(Received for publication, July 12, 1995; and in revised form, December 15, 1995)
From the
Bcl-x, a member of the Bcl-2 family, has two alternatively
spliced forms, Bcl-x and Bcl-x
.
Bcl-x
, like Bcl-2, is able to protect cells from a wide
variety of apoptotic stimuli. Bcl-x
, as a result of
alternative splicing, lacks 63 amino acids that comprise the region of
greatest amino acid identity between Bcl-x
and Bcl-2. These
amino acids contain the highly conserved BH1 and BH2 regions, which
have been used to define the Bcl-2 family. We show that both
Bcl-x
and Bcl-x
are able to regulate cell
survival in a dose-dependent fashion. Bcl-x
is able to
increase the cellular apoptotic threshold and is able to form stable
complexes with Bax both in vitro and in vivo. In
contrast, Bcl-x
can effectively inhibit the protective
effects of Bcl-x
following growth factor withdrawal and
chemotherapeutic drug treatment. However, compared with Bax,
Bcl-x
binds to Bcl-x
weakly when assessed by in vitro binding assays. Coimmunoprecipitation from mammalian
cells demonstrates that Bcl-x
does not show an observable
ability to form heterodimers with other Bcl-2 family members. In
addition, overexpression of Bcl-x
does not alter the
ability of Bax to heterodimerize with Bcl-x
in
vivo. Thus, Bcl-x
does not appear to function by
competitively disrupting the formation of dimers composed of other
Bcl-2 family members. This suggests that Bcl-x
can enhance
cellular sensitivity to apoptosis via a mechanism of action distinct
from other Bcl-2 family members that promote apoptosis.
Apoptosis is an active cellular suicide process that is characterized by distinct biochemical and morphological changes such as DNA fragmentation, plasma membrane blebbing, and cell volume shrinkage (1, 2) . Apoptosis is recognized as an important physiological event involved in development, in organismal homeostasis, and possibly even in the prevention of neoplastic transformation(3, 4, 5, 6, 7, 8) . Recent data suggest that disruption or dysregulation of the genes that control apoptosis affect neuronal development, disturb lymphocyte homeostasis, initiate tumor progression, and enhance resistance to present cancer treatment modalities(9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) .
A number of genes have been described that function to regulate
susceptibility to apoptosis. We have recently identified a gene, bcl-x, which is a member of the growing Bcl-2
family(20) . bcl-x has two alternatively spliced forms
that encode two different protein products. Bcl-x has the
larger open reading frame of 233 amino acids and, like Bcl-2, functions
to inhibit apoptosis induced by a variety of stimuli. As a result of
alternative splicing, Bcl-x
lacks an internal 63 amino
acids that comprise the region of greatest conservation between
Bcl-x
and Bcl-2. Although Bcl-x
has been shown
to inhibit the ability of Bcl-2 to protect from growth factor
withdrawal, the ability of Bcl-x
to inhibit function or
competitively bind to other family members has not been examined.
Within the internal 63-amino acid domain deleted in
Bcl-x, there exists two homology regions that define
members of the Bcl-2 family. These regions are referred to as BH1 and
BH2 and have been suggested to be important in mediating protein
interactions between Bcl-2 family members. Mutations in the BH1 or BH2
regions of Bcl-2 or Bcl-x
disrupt their ability to
heterodimerize with Bax, a Bcl-2 family member that accelerates cell
death when overexpressed in a growth factor-dependent cell line.
Moreover, these mutations abrogate the ability of Bcl-2 and Bcl-x
to protect cells from
apoptosis(21, 22, 23) . Another family
member, Bad, preferentially heterodimerizes with Bcl-x
through BH1 and BH2 interactions and functions to antagonize the
anti-apoptotic properties of Bcl-x
by disrupting
Bax/Bcl-x
heterodimers(24) . The absence of BH1 and
BH2 regions in Bcl-x
suggests that Bcl-x
may
promote cell death by a mechanism distinct from Bax or Bad. However,
analysis examining protein interactions using the yeast two-hybrid
system has shown that Bcl-x
retains the ability to
associate with Bcl-2 and Bcl-x
and argues that Bcl-x
may inhibit Bcl-2 function through a mechanism based on
competitive dimerization(22, 25) . The demonstration
that Bcl-2 family members are able to selectively interact with each
other in both the yeast two-hybrid system and in mammalian cells
suggests that protein interactions between these molecules may be an
important mechanism to regulate the apoptotic threshold of a cell.
