From the Abramson Family Cancer Research Institute
and Department of Cancer Biology, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania 19104 and the § Center
for Apoptosis Research and the Department of Microbiology and
Immunology, Kimmel Cancer Institute, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107
Received for publication, February 27, 2003
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ABSTRACT |
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Proteolytic activation of initiator procaspases
is a crucial step in the cellular commitment to apoptosis. Alternative
models have been postulated for the activation mechanism, namely the oligomerization or induced proximity model and the allosteric regulation model. While the former holds that procaspases become activated upon proper oligomerization by an adaptor protein, the latter
states that the adaptor is an allosteric regulator for procaspases. The allosteric regulation model has been applied for the
activation of procaspase-9 by apoptotic protease-activating factor
(Apaf-1) in an oligomeric complex known as the apoptosome. Using approaches that allow for controlled oligomerization, we show
here that aggregation of multiple procaspase-9 molecules can induce
their activation independent of the apoptosome. Oligomerization-induced procaspase-9 activation, both within the apoptosome and in
artificial systems, requires stable homophilic association of the
protease domains, raising the possibility that the function of Apaf-1
is not only to oligomerize procaspase-9 but also to maintain the interaction of the caspase-9 protease domain after processing. In
addition, we provide biochemical evidence that other apoptosis initiator caspases (caspase-2 and -10) as well as a procaspase involved
in inflammation (murine caspase-11) are also activated by
oligomerization. Thus, oligomerization of precursor molecules appears
to be a general mechanism for the activation of both apoptosis initiator and inflammatory procaspases.
Apoptosis is a physiological form of cell death critical for
development and the maintenance of homeostasis in multicellular organisms. The central component of the apoptosis machinery is a group
of cysteine proteases called caspases that cleave proteins after
aspartic acid residues (1-3). These proteases exist in healthy cells
as inactive procaspases, which comprise an NH2-terminal prodomain of variable length and a COOH-terminal protease domain that
can be further divided into the large and small subunits, the
constituent units of mature caspases. Conversion of procaspases to
mature caspases involves at least one cleavage event that separates the
large and small subunits but often also another cleavage event that
separates the prodomain and large subunit. During apoptosis, caspase activation occurs sequentially with long prodomain-containing caspases (initiator caspases, including caspase-2, -8, -9, and -10)
being activated first that then cleave and activate short prodomain-containing caspases (effector caspases, including caspase-3, -6, and -7). Mature effector caspases cleave a wide range of
intracellular structural and regulatory proteins, leading to a set of
stereotypic changes in cell morphology and eventual cell death.
Mammalian caspases also play a critical role in inflammation. The
maturation of proinflammatory cytokine interleukin-1 Activation of the initiator caspases is a critical step in the
activation of the caspase cascade and apoptosis. This activation is an
autocatalytic process mediated by the interaction of an initiator
caspase, via its prodomain, with its adaptor protein. How exactly
initiator caspases become activated remains a matter of debate (6, 7).
Two alternative models have been described: the oligomerization model
(also known as the induced proximity model) and the allosteric
regulation model. The oligomerization model was originally proposed for
the activation of caspase-8 (8-10), which is engaged by a group of
cell surface death receptors in the tumor necrosis factor
receptor family (11). Binding of these receptors to their
corresponding trimeric ligands or agonistic antibodies leads to the
recruitment, via adaptor proteins such as Fas-associated death
domain, of multiple procaspase-8 molecules to a
membrane-associated death-inducing signaling complex. The increased
local concentration of procaspase-8 in the death-inducing signaling
complex likely leads to cross-cleavage among them. The oligomerization
model has also been proposed for the activation of CED3 (12), a caspase
required for developmental cell death in the nematode
Caenorhabditis elegans, and the mammalian counterpart of
CED3, caspase-9 (13, 14).
More recently an alternative model has been proposed for the activation
of caspase-9 (15). Caspase-9 is the initiator caspase for the
mitochondrial apoptosis pathway that is activated by various intracellular lethal signals, including developmental lineage information, oncogenic transformation, and severe DNA damage. These
signals lead to the release of mitochondrial cytochrome c to
the cytosol where it binds to an adaptor protein
Apaf-11 to form an oligomeric
complex termed the apoptosome (16). This apoptosome recruits
caspase-9, leading to its autoproteolytic processing (13, 14).
