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Molecular and Cellular Biology, October 1999, p. 6673-6681, Vol. 19, No. 10
Gwen Knapp Center for Lupus and Immunology
Research,1 Department of
Medicine,2 Howard Hughes Medical
Institute,3 and Department of Molecular
Genetics and Cell Biology,4 The University of
Chicago, Chicago, Illinois 60637, and Pharmaceutical
Discovery Division, Abbott Laboratories, Abbott Park, Illinois
606645
Received 8 December 1998/Returned for modification 23 February
1999/Accepted 6 July 1999
bcl-x is a member of the bcl-2 family of
genes. The major protein product, Bcl-xL, is a
233-amino-acid protein which has antiapoptotic properties. In contrast,
one of the alternatively spliced transcripts of the bcl-x
gene codes for the protein Bcl-xS, which lacks 63 amino
acids present in Bcl-xL and has proapoptotic activity.
Unlike other proapoptotic Bcl-2 family members, such as Bax and Bak, Bcl-xS does not seem to induce cell death in the absence of
an additional death signal. However, Bcl-xS does interfere
with the ability of Bcl-xL to antagonize Bax-induced death
in transiently transfected 293 cells. Mutational analysis of
Bcl-xS was conducted to identify the domains necessary to
mediate its proapoptotic phenotype. Deletion mutants of
Bcl-xS which still contained an intact BH3 domain retained
the ability to inhibit survival through antagonism of
Bcl-xL. Bcl-xS was able to form heterodimers
with Bcl-xL in mammalian cells, and its ability to inhibit
survival correlated with the ability to heterodimerize with
Bcl-xL. Deletion mutants of Bax and Bcl-2, which lacked BH1
and BH2 domains but contained a BH3 domain, were able to antagonize the
survival effect conferred by Bcl-xL. The results suggest
that BH3 domains from both pro- and antiapoptotic Bcl-2 family members,
while lacking an intrinsic ability to promote programmed cell death,
can be potent inhibitors of Bcl-xL survival function.
Like several other Bcl-2 gene family
members, including bcl-2 and bax, the
bcl-x gene can encode several protein products as a result
of alternative splicing. The longest reading frame encodes
Bcl-xL, a protein with high homology to Bcl-2
(1). Like Bcl-2, Bcl-xL has four bcl-2 homology
(BH) domains, a region of low homology between BH3 and BH4, the loop
domain, and a transmembrane domain at the C terminus. Three other
transcripts, known as bcl-x Bcl-xS exhibits a more limited tissue distribution than
Bcl-xL, which appears to be the most ubiquitously expressed
isoform. Bcl-xS has been detected by Western blot analysis
in human tonsil, prostate, testis, ovary, and oviduct, as well as in
murine basal ganglia (21). bcl-xS
mRNA levels have been reported to increase prior to apoptosis in
involuting mammary epithelial cells and in ischemic rat brains (8,
15).
Bcl-xS has been shown, in several experimental systems, to
be capable of promoting apoptosis. Coexpression of Bcl-xS
with Bcl-xL or Bcl-2 reversed the protected phenotype of
these transfected cells in response to growth factor deprivation and
chemotherapeutic treatment (1, 24, 34). Transgenic mice
overexpressing Bcl-xS in keratinocytes demonstrated an
increased susceptibility to apoptosis in the skin induced by UV
irradiation (28). Infection with an adenoviral vector
engineered to encode Bcl-xS resulted in an increased susceptibility of tumor cells to apoptosis both in vitro and in vivo
(5, 9, 10). These data all demonstrate the efficacy of
Bcl-xS as a negative modulator of cell survival.
Bcl-xS lacks the BH1 and BH2 domains, which have been shown
to be important in mediating the antiapoptotic function of
Bcl-xL (38). These two domains are important not
only for heterodimerization with proapoptotic members, such as Bax and
Bak, but also for pore-forming properties (30, 31). Like
many other proapoptotic family members, Bcl-xS maintains an
intact BH3 domain. Unlike any other proapoptotic family member,
Bcl-xS also contains a BH4 domain, which has been shown to
be important for the antiapoptotic properties of Bcl-2 and
Bcl-xL (16, 17).
To investigate the domain(s) of Bcl-xS necessary to
potentiate apoptosis, deletion mutants of Bcl-xS were
generated and tested for function in a transient-transfection system.
