(Received for publication, March 13, 1997, and in revised form, May 30, 1997)
From the Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan 48109
In the nematode Caenorhabditis elegans, three genes, ced-3, ced-4, and ced-9, play critical roles in the induction and execution of the death pathway. Genetic studies have suggested that ced-9 controls programmed cell death by regulating ced-4 and ced-3. However, the mechanism by which CED-9 controls the activities of CED-4 and the cysteine protease CED-3, the effector arm of the cell-death pathway, remains poorly understood. Immunoprecipitation analysis demonstrates that CED-9 forms a multimeric protein complex with CED-4 and CED-3 in vivo. Expression of wild-type CED-4 promotes the ability of CED-3 to induce apoptosis in mammalian cells, which is inhibited by CED-9. The pro-apoptotic activity of CED-4 requires the expression of a functional CED-3 protease. Significantly, loss-of-function CED-4 mutants are impaired in their ability to promote CED-3-mediated apoptosis. Expression of CED-4 enhances the proteolytic activation of CED-3. We also show that CED-9 inhibits the formation of p13 and p15, two cleavage products of CED-3 associated with its proteolytic activation in vivo. Moreover, CED-9 inhibits the enzymatic activity of CED-3 promoted by CED-4. Thus, these results provide evidence that CED-4 and CED-9 regulate the activity of CED-3 through physical interactions, which may provide a molecular basis for the control of programmed cell death in C. elegans.
Programmed cell death
(PCD)1 is critical during
organ development and tissue homeostasis (1). In the nematode
Caenorhabditis elegans, 131 of the 1090 somatic cells
generated during development undergo PCD (2). Three genes have been
identified in the nematode that play critical roles in the induction
and execution of PCD (3). The ced-9 gene protects cells that
normally survive from PCD during worm development (4, 5).
ced-9 encodes a protein with significant homology to the
mammalian Bcl-2 and Bcl-XL survival proteins (4, 5).
Furthermore, bcl-2 can partially substitute for
ced-9 in C. elegans, suggesting that
bcl-2 is a homolog of ced-9 (6, 7). In contrast,
two nematode genes, ced-3 and ced-4, are required
for the execution of the cell death program. Thus, loss-of-function
mutations of ced-3 and ced-4 cause all 131 somatic cells that normally die to survive (3). The ced-3 product is homologous to the mammalian interleukin-1-converting enzyme (ICE), which is a member of a family of cysteine proteases (designated caspases) (7, 8). CED-3 and related caspases are thought to
act as effectors of the nematode and mammalian PCD pathway (7, 8).
Genetic experiments have suggested that ced-9 protects cells from undergoing PCD by preventing the death-promoting activity of ced-3 and ced-4 (4, 9). Consistent with genetic experiments in C. elegans, Bcl-2 and Bcl-XL, two mammalian homologs of CED-9, can inhibit the activation of ICE-like proteases and therefore appear to act upstream of the death proteases in the mammalian apoptotic pathway (10, 11). Overexpression of ced-4 in the nematode ALM neurons causes cell death that requires ced-3 activity for efficient killing, suggesting that ced-4 acts upstream of ced-3 (9). Furthermore, protection against ced-3-induced cell death by ced-9 requires ced-4 activity, suggesting that ced-9 controls ced-3 by acting at least in part through ced-4 (9). Although genetic analysis has been essential for the identification and initial characterization of the nematode PCD pathway, the biochemical basis by which CED-3, CED-4, and CED-9 regulate cell death has remained elusive. Recent studies, however, indicate that CED-9 interacts with CED-4 suggesting that CED-9 regulates cell death by binding to and inactivating CED-4 (12-14). In the present studies, we performed further functional and biochemical analyses of CED-3, CED-4, and CED-9 interactions using mammalian cells as a model system to gain insight into the regulation and molecular basis of the PCD pathway.
The expression plasmids producing
epitope-tagged CED-4 and CED-9 have been described (13). Plasmids
encoding Myc-tagged CED-4 mutants were generated by PCR amplification
of ced-4S template DNA using 3 primers that included the
natural translation termination sequences as described (15).
