* Molecular Cell Biology Laboratory, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland; Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121; § Department of
Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037;
The Burnham Institute, La
Jolla, California 92037; ¶ Departments of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research,
Pointe Claire-Dorval, Quebec, H9R 4P8, Canada; and ** Center for Apoptosis Research and The Kimmel Cancer Institute,
Jefferson Medical College, Philadelphia, Pennsylvania 19107
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Abstract |
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Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.
Key words: Apaf-1; apoptosis; caspases; cell-free; cytochrome c ![]() |
Introduction |
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NUMEROUS studies have implicated caspases (cysteine aspartate-specific proteases) as the molecular instigators of apoptosis (Yuan et al., 1993;
Gagliardini et al., 1994
; Kumar et al., 1994
; Lazebnik et al.,
1994
; Wang et al., 1994
; Nicholson et al., 1995
; Tewari et
al., 1995
; Kuida et al., 1996
). Caspases are a family of human proteases that cleave their substrates after aspartic acid residues, an uncommon substrate preference (Jacobson and Evan, 1994
; Martin and Green, 1995
; Alnemri et
al., 1996
; Chinnaiyan and Dixit, 1996
; Henkart, 1996
; Alnemri, 1997
; Salvesen and Dixit, 1997
). Caspases are typically constitutively present within cells as inactive zymogens that require proteolytic processing to achieve their active, two-chain configurations (Thornberry et al.,
1992
; Walker et al., 1994
; Darmon et al., 1995
; Gu et al.,
1995
; Duan et al., 1996
; Schlegel et al., 1996
; MacFarlane et
al., 1997
). In vitro, caspases are known to cleave a number
of structural as well as RNA splicing and DNA repair-associated proteins and can also process other caspases
(Casciola-Rosen et al., 1994
, 1995
; Brancolini et al., 1995
;
Emoto et al., 1995
; Martin et al., 1995a
; Tewari et al., 1995
;
Casiano et al., 1996
; Fernandes-Alnemri et al., 1996
; Hsu
and Yeh, 1996
; Kayalar et al., 1996
; Takahashi et al., 1996
;
Weaver et al., 1996
). The consequences of these cleavage
events are now emerging and suggest that they are responsible for many of the phenotypic changes that occur during
apoptosis. In addition, the observation that caspases can
process other caspases suggests that there is likely to be a
stepwise activation of caspases during apoptosis, similar to
the clotting or complement cascades (Martin and Green,
1995
).
Several studies suggest that receptor-associated adaptor
proteins, such as FADD/MORT-1, that facilitate close
association of certain caspases promote caspase autoprocessing (Boldin et al., 1996; Fernandes-Alnemri et al., 1996
;
Muzio et al., 1996
; Ahmad et al., 1997
; Duan and Dixit,
1997
; Yang et al., 1998
). Similar adaptor molecules, such
as the recently described CED-4 homologue, Apaf-1, may
play key roles in promoting apoptosis by clustering caspases
at intracellular sites. Current evidence suggests that there
are several distinct routes to caspase activation depending upon the stimulus that initiates the death program.
Many studies have shown that cytochrome c enters the
cytosol during apoptosis, probably as a result of loss of this
protein from mitochondria rather than as a consequence
of failed import (Liu et al., 1996; Kluck et al., 1997a
,b;
Reed, 1997
; Yang et al., 1997
; Bossy-Wetzel et al., 1998
).
Cell death initiator or repressor proteins such as Bid and
Bcl-2 have been shown to regulate this event, suggesting
that this is a critical step in the death signaling cascade
(Kluck et al., 1997a
; Yang et al., 1997
; Li et al., 1998
; Luo
et al., 1998
). Studies using cell-free systems have shown
that cytochrome c, in association with dATP, is capable of
initiating apoptosis-like changes in cytosols derived from a
variety of cell types (Liu et al., 1996
; Kluck et al., 1997a
,b; Deveraux et al., 1998
; Pan et al., 1998a
). The apoptosis-promoting activity of cytochrome c is due to its ability to
interact with the CED-4 homologue Apaf-1 (Zou et al.,
1997
). Binding of cytochrome c to Apaf-1 enables this protein to recruit caspase-9 and to stimulate processing of the
inactive caspase-9 zymogen to its active form (Li et al.,
1997
; Srinivasula et al., 1998
). Once active, caspase-9 then
presumably triggers a cascade of caspase activation events leading to apoptosis.
