulkier alanine in the G138A mutant may block Bak or Bax from entering the binding pocket (25).
To obtain further information regarding the structural requirements for Bax-Bcl-2 and Bax-Bcl-xL heterodimer formation and to obtain new reagents to probe the functional importance of such dimerization, we utilized random mutagenesis and yeast two-hybrid analysis to identify novel Bax mutants that have acquired the ability to bind to the G145A mutant of Bcl-2. This approach was based on the strategy that was successfully used to identify mutants of Raf-1 that bind Ras(E37G), a Ras mutant with which wild-type Raf-1 does not interact (27). Our results indicate that the G145A and G138A mutants of Bcl-2 and Bcl-xL retain intact binding sites for dimerization partners, but suggest that structural constraints in full-length Bax prevent it from occupying those sites. Our results further indicate that Bcl-2 family members can inhibit cell death directly as a result of binding death-promoting family members.
Plasmid Constructs
Yeast Two-hybrid PlasmidsHuman Bax minus its transmembrane
domain (Bax(TM)) was PCR1-amplified to
add EcoRI and XhoI restriction sites and then
subcloned into vector pJG4-5 (28). Constructs containing human
Bcl-2(
TM) and Bcl-xL(
TM) in the yeast two-hybrid vector
pJG4-5 were described previously (16). They were excised as
EcoRI/XhoI fragments and ligated into the bait
vector pGilda, which produces a LexA fusion protein. Bcl-2(G145A)(-TM)
was PCR-amplified from plasmid M1-3 (15) and subcloned into the vectors
pGilda and pJG4-5. Site-directed mutagenesis was performed on
Bcl-xL (in pBluescript (Stratagene)) to generate mutants
F131V,D133A and G148E,G187A using the Muta-Gene M13 Kit (Bio-Rad). The
mutants were subsequently PCR-amplified and subcloned into pGilda as
EcoRI/XhoI fragments.
Bcl-2, Bcl-2(G145A), and Bax,
including their transmembrane-spanning domains, were subcloned into the
mammalian expression vector pCIneo (Promega) to yield pCIBcl-2,
pCIBcl-2(G145A), and pCIBax, respectively. To generate an expression
construct for BaxC with a transmembrane domain (Bax
C+TM), two
separate PCRs were performed with Bax(wt) (where wt is wild-type) in
pCIneo as a template to generate two fragments of 369 and 94 base
pairs, respectively. The following primers were used: 5
-ATC AGT GAA TTC ACT ATG GAC GGG GAG-3
, 5
-CGC CAC AAA GAT GGT CAC GTT GAA GTT GCC
GTC AGA AAA CAT GTC-3
, 5
-TCT GAC GGC AAC TTC AAC GTG ACC ATC TTT GTG
GCG GGA GTG-3
, and 5
-ATC GAT CTC GAG TCA GCC CAT CTT CTT CCA GAT-3
.
Bax
C+TM was generated by annealing these two PCR products together
(95 °C, 5 min; 65 °C, 5 min; and cooling slowly to <30 °C)
and amplifying the resulting fragment. The final 463-base pair fragment
was gel-isolated, digested with EcoRI and XhoI,
and ligated into EcoRI/SalI-digested pCIneo.
For GST and 6-histidine fusion proteins, Bcl-2(G145A), Bcl-xL, Bcl-xL(G138A), Bcl-xL(F131V,D133A), Bcl-xL(G148E,G187A), and Bax were cloned into the bacterial expression vector pGEX-4T-1 (Pharmacia Biotech Inc.) or pET15b (Novagen) and expressed in bacteria. In each case, the C-terminal membrane-spanning domain was deleted. The construct for expression of GST-Bcl-2 coding for amino acids 1-218 of human Bcl-2 was a generous gift from Dr. John Reed.