Subsequently, the ced-4 constructs were ligated into the
KpnI site of pcDNA3 (Invitrogen). An HA- or Flag-tagged
ced-3 insert was constructed by introducing each epitope tag
at the COOH terminus of CED-3 by PCR. Inserts to express mutant CED-3
proteins were generated by PCR amplification of ced-3 template using a 3
primer that included a translation termination codon (D220 CED-3) or by two-step mutagenesis to generate G360S CED-3.
The HA-, Flag-, or Myc-tagged inserts were ligated into the
BamHI site of pcDNA3. Orientation of the inserts was
determined by restriction mapping. Authenticity of all tagged
constructs was confirmed by dideoxy sequencing.
Culture dishes containing 2-5 × 106 human embryonic kidney 293T cells were transfected with the indicated amount (see figure legends) of plasmid DNA by the calcium phosphate method. The expression of HA-CED-9, Myc-CED-4, and Flag-tagged CED-3 was determined in total lysates by immunoblotting as described previously (16). For immunoprecipitations, cells from each dish were lysed in 1 ml of Nonidet P-40 isotonic lysis buffer at 14-20 h after transfection, and soluble lysates were incubated with 1 µg/ml rabbit anti-Myc, anti-Flag, or anti-HA antibody (Santa Cruz) or anti-AU1 antibody (Babco) or normal rabbit IgG overnight at 4 °C with 5% (v/v) of protein A-Sepharose 4B (Zymed Laboratories). Immune complexes were centrifuged, washed with excess cold Nonidet P-40 isotonic lysis buffer at least four times, separated on a 15% SDS-polyacrylamide gel, and immunoblotted with monoclonal anti-HA (clone 12CA5, Boehringer Mannheim), monoclonal anti-Flag (clone M2, Kodak), or rabbit anti-Myc antibody (Santa Cruz). The proteins were detected using an enhanced chemiluminescence (ECL) system (Amersham Corp.).
Cleavage of Fluorogenic Substrate293T cells (5 × 106) in 100-mm plates were transiently co-transfected with 5 µg of pcDNA3-ced-4-Myc and/or pcDNA3-ced-3-Flag in the presence or absence of 5 µg of pcDNA3-HA-ced-9 by calcium phosphate method. The total amount of transfected plasmid DNA was always 15 µg/well and was adjusted by adding vector pcDNA3 DNA. At 14 h, cells were lysed in buffer A (25 mM HEPES, 1 mM EGTA, 5 mM MgCl2, pH 7.6, containing protease inhibitors and 0.2% Nonidet P-40). 150 µg of protein lysate was immunoprecipitated with 10 µg of anti-Flag antibody or control in 200 µl of buffer A and incubated at room temperature for 1 h. CED-3 complexes were immunoprecipitated with 20 µl of protein A-Sepharose and washed with buffer A four times. 100 µM of the fluorogenic substrate Ac-DEVD-AMC (Alexis, San Diego, CA) was added in buffer A and incubated at 37 °C for 1, 3, and 6 h in triplicate. Cleavage of substrate emitted a fluorescent signal that was quantified in a fluorometer at excitation 340 nm and emission 460 nm. Statistical significance was determined by 1-way ANOVA followed by Student-Neuman-Keuls post-hoc comparisons.
Apoptosis Assay293T cells (5 × 105) in
each well of 6-well plate were transiently co-transfected in triplicate
with 0.33 µg of a reporter plasmid pcDNA3--gal plus 0.66 µg
of pcDNA3-ced-4-Myc and/or pcDNA3-ced-3-Flag or mutant
tagged-ced-3 constructs in the presence or absence of 0.66 µg of
pcDNA3-HA-ced-9 by calcium phosphate method. The total amount of
transfected plasmid DNA was always 2 µg/well and was adjusted by
adding vector pcDNA3 DNA. Apoptosis was determined at 24 h
after transfection by analysis of at least 300 cells expressing
-galactosidase as described (8).