To explore more fully the range of caspase activation events that are triggered by cytochrome c, we have used a human cell-free system based on Jurkat postnuclear extracts. Here, we show that cytochrome c is capable of initiating processing of multiple caspases (-2, -3, -6, -7, -8, -9, and -10) in cell-free extracts, as well as a range of biochemical and morphological events characteristic of apoptosis. In contrast, activation of caspases-1, -4, and -5 was not observed in response to cytochrome c, suggesting that these caspases do not participate in apoptosis or do so upstream of the point of entry of cytochrome c into the cytosol. Strikingly, depletion of caspase-9 from cell extracts rendered all of the other caspases examined unresponsive to cytochrome c, suggesting that all of these caspase activation events lie on the same pathway, with caspase-9 at the apex of the cascade. Based on data generated by immunodepletion of specific caspases, we propose an order of the caspase activation events that lie downstream of caspase-9 in the cytochrome c-inducible pathway.
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Materials and Methods |
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Materials
Anti-caspase-3 and anti-caspase-9 polyclonal antibodies were generated
by immunizing rabbits with GST-caspase-3 fusion protein or purified
recombinant caspase-9, respectively; anti-caspase-3 and anti-caspase-7
mouse mAbs were purchased from Transduction Laboratories; purified
rabbit polyclonal anti-caspase-6 antibody was purchased from Upstate
Biotechnology; rabbit polyclonal anti-caspase-1 (ICE) was kindly provided by Dr. Douglas K. Miller; anti-U1snRNP and anti-PARP autoantibodies were derived from human subjects, as previously described (Casiano et al., 1996); anti-
-fodrin (nonerythroid spectrin) was purchased
from Chemicon International; and anti-
-actin antibody was purchased
from ICN. Ac-YVAD-CHO and Ac-DEVD-CHO peptides were purchased from BACHEM Bioscience; YVAD-pNA and DEVD-pNA peptides were purchased from Biomol Ltd. GST-CrmA fusion protein was
kindly provided by Dr. David Pickup. Bovine heart cytochrome c was purchased from
GST-Apaf-11-97 and GST-Apaf-11-401 fusion proteins were produced by
PCR-mediated amplification of the relevant coding sequences from the
full-length Apaf-1 cDNA (kindly provided by Dr. Xiaodong Wang), followed by subcloning of the resulting PCR products in-frame with the GST
coding region of pGEX4TK2 (). Plasmids encoding GST and
GST fusion proteins were transformed into Escherichia coli DH5 and
bacteria were induced to express the recombinant proteins in the presence
of 100 µM IPTG for 4 h at 30°C. GST and GST fusion proteins were subsequently purified using glutathione Sepharose () according to
standard procedures.
In Vitro Association Assays
The ability of caspases-1, -2, -3, -6, -7, -8, -9, and -10 to interact with GST-Apaf-1 fusion proteins was assessed as follows. [35S]Methionine-labeled caspases (5-15-µl aliquots of translation reactions) were brought to 200 µl in GST buffer (50 mM Tris, pH 7.6, 120 mM NaCl, 0.1% CHAPS, 100 µM PMSF, 10 µg/ml leupeptin, and 2 µg/ml aprotinin). 2-µl aliquots (~6 µg protein) of glutathione Sepharose-immobilized GST or GST-Apaf-1 fusion proteins were then added, followed by incubation for 2 h at 4°C under constant rotation. Bead complexes were then washed several times in GST buffer and bound caspases were detected by SDS-PAGE/fluorography.