Protein Expression and Solid-phase Protein-Protein Binding Assays
Protein expression was induced by the addition of
isopropyl-1-thio--D-galactopyranoside to bacteria
transformed with the appropriate bacterial expression vector and grown
in 3-liter batches in shaker flasks. Expressed proteins were purified
by one-step affinity chromatography using His-Bind® metal
chelation resin (Novagen) (for 6-histidine fusion proteins) or
glutathione-Sepharose columns (Pharmacia) on a Pharmacia FPLC system
(21) (for GST fusion proteins), and their purity was assessed by
SDS-polyacrylamide gel electrophoresis. Protein concentrations were
determined using a bicinchoninic acid method (Pierce) with bovine serum
albumin as the standard. The solid-phase binding assays were performed
as described previously (21). Briefly, 6-histidine-tagged proteins were
coated onto microtiter wells, which were then incubated with a
GST-tagged binding partner. Binding was then detected with an anti-GST
monoclonal antibody and a conjugated secondary antibody.
Yeast Two-hybrid Interaction Assays
To generate a library of mutant Bax plasmids, pJG4-5-Bax was
transformed into Escherichia coli strain XLRed (Stratagene), and DNA was purified from 1 × 1010 cells.
Saccharomyces cerevisiae strain EGY48 (MAT
ura3 trp1 his3 LEU2::pLexAop3-LEU2) was
transformed with the following plasmids: pGilda-Bcl-2(G145A), p18-34
(28), and 10 µg of the pJG4-5-Bax library DNA. 500,000 transformants
were screened for growth in the absence of leucine and assayed for
-galactosidase activity. The library plasmid was recovered from
selected clones, and DNA sequence analysis was performed using an
Applied Biosystems Model 377 sequencer.
For quantitative -galactosidase activity assays, each bait vector
was cotransformed with its respective prey plasmid and the reporter
plasmid p18-34 into yeast strain EGY191 (MAT
ura3 trp1
his3 LEU2::pLexAop1-LEU2). Yeast cells were grown
in synthetic complete medium lacking uracil, tryptophan, and histidine
as necessary to select for the presence of various plasmids and
containing 2% raffinose to A600 = 0.2. Cultures
were induced with galactose to a final concentration of 2% for 6 h. 100 µl of the cells were lysed in 0.1 ml of Z-buffer (60 mM Na2HPO4, 40 mM
Na2PO4, 10 mM KCl, 1 mM
MgSO4, and 50 mM 2-mercaptoethanol) and 3.5 units of lyticase (Boehringer Mannheim) for 1 h in 96-well plates.
To assay
-galactosidase activity, 50 µl of
o-nitrophenyl-
-D-galactopyranoside (4 mg/ml)
in Z-buffer were added to the reaction, and the reaction was stopped by
the addition of 50 µl of 1 M NaOH. The absorbance of the
reaction was measured at A420. Three colonies
for each transformation were assayed in duplicates.
Mammalian Transfection Assays
For single transfections, mammalian 293 cells (2 × 105 cells/35-mm well) were transfected either with carrier
DNA or with 3.5 µg of pCIneo, or pCIBax, or pCIBaxC+TM and 0.7 µg of CMV-
-gal (where CMV is cytomegalovirus and
-gal is
-galactosidase). For cotransfection experiments, cells were, in
addition, transfected with either pCIBcl-2 or pCIBcl-2(G145A). The
transfected cells were incubated for 6 h at 37 °C, washed twice
with phosphate-buffered saline, refed with fresh complete medium, and
incubated overnight. 24 h after transfection, the cells were
rinsed with phosphate-buffered saline, fixed in phosphate-buffered
saline containing 2% formaldehyde and 0.2% glutaraldehyde, and
stained with 1 mg/ml X-gal solution. Blue cells were counted as either
viable (flat with normal epithelial shape) or dead (rounded and
shrunken).