The present experiments were undertaken to further characterize the
functional and biochemical properties of Bcl-x in mammalian
cells. Here we show that Bcl-x
is able to antagonize the
protective effects of Bcl-x
in a dose-dependent manner.
However, in comparison to Bcl-x
, Bcl-x
has a
substantially reduced ability to form immunoprecipitatable dimers with
other Bcl-2 family members. These data suggest that Bcl-x
does not reduce resistance to apoptosis simply by acting as a
competitive dimerization substrate.
Clones were categorized into two groups, intermediate
Bcl-x expressors and high Bcl-x
expressors.
Clones in each of the two groups varied in their expression of
Bcl-x
(Fig. 1). In the absence of Bcl-x
expression, Bcl-x
protected FL5.12 cells from
apoptosis induced by IL-3 withdrawal in a dose-dependent fashion. Fig. 2A shows that clone 22, which expresses a high
amount of Bcl-x
, had a dramatically enhanced viability
after growth factor removal when compared with the control transfected
cells. Clone 7, in accord with its lower Bcl-x
expression
levels, displayed a viability intermediate between the Neo control
transfectants and clone 22.
Figure 1:
Expression levels of Bcl-x and Bcl-x
. FL5.12 cells were cotransfected with equal
molar amounts of pSFFV-Neo-Bcl-x
and
pSFFV-Neo-Bcl-x
. Single cell clones were derived by
limiting dilution and screened by immunoblotting with 2A1, a mouse
monoclonal antibody to Bcl-x. Bcl-x
migrates with an
apparent molecular mass of 30 kDa, whereas Bcl-x
migrates
with an apparent molecular mass of 21 kDa. Clones were grouped into
intermediate Bcl-x
expressors (2, 14, 21, 7) and high Bcl-x
expressors(9, 19, 10, 22) .
The lane labeled N represents a Neo control transfectant.
Molecular masses are indicated in kilodaltons at the right of
the gel.
Figure 2:
Bcl-x protects cells from
apoptosis in a dose-dependent fashion and is antagonized by
Bcl-x
. The clones shown in Fig. 1were washed 3
times in IL-3-free medium and resuspended at 5
10
cells/ml. Viability was determined over the course of 6 days by
propidium iodide exclusion. A, comparison of viability between
an intermediate Bcl-x
expressor (clone 7), a high
Bcl-x
expressor (clone 9), and a Neo control transfectant. B, viability of intermediate Bcl-x
expressors in
the presence of various levels of Bcl-x
. C,
viability of high Bcl-x
expressors in the presence of
various levels of Bcl-x
. These results are representative
of at least three independent experiments.
Fig. 2B demonstrates
that when Bcl-x was expressed in conjunction with
intermediate levels of Bcl-x
, it took relatively little
Bcl-x
to almost completely reverse the protective effects
of Bcl-x
from IL-3 withdrawal. After exhibiting significant
protection at 24 h, clones 2, 14, and 21 precipitously lost viability
between 24 and 48 h and reached levels at or below the control
transfectants (compare clones 2, 14, and 21 in Fig. 2B with the Neo clone from Fig. 2A). In the presence
of high levels of Bcl-x
expression, it took correspondingly
more Bcl-x
to see significant antagonism of Bcl-x
(Fig. 2C). The level of antagonism increased with
increasing amounts of Bcl-x
(compare clones 10, 19, and 9).
To further confirm that Bcl-x is able to antagonize the
ability of Bcl-x
to protect FL5.12 cells from growth factor
removal, we also supertransfected a Bcl-x
expressing clone
with the Bcl-x
expression vector. Additionally, we employed
a transient transfection assay to determine the effects of Bcl-x
on viability. In both cases, similar results to the
cotransfectants were obtained (data not shown).