Depletion of Caspases from Cell Extracts
Caspase-9 was depleted from cell extracts using either glutathione Sepharose-immobilized GST-Apaf-11-97 or protein A/G agarose-immobilized anti-caspase-9 antibody, as follows. For GST-Apaf-11-97 depletions, 40 µl of a 50% slurry of GST-Apaf-1 or GST was added to 100-µl aliquots of Jurkat cell extract which were incubated overnight at 4°C under constant rotation. Beads were then pelleted and extracts were used immediately. For antibody depletions, 40-µl aliquots of protein A/G agarose () were precoated with anti-caspase-9 rabbit polyclonal antibody by incubation with 50 µl of either anti-caspase-9 antiserum or a control (anti-RelA; ) rabbit polyclonal in a total volume of 300 µl in PBS, pH 7.2, for 3 h at 4°C under rotation. Antibody-coated beads were then washed three times before addition to Jurkat cell extracts (100 µl) which were incubated overnight under constant rotation at 4°C. Beads were then removed from the extracts before use. Caspase-3, -6, and -7 immunodepletions were performed in a similar manner with the exception that 5 µg of each antibody was used to precoat 40-µl aliquots of protein A/G agarose before depletion.
Preparation of Cell-free Extracts
Cell-free extracts were generated from Jurkat T lymphoblastoid cells or
MCF-7 cells as previously described (Martin et al., 1995b, 1996
), with the
following modifications. Cells (2-5 × 108) were pelleted and washed twice
with PBS, pH 7.2, followed by a single wash with 5 ml of ice-cold cell extract buffer (CEB;1 20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 100 µM PMSF,
10 µg/ml leupeptin, 2 µg/ml aprotinin). Cells were then transferred to a
2-ml Dounce-type homogenizer, were pelleted, and two volumes of ice-cold
CEB was added to the volume of the packed cell pellet. Cells were allowed to swell under the hypotonic conditions for 15 min on ice. Cells were then disrupted with 20 strokes of a B-type pestle. Lysis was confirmed by examination of a small aliquot of the suspension under a light
microscope. Lysates were then transferred to Eppendorf tubes and were
centrifuged at 15,000 g for 15 min at 4°C (S15 or postnuclear extracts). The
supernatant was removed while taking care to avoid the pellet. Supernatants were then frozen in aliquots at
70°C until required.
Cell-free Reactions
Cell-free reactions were typically set up in 10- or 100-µl reaction volumes.
For 100-µl scale reactions, 50 µl of cell extract (~5 mg/ml) and 10 µl of rat
liver nuclei were brought to a final volume of 100 µl in CEB, with or without peptides or proteins solubilized in the same buffer. Apoptosis was typically induced by addition of bovine heart cytochrome c to extracts at a final concentration of 50 µg/ml. Where necessary, dATP was also to a final
concentration of 1 mM, although many extracts did not require addition
of this nucleotide triphosphate. To initiate apoptosis, extracts were incubated at 37°C for periods of up to 3 h. At time points indicated in the text,
2-µl aliquots were removed for determination of percentages of apoptotic
nuclei using Hoechst 33342 staining, as previously described (Martin et al.,
1995b, 1996
). Samples of extract (10-20 µl) were also removed at times indicated in the text and frozen at
70°C for subsequent SDS-PAGE/Western blot or fluorographic determination of substrate cleavage profiles or
caspase activation.
Coupled In Vitro Transcription/Translations
[35S]Methionine-labeled caspases were in vitro transcribed and translated
using the TNT kit (), as previously described (Martin et al.,
1996). For use in coupled in vitro transcription/translation experiments,
plasmids encoding each of the caspases used were grown in E. coli DH5
strain and were purified using tip-100 Qiagen columns. Typically, 1 µg of
plasmid was used in a 50 µl transcription/translation reaction containing
4 µl of translation grade [35S]methionine (1,000 µCi/ml; ICN).
