From the Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bap31 is a polytopic integral membrane protein of
the endoplasmic reticulum and forms a complex with
Bcl-2/Bcl-XL and procaspase-8 (Ng, F. W. H., Nguyen, M., Kwan, T., Branton, P. E., Nicholson, W. D.,
Cromlish, J. A., and Shore, G. C. (1997) J. Cell Biol. 139, 327-338). In co-transfected human cells, procaspase-8 is capable of interacting with Ced-4, an important adaptor molecule in
Caenorhabditis elegans that binds to and activates the
C. elegans procaspase, proCed-3. Here, we show that the
predicted death effector homology domain within the cytosolic region of
Bap31 interacts with Ced-4 and contributes to recruitment of
procaspase-8. Bcl-XL, which binds directly but weakly to
the polytopic transmembrane region of Bap31, indirectly and
cooperatively associates with the Bap31 cytosolic domain, dependent on
the presence of procaspase-8 and Ced-4. Ced-4c does not interact
with Bcl-XL but rather displaces it from Bap31, suggesting
that an endogenous Ced-4-like adaptor is a normal constituent of the
Bap31 complex and is required for stable association of
Bcl-XL with Bap31 in vivo. These findings indicate that Bap31 is capable of recruiting essential components of a
core death regulatory machinery.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genetic studies in the nematode Caenorhabditis elegans have identified and ordered a core machinery for regulation of apoptotic programmed cell death in which the Bcl-2 homolog, Ced-9, prevents Ced-4 from activating the caspase, Ced-3, and thus blocks ensuing cell death (1-4). Recent reconstitution of these events both in vitro and in heterologous yeast and human cells has revealed that Ced-9 directly binds and sequesters Ced-4 (5-9), which in turn remains associated with Ced-3 (6, 9). This presumably prevents the Ced-4 adaptor from triggering autocatalytic processing and activation of Ced-3 (10, 11). Of note, when expressed in human cells, Ced-4 is also capable of mechanically linking the Bcl-2 family protein, Bcl-XL, to procaspase-8 (6). Ced-4 likely achieves this by substituting for an endogenous Ced-4-like adaptor molecule that otherwise would connect Bcl-XL and procaspase-8 (6). Caspase-8 (12-14) belongs to the initiator class of caspases (14-16) whose members appear to function upstream of mitochondria (17) to activate the death pathway. Significantly, Ced-4 itself does not associate with procaspase-3 (6), a downstream effector caspase whose activation may depend on a combination of mitochondrial-released factors and the Ced-4-like cytosolic protein, Apaf-1 (Ref. 18 and reviewed in Ref. 19). A major question, however, is the mechanism by which these Ced-4 controlled events are linked to the plethora of signals that result in activation of caspases and subsequent cell death.
Recently, we identified a Bcl-2/Bcl-XL and procaspase-8 associated protein in the endoplasmic reticulum (ER),1 p28 Bap31 (20). Bcl-2 family proteins are located in the ER/nuclear envelope and mitochondrial outer membrane (21-23) and, in the latter location, appear to prevent activation of downstream effector caspases such as procaspase-3 in response to diverse death signals (see Refs. 19 and 24). Initiator caspases such as procaspase-8, on the other hand, are well characterized constituents of the Fas and TNFR1 apoptosis signaling complexes in the plasma membrane (25-27). These complexes, however, are highly restricted in the death signals to which they respond and are not directly influenced by Bcl-2 family proteins. The ability of Bap31 to associate with both procaspase-8 and Bcl-2/Bcl-XL, therefore, raises the possibility that the Bap31 complex in the ER might cooperate with events in the mitochondrion to control proximal and distal steps in a Bcl-2-regulated caspase cascade. If so, the ability of Ced-4 to bridge Bcl-XL and procaspase-8 (6) predicts that a Ced-4-like adaptor molecule may also be a part of the Bap31 complex.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmids--