Processed caspase-9 is unusual among caspases in that its prodomain is
not separated from the large subunit. More interestingly the processed
caspase-9 remains minimally active unless it is associated with Apaf-1
in the apoptosome, which enhances the activity of caspase-9 by about
1,000-fold (15). This observation has led to the allosteric regulation
model, which postulates that Apaf-1 is a cofactor of procaspase-9
capable of inducing conformational changes that render the zymogen
proteolytically competent (15).
In this study, we examine the activation of caspase-9 using approaches
that allow for controlled oligomerization independent of the
apoptosome. Our data confirm that caspase-9 is activated by
oligomerization. Through mutagenesis analysis, we showed that formation
of a stable intermediate by two procaspase-9 molecules through their
protease domains is a requisite step in caspase-9 activation. We also
showed that other initiator caspases as well as inflammatory caspases
are activated by induced proximity. Thus, oligomerization appears to be
the general mechanism for the activation of both initiator and
inflammatory caspases.
Reagents and Expression Constructs--
HeLa and 293 cells were
obtained from ATCC. The following reagents were purchased from the
indicated sources: protein A-agarose (Invitrogen),
glutathione-Sepharose (Amersham Biosciences), and rabbit cytochrome
c and anti-FLAG M2-agarose beads (Sigma). AP20187 was kindly
provided by ARIAD Pharmaceuticals. Plasmids for mammalian cell
expression and in vitro translation were made in pRK5. The Fv-caspase fusion plasmids contained an NH2-terminal c-Src
myristoylation signal, a COOH-terminal FLAG epitope tag, and a
five-amino acid stretch (GGGGS) between the Fv domain and the protease
domain. Expression plasmids for various full-length caspase-9 proteins contained a COOH-terminal FLAG or HA tag as indicated, while plasmids for caspase-9 protease domains and caspase-2 contained an
NH2-terminal FLAG or HA tag as indicated. All point
mutations used in this study were made by overlap PCR.
Production of Recombinant Apaf-1--
Recombinant Apaf-1 with a
COOH-terminal His9 tag was expressed in insect Sf-9
cells through baculoviral infection and affinity purified with
nickel-nitrilotriacetic acid-agarose (Qiagen) using 300 mM
imidazole for elution as described previously (17). The Apaf-1 protein
was further purified on a Mono-Q HT5/5 column driven by a fast liquid
chromatography system (Amersham Biosciences). The Mono-Q column was
equilibrated with CEB buffer (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride,
and 1 mM dithiothreitol) and subsequently eluted with a
gradient of 0-500 mM NaCl. The peak fractions containing
Apaf-1 were collected and concentrated by Centricon-10 Plus
(Millipore). The purified Apaf-1 was stored at In Vitro Transcription/Translation and Caspase
Processing--
In vitro transcription and translation were
done using the TNT SP6 reticulocyte lysate system (Promega)
in the presence of either non-radioisotope-labeled methionine or
[35S]methionine according to the manufacturer's
instruction. Processing of Fv-caspase fusion proteins and FLAG-tagged
caspase-2/-9 was performed in the presence of 100 nM
AP20187 and the indicated concentration of M2, respectively, at
30 °C for 4-8 h. Processing of full-length caspase-9 proteins by
Apaf-1 was performed using the indicated amounts of recombinant Apaf-1
and 1.2 µl of in vitro translated, 35S-labeled
caspase-9 proteins in a final volume of 6 µl.
Immunoprecipitation Assay--
Three microliters of each
in vitro translated protein was added to 500 µl of lysis
buffer (12). The proteins were precleared with 20 µl of
glutathione-Sepharose beads for 1 h and then immunoprecipitated with anti-FLAG antibody conjugated on agarose beads. After washes, the
beads were analyzed by SDS-PAGE and autoradiography.
Cell Death Assay--
HEK293 and HeLa cells seeded in six-well
plates were transfected the 2nd day with the caspase-9 plasmids plus a
green fluorescence protein expression plasmid (pEGFP)
(Clontech) via the calcium phosphate precipitation
method. Cells were fixed at 24 h after transfection. The
percentage of apoptosis was determined by the number of fluorescent
cells with apoptotic morphology divided by the total number of
fluorescent cells. Data shown are averages and standard deviations of
three independent experiments, and for each experiment, more than 300 blue cells from randomly chosen fields were counted.