YVAD-pNA and DEVD-pNA Cleavage Assay
At times indicated in the text, 10-µl aliquots of cell-free reactions were removed and were diluted to 100 µl by the addition of ice-cold protease reaction buffer (PRB; 50 mM Hepes, pH 7.4, 75 mM NaCl, 0.1% CHAPS, 2 mM dithiothreitol). Samples were held on ice until completion of the experiment and were then divided into two separate 50-µl portions for the separate assessment of YVAD-p-nitroanalide (YVAD-pNA) and DEVD-pNA cleavage activity, respectively. To each 50-µl aliquot, 5.5 µl of a 10× stock of each peptide (500 µM) was added such that the final concentration of either peptide in the reaction was 50 µM. Reactions were then incubated for 30 min at 37°C, followed by addition of 950 µl ice-cold dH2O to stop the reaction. OD400 readings of each sample were then taken against a blank containing buffer and peptide alone (i.e., no extract).
SDS-PAGE and Western Blot Analysis
Proteins were subjected to standard SDS-PAGE at 60-70 V and were
transferred onto 0.45 µM PVDF membranes (Bio-Rad) for 3 h at 50-75
mA, followed by probing for various proteins using the polyclonal antibodies described under materials. Bound antibodies were detected using
appropriate peroxidase-coupled secondary antibodies (), followed by detection using the Supersignal chemiluminescence system
(Pierce), all as previously described (Martin et al., 1996).
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Results |
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Cytochrome c Initiates Multiple Features of Apoptosis in Jurkat Cell Extracts
Addition of purified cytochrome c to postnuclear (15,000 g;
S15) extracts of Jurkat T lymphoblastoid cells was sufficient to initiate the whole spectrum of events characteristic of apoptosis in these extracts. Nuclei incubated in the
extracts in the presence of cytochrome c rapidly exhibited
apoptotic features (chromatin margination and nuclear
fragmentation; Fig. 1 A) and chromatin also underwent
fragmentation into ~200-bp multiples (data not shown).
Proteolysis of several caspase substrates (-fodrin, U1sn- RNP, PARP) was also observed in response to cytochrome c (Fig. 1 B). Interestingly, although previous reports have shown that addition of dATP (or ATP) to cell
extracts is required for the proapoptotic activities of cytochrome c, many extracts did not require addition of exogenous nucleotide triphosphates, presumably due to sufficiently high levels of ATP or dATP endogenous to these extracts.
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Cytochrome c-initiated Apoptosis Is Associated with Proteolytic Processing of Caspase-3, but Not Caspase-1
Previous studies have shown that caspases-3 and -9 are activated in response to cytochrome c (Liu et al., 1996; Li
et al., 1997
; Kluck et al., 1997b
; Zou et al., 1997
; Pan et al.,
1998a
). We initially confirmed these observations before
assessing the activation of other caspases in this context.
Fig. 2 demonstrates that caspase-3 endogenous to Jurkat
cell extracts was rapidly converted from the 36-kD proenzyme to the p17/p12 mature form in the presence of cytochrome c. Processing occurred in a two-step manner, with
the initial appearance of a p24/p12 intermediate in the
extracts, followed by accumulation of the mature p17/
p12 form of the enzyme (Fig. 2, A and C), reminiscent of
the mechanism of activation of caspase-3 in response to
granzyme B (Martin et al., 1996
). This was further confirmed by addition of [35S]methionine-labeled caspase-3 to
the extracts, which enabled detection of the caspase-3-p12
chain that was not recognized by the anti-caspase-3 polyclonal antibody used (Fig. 2 B). In direct contrast, conversion of caspase-1 (ICE) to its mature form was not detected in the same extracts over an identical time course (Fig. 2 A). To further confirm that processed caspases
were active, we used synthetic tetrapeptide substrates that
are preferentially cleaved by caspase-1-like (YVAD-pNA)
or caspase-3-like (DEVD-pNA) proteases to assess the
induction of caspase-1- or caspase-3-like proteolytic activity in response to cytochrome c. We observed a striking induction of DEVD-pNA cleaving activity within 15 min of
addition of cytochrome c to the extracts, whereas YVAD-pNA cleaving activity did not rise above basal levels during the same time course (Fig. 2 D), in agreement with our
observations on the absence of caspase-1 processing in this
context (Fig. 2 A).