cDNAs encoding proteins tagged with specific
epitopes were constructed in expression vectors. Flag epitope was
inserted toward the C terminus of Bap31, immediately upstream of the
KKEE ER retrieval signal; Myc and HA epitopes were placed at the C
termini of Bcl-XL and proFLICE, respectively. Details are
provided in Ref. 20. Standard recombinant DNA methodology was employed
to create pCDNA3.1 vectors (Invitrogen) encoding Bap31N119,
lacking amino acids 2-119, and Bap31
167-240, lacking amino acids
167-240. Authenticity of all constructs was validated by sequence
analysis. Similarly, pGEX 2T vectors (Invitrogen) encoding GST and
GST-Bap31
N119 were employed to express the proteins in bacteria,
which were purified as described in refs. 20 and 30. The coding
region of Ced-4S (28) was obtained by reverse transcription-polymerase
chain reaction using total C. elegans RNA and
5
-CGAGGTACCATGCTCTGCGAAATCGA-3
as the sense primer and
5
-CGAGGTACCTCAAGCGTAATCTGGAACATCGTATGGGTAACAGCATGCAAAATTTTTG-3
as
antisense primer, and the construct was inserted into pCDNA3.1 by
standard manipulations. Reverse transcription-polymerase chain reaction
was employed to delete the region encoding amino acids 303-548, using
5
-CTAGAATACCCATACGATGTTCCAGATTACGCTTAAGGTACCGC-3
as sense primer and
5
-GGCCGCGGTACCTTAAGCGTAATCTGGAACATCGTATGGGTATT-3
as antisense
primer.
Transfections and Immunoprecipitation-- Details concerning transfection of expression vectors into human 293T cells, subsequent preparation of cell lysates, and analysis of products by immunoprecipitation followed by transfer to nitrocellulose for immunoblot analysis are given in Ref. 20.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The predicted death effector homology domain in Bap31 (amino acids
165-238) is flanked on either side by sites that are cleaved by
caspase-8 or a related caspase during adenovirus E1A-induced apoptosis
(20) (summarized in Fig. 1A).
The resulting p20 Bap31 product is a potent inducer of apoptosis when
expressed ectopically in otherwise normal cells, presumably because it
has a dominant-negative effect on endogenous Bap31 (20). In
vitro mapping has revealed that Bcl-2 proteins bind directly to
the N-terminal domain of Bap31, which includes the polytopic
membrane-associated region, and weakly if at all with the cytosolic
domain (20). As expected, therefore, full-length Flag-tagged Bap31, but
not the Bap31 cytosolic domain alone (Bap31N119), interacted with
Bcl-XL in co-transfected human 293T cells, as judged by
co-immunoprecipitation with anti-Flag antibody (Fig. 1B,
compare lanes 3 and 4). However, when
Bap31
N119 was coexpressed with both Bcl-XL and
procaspase-8 (proFLICE), a strong association between
Bcl-XL and the Bap31 cytosolic domain was recorded (lane
5). Thus, Bcl-XL may be tethered at the N terminus of Bap31
and associate indirectly and cooperatively with the Bap31 cytosolic
domain, dependent on the presence of procaspase-8. Failure to achieve
reconstitution of these interactions in vitro (not shown),
however, suggested that an adaptor molecule might be required.
|
As a preliminary step indicating that Ced-4 might associate with Bap31,
a transcription-translation product of the proapoptotic short
splice form of Ced-4 (28) was found to interact in vitro with a glutathione S-transferase fusion protein containing
the Bap31 cytosolic domain (GST-Bap31N119) (Fig.
2). Furthermore, when Ced-4 was
co-expressed with Bap31-Flag in 293T cells and total cell extracts were
incubated with anti-Flag antibody, Ced-4 co-immunopreciptated with
Bap31-Flag but not with Control-Flag (Fig.