In
clones that expressed high amounts of Bcl-x, high levels of
Bcl-x
were required to significantly antagonize Bcl-x
(Fig. 3, B and D). Only in clone 9 did
we see significantly enhanced sensitivity to either etoposide or
vincristine treatment when compared with clone 22.
Figure 3:
Bcl-x is able to antagonize
the multidrug resistance conferred by Bcl-x
. The clones
from Fig. 1were resuspended in medium without G418 at a
concentration of 5
10
cells/ml. Either 10 µg/ml
of etoposide (A and B) or 0.1 µg/ml of
vincristine (C and D) was added, and viability was
determined over the course of 5 days by propidium iodide exclusion.
These results are representative of at least three independent
experiments.
Figure 4:
Bcl-x and Bcl-x
exhibit similar immunofluorescent staining patterns. A polyclonal
population of FL5.12 cells cotransfected with HA-Bcl-x
and
FLAG-Bcl-x
were immunofluorescently stained with an anti-HA
antibody and an anti-FLAG antibody. Specimens were analyzed by
laser-scanning confocal microscopy. Top, Bcl-x
staining alone. Middle, the same field showing
Bcl-x
staining. Bottom, composite image of
Bcl-x
and Bcl-x
staining.
The
cotransfected cells used in the immunofluorescent staining experiment
were from a bulk transfection using equal molar amounts of each plasmid (Fig. 4). Because Bcl-x increases sensitivity to
cell death, cells that express high levels may show a survival
disadvantage. Consistent with this prediction, the immunofluorescent
staining of the polyclonal population of transfected cells showed that
cells that expressed detectable Bcl-x
in the absence of
Bcl-x
were rare. In contrast, cells that selectively
express Bcl-x
(red) were easily detectable. For
example, the bottom panel of Fig. 4shows several cells that
expressed Bcl-x
only (red) but no cells that
expressed Bcl-x
only (green). This pattern of
Bcl-x
expression is in accord with the results we obtained
from screening clones from various Bcl-x
and Bcl-x
cotransfections.
Cells expressing both HA-Bcl-x and FLAG-Bcl-x
were metabolically labeled, and
coimmunoprecipitation studies were performed. Fig. 5A (first lane) shows that an anti-Bcl-x monoclonal antibody
that recognizes both HA-Bcl-x
and FLAG-Bcl-x
immunoprecipitated both of these proteins, and furthermore, both
were expressed at relatively high levels. As previously reported, Bax
was coimmunoprecipitated with Bcl-x
and thus is also
present in the first lane(24) . An additional band at
approximately 26 kDa is also seen in the first lane. Based on its
migration, this band appears to be endogenous Bcl-2. This was
subsequently confirmed by Bcl-2 immunoblotting of Bcl-x
immunoprecipitations (data not shown). Immunoprecipitation with the
anti-HA monoclonal antibody precipitated HA-Bcl-x
and the
associated proteins Bax and Bcl-2; however, FLAG-Bcl-x
was
not coprecipitated (Fig. 5A, second lane).
Conversely, the anti-FLAG antibody only immunoprecipitated
FLAG-Bcl-x
(Fig. 5A, third lane).
The failure of Bcl-x
to interact with Bcl-x
was
also confirmed by immunoblotting with an anti-Bcl-x antibody after
immunoprecipitating with an anti-HA antibody from FL5.12 cells
cotransfected with HA-Bcl-x
and untagged Bcl-x
(data not shown).
Figure 5:
Protein interactions between Bcl-2 family
members in vivo. Cells were metabolically labeled overnight
with [S]methionine and immunoprecipitated with
antibodies against Bcl-x, the HA epitope, the FLAG epitope, or Bcl-2,
as indicated at the top of each gel. A,
immunoprecipitation from FL5.12 cells cotransfected with HA-Bcl-x
and FLAG-Bcl-x
. B, immunoprecipitations from
FL5.12 cells cotransfected with Bcl-2 and FLAG-Bcl-x
or
cells cotransfected with HA-Bcl-x
and Bcl-x
.
The cell type used for the immunoprecipitation is indicated at the bottom of each gel, the positions of each Bcl-2 family member
is indicated at the right of each gel, and the molecular
masses are shown in kilodaltons at the left of each
gel.