Genetic studies have suggested that CED-4
regulates the activity of CED-3 in the C. elegans death
pathway (9). To determine if CED-4 interacts with CED-3, we transiently
co-transfected human 293T cells with expression plasmids producing
Flag-tagged CED-3, HA-tagged CED-9, and Myc epitope-tagged CED-4.
Because CED-3 induces apoptosis in mammalian cells, we used a
catalytically inactive mutant of CED-3 (CED-3-Flag-G360S) to study the
interaction of CED-3, CED-4, and CED-9. Immunoprecipitates were
prepared with rabbit anti-Myc antibody and subjected to
SDS-polyacrylamide gel electrophoresis. Immunoblotting with a
monoclonal antibody to Flag revealed that CED-4 co-immunoprecitated
CED-3 in the presence or absence of CED-9 (Fig.
1A). Furthermore,
immunoprecpitation with anti-HA followed by immunoblotting with
anti-Flag revealed that CED-9 co-immunoprecipitated CED-3 (Fig.
1A). To further verify these results, we performed
reciprocal experiments. Immunoprecipitation of CED-3 with anti-Flag
antibody co-imunoprecipitated CED-9 (Fig. 1B). CED-4
co-immunoprecipitated both CED-3 and CED-9 (Fig. 1A). More
importantly, CED-3 was unable to interact with CED-9 in the absence of
CED-4 (Fig. 1, A and B) indicating that CED-3
associates with CED-9 through CED-4. These results confirm observations
recently reported by Chinnaiyan et al. (12).
Interaction of CED-4 Mutants with CED-3 and CED-9
To further
characterize the specificity and functional significance of the
interaction between CED-3 and CED-4, we determined the ability of CED-3
to associate with wild-type CED-4 and three natural CED-4 mutants that
exhibit a loss of function phenotype in C. elegans (Fig.
2A). Two point mutations,
n1948 and n1894, introduce a single amino acid change (Ile to Asn) at
position 258 and a stop codon at residue 401 of the CED-4 protein,
respectively (15). We engineered another mutation, G328, to insert a
stop codon at amino acid 328 to recapitulate the loss of function
mutation n1416, which results from a Tc4 insertion at residue 328 of
CED-4 (15). To assess the interaction of CED-3 with CED-4 proteins, 293T cells were transiently co-transfected with plasmids producing Flag-tagged CED-3 and Myc-tagged wild-type or mutant CED-4 proteins. Immunoprecipitation analysis revealed that CED-3 co-immunoprecipitated wild-type CED-4 (Fig. 2B), confirming our results presented
in Fig. 1. Significantly, W401 and G328, two loss of function CED-4 mutants resulting from C-terminal truncations retained their ability to
associate with CED-3 (Fig. 2B). Similarly, the missense,
loss of function I258N mutant resulting from a single amino acid
substitution at position 258 of CED-4 retained its ability to interact
with CED-3 (Fig. 2C). Because CED-4 also interacts with
CED-9 (12-14), we next determined the ability of the CED-4 mutants to
associate with CED-9. Immunoprecipitation analysis revealed that all
three loss of function CED-4 mutants retained their capacity to
interact with CED-9 although the binding of the CED-4 mutants,
particularly G328 and I258N, was reduced when compared with wild-type
CED-4 Fig. 2D).
CED-4 Associates with the Prodomain of CED-3 and with a Loss of Function CED-3 Mutant
To further define the interaction of CED-4 with CED-3, the ability of CED-4 to interact with two CED-3 mutants was investigated. We engineered CED-3 D220 to express a truncated CED-3 mutant (residues 1-220) containing the prodomain of CED-3 and CED-3 G360S, a natural loss of function CED-3 mutant (17) with a single amino acid change (Gly to Ser) at position 360 located in the conserved pentapeptide QACRG of CED-3 (Fig. 2A). CED-4 coimmuprecipitated with both CED-3 mutants (Fig. 2E) The results indicate that CED-4 can interact with the prodomain of CED-3 and with CED-3 containing a single amino acid change in its catalytic domain, which is in agreement with a recent report (12).