Previous experiments with the BH1 mutant
Bcl-2(G145A) demonstrated that while it is functionally inactive and
fails to bind Bax, it can bind wild-type Bcl-2 (15). We have recently
demonstrated that Bcl-2 utilizes a common binding site to form either a
homodimer with the BH3 domain of another Bcl-2 molecule or a
heterodimer with the BH3 domain of Bax (21). Thus, the ability of
Bcl-2(G145A) to bind wild-type Bcl-2 could simply reflect an intact
binding site in the wild-type molecule binding to the intact BH3 domain in Bcl-2(G145A). To determine whether Bcl-2(G145A) itself contains an
active binding site, we used a yeast two-hybrid assay to measure its
interactions with different family members. The results confirm that
wild-type Bcl-2 can dimerize with Bax, whereas Bcl-2(G145A) cannot, and
that Bcl-2(G145A) can bind to wild-type Bcl-2 (Fig. 1A). More important, the results also show
that Bcl-2(G145A) is capable of dimerizing with another molecule of
Bcl-2(G145A) and that the strength of this interaction in this assay is
similar to that seen with wild-type Bcl-2 (Fig. 1A). The
ability of Bcl-2(G145A) to form a homodimer was confirmed in a
solid-phase binding assay using purified recombinant components (Fig.
1B). These data indicate that while the binding site in
Bcl-2(G145A) is altered so that it no longer binds Bax, it is still
capable of binding another Bcl-2(G145A) molecule.
Isolation of Bax Mutants That Bind Bcl-2(G145A)
Given the
observation that Bcl-2(G145A) contains an active binding site for at
least some ligands, we were interested in probing the structural basis
of the failure of this mutant to bind Bax. Our strategy was to randomly
mutagenize Bax, screen for mutants that bind Bcl-2(G145A) in a yeast
two-hybrid system, and then characterize the resulting mutant Bax
clones (Fig. 2). Bax mutants were prepared by growing a
yeast two-hybrid expression plasmid encoding a B42-Bax fusion protein
in an E. coli strain deficient in DNA repair (E. coli XLRed). A library of Bax mutants was cotransformed with a
LexA-Bcl-2(G145A) plasmid into S. cerevisiae strain EGY48. 500,000 mutants were screened for interaction with Bcl-2(G145A), resulting in 500 colonies that formed on selective medium. 120 of these
were further analyzed for -galactosidase activity by filter assay.
All 120 colonies were positive for
-galactosidase, and all were
subsequently analyzed for Bax expression by SDS-polyacrylamide gel
electrophoresis and Western blot analysis with an anti-Bax antibody.
Each of the 120 samples contained an immunoreactive band; the bands ran
with differing mobilities, but all ran faster than full-length
unmutagenized Bax (data not shown). We isolated and sequenced the DNA
from 13 clones, and the sequences of five of the clones are presented
in Fig. 3. Each of the 13 mutations resulted in a
truncated Bax protein with an intact N terminus including the BH3
domain, but with the BH2 domain and at least part of the BH1 domain
deleted (Fig. 3). The mutations were caused by frameshifts due either
to single base pair insertions (Bax mutants 5 and 11) or deletions (Bax
mutants 38 and 48) or to a single base pair substitution that
introduced a stop codon in the BH1 domain (Bax mutant 31) (Fig. 3). Bax
mutant 31, containing no extraneous amino acid sequence, was chosen for
the most extensive analysis and is referred to as Bax
C.
Binding of Bcl-2 and Bcl-2(G145A) to Bax
The binding of
Bcl-2 and Bcl-2(G145A) to wild-type and mutant Bax was analyzed using
quantitative yeast two-hybrid assays and a quantitative solid-phase
binding assay. As predicted from the results of the nonquantitative
screen, Bcl-2(G145A) bound avidly to BaxC in the quantitative yeast
two-hybrid assay. Wild-type Bcl-2 also bound to the Bax
C mutant
(Fig. 4A). To exclude the possibility that
Bax
C nonspecifically binds to LexA fusion proteins or directly
activates
-galactosidase expression by itself, we tested it in the
yeast two-hybrid assay against three control plasmids: an empty LexA
vector, LexA-Bad, and LexA-Ced-3. No
-galactosidase reaction product
was observed in any of these controls (Fig. 4A and data not
shown). The binding observed in the yeast two-hybrid assays was
confirmed by solid-phase assays using purified recombinant proteins.