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Cytochrome c induced apoptotic changes in nuclei added to the extracts at concentrations of as little as 1-5 µg/ml, but had no direct effects on nuclei in the absence of cell extract (Fig. 3 A). Interestingly, at cytochrome c concentrations where only partial caspase-3 activation was observed (5 µg/ml), substrates such as fodrin, PARP, and U1snRNP were almost completely cleaved (Fig. 3 B), suggesting that only a small amount of the total caspase-3 pool is required in order to effect complete proteolysis of these substrates. An alternative explanation is that cytochrome c activates other caspases in the extracts that are capable of cleaving these substrates. Cytochrome c failed to trigger caspase-1 processing at any of the concentrations tested (Fig. 3 B).
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Cytochrome c Initiates a Cascade of Protease Activation Events Involving Caspases-2, -3, -6, -7, -8, -9, and -10
Recent studies have shown that caspase-9 is activated in
response to cytochrome c due to clustering of caspase-9 by
Apaf-1 and that this results in activation of Caspases-3 (Li
et al., 1997; Pan et al., 1998a
; Srinivasula et al., 1998
). To
explore the full range of caspase activation events in the
cytochrome c-initiated proteolytic cascade, we introduced
[35S]methionine-labeled caspases-1, -2, -3, -4, -5, -6, -7, -8, -9, and -10 into Jurkat cell-free extracts and monitored
processing of these proteases to their mature forms. Fig. 4
demonstrates that cytochrome c/dATP triggered maturation of caspases-2, -3, -6, -7, -8, -9, and -10, whereas none of
the ICE subfamily proteases (caspases-1, -4, and -5) were
processed under the same conditions. Significantly, cytochrome c failed to activate any of the caspases in the absence of cell extract, suggesting that a cytosolic factor such
as Apaf-1 was required for all of these activation events. Caspases with long prodomains such as caspases-2, -8, and
-10 are generally considered to be upstream or signaling
caspases in the cell death pathway due to their ability to
associate with cell surface death receptor molecules such
as Fas/CD95 or TNFR1. Therefore, it was somewhat surprising that these caspases became activated in the presence of cytochrome c. However, it has been reported
recently that thymocytes from APAF-1 null mice are impaired with respect to caspase-2 and caspase-8 activation
in response to several proapoptotic stimuli (Yoshida et
al., 1998
). Similarly, dexamethasone-induced processing of
caspases-2 and -8 was found to be impaired in mice deficient for caspase-9 (Hakem et al., 1998
). These data suggest that these caspases are indeed activated in the Apaf-1
pathway in vivo.
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To explore the range of cytochrome c-inducible caspase activation events in more detail, we monitored the kinetics of activation of all caspases relative to each other in this system. Fig. 5 shows that detectable activation of most caspases, with the exceptions of caspases-8 and -10, appeared to occur contemporaneously, typically within 30 min of addition of cytochrome c to the extracts. In contrast, processing of caspases-8 and -10 were noticeably delayed relative to the other caspases, suggesting that these caspases might be activated late in this pathway.
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APAF-1 Selectively Binds to Caspase-9
The observation that multiple caspases were activated in response to cytochrome c suggested either that all of these caspase activation events occurred downstream of caspase-9 (the only caspase known to directly associate with Apaf-1/ cytochrome c), or that a number of distinct caspase activation pathways could be instigated by cytochrome c, independent of caspase-9. To discriminate between these possibilities, we first explored whether Apaf-1 could directly bind caspases other than caspase-9. Fig. 6 demonstrates that a GST fusion protein comprising the CED-3 homology region of Apaf-1 (amino acid [aa] residues 1-97) selectively bound caspase-9 but did not bind any of the other caspases (-1, -2, -3, -6, -7, -8, and -10) tested. Similar results were obtained using a different GST fusion that spanned the CED-3 as well as the CED-4 homology regions of Apaf-1 (aa 1-412; Slee, E.A., and S.J. Martin, data not shown). These data demonstrate that Apaf-1 is highly selective for caspase-9, although they do not rule out the possibility that other caspases may become recruited to Apaf-1 via suitable adaptor molecules.