CED-4 Promotes the Ability of CED-3 to Kill Mammalian CellsGenetic analysis in C. elegans has suggested
that CED-4 regulates the activity of CED-3 and requires CED-3 to
activate cell death. To further explore the function of CED-4 in
apoptosis, we determined if CED-4 could regulate the cell
death-promoting ability of CED-3 in mammalian cells. To do this, 293T
cells were transiently transfected with expression constructs producing
CED-4, CED-3, and empty vector. Expression of CED-4 did not induce
apoptosis in 293T cells above the levels observed with control plasmid
(Fig. 3A). Transfection of
CED-3 induced a modest but significant level of apoptosis in 293T cells
(Fig. 3A). More importantly, the great majority of the cells
underwent apoptosis when co-transfected with CED-3 and CED-4 (Fig.
3A), indicating that CED-4 potentiates the ability of CED-3
to induce apoptosis. Significantly, expression of CED-9 blocked the
ability of CED-4 and CED-3 to induce apoptosis (Fig.
3A). Together with genetic experiments in C. elegans (3, 9), these results suggest that CED-4 interacts with
and activates the death-promoting ability of CED-3, which can be
inhibited by the interaction of CED-9 with CED-4. To further assess the
function of CED-4, we tested the ability of loss of function CED-4
mutants to promote CED-3-mediated apoptosis. Functional analysis
revealed that the loss of function CED-4 mutants were defective in
their ability to potentiate CED-3-mediated apoptosis when compared with wild-type CED-4 (Fig. 3A). The levels of wild-type and
mutant W401 and G328 CED-4 proteins expressed in 293T cells were
similar, ruling out the trivial explanation that the differential
ability of these proteins to promote apoptosis is due to differences in protein expression (Fig. 2 and data not shown). However, the expression of the I258N CED-4 mutant is reduced and appears unstable, which agrees
with previous results (14).
A Functional CED-3 Protein Is Required for CED-4 to Promote Apoptosis
To further determine the mechanism by which CED-4 potentiates CED-3-mediated apoptosis, we examined whether the pro-apoptotic activity of CED-4 requires a "functional" CED-3 protease. In contrast to the killing observed with wild-type CED-3, CED-4 failed to promote apoptosis mediated by either a truncated CED-3 mutant containing the prodomain of CED-3 or G360S CED-3, a natural loss of function CED-3 mutant with a single amino acid change (Gly to Ser) at position 360 located in the conserved active pentapeptide QACRG of CED-3 (Fig. 3B). Because the CED-3 (G360S) mutant protein is known to be inactive as a cysteine protease in vitro (17), we conclude that the protease activity of CED-3 is required for CED-4 to promote apoptosis in mammalian cells. These results are consistent with genetic studies which have shown that the CED-3 protease activity is crucial for the death-promoting function of CED-4 in C. elegans (9).
CED-4 Enhances the Proteolytic Activation of CED-3 and CED-9 Inhibits the Processing of CED-3 in Mammalian CellsTo assess if
the survival protein CED-9 regulates the activation of the CED-3
protease in vivo, cellular lysates from 293T cells
transiently transfected with plasmids expressing CED-3, CED-4, and
CED-9 were analyzed for the presence of p13 and p15, two cleavage
products of CED-3 associated with its proteolytic activation (17, 18).
As shown in Fig. 4A,
expression of CED-3 resulted in partial processing of the immature
56-kDa CED-3-FLAG protein as determined by the detection of the mature
p13 and p15 products of CED-3 (17). Significantly, processing of CED-3
increased dramatically in the presence of CED-4 when compared with
cells expressing CED-3 alone (Fig. 4A). Moreover, expression
of CED-9 inhibited the processing of CED-3 in cells transiently
transfected with CED-3 plus CED-4 as determined by the absence of the
p13 and p15 proteolytic products of CED-3 (Fig. 4A). To
verify these results, we determined the caspase enzymatic activity of
CED-3 in lysates from cells transiently transfected with CED-3, CED-3 plus CED-4, and CED-3 plus CED-4 plus CED-9. To measure the protease activity associated with CED-3, aliquots of the same cellular lysates
used in Fig. 4B were incubated with anti-Flag antibody to
immunoprecipitate CED-3 protein complexes, and the immunoprecipitates were assayed for enzymatic activity using the Ac-DEVD-AMC fluorogenic substrate (19). In agreement with results shown in Fig. 4A, lysates from cells expressing CED-3 or CED-3 plus CED-4 exhibited significant enzymatic activity when compared with control lysates (Fig.