3A, lanes 3 and
4). This association was significantly reduced using a Bap31
construct lacking the putative death effector homology domain
(Bap31
167-240-Flag, lane 2). In fact, the residual
amount of Ced-4 that was recovered in association with the Bap31
deletion mutant (lane 2) may have resulted because
immunoprecipitation of Bap31
167-240-Flag with anti-Flag antibody
also precipitated endogenous Bap31 (lanes 7). The latter is
consistent with our findings that Bap31 forms homooligomers both
in vitro and in
vivo.2 Of note,
expression of Ced-4 did not lead to significant
caspase-dependent cleavage of Bap31. This is concordant
with the observation that Ced-4 does not independently induce
apoptosis in 293T cells (9).
|
|
Importantly, the presence of Bcl-XL did not influence the ability of Ced-4 to associate with Bap31 in 293T cells (Fig. 3A, lanes 4 and 6), suggesting that Bcl-XL does not function to prevent Ced-4 from engaging the Bap31 complex. Moreover, this was also extended to procaspase-8, where it was found that Ced-4 and procaspase-8, when expressed individually in co-transfected 293T cells, associated with Bap31-Flag (Fig. 3B, lanes 3 and 5) to the same extent as in the situation where Ced-4 and procaspase-8 were expressed together (lane 7). Again, as was the case for Ced-4 (Fig. 3A), the ability of procaspase-8 to associate with Bap31 was significantly reduced by deleting the putative Bap31 death effector homology domain (Fig. 3C).
One obvious explanation to account for the ability of Ced-4 to
associate with the Bap31 complex is that Ced-4 may be substituting for
an endogenous Ced-4-like adaptor molecule. Earlier studies identified a
means of testing this hypothesis (Ref. 6, see schematic in Fig.
4). A mutant Ced-4 lacking the C-terminal
248 amino acids, Ced4c, failed to interact with Ced-9 or
Bcl-XL but retained the ability to associate with Ced-3 or
procaspase-8 in co-transfected 293T cells. Thus, it competes in
vivo with an endogenous adaptor that bridges Bcl-XL
and procaspase-8 and prevents precipitation of procaspase-8 by an
antibody directed to Bcl-XL (6). Similarly, we found that
Ced-4
c lacking the C-terminal 246 amino acids also competed for the
ability of Bcl-XL to bind to Bap31 in vivo (Fig. 4, lanes 1 and 3, upper panel),
strongly implying that it does so by displacing an endogenous adaptor
that otherwise would make contact with Bcl-XL. Wild-type
Ced-4 also reduced somewhat the level of Bcl-XL that was
recovered with Bap31 (lanes 1 and 2), suggesting
that Ced-4 is less efficient than the endogenous adaptor molecule at
contributing to the association of Bcl-XL with the Bap31
complex. Taken together, the results in Fig. 4 and those in Fig.
1B demonstrate that cooperative interactions between
Bcl-XL and both Ced-4 and procaspase-8 contribute to the
stable binding of Bcl-XL to Bap31 in vivo.
|
In conclusion, the ability of Ced-9 to bind to and prevent Ced-4 from activating Ced-3 provides a simple explanation to account for the molecular control over apoptotic cell death. Nevertheless, it remains to be explained how the corresponding molecular complexes in mammalian cells are linked to the multitude of signals that can trigger activation of procaspases and how they interface with the numerous proapoptotic and antiapoptotic regulators that modulate these signals (24, 29). The cooperative associations between Bcl-XL, Ced-4, procaspase-8, and the Bap31 cytosolic domain observed here, on the other hand, suggest that Bap31 may provide one such bridge and predict that an endogenous Ced-4-like adaptor molecule may be a normal constituent of the Bap31 complex.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Mai Nguyen, Phil Branton, John Bergeron, and Josée Lavoie for discussions and to Siegfried Hekimi for providing C. elegans RNA.
![]() |
FOOTNOTES |
---|
* This work was supported by operating grants from the National Cancer Institute and the Medical Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 514-398-7282;
Fax: 514-398-7384; E-mail: shore{at}medcor.mcgill.ca.
1 The abbreviations used are: ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; HA, hemagglutinin.
2 F. W. H. Ng and G. C. Shore, unpublished observation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|