The BH3 domain of Bcl-xS was found to be necessary for the
inhibition of the antiapoptotic function of Bcl-xL. The BH3
domains of several other Bcl-2 family proteins were tested and found to
share the ability to suppress the survival function of
Bcl-xL. These data suggest that the ability to inhibit the
antiapoptotic action of proteins such as Bcl-xL is a
feature shared by all BH3 domains, including BH3 domains which reside
in proteins with antiapoptotic function.
Plasmid construction.
Deletion mutations were generated by
PCR mutagenesis and confirmed by sequencing. All Bcl-xS
wild-type and deletion constructs had either a FLAG epitope (MDYKDDDDK)
or a hemagglutinin (MDYPYDVPDYA) epitope added to the 5' end by
PCR. These constructs were cloned into the plasmid pBluescript II SK(+)
(Stratagene) and subcloned into the EcoRI restriction site
of the mammalian expression plasmids pSFFV-Neo and pCDNA 3 (Invitrogen).
Cell culture and transfection.
Human embryonic kidney 293 cells were cultured at 37°C in 5% CO2 in Dulbecco's
modified Eagle's media supplemented with 10% fetal calf serum, 2 mM
glutamine, 100 U of penicillin per ml, and 100 µg of streptomycin per
ml. Transfections were carried out in either six-well plates or
10-cm-diameter plates. All death assays were conducted in six-well
plates. Cells were seeded at approximately 30% confluency and
transfected 24 h later by standard calcium phosphate protocols
with the indicated amounts of DNA plus 50 ng of the pEGFP plasmid
(Invitrogen), which expresses enhanced green fluorescent protein
(EGFP). The quantity of DNA transfected was kept constant for each well
or dish transfected throughout each individual experiment. Following
transfection, cells were harvested at 15 h for Western blot
analysis or 24 h for viability assays. Both adherent and
nonadherent cells were collected for cell harvesting. Adherent cells
were detached from the plates by incubation with a sterile solution of
5 mM EDTA in phosphate-buffered saline (PBS).
Viability assays.
Supernatants and cells were harvested
24 h following transfection. Cells were pelleted and fixed in 1 mM
paraformaldehyde for 10 min. Following fixation, cells were repelleted
and resuspended in 75% cold ethanol. After a minimum of 1 h of
incubation at 4°C, fixed cells were pelleted and resuspended in
propidium iodide (PI) staining buffer (3.8 mM sodium citrate, 0.125 mg
of RNase A per ml, and 0.01 mg of PI per ml) and incubated at 4°C for
1 h. Cells were then analyzed by flow cytometry to measure PI
staining of the EGFP-positive cells (22).
Western blotting and immunoprecipitation.
Cells were
harvested 15 h posttransfection. Cells in suspension and adherent
cell fractions were combined and pelleted. Cells were lysed in either
radio immunoprecipitation assay buffer (1% Nonidet P-40, 1%
deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) or NET-N (100 mM
NaCl, 1 mM EDTA, 20 mM Tris, 0.2% Nonidet P-40) for
immunoprecipitations. Lysates were supplemented with 8 µg of
aprotinin per ml, 2 µg of leupeptin per ml, and 170 µg of
phenylmethylsulfonyl fluoride per ml. After cellular debris was removed
by centrifugation, protein concentrations were assayed by colorimetric
bicinchoninic acid analysis (Pierce). Western blot analyses were
performed as described previously (2). For FLAG epitope
Western blots, membranes were incubated with 1 µg of M2 mouse
monoclonal anti-FLAG antibody (Kodak) per ml. Bcl-x expression was
assayed with either the S-18 rabbit polyclonal antibody (Santa Cruz) at
250 ng/ml or the 13.6 polyclonal antibody at a 1:5,000 dilution. Bax
was probed with the N-20 rabbit polyclonal antibody (Santa Cruz) at 250 ng/ml. The 12CA5 mouse monoclonal antibody (Boehringer Mannheim) was used at 1 µg/ml to detect hemagglutinin (HA) epitope tags.
Immunoprecipitations were carried out as described previously
(2). Briefly, cells were harvested and lysed in NET-N buffer
(pH 8.0). Lysates were precleared and incubated with antibody and then
incubated with protein G-agarose. Immunoprecipitated proteins were then
separated by SDS-polyacrylamide gel electrophoresis (PAGE). For every
100 µg of protein, FLAG immunoprecipitations were carried out with 1 µg of M2 antibody per ml, and HA immunoprecipitations utilized 12CA5
antibody (10 µg/ml).