GST fusions of both wild-type Bcl-2 and Bcl-2(G145A) demonstrated
saturable binding to immobilized 6×His-Bax
C (Fig. 4B).
In contrast, only wild-type Bcl-2 showed strong binding to wild-type
Bax in this assay (Fig. 4B).
Bax
Since BaxC was
found to bind to mutant Bcl-2, we sought to determine whether this
truncated Bax molecule would also bind to mutants of
Bcl-xL. Site-directed mutagenesis of Bcl-xL has yielded a series of mutants that fail to bind wild-type Bax, but that
differ in their functional effects on cell death (17, 24). For example,
immunoprecipitation studies have shown that Bcl-xL(G138A) does not bind wild-type Bax and is functionally inactive, whereas the
double mutants Bcl-xL(F131V,D133A) and
Bcl-xL(G148E,G187A) do not bind wild-type Bax, but
retain substantial cell survival activity (17, 24). The failure of
these mutant Bcl-xL molecules to bind wild-type Bax was
confirmed in yeast two-hybrid assays (Fig. 5,
A and B). However, when tested for binding to
Bax
C, all of the Bcl-xL mutants bound avidly (Fig. 5,
A and B). The Bcl-xL(G138A) and
Bcl-xL(F131V,D133A) mutants were further analyzed in
solid-phase binding assays. As in the yeast two-hybrid assay, these
Bcl-xL mutants failed to bind His6-Bax(wt), but
showed clear saturable binding to 6×His-Bax
C (Fig. 5, D
and E). Wild-type Bcl-xL bound to both wild-type
and truncated Bax molecules, with an apparent affinity that was greater
for truncated Bax (Fig. 5C).
Bax
Since BaxC was
found to bind more avidly to Bcl-2 and Bcl-xL than did
wild-type Bax, we compared these two Bax molecules for their ability to
induce apoptosis in the human embryonic kidney 293 cell line. The
efficiency with which wild-type Bax induces apoptosis is dependent
in part on the presence of its membrane anchor
sequence,2 as was seen previously for
certain fragments of Bak (22). Thus, to compare the death-inducing
effects of truncated Bax with wild-type Bax, we constructed an
expression plasmid with the Bax transmembrane domain fused to the C
terminus of Bax
C, denoted Bax
C+TM. 293 cells were transiently
transfected with the relevant Bax construct or empty expression vector
or no vector, together with a
-galactosidase expression construct.
After 24 h, cells were stained with X-gal, and blue cells were
evaluated for survival. The Bax
C+TM construct killed as efficiently
as did wild-type Bax (Fig. 6), although the expression
level of the Bax
C+TM protein was at least 20-fold lower than that of
wild-type Bax by Western blot analysis (data not shown). Thus, enhanced
death-promoting activity correlates with the enhanced binding avidity
of Bax
C.
Bcl-2(G145A) Inhibits Bax
Expression of wild-type Bcl-2 inhibited the cell death
induced by Bax expression in 293 cells (Fig. 7). In
contrast, Bcl-2(G145A), which cannot bind wild-type Bax, did not
protect against Bax-induced death in 293 cells (Fig. 7). These results
are in accord with earlier studies demonstrating that wild-type Bcl-2
inhibits cell death induced by interleukin-3 withdrawal in FL5.12
cells, but that Bcl-2(G145A) is inert (15). Since Bcl-2(G145A) binds
BaxC, we tested to see if this Bcl-2 mutant would specifically
inhibit Bax
C+TM-induced apoptosis. When cotransfected with
Bax
C+TM, the normally inactive Bcl-2(G145A) protein was as effective
as wild-type Bcl-2 in inhibiting cell death (Fig. 7). These results indicate that apoptosis can be suppressed by molecules that bind Bax-like proteins but that have no other intrinsic death-inhibiting activity.