4B). Importantly, the caspase activity of CED-3 from cells expressing CED-3 and CED-4 was inhibited by CED-9 (Fig. 4B).
In control experiments, there was no significant protease activity when
the same cellular lysates were immunoprecipitated with control antibody
(data not shown). Because the cellular expression of CED-3 was
diminished in the presence of CED-4 and enhanced in the presence of
CED-9 when compared with cells expressing CED-3 alone (Fig. 4), we
adjusted the values of enzymatic activity obtained with the Ac-DEVD-AMC
substrate to normalize for the amount of CED-3 protein (immature plus
mature). After normalization, we found that the enzymatic activity of
CED-3 was enhanced significantly by co-expression of CED-4 (Fig.
4C). In contrast, CED-9 inhibited the catalytic activity of
CED-3 in the presence of CED-4 (Fig. 4C).
These results demonstrate that CED-4 interacts with both CED-3 and CED-9 in mammalian cells and presumably in C. elegans cells. Our studies confirmed and extended the reported interaction of CED-4 with CED-3 and CED-9 (14). However, our functional results differ from those recently reported (14), which demonstrated that CED-4 was capable of inducing apoptosis in 293T cells even in the absence of CED-3. In addition to 293T cells, we found that expression of CED-4 failed to kill human MCF-7 breast carcinoma cells and COS-7 monkey cells.2 In agreement with our results, another group has found that expression of CED-4 fails to kill Rat-1 and HeLa cells.3 Thus, we do not have an explanation to account for the discrepancy in the results. In our studies, CED-4 functioned only to promote apoptosis in mammalian cells, which was dependent upon the expression of enzymatically active CED-3. These observations support previous genetic experiments in C. elegans in which efficient killing by CED-4 was shown to be dependent upon CED-3 activity (9). Together with the results from other investigators (14), these studies argue that CED-4 promotes cell death by interacting with and activating CED-3. However, the mechanism by which CED-4 activates CED-3 is unclear and needs to be further investigated. Because three loss of function CED-4 mutants retained their ability to associate with CED-3, our studies suggest that the interaction between CED-4 and CED-3 is not sufficient for death-promoting function of CED-4. Consistent with the analysis in C. elegans, the loss of function CED-4 mutants were impaired in their ability to potentiate CED-3-mediated apoptosis in mammalian cells. Expression of CED-9 inhibited the killing activity of CED-4 plus CED-3, and in another study, it inhibited killing induced by CED-4 alone (14). Because CED-9 can form a protein complex with CED-3 through CED-4, the protective function of CED-9 could be mediated by preventing the activation of CED-3 through CED-4. Indeed, we show for the first time that CED-4 enhances the processing and conversion of CED-3 into the active CED-3 form in vivo. Furthermore, we show that CED-9 inhibits the proteolytic activation of CED-3 in cells that co-express CED-4. Since a C. elegans cell culture is not available, the biochemical analysis of the death regulators CED-3, CED-4, and CED-9 was performed in mammalian cells. The biochemical results that we have obtained with CED-3, CED-4 and CED-9 are consistent with the genetic analysis in C. elegans (9). However, we cannot formally rule out that these worm proteins behave differently in C. elegans cells. These studies predict that Bcl-2 and Bcl-XL, the mammalian CED-9 homologs, may regulate apoptosis by interacting with caspases, the cysteine proteases of the ICE/CED-3 family through a mammalian CED-4 counterpart.
We thank R. Horvitz for plasmids, M. Clarke and R. Ellis for stimulating discussions, and M. Benedict, D. Ekhterae, M. González-Garcìa, and Y. Hu for critical review of the manuscript.