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Depletion of Caspase-9 from Jurkat Extracts Abrogates Processing of all Caspases in Response to Cytochrome c
To determine whether caspase-9 was required for activation of all other caspases in this context, we depleted this protease from Jurkat extracts before the addition of cytochrome c. Depletion of caspase-9 using Sepharose- immobilized GST-Apaf-11-97 (Fig. 7 A), or anti-caspase-9 polyclonal antibody (Fig. 7 B), rendered all caspases unresponsive to cytochrome c. In contrast, mock depletions performed using Sepharose-GST or control polyclonal antibody did not interfere with cytochrome c-induced activation of any of the caspases examined (Fig. 7, A and B). These data suggest that caspase-9 is critical for cytochrome c-initiated caspase activation events and cannot be substituted for by the other caspases present in the extracts. They also provide further support for the idea that Apaf-1 is selective for caspase-9 (Fig. 6) and fails to initiate the apoptotic program in its absence.
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Ordering the Cytochrome c-inducible Caspase Cascade Downstream of Caspase-9
As a preliminary approach to ordering the sequence of caspase activation events triggered by cytochrome c, we investigated the effects of the tetrapeptide caspase inhibitors YVAD-CHO and DEVD-CHO, as well as the cowpox virus-derived caspase inhibitor CrmA, on the processing of all caspases downstream of caspase-9. The caspase-1-selective inhibitor YVAD-CHO had similar effects on all caspases, exhibiting little inhibition of caspase activation except at the highest concentration tested (Fig. 8). These data are consistent with YVAD-CHO directly inhibiting caspase-9 at high concentrations and terminating activation of all downstream caspases, in agreement with the results obtained by depletion of caspase-9 from the extracts (Fig. 7). Broadly similar effects on all caspases was also observed using GST-CrmA, with the exception that activation of caspases-8 and -10 were blocked at all concentrations of this inhibitor, whereas processing of all of the other caspases was seen at the lowest concentration tested (0.2 µM). This suggests that caspase-8 processing is downstream of the other caspases in the context of cytochrome c.
|
Strikingly, very different inhibitory effects were observed using the caspase-3- and caspase-7-selective inhibitor DEVD-CHO. Whereas processing of caspases-2, -6, -8, and -10 was completely blocked at all concentrations of DEVD-CHO tested, very significant processing of caspases-3 and -7 was observed in the presence of 1-10 µM of this inhibitor. These data suggest that, after caspase-9, caspases-3 and -7 are the next caspases to become activated in the cytochrome c-initiated caspase cascade. Inhibition of caspase-3 and -7 activities by DEVD-CHO prevents further activation of any other caspases downstream of this point.
Fig. 8 also demonstrates that maturation of caspase-3 in
the presence of 1-10 µM DEVD-CHO was arrested at the
p24/p12 intermediate. This confirms our earlier observation (Fig. 2, A-C) that cytochrome c-initiated caspase-3
processing was achieved via two distinct processing steps,
with the first step involving a single cleavage of the proenzyme between the large and small subunits to produce a p24/p12 intermediate which then undergoes further
processing via a distinct protease. The observation that
caspase-3 maturation was completely arrested at the p24/
p12 intermediate in the presence of DEVD-CHO suggests
that the second step (i.e., removal of the prodomain) is autocatalytic. A similar two-step processing mechanism has
been reported for granzyme B-mediated activation of
caspase-3 (Fernandes-Alnemri et al., 1996; Martin et al.,
1996
).