We also immunoprecipitated Bcl-2 from FL5.12
cells cotransfected with Bcl-2 and FLAG-Bcl-x. Fig. 5B (first lane) shows that the anti-Bcl-2
antibody coprecipitated Bax but not FLAG-Bcl-x
. The
anti-FLAG antibody only immunoprecipitated FLAG-Bcl-x
(Fig. 5B, second lane). Independent
experiments using cells cotransfected with untagged Bcl-x
and Bcl-2 were used in immunoprecipitation/immunoblotting
experiments to confirm the results seen in the first and second lanes
of Fig. 5B. Neither immunoprecipitation of Bcl-2
followed by Bcl-x immunoblotting nor Bcl-x immunoprecipitation followed
by Bcl-2 immunoblotting showed any evidence for Bcl-x
interacting with Bcl-2 (data not shown).
Finally, we
immunoprecipitated with the anti-HA antibody from cells cotransfected
with HA-Bcl-x and untagged Bcl-x
to determine
if Bcl-x
homodimerizes. As seen in Fig. 5B (third and fourth lanes), HA-Bcl-x
did not associate with untagged Bcl-x
, suggesting
that Bcl-x
does not preferentially homodimerize.
Figure 6:
Bcl-x preferentially interacts
with Bax over Bcl-x
in vitro. cDNA-containing
plasmids for HA-Bcl-x
, FLAG-Bcl-x
, and/or Bax
were in vitro transcribed/translated and immunoprecipitated as
described under ``Materials and Methods.'' A,
HA-Bcl-x
, FLAG-Bcl-x
, and Bax were translated
together and immunoprecipitated with an anti-HA antibody. Lane 1 represents one-tenth of the input volume used for the
immunoprecipitation. Lane 2 represents the
immunoprecipitation. Quantitation of the FLAG-Bcl-x
and the
Bax signal intensities using a PhosphorImager (Molecular Dynamics)
showed that after normalization, Bax coprecipitates with HA-Bcl-x
3.5-fold better than FLAG-Bcl-x
. B,
HA-Bcl-x
was cotranslated with either FLAG-Bcl-x
(lanes 1, 3, and 5) or Bax (lanes
2, 4, and 6) and immunoprecipitated with an
anti-HA antibody. Lanes 1 and 2 represent one-tenth
of the input volume used for the immunoprecipitations. Lanes 3 and 4 are the immunoprecipitations, and lanes 4 and 5 are a 4-fold longer exposure of lanes 3 and 4.
In vitro binding studies also revealed that Bax and Bcl-x have
different requirements for binding to Bcl-x
. Bcl-x
and Bcl-x
have a conserved region contained within 25
amino acids located at the NH
terminus of both proteins
that is absent in Bax. As seen in Fig. 7, when these 25 amino
acids were deleted from the NH
terminus of
Bcl-x
, the truncated protein had an enhanced ability to
bind to FLAG-Bcl-x
. In contrast, the
NH
-terminal truncation of Bcl-x
abolished
binding to Bax. When the conserved NH
-terminal region was
removed from Bcl-x
, the binding to HA-Bcl-x
was
also abolished (data not shown). Thus, in order for Bax to bind to
Bcl-x
, the NH
-terminal region must be present
in Bcl-x
. In contrast, in order for Bcl-x
to
bind to Bcl-x
, the NH
-terminal region need only
be present in Bcl-x
. Removal of the NH
-terminal
region from Bcl-x
actually enhances binding to
Bcl-x
.
Figure 7:
In vitro binding to
Bcl-x by Bax but not Bcl-x
requires a 25-amino
acid region within the NH
terminus of Bcl-x
. A
deletion mutant of Bcl-x
was constructed that lacked the
first 25 NH
-terminal amino acids (
N
Bcl-x
). cDNA-containing plasmids of
N Bcl-x
and FLAG-Bcl-x
(lanes 1 and 2) or
N Bcl-x
and Bax (lanes 3 and 4) were in vitro transcribed/translated and immunoprecipitated with an
anti-FLAG antibody (lane 2) or an anti-Bcl-x antibody (lane 4). Lanes 1 and 3 represent one-tenth
of the input volume used for immunoprecipitation. The band that
migrates slightly below Bax (*) in lanes 1-4 is a
premature termination product of the NH
-terminal deleted
Bcl-x
, as it is observed when
N Bcl-x
is
translated by itself and after immunoprecipitation by the anti-Bcl-x
antibody.