Activation of Procaspase-9 by Apoptosome-independent
Oligomerization--
Given that both the oligomerization and the
allosteric regulation models have been proposed for the activation of
caspase-9, we wished to examine whether induced oligomerization of
caspase-9 outside the apoptosome complex could lead to its activation.
Two approaches that allow for controlled oligomerization were used. The
first one was based on the FK506-binding protein (FKBP) and a divalent
FKBP ligand. A derivative of FKBP termed Fv (18) was fused to the
protease domain of caspase-9 in place of the protein interaction motif
caspase recruitment domain (CARD). A divalent Fv ligand, AP20187, binds
to Fv with high affinity but not to endogenous FKBP, thus minimizing
the interference from endogenous FKBP and allowing for the effective
dimerization of Fv fusion proteins. When Fv-caspase-9 fusion protein
was treated with AP20187, the zymogen underwent proteolytic processing
between the small subunit and the rest of the protein, a pattern that recapitulates the processing of the full-length caspase-9 in the apoptosome (Fig. 1A,
lane 2). To confirm that the cleavage did occur at one of
the previously determined cleavage sites (13, 19), we mutated the two
major cleavage sites, Asp-315 and Asp-330, as well as a minor cleavage
site, Glu-306, to Ala. The resulting mutant failed to undergo
processing upon dimerization (Fig. 1A, lane 6).
In addition, the processing was autoproteolytic as shown by the
corresponding active site Cys-287 to Ala mutant that failed to be
processed (Fig. 1A, lane 4).
To ascertain that oligomerization of full-length procaspase-9 is
sufficient to trigger its activation independent of Apaf-1, as the
second approach, we used antibody-mediated oligomerization of a tagged
full-length caspase-9 protein. FLAG-tagged caspase-9 was induced to
dimerize/oligomerize by the anti-FLAG monoclonal antibody M2. Notably
the processing of the full-length zymogen was evident upon M2
treatment, generating fragments (p10 and p35) characteristic of mature
caspase-9 (Fig. 1B, lanes 2-4). The specificity of the reaction was confirmed by the observation that an HA-tagged caspase-9 fusion failed to be activated upon M2 treatment (Fig. 1B, lane 6).
Similar to Fv-mediated activation of caspase-9, the M2-induced
processing of full-length procaspase-9 was also an autocatalytic event,
and it occurred at one of the previously identified cleavage sites
(Fig. 1B, lanes 8 and 10). We
therefore conclude that mere oligomerization of procaspase-9 induces
its proteolytic processing.
Formation of an Intermediate by Stable Interaction of the Protease
Domain Is a Requisite Step in Caspase-9 Activation--
How may
oligomerization lead to caspase-9 activation? We recently found that
the formation of an active intermediate by two precursor molecules
through their protease domains is a requisite step in the activation of
caspase-8 (20). A previous study also showed that procaspase-9 is
mainly an inactive monomer at its physiological concentration but forms
a dimer at the high concentration used for crystal formation (21). To
determine whether the homophilic association of the protease domain is
required for the activation of caspase-9 in the apoptosome, we
generated mutations in the procaspase-9 protease domain that abolished
its self-association and examined the effects of these mutations on
Apaf-1-dependent caspase-9 activation. Sequence alignment
of caspase-9 with other caspases, in combination with structural
information of several mature caspases, revealed amino acids on
procaspase-9 that are potentially important for the protease domain
interaction. We mutagenized three single or paired residues to those of
opposite charge for charged amino acids and to alanines for the others (Fig. 2A). The caspase-9 protease domain harboring each of
these mutations failed to associate with itself (Fig. 2B,
lanes 3-5).
Next we introduced these mutations into full-length procaspase-9 and
tested their effect on procaspase-9 activation. When the wild type
procaspase-9 protein was treated with recombinant Apaf-1, it became
activated in the presence of cytochrome c and dATP as
predicted (Fig. 3A, lane
1). However, when the interaction-deficient mutants were treated
with Apaf-1 under the same conditions, the processing was either
abolished or severely impaired (Fig. 3A, lanes
6-14). Because caspase-9 can become activated without being processed (19), we wished to determine whether these
interaction-deficient mutants lost their cell killing activity. When
transfected into 293 or HeLa cells, each of these mutants failed to
induce cell death (Fig. 3B, lanes 4-6 and data
not shown). FLAG-tagged procaspase-9 harboring each of these mutations
also failed to be activated upon M2 treatment (data not shown). The
correlation between the loss of self-interaction of the protease domain
and the loss of caspase-9 activation indicates that the association of
the protease domain is a requisite step for the activation of
caspase-9.