Depletion of Caspase-3 from Cell Extracts Ablates Cytochrome c-induced Processing of Caspases-2, -6, -8, and -10 and Reveals a Feedback Loop Involving Caspase-9
To confirm that caspase-3 was activated upstream of caspases-2, -6, -8, and -10 in response to cytochrome c and to ask whether caspase-3 was required for processing of any of the other caspases in this context, we immunodepleted caspase-3 from cell extracts (Fig. 9). Caspase-3-depleted extracts were compared with mock-depleted extracts for their ability to support cytochrome c-induced processing of caspases-2, -3, -6, -7, -8, -9, and -10. Strikingly, removal of caspase-3 from the extracts abrogated processing of caspases-2, -6, -8, and -10 but had only a marginal effect on the processing of caspase-7 in the presence of cytochrome c (Fig. 9 A). These data suggest that caspases-3 and -7 are directly processed downstream of caspase-9 in the cytochrome c/Apaf-1-inducible caspase cascade and that caspase-3 is necessary for the activation (either directly or indirectly) of caspases-2, -6, -8, and -10.
|
Interestingly, although caspase-3 was activated downstream of caspase-9 in response to cytochrome c as expected (Fig. 7), partial inhibition of caspase-9 processing
was observed in caspase-3-depleted, but not in mock-
depleted, extracts (Fig. 9 B). Consistent with this, reintroduced 35S-labeled caspase-3 was processed less efficiently
in caspase-3-depleted extracts as compared with mock-
depleted extracts, suggesting that caspase-9 activation was
less efficient in these extracts (Fig. 9 A). In normal or
mock-depleted extracts, the processed form of caspase-9
could be resolved into two major bands migrating at ~37
and ~35 kD, as recently reported (Srinivasula et al., 1998). However, in caspase-3-depleted extracts the 37-kD cleavage product was not produced (Fig. 9 B), suggesting that
caspase-9 activation in response to cytochrome c is partially achieved via a feedback loop involving caspase-3. To
confirm this, we compared Jurkat postnuclear extracts
with similar extracts prepared from MCF-7 cells which are
devoid of caspase-3 due to a deletion in exon 3 of the
CASP-3 gene (Jänicke et al., 1998
). Using MCF-7 cell extracts we confirmed that, in the absence of caspase-3,
caspase-9 is processed to a single 35-kD cleavage product,
whereas both the 35- and 37-kD cleavage products were
produced in Jurkat extracts (Fig. 9 B). These data are consistent with the interpretation that cytochrome c/Apaf-1-triggered processing of caspase-9 is initially autocatalytic, producing the p35 form via cleavage at Asp-315, but
upon activation of caspase-3 is also achieved via a feedback loop in which caspase-3 processes caspase-9 at Asp-330 (Fig. 9 C; Srinivasula et al., 1998
).
Caspase-6 Is Required for Cytochrome c-induced Processing of Caspases-8 and -10
We next depleted caspase-6 or caspase-7 from Jurkat extracts to ask whether either of these caspases was required for any of the other caspase activation events seen in the presence of cytochrome c (Fig. 10). Immunodepletion of caspase-6 failed to have any effect on the processing of caspases-3, -7, and -9 in response to cytochrome c, consistent with caspase-6 activation being downstream of the latter caspases (Fig. 10 A). Caspase-2 processing was also unaffected in the absence of caspase-6, suggesting that caspase-2 is directly processed, along with caspase-6, upon activation of caspase-3 in the extracts. However, processing of caspases-8 and -10 was largely abrogated in extracts devoid of caspase-6 (Fig. 10 A). These data suggest that, upon activation by caspase-3, caspase-6 in turn promotes the processing of caspases-8 and -10 further down the cascade.
|
In contrast to the effects seen after depletion of caspases-9, -3, or -6, immunodepletion of caspase-7 from the extracts failed to have any detectable inhibitory effects on any of the caspase processing events observed in the presence of cytochrome c (Fig. 10 B). Taken together, these data suggest an order of caspase activation events that take place distal to entry of cytochrome c into the cytosol (Fig. 11).