Figure 8:
Bcl-x does not alter the
amount of Bax not heterodimerized with Bcl-x
. Clones 2, 7,
9, and 22 were lysed in 0.2% Nonidet P-40. Two-thirds of the lysate was
immunoprecipitated twice with 7B2, a mouse monoclonal antibody to
Bcl-x. The one-third left from the original lysate and the supernatant
left from the immunoprecipitation were saved. Top, one-third
of the original lysate, one-half of the 7B2 immunoprecipitation, and
one-half of the supernatant left from the immunoprecipitation were
analyzed by 12% SDS-PAGE and immunoblotting with an anti-Bax antibody.
The amount of Bax in the lysate represents the relative amount of total
Bax (labeled T). The amount of Bax in the 7B2
immunoprecipitation represents the relative amount of Bax
heterodimerized to Bcl-x (labeled H). The amount of Bax left
in the supernatant represents the relative amount of Bax not
heterodimerized with Bcl-x (labeled U). Also shown is the
control antibody immunoprecipitation and its supernatant (last two
lanes). The 28-kDa band that is present in the H lanes
probably represents light chain from the Bcl-x antibody. Bottom, the remaining one-half of the 7B2 immunoprecipitation
and the remaining one-half of the supernatant were analyzed by 12%
SDS-PAGE and immunoblotting with 13.4, a rabbit anti-Bcl-x polyclonal
antisera. The lanes labeled I represent the 7B2
immunoprecipitation, and the lanes labeled S represent the
supernatants from these immunoprecipitations. The last two lanes represent a control antibody immunoprecipitation and its
supernatant.
The top
panel of Fig. 8shows that there was little difference
between the amount of Bax not heterodimerized in clone 2, which
expresses Bcl-x and Bcl-x
, compared with clone
7, which expresses Bcl-x
alone (compare first and third lanes to fourth and sixth lanes).
Densitometry scanning of three independent experiments showed that the
fraction of Bax not heterodimerized to Bcl-x in clone 2 was 0.37
± 0.07, and in clone 7 the value was 0.31 ± 0.15. The
failure to see a substantial increase in the amount of Bax not
heterodimerized was also evident when comparing clone 9 and clone 22
(compare seventh and ninth lanes to tenth and twelfth lanes). In two independent experiments, the
amount of Bax not heterodimerized to Bcl-x was less than 0.10 for both
clones.
The bottom panel of Fig. 8demonstrates that
the immunoprecipitations with the anti-Bcl-x antibody effectively
removed nearly all of the Bcl-x. It should be noted that this
immunoblot was done with 13.4, a rabbit Bcl-x-specific polyclonal
antisera, to eliminate reaction with Ig light chain present in the
immunoprecipitation. This antibody seems to preferentially recognize
Bcl-x, which is why the ratios between Bcl-x
and Bcl-x
observed in this immunoblot differ from the
immunoblot presented in Fig. 1.
In this study we demonstrate that Bcl-x can
inhibit the ability of Bcl-x
to protect cells from
apoptosis induced by growth factor withdrawal or chemotherapeutic
drugs. A previous study demonstrated that Bcl-x
can also
block the protective effects of Bcl-2. The inhibitory property of
Bcl-x
does not appear to involve observable in vivo heterodimerization with Bcl-x
, Bcl-2, or Bax.
Bcl-x
can weakly interact with Bcl-x
in
vitro, but this interaction seems to be fundamentally different
from the Bcl-x
/Bax association in both strength and
structural requirements. Consistent with these binding properties,
Bcl-x
does not significantly decrease the amount of Bax
heterodimerized with Bcl-x
in vivo.