Activation of the Other Initiator Caspases and Inflammatory
Caspases by Oligomerization--
Besides caspase-8 and -9, the other
human initiator caspases include caspase-2 and -10. Procaspase-2 is
structurally similar to procaspase-9 in that both contain a CARD motif
in their prodomains (22). Recent studies have shown that caspase-2 is
an upstream initiator caspase for DNA damage-induced apoptosis and
induction of cytochrome c release from the mitochondria
(23-25). To examine whether caspase-2 can be activated by
oligomerization, we tagged procaspase-2 with a FLAG epitope at the
NH2 terminus and treated the protein with M2. Similar to
procaspase-9-FLAG, FLAG-procaspase-2 underwent self-processing upon M2
treatment, generating fragments typical of mature caspase-2 (Fig.
4A). Interestingly the
processing of caspase-2 mirrored that of caspase-9 with only one
cleavage event that separates the small subunit from the rest of the
protein.
Procaspase-10 is structurally similar to procaspase-8 with two tandem
death effector domains in its prodomain (26) and is engaged by the
death receptors to deliver lethal signals to cells. When the protease
domains of multiple caspase-10 molecules were brought into close
proximity through Fv-mediated oligomerization, they underwent
self-processing (Fig. 4B).
Murine caspase-11 is required for the activation of procaspase-1 (5),
which processes the precursor of proinflammatory cytokine
interleukin-1
Understanding the mechanism of caspase activation is central to our
understanding of apoptosis regulation as well as inflammatory responses. Based on this study and previous studies by ourselves and
others, we propose that oligomerization is a unifying theme for the
activation of long prodomain-containing caspases, including both
apoptosis initiator and inflammatory caspases. However, there are
notable variations among initiator caspases. For example, while
processed caspase-8 appears to be completely functional, processed
caspase-9 needs to be associated with Apaf-1 to gain its full potential
(15). In light of the fact that caspase-9 activation requires
interaction of its protease domain (Fig. 2, 3), it is possible that the
processed caspase-9 subunits cannot form stable tetrameric complexes on
their own, and the function of Apaf-1 is to hold these subunits in
place as opposed to inducing conformational changes in them. This
hypothesis is consistent with the observation that processed caspase-9
is mainly a monomer in solution (21). As for procaspase-2, although the
protein complex that promotes its activation remains elusive, the
similarity in the structure and processing patterns between caspase-2
and -9 suggest that caspase-2 may also require an association with its
adaptor protein to be fully active. Finally, in addition to apoptosis-inducing initiator caspases, we found that
dimerization-mediated caspase activation is also applicable to the
inflammatory caspases caspase-1 and -11. To date, the regulation of
inflammatory caspase activation is not well understood, but upstream
adaptor proteins may exist to facilitate the dimerization and
autoactivation of these caspases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, for example,
requires both caspase-1 and caspase-11 (4, 5). Similar to initiator caspases, the inflammatory caspases contain a relatively long prodomain.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
80 °C.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Oligomerization induces procaspase-9
activation independent of the apoptosome. A,
procaspase-9 self-processing induced by FKBP-mediated dimerization.
In vitro translated, [35S]methionine-labeled
Fv fusions of the caspase-9 (C9) protease domain (amino
acids 124-416) were incubated with 100 nM AP20187
(AP) for 4 h. Reaction products were resolved by
SDS-PAGE, and 35S-labeled products were detected by
autoradiography. WT, wild type caspase-9 protease domain;
C/A, the active site Cys-287 to Ala
mutation; 3A, the processing site Glu-306, Asp-315, and
Asp-330 to Ala mutations. The deduced domain structures of the
indicated bands are shown on the left. FL,
full-length fusion protein. Molecular mass standards are shown
on the right. B, activation of full-length
caspase-9 by antibody-mediated oligomerization. In vitro
translated, 35S-labeled full-length wild type or mutant
caspase-9 proteins (with either a COOH-terminal FLAG (F) or
HA tag) were incubated with the indicated concentration of M2 for
4 h. The reaction mixtures were analyzed as in A.