Immunofluorescence staining.
Cells were harvested and fixed
with 1% paraformaldehyde at 15 h posttransfection. Fixed cells
were washed with a 0.03% saponin solution in PBS. M2 antibody staining
was conducted with 1 µg/106 cells in a 0.3% saponin
solution in PBS supplemented with 20% goat serum. Cells were stained
with M2 antibody for 1 h at 4°C, washed with 0.03% saponin, and
then incubated with an anti-mouse fluorescein isothiocyanate-conjugated
secondary antibody in 0.3% saponin for 1 h at 4°C. Following
secondary-antibody incubation, cells were washed twice with a 0.03%
saponin solution, followed by a wash with a fluorescence-activated cell
sorter buffer (PBS with 1% bovine serum albumin and 0.01% sodium
azide). Cells were then analyzed by flow cytometry for fluorescein
isothiocyanate expression.
In vitro fluorescence titration assay.
Recombinant BH3
peptide was purchased from PeptidoGenic Research & Co. (Livermore,
Calif.) and purified by reverse-phase high-performance liquid
chromatography on a C8 column. For titration, the
fluorescence emission of the Trp residues of Bcl-xL was
monitored as a function of increasing peptide concentration. The
fluorescence measurements were done on a Shimadzu RF5000U
spectrofluorometer with excitation and emission wavelengths of 290 and
340 nm, respectively. The Trp fluorescence intensity was fitted with
the equation Yeast culture and Bax toxicity assay.
Yeast studies were
carried out with Saccharomyces cerevisiae W303 (ade2-1
can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1). The yeast cells
were maintained on selective synthetic complete medium (1.5 g of U.S.
Biological yeast nitrogen base per liter, 5 g of ammonium sulfate
per liter, 2 g of amino acid powder mix lacking histidine per
liter). Human cDNAs for Bax, the Bax Bcl-xS fails to promote cell death but can reverse
Bcl-xL protection.
In order to investigate the
proapoptotic function of Bcl-xS, the human embryonic 293 kidney epithelial cell line was transiently transfected by using
calcium phosphate with expression constructs of various Bcl-2 family
members. Cells were cotransfected with an EGFP expression plasmid as a
marker for transfection. Twenty-four hours posttransfection, cells were
harvested, and the DNA content of the EGFP-positive cells was measured
by staining them with PI, followed by flow cytometric analysis
(22). For each transfection, 1 µg of each expression
construct was used and a quantity of a control vector (Neo) was added
to maintain total DNA quantity at 3 µg per transfection. At the
concentrations used, Bax induced a sixfold-higher percentage of
subdiploid cells than the Neo control vector (Fig.
1A). Equal or greater amounts of
Bcl-xS (up to 3 µg per transfection) had no effect on
cell viability. The ability of 1 µg of Bax to induce apoptosis was
blocked by cotransfection with an equal amount of Bcl-xL.
When all three plasmids were cotransfected at 1 µg each, the presence
of Bcl-xS reversed the ability of Bcl-xL to
inhibit Bax-induced apoptosis. Cotransfection of Bcl-xS
with Bax did not enhance Bax-induced apoptosis, and the transfection of
Bcl-xL and/or Bcl-xS in the absence of Bax
transfection was indistinguishable from that of the Neo control (data
not shown). To confirm that the alterations in cell viability
correlated with the expression of the transfected proteins, cell
lysates of the transfectants were analyzed for protein expression (Fig.
1B). Western blotting confirmed that the expected proteins were
observed following transfection and that the levels of individual
proteins were similar whether transfected singly or in combination.
Although the levels of Bax relative to those of Bcl-xL and
Bcl-xS are difficult to estimate since those proteins are
detected with distinct antibodies, the levels of Bax transfected alone
consistently induced death in 293 cells. In addition, we have found
that the levels of Bcl-xS detected by Western blotting with
anti-Bcl-x polyclonal antisera relative to those Bcl-xL are
consistently underestimated (see below).