By in
vitro analysis, we found that removing 25 amino acids from the
NH terminus of Bcl-x
enhanced the ability of
Bcl-x
to interact with Bcl-x
. In contrast, the
removal of these amino acids from Bcl-x
eliminates the
ability of Bcl-x
to heterodimerize with Bax. The amino
terminus of Bcl-x
most likely interacts with its own
BH1/BH2 region as suggested by the yeast two-hybrid studies (25) and our unpublished data. (
)Thus, it is likely
that removal of the amino terminus of Bcl-x
allows the
identical amino-terminal region in Bcl-x
to more readily
interact with the BH1/BH2 region of Bcl-x
. Consistent with
this explanation, truncation of the amino terminus from Bcl-x
prevents it from interacting with Bcl-x
. Because
intramolecular interactions are more favorable than intermolecular
interactions, this would also explain why the
Bcl-x
/Bcl-x
association is weak. The failure to
see Bcl-x
homodimerize in vivo and in vitro (data not shown) can also be explained by this intramolecular versus intermolecular competition. Finally, a potential
explanation for why studies using the yeast two-hybrid system showed
strong associations between Bcl-x
and either Bcl-x
or Bcl-2 is because the yeast two-hybrid system involves the use
of fusion proteins that may disrupt the intramolecular association
between the amino terminus and the BH1/BH2 region. If the amino
terminus of Bcl-x
competes with the amino terminus of
Bcl-x
for binding to the BH1/BH2 region of
Bcl-x
, disrupting these intramolecular interactions within
Bcl-x
would facilitate Bcl-x
binding.
We
cannot be sure whether the weak association between Bcl-x and Bcl-x
observed in vitro is a
physiologically relevant interaction or an in vitro artifact.
One possibility is that Bcl-x
/Bcl-x
interaction
occurs but simply cannot be detected using the in vivo methods
employed due to the weakness of the interaction. Overexpression of
Bcl-x
relative to Bcl-x
may facilitate seeing
association in vivo; however, we found it difficult to obtain
such clones. Another possibility is that if the Bcl-x
association with Bcl-x
is mediated by an
intermolecular interaction involving the amino terminus of
Bcl-x
, the in vitro associations observed may be
an artifact of a misfolded Bcl-x
aggregating with
Bcl-x
. In vivo, chaperones may prevent such
products from forming. Finally, even if the interaction between
Bcl-x
and Bcl-x
is physiologically relevant, it
is unlikely to be the mechanism by which Bcl-x
promotes
cell death not only because the interaction is weak but also because
Bcl-x
fails to interact with Bcl-2 either in vivo or in vitro, despite being a potent antagonist of Bcl-2
function.
Regardless of the significance of the
Bcl-x/Bcl-x
association, Bcl-x
does
not appear to antagonize Bcl-x
by directly or indirectly
liberating Bax. Increasing the amount of Bax homodimers has been
postulated to be a general mechanism for enhancing sensitivity to cell
death(28) . Bad is a recently cloned antagonist of Bcl-x
that may work by effectively competing with Bax for Bcl-x
binding, resulting in an increase in free or homodimerized
Bax(24) . However, we did not observe any significant changes
in the amount of Bax not heterodimerized to Bcl-x
when
Bcl-x
was present. Our results suggest that increasing the
sensitivity to apoptosis does not absolutely require an increase in
free or homodimerized Bax.
If Bcl-x does not liberate
Bax or antagonize Bcl-x
/Bcl-2 by direct association, how
might Bcl-x
work? Bcl-x
could promote cell
death by binding to a yet unidentified factor(s) that is regulated by
Bcl-x
and Bcl-2 to promote survival. The binding of this
factor by Bcl-x
sequesters the factor into a complex that
now accelerates cell death. Another possibility is that Bcl-x
does not act as a competitor in protein-protein interactions,
rather Bcl-x
and Bcl-2 are active effectors of cell
survival, and Bcl-x
counters these molecules by performing
the opposite effector function. Irrespective of how these molecules are
working to influence cell survival, it is becoming clear that at least
two independent domains, the NH
-terminal 25 amino acids and
the internal BH1/BH2 domain found in both Bcl-x
and Bcl-2,
are required to produce a protein that increases the apoptotic
threshold of a cell. Production of a protein containing only one of
these domains such as Bcl-x
, which lacks the BH1/BH2
domain, or Bax, which lacks the NH
-terminal homology
domain, results in a transdominant inhibitor.