We have utilized a strategy of random mutagenesis and yeast two-hybrid analysis to identify mutants of Bax that regain the ability to bind the inactive Bcl-2(G145A) protein. A similar strategy had previously been used to isolate mutants of Raf-1 that complement inactivating Ras mutations (27). These methods represent a general approach for probing the structural requirements for protein-protein interactions.
A panel of Bax mutants that bound to Bcl-2(G145A) was isolated, and
they all coded for truncated Bax proteins that included the BH3 domain
but lacked complete BH1 and BH2 domains. These results are in accord
with previous studies indicating that the BH3 domain in Bax is
responsible for the binding of Bax to Bcl-2 (23). The fact that
full-length Bax fails to bind Bcl-2(G145A) but that truncated Bax does
bind has interesting structural implications. The results demonstrate
that the G145A mutation in Bcl-2 does not destroy the binding site for
BH3 domains. This conclusion is further supported by our observation
that Bcl-2(G145A) can form a homodimer (Fig. 1, A and
B) since our previous work has shown that Bcl-2
homodimerization is a BH3-dependent interaction (21). Thus,
the data suggest a model in which the BH3 domain in wild-type Bax is
constrained in such a way that it cannot fit into the binding groove of
the Bcl-2(G145A) or Bcl-xL(G138A) mutant. In this model,
when conformational constraints are released by C-terminal Bax
truncation, binding can occur (Fig. 8). Similarly, other
mutations such as Bcl-xL(F131V,D133A) and
Bcl-xL(G148E,G187A) cannot bind the constrained BH3 domain
of wild-type Bax, but are capable of binding the presumably more
flexible truncated molecule.
The Bax mutants isolated in our screen all contained intact BH3
domains, but lacked most or all of the BH1/BH2 region. Interestingly, two naturally occurring proteins, Bik (22, 29, 30) and Bid (31), that
also contain only the BH3 domain have been described. Like Bax and
BaxC+TM, Bik and Bid are also inducers of cell death (29-31). The
existence of Bax-like molecules lacking BH1 and BH2 domains presents
the possibility that these molecules may have binding properties that
are distinct from those of wild-type Bax and that resemble the mutant
truncated Bax molecules described here. If so, cell survival functions
retained by mutants Bcl-xL(F131V,D133A) and
Bcl-xL(G148E,G187A) (24) could be due in part to their
ability to bind death-inducing family members that contain only BH3
domains, even though these mutants do not interact with Bax or Bak.
It has not been clearly established whether the cell-protective Bcl-2
family members or the death-inducing family members are the key
molecules that interface with the downstream death effector elements in
the cell. Data exist to support the hypothesis that the pro-survival
members have intrinsic anti-apoptotic activity that is antagonized by
the death-promoting members (24). Such anti-apoptotic activity may be
related to the binding of death effector molecules (32-34) or to
effects on ion homeostasis (35). However, other data support the idea
that the death-inducing family members actively promote apoptosis and
that this activity is antagonized by the pro-survival members (31, 36).
Additional data can be interpreted to support the heterodimer as the
active molecular species (37). We have demonstrated that the normally
inactive Bcl-2(G145A) mutant can suppress cell death induced by
BaxC+TM. Since this Bcl-2 mutant has no intrinsic anti-apoptotic
activity, its observed cellular effect must be due to binding and
inactivating the Bax
C+TM molecule. Thus, a Bcl-2 family member can
have cell survival effects strictly by passive sequestration of a
death-promoting family member.
We thank Dr. Erica Golemis (Fox Chase Cancer Center) for providing S. cerevisiae strains EGY48 and EGY191 and the anti-LexA antibody and Dr. Chris Kaiser (Massachusetts Institute of Technology) for plasmid pGilda. We thank Dr. John Reed (Burnham Institute) for plasmids pJG4-5-Bcl-2 and pJG4-5-Bcl-xL, GST-Bcl-2, and the anti-GST monoclonal antibody (7E5A6); Dr. Stan Korsmeyer for Bcl-2(G145A) cDNA; and Lisa Trout for helping with the artwork and the manuscript.