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The BH3 Domain of Bcl-xS Is Required
for Inhibition of the Antiapoptotic Function of
Bcl-xL
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TM, bcl-x
, and
bcl-x
, are similar to bcl-xL
except that these transcripts lack a transmembrane domain at the C
terminus (11, 13, 37). These alternatively spliced
transcripts produce proteins that are also antiapoptotic. In contrast,
the smallest transcript, bcl-xS, which differs
from bcl-xL by having 189 bases spliced out,
encodes a proapoptotic protein. As a result of this deletion, Bcl-xS lacks BH1 and BH2 domains but still contains BH3,
BH4, loop, and transmembrane domains.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Iobs = Xb × I(b 
f), where Iobs is the difference between the observed intensity and the fluorescence intensity of the free protein, Xb is the
mole fraction of the bound state, and I(b f) is the fluorescence difference between the bound and
free forms of the protein. Mole fractions were calculated from a
dissociation constant, Kd, by using the known
initial concentrations of the protein and peptide. A least-squares
analysis was then performed with systematic variations of
Kd and I(b f). A linear correction for peptide fluorescence was
applied to the BH3 peptide prior to data analysis.
2 domain, Bcl-xS,
and the Bcl-xS 2 domain, all with an N-terminal HA tag, were cloned into the multicopy expression plasmid p423 GALL (histidine selection) controlled by a galactose-inducible promoter
(26). Yeast cells were transformed with the p423 GALL
expression plasmids by the lithium acetate method and selected on
histidine-deficient plates (12). For the Bax toxicity assay,
yeast cells were grown at 30°C for 24 h on histidine-deficient
plates containing 2% dextrose as a carbon source, after which they
were resuspended in histidine-deficient liquid media containing 2%
raffinose. The yeasts were then normalized to an optical density of 0.3 at 600 nm, spotted at 10-fold serial dilutions on both
histidine-deficient dextrose and histidine-deficient 2% galactose-2%
raffinose plates, and allowed to grow at 30°C for 3 to 4 days.
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
Bcl-xS can prevent Bcl-xL rescue
of Bax-induced cell death. (A) 293 cells, plated in six-well dishes,
were transiently transfected with the indicated combinations of
constructs, each at 1 µg per transfection. The amount of total DNA
per transfection was maintained at 3 µg. Cells were harvested 24 h posttransfection, and the subdiploid percentage of the subpopulation
of cells expressing the marker for transfection, EGFP, was measured.
Means and standard deviations of at least three independent experiments
are shown. (B) Transfections were conducted as for panel A, but cells
were harvested at 15 h posttransfection. Cells were lysed, and
proteins were separated by SDS-PAGE. Proteins were blotted with a
polyclonal anti-Bcl-x antibody and a polyclonal anti-Bax antibody.
The BH3 domain is necessary for the proapoptotic phenotype of
Bcl-xS.
To identify the regions of Bcl-xS
important for its inhibition of Bcl-xL function, a series
of deletion mutants of Bcl-xS were generated and tested for
function in the 293 assay (Fig. 2). With
the previously solved structure of Bcl-xL used as a
reference, the deletion mutants of Bcl-xS were designed to
test the significance of secondary-structure elements found in
Bcl-xS. The 63 amino acids in Bcl-xL that are
not found in Bcl-xS encompass the core hydrophobic
alpha-helices 5 and 6 in Bcl-xL. The primary sequences of
the other amphipathic helices are little affected by this deletion and
are predicted to maintain their secondary structure in
Bcl-xS. The deletion mutants of Bcl-xS were
designed to test the contribution of the remaining alpha-helices (1,
2, 3, and 4) to proapoptotic function. All Bcl-xS
constructs contained an N-terminal FLAG epitope tag (MDYKDDDDK) and the
wild-type transmembrane domain. Previous work with the
Bcl-xL homologue Bcl-2 demonstrated that the C-terminal hydrophobic domain was necessary and sufficient for proper membrane targeting (27). When transfected in the absence of other
Bcl-2 family members (at 1 µg per transfection), as in the case of
wild-type Bcl-xS, none of the deletion mutants were able to
induce apoptosis (data not shown). The Bcl-xS mutants
were then tested for their ability to inhibit cell survival in the
presence of Bcl-xL and Bax. One microgram each of Bax,
Bcl-xL, and the various Bcl-xS deletion mutants
was transiently transfected into 293 cells. Analysis of the
Bcl-xS deletion mutants revealed that most of the
Bcl-xS molecule is dispensable for its proapoptotic
function, including the BH4 domain and the loop domain (Fig.