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Fig. 2.
Mutations that abolished the homophilic
interaction of the caspase-9 protease domain. A,
summary of the caspase-9 mutations. Amino acid substitutions, amino
acids at the domain boundaries, the CARD, and the small (p10) subunits
are marked. p35 and p37 are two alternative processing products.
B, defective self-association of the mutant caspase-9
protease domain. Results of the immunoprecipitation assay of the
caspase-9 protease domain proteins with the indicated combination of
radiolabeled and unlabeled HA- and FLAG-tagged proteins are shown.
Proteins were precipitated with anti-FLAG beads, and the bound proteins
were analyzed by SDS-PAGE and autoradiography. C9,
caspase-9; PD, protease domain; IP,
immunoprecipitation; WT, wild type; M1, D150K;
M2, F296A/V298A; M3, K409D/K410D.
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Fig. 3.
Loss of homophilic interaction of the
protease domain led to defective caspase-9 activation.
A, mutations in the protease domain abolished procaspase-9
processing. In vitro translated, 35S-labeled
procaspase-9 proteins were incubated with 0.2 (+) or 0.6 µg (++) of
Apaf-1 or without Apaf-1 ( ) in the presence of 100 ng/ml cytochrome
c for 4 h. Reaction mixtures were then subjected to
SDS-PAGE and autoradiography analyses. B, the cell death
activity of various procaspase-9 mutants. Two micrograms of each
plasmid was transfected into 293 cells together with pEGFP.
GFP-positive cells were scored for apoptosis 24 h
post-transfection. Data shown (mean ± S.D.) are representative of
three independent experiments done in duplicates. Similar results were
obtained using HeLa cells (not shown). C9, caspase-9;
WT, wild type; C/S, Cys to Ser
mutation; M1, D150K; M2, F296A/V298A;
M3, K409D/K410D; FL, full-length fusion
protein.
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Fig. 4.
Dimerization-induced autoprocessing of
procaspase-2, -10, and -11. A, activation of
procaspase-2 by antibody-mediated oligomerization.
35S-Labeled FLAG-tagged procaspase-2 (C2) was
treated with increasing concentrations of M2 (8.75, 17.5, and 35 ng/µl) or without M2 ( ) for 12 h. The reaction mixtures were
analyzed by SDS-PAGE and autoradiography. B, dimerization
induces activation of procaspase-10 and -11. In vitro
translated, [35S]methionine-labeled Fv-caspase fusions
and their corresponding active site Cys to Ser mutants
(C/S) were treated with dimerizer AP20187 (100 nM) (+) or left untreated (
) for 4 h. The reaction
mixtures were then analyzed as in A. The p12 subunit of
Fv-caspase-10 (C10) could barely be detected due to the low
content of methionine in this fragment. C11, caspase-11;
WT, wild type; FL or F.L., full-length
fusion protein; AP, AP20187.
(4). Our previous study has shown that procaspase-1 is
activated by oligomerization (8, 27, 28). To examine whether caspase-11
is activated in a similar mechanism, we used Fv-mediated dimerization.
As shown in Fig. 4B, an Fv fusion of the wild type
caspase-11 protease domain, but not a corresponding fusion of the
catalytically inactive mutant, was converted to the mature enzyme by
AP20187-induced oligomerization. Thus, inflammatory caspases are also
likely to be activated by oligomerization.
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ACKNOWLEDGEMENTS |
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We thank Rachael Stratt for excellent help with experiments and manuscript preparation, Dr. Junying Yuan for caspase-2 and caspase-11 cDNAs, Dr. Shimin Hu for reagents, and ARIAD Pharmaceuticals for the FKBP dimerization system.
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FOOTNOTES |
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* This study was supported by an NCI, National Institutes of Health training grant (to D. W. C.) and by National Institutes of Health Grants GM60911 (to X. Y.) and AG14357 (to E. S. A.).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.: 215-573-6739; Fax: 215-573-8606; E-mail: xyang@mail.med.upenn.edu.
Published, JBC Papers in Press, March 13, 2003, DOI 10.1074/jbc.C300089200
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ABBREVIATIONS |
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The abbreviations used are: Apaf-1, apoptotic protease-activating factor; FKBP, FK506-binding protein; HA, hemagglutinin; GFP, green fluorescent protein; CARD, caspase recruitment domain.
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