3A). The only region essential for
function appeared to be the 2 helix, which encompasses the BH3
domain. Furthermore, a point mutant of Leu 90 in the BH3 domain, which
is completely conserved in the BH3 domains of all mammalian Bcl-2
family proteins, is sufficient to inactivate construct 2347. To
ensure that differential expression of the deletion constructs was not
responsible for the results, the transfectants were intracellularly
stained with an anti-FLAG antibody. No significant differences were
detected in the expression levels of the different mutants (Fig. 3B).
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Bcl-xS heterodimerizes with Bcl-xL through
its BH3 domain.
To determine whether heterodimerization with
Bcl-xL correlated with the phenotype of the various
Bcl-xS mutants, equal amounts of Bcl-xL and
various Bcl-xS constructs were cotransfected into 293 cells. In addition to wild-type Bcl-xS, three deletion
mutants of Bcl-xS were chosen for this assay. Constructs
2347 and 2347 L90A differ only by a point mutation that abolishes
2347 function. The mutant 2, containing the BH3 domain, is the
minimal construct which still facilitates apoptosis when coexpressed
with Bax and Bcl-xL. Cells were harvested 15 h
posttransfection, and the Bcl-xS mutants were
immunoprecipitated with the anti-FLAG antibody against the FLAG epitope
present on these constructs. Western blot analyses were performed on
both whole-cell lysates and immunoprecipitated proteins to examine the
degree of coassociation of Bcl-xL with the
Bcl-xS mutants (Fig. 4). In
contrast to the results shown in Fig. 1, Fig. 4 shows that the
anti-Bcl-x antibody S-18 recognizes Bcl-xS and
Bcl-xL equivalently, confirming that roughly equivalent levels of Bcl-xL and Bcl-xS are achieved under
these transfection conditions. Unfortunately, this antibody also
interacts nonspecifically with a 32-kDa protein. In Fig. 4, the amount
of Bcl-xL expressed is constant in all lanes. Since the
anti-Bcl-x antibody, S-18, used in these experiments binds to the 1
helix of Bcl-x, it did not recognize the deletion mutants of
Bcl-xS. The Western blot analysis with anti-FLAG antibody
demonstrated that all the Bcl-xS constructs are expressed
roughly equivalently with the exception of 2, which is expressed at
a lower level. Immunoprecipitations with the anti-FLAG antibody,
followed by Western blotting with the same antibody, demonstrated that
the three deletion mutants of Bcl-xS were
immunoprecipitated in the same relative ratio as expressed in the
whole-cell lysates. As shown in Fig. 4D, Bcl-xS migrated
alongside the immunoglobulin light chain of the anti-FLAG antibody used
for the immunoprecipitation. This prevented the visualization of
Bcl-xS in the anti-FLAG Western blot. Western blot analysis
with the S-18 antibody demonstrated the presence of Bcl-xS
in the immunoprecipitation. The two functional mutants of
Bcl-xS, 2347 and 2, as well as wild-type
Bcl-xS, heterodimerized with Bcl-xL, whereas
the nonfunctional 2347 L90A mutant did not. Interestingly, the
minimal 2 construct, even though underexpressed, heterodimerized
with significantly more Bcl-xL than did the other two
functional constructs.
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2 construct (minus the transmembrane domain) was synthesized and
tested in the in vitro fluorescence titration assay. Titrations with a
25-mer peptide encompassing the BH3 domain of Bcl-x (residues 80 through 104) bound to Bcl-xL with a
Kd of 0.49 ± 0.28 µM, which is of a
magnitude comparable to the binding constant of the 16-mer peptide of
Bak BH3 to Bcl-xL. This value is 3 orders of magnitude greater than that of the Bcl-x 16-mer peptide composed of residues 84 to 99, which had a Kd of 325 µM. Thus, as was
observed with the BH3 domain of Bad, although a minimal BH3 peptide of
Bcl-x did not heterodimerize with Bcl-xL, extension of the
peptide to encompass the entire alpha-helical domain (2) resulted in
high-affinity binding.
It is possible that Bcl-xS interacts with other Bcl-2
family members, particularly Bax. Bid, another BH3-only family member, has been reported to heterodimerize with Bcl-2 and Bax, leading to the
inactivation of Bcl-2 and the potentiation of Bax-induced death
(35). To test whether Bcl-xS and Bax interacted,
293 cells were transfected with either FLAG-Bcl-xL or
FLAG-Bcl-xS, and the lysates were immunoprecipitated with
an anti-FLAG antibody and Western blotted with either anti-Bcl-x
antibody or anti-Bax antibody. Bcl-xL interacted with
endogenous Bax, as shown previously. However, no significant amount of
Bax was detectable in the Bcl-xS immunoprecipitation (data
not shown), supporting the yeast two-hybrid data that demonstrated the
interaction of Bcl-xS with Bcl-xL and Bcl-2 but
not with Bax (32).
BH3 domains from both pro- and antiapoptotic Bcl-2 proteins exhibit
an intrinsic antisurvival phenotype.
Most studies of the function
of BH3 domains have focused on the BH3 domains found in proapoptotic
proteins, such as Bak, Bax, and Bad. The studies presented here
demonstrate that the BH3 domain found in Bcl-xS, which is
identical to the BH3 domain of Bcl-xL, can also antagonize
survival through the inactivation of Bcl-xL. This suggested
that all BH3 domains might have an intrinsic ability to inhibit
survival through heterodimerization with antiapoptotic Bcl-2 family
members, resulting in their inactivation. To test this possibility, we
generated deletion mutants of two additional Bcl-2 family members, Bax
and Bcl-2. Relying on the homology between Bcl-xL and these
two proteins, the new mutants were designed both to be analogous to
2 (the smallest Bcl-xS deletion mutant which still
retained function) and, like Bcl-xS 2, to contain the
BH3 domain as well as the putative C-terminal transmembrane domain (Fig. 5). These mutants, designated Bax
2 and Bcl-2 2, were tested for their ability to antagonize the
ability of Bcl-xL to block Bax-induced death in 293 cells
(Fig. 5A). As demonstrated previously, wild-type Bcl-xS and
the Bcl-xS 2 construct prevented Bcl-xL from
protecting against Bax-induced apoptosis. As expected, the
2-containing deletion construct of Bax also prevented
Bcl-xL from exerting its survival properties in the
presence of Bax. Surprisingly, however, the 2-containing deletion
mutant of Bcl-2, an antiapoptotic protein, was also able to block
Bcl-xL function under these conditions. As observed with
the deletion mutants of Bcl-xS, none of the transfected
2 mutants of Bax and Bcl-2 led to significant apoptosis in the
absence of cotransfected Bax, even when the concentration of
transfected plasmid was increased threefold (Fig. 5B and data not
shown).
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2 constructs of Bax or Bcl-xS under similar conditions had no effect on the growth of yeast (Fig.
5D). These data support the results obtained in 293 cells, which
suggest that BH3-only-containing constructs lack the ability to
directly promote cell death.
BH3 domain of Bax shows higher affinity for Bcl-xL but
cannot promote death independently.
As noted previously, the
immunoprecipitation of the 2 construct of Bcl-xS
coprecipitated a significantly greater amount of Bcl-xL
than did either Bcl-xS or 2347, particularly when the lower levels of expression of 2 relative to the other constructs are
taken into account. This fact suggested that the minimal 2 construct
might have a higher affinity for Bcl-xL, perhaps due to the
lack of steric hindrance which might otherwise be imposed upon the BH3
domain by other domains present in Bcl-xS. To investigate whether increased binding by minimal BH3 domains is a general characteristic of Bcl-2 family proteins, we compared binding affinities of Bax and Bax 2 for Bcl-xL. Both Bax and Bax 2 were
N-terminally tagged with the HA epitope. Equal quantities of
Bcl-xL and either Bax or Bax 2 expression constructs
were cotransfected. Cells were harvested, lysed, and immunoprecipitated
with the anti-HA antibody 12CA5. A nonspecific band comigrated with HA
Bax to about 21 kD. Similar to the 2 construct of
Bcl-xS, the Bax 2 protein was underexpressed relative to
wild-type Bax (Fig. 6). The Bax 2
protein heterodimerized with more Bcl-xL than did the
wild-type Bax protein. Despite this increased ability to associate with Bcl-xL, Bax 2 lacked the independent cell-killing
function in 293 cells displayed by wild-type Bax (Fig. 5). Thus, in
this assay, the ability of proapoptotic proteins to heterodimerize with
Bcl-xL correlated with an ability to reverse the functional
effects of Bcl-xL, but did not appear to reflect a direct
proapoptotic function. In contrast, full-length Bax did not require the
presence of Bcl-xL to promote death.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The proapoptotic product of the bcl-x gene, Bcl-xS, is a potent antagonist of the antiapoptotic function of Bcl-xL and Bcl-2. Although much work has focused on elucidating the role of the major product of the bcl-x gene, Bcl-xL, relatively little attention has been directed towards Bcl-xS. The studies described here have examined the mechanism of action of Bcl-xS and have identified the BH3 domain as necessary for its function. Furthermore, it was found that BH3 domains, in general, have an ability to block Bcl-xL from functioning. This ability, however, is not sufficient to account for the proapoptotic, death-inducing action of Bax.
It is interesting that Bcl-xS did not induce apoptosis whereas Bax was able to do so. Previous reports have suggested that Bcl-xS can induce apoptosis (5). Furthermore, other family members which lack BH1 and BH2 domains, such as Bik and Hrk, were shown to be able to induce apoptosis in other cell lines by a similar transient transfection protocol (14, 18). However, the overexpression of Bcl-xS at levels that completely inhibit Bcl-xL did not induce apoptosis on its own. This discrepancy in apoptotic induction could be due to many different factors. The simplest explanation might be the differing levels of endogenous apoptosis-regulatory factors in different cell lines. The 293 cells, which were established by adenoviral transformation, express the viral homologue of Bcl-2, the E1B 19,000-molecular-weight (19K) protein. E1B 19K has previously been shown to be resistant to inhibition by Bcl-xS (23). This, however, cannot be the only explanation for the failure of Bcl-xS to cause death independently. Unlike Bax, which contains BH1 and BH2 domains in addition to a BH3 domain, Bcl-xS is unable to induce cell death when overexpressed in 293 cells or when expressed in yeast.
We have shown that the association of Bcl-xS with Bcl-xL is dependent on an intact BH3 domain. The other domain present in Bcl-xS, BH4, is dispensable for its proapoptotic phenotype. As in the case of BH3-only family members, such as Bad, Bik, and Hrk, which lack BH1 and BH2 domains, the proapoptotic function of Bcl-xS appears to be dependent on its ability to heterodimerize and inactivate antiapoptotic proteins, such as Bcl-xL.
The ability of Bcl-xS to heterodimerize had, hitherto, been in dispute. Although the yeast two-hybrid data suggested that Bcl-xS could heterodimerize with Bcl-xL, in vivo experiments failed to show evidence of such interactions (24). There are several significant differences between the experiments described in this study and those previously described. Previous studies on interactions were carried out with the FL5.12 cell line and stable transfectants. The present studies used both a different cell line and a transient-transfection assay, which may have allowed the introduction of higher levels of expression than can be maintained by stable expression. In the FL5.12 cell line, clones expressing high levels of Bcl-xS could not be isolated, whereas clones with highly expressed Bcl-xL were easily generated, suggesting that high levels of Bcl-xS put cells at a growth disadvantage even in the absence of growth factor withdrawal, perhaps due to increased susceptibility to environmental stresses. Furthermore, FL5.12 cells express relatively high levels of endogenous Bax protein, a strong binder of Bcl-xL, which could serve as a possible competitive inhibitor for Bcl-xL-Bcl-xS interactions. In addition, interactions could be influenced by C terminus-dependent targeting. Recent evidence has suggested that the ability of the carboxy terminus to induce the membrane localization of Bax can be regulated in at least some cell lines (36). Finally, the interactions detected in 293 cells were weak in comparison with Bax-Bcl-xL interactions assayed in 35S-labeled FL5.12 cells.
Previous findings demonstrated that Bad, a BH3-only-containing Bcl-2 family member, lacked heterodimerization-independent cell death-promoting activity. Neither wild-type E1B 19K, which does not heterodimerize with Bad, nor functional point mutants of Bcl-xL that failed to bind to Bad were inhibitable by the overexpression of Bad (3, 19). Mutants of Bcl-xS lacking an ability to heterodimerize with Bcl-xL could not reverse the survival function of Bcl-xL, suggesting that heterodimerization is the mechanism through which BH3-only proteins exert their antisurvival properties. Thus, BH3-only proteins may lack independent proapoptotic activity.
Unlike Bcl-xS, proapoptotic proteins with BH1 and BH2
domains, such as Bax, can induce apoptosis irrespective of their
ability to associate with antiapoptotic proteins (33).
Bcl-xS failed to induce death in 293 cells even when
transfected at high levels. Although Bcl-xS and Bax can
heterodimerize with and inactivate Bcl-xL, only Bax has the
ability to induce apoptosis independently in 293 cells and to cause a
growth defect in yeast. Furthermore, the Bax deletion mutant, Bax 2,
which lacks the BH1 and BH2 domains of Bax but contains a BH3 domain,
has an even greater ability to heterodimerize with Bcl-xL
than with wild-type Bax but is unable to induce apoptosis in 293 cells
or delayed growth in yeast when expressed alone. Bax 2 is still a
functionally active proapoptotic entity, as it was able to antagonize
the ability of Bcl-xL to inhibit Bax. These data clearly
demonstrate that the death-inducing activity of Bax requires more than
heterodimerization to Bcl-xL. One possibility is that the
heterodimerization-independent activity and the death-inducing activity
of Bax are both dependent on pore formation. Since pore formation is
predicted to be dependent on the 5 and 6 helices encompassing the
BH1 and BH2 domains (31), which are absent in
Bcl-xS and Bax 2, these two constructs would be
predicted to lack the ability to form channels in lipid bilayers. Recent data suggest that point mutations in the 5 and 6 helices affect pore-forming properties which can, in turn, influence apoptotic regulation by these proteins (25). Thus, it is reasonable to conclude that the deletion of these two helices can have significant effects on the ability of these proteins to independently regulate cell death.
The ability of the BH3 domains from Bax, Bcl-x, and Bcl-2 to facilitate
apoptosis to a similar degree suggests that all BH3 domains have an
intrinsic ability to inactivate inhibitors of apoptosis
(20). An earlier finding demonstrated that the BH3 domains
of the proapoptotic proteins Bak, Bax, and Bid, but not that of Bcl-2,
could promote apoptosis in Xenopus egg extracts (7). However, the BH3 peptides used in that system were
15-mers, while the minimal 2 constructs used in our assays contained
a sequence flanking the 2 helix. As the fluorescence-quenching experiments with Bad and Bcl-x peptides of various lengths demonstrate, the ability of BH3-containing peptides to bind to Bcl-xL
can depend on the sequence outside the 2 helix (19).
Whether this additional sequence is necessary for specific binding
interactions or simply for the stabilization of the core 2 helix is unclear.
The ability of the BH3 domain of Bcl-xS and Bcl-2 to inhibit cell survival in the presence of Bax and Bcl-xL presents an interesting problem. The BH3 domain of Bcl-xS is identical to the BH3 domain of Bcl-xL. Furthermore, unlike Bcl-xS, Bcl-2 has no known alternative-splicing variants which are proapoptotic, yet it still contains a proapoptotic BH3 domain. Clearly, this proapoptotic function of BH3 is subordinate to the antiapoptotic effects of intact Bcl-2. The conversion by caspase cleavage of Bcl-2 and Bcl-xL from antiapoptotic to proapoptotic proteins, as recently reported, could be due to enhancement in function or the availability of the BH3 domain resulting from a confirmational change (4, 6). Alternatively, a BH3 domain-only construct may act as a dominant negative. For example, it may be that the BH3 domain is critical for dimerization but additional regions of the protein, including BH1 and BH2, are necessary for prosurvival function. The inactivation of the survival function of Bcl-2 by point mutations in BH1 and BH2 support this possibility (38). BH1 and BH2 domains have been shown to play a critical role in the formation of the hydrophobic binding pocket of Bcl-xL (30). In addition, these domains are necessary for the pore-forming properties of Bcl-xL. Further experiments will need to be conducted to determine if one or both of these functional properties play an important role in the proapoptotic function of Bax or the antiapoptotic function of Bcl-xL.
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ACKNOWLEDGMENTS |
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B.S.C. and A.K. made equal contributions to this work.
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FOOTNOTES |
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* Corresponding author. Present address: Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104. Phone: (215) 746-5534. Fax: (215) 746-5511. E-mail: craig{at}mail.med.upenn.edu.
Present address: Abramson Family Cancer Research Institute,
University of Pennsylvania, Philadelphia, PA 19104.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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