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Discussion |
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In this study, we have shown that cytochrome c can initiate a complex series of caspase activation events, ultimately resulting in apoptotic changes (nuclear condensation and fragmentation, degradation of several caspase substrates, DNA fragmentation) in cell extracts. Surprisingly, cytochrome c initiated activation of several of the so-called signaling (caspases-2, -8, -9, and -10) as well as effector (-3, -6, -7) caspases. In contrast, no detectable processing of the ICE subfamily caspases (-1, -4, -5) was observed in this context. All of these events were abrogated by removal of caspase-9 from the extracts, confirming that this protease is indispensable for cytochrome c-initiated triggering of the death program and occupies an apical point in the caspase cascade. In line with these observations, the CARD domain of Apaf-1 was shown to bind selectively to caspase-9. Inhibitory profiles generated with the caspase inhibitor DEVD-CHO suggested that caspases-3 and -7 were activated downstream of caspase-9 and that these caspases then went on to propagate the caspase cascade by activating caspases-2, -6, -8, and -10. This interpretation was confirmed and extended by immunodepleting caspases-3, -6, or -7 from the extracts and assessing the impact of their removal on the other caspase activation events. Interestingly, removal of caspase-3 revealed that this caspase was required for four other caspase activation events and also revealed a feedback loop in this pathway involving caspase-9.
In this study we have assessed caspase activation events in most cases (with the exception of caspases-1, -3, and -9) by adding 35S-labeled in vitro transcribed and translated caspases to the cell extracts. Clearly, this raises the issue of whether caspases endogenous to the extracts behave in the same way as their exogenously added counterparts. Where antibodies were available to us (caspases-2, -6, -7, and -8), we confirmed that the order and kinetics of endogenous caspase processing was essentially identical to that observed using exogenously added caspases (Slee, E.A., and S.J. Martin, data not shown). However, the use of radiolabeled caspases enabled us to track complete processing of each caspase whereas many of the available anticaspase antibodies recognized processed forms inefficiently or not at all.
Although the initial report that cytochrome c could trigger caspase-3 processing was surprising (Liu et al., 1996),
much evidence has accumulated to suggest that release of
cytochrome c from mitochondria is an important control
point in apoptosis (reviewed by Reed, 1997
). It is still unclear exactly how cytochrome c release is achieved, although recent reports suggest that death-promoting members of the Bcl-2 family such as Bax or Bid may play a role in this, possibly due to their ability to form ion or small
protein channels (Jurgensmeier et al., 1998
; Li et al., 1998
;
Luo et al., 1998
). Although opening of a permeability transition pore was proposed previously as one possible means
of enabling cytochrome c escape to the cytosol (Kroemer
et al., 1997
), cytochrome c release has been observed in situations where either no loss in mitochondrial transmembrane potential was observed or where changes in transmembrane potential occurred after cytochrome c efflux
(Kluck et al., 1997a
; Yang et al., 1997
; Bossy-Wetzel et al.,
1998
).
Irrespective of the exact mechanism of release, much evidence now exists to suggest that cytochrome c plays an
important role as an initiator of the death machinery in
cases where cellular damage is general (i.e., radiation, heat
shock, cytotoxic drugs), or as an amplifier of death signals
in cases where caspase activation is initiated by a membrane receptor such as Fas (Kuwana et al., 1998; Li et al.,
1998
; Luo et al., 1998
; Scaffidi et al., 1998
). Perhaps the
most compelling argument for a central role for cytochrome c in apoptosis is the finding of Wang and colleagues that cytochrome c binds to and activates Apaf-1,
the first human CED-4 homologue to be discovered (Zou
et al., 1997
). Gene targeting experiments in mice have revealed that Apaf-1 plays a critical role in developmental-related cell death in the brain, as well as in cytotoxic drug-induced cell death in other cell lineages (Cecconi et al.,
1998
; Yoshida et al., 1998
). At present, cytochrome c is the
only known activator of Apaf-1, although it is possible that
other pathways may harness the caspase-activating properties of this molecule.
Although numerous studies have appeared documenting the activation of individual caspases in the context of
many different death-promoting stimuli, it is still unclear
whether caspases are activated sequentially or in parallel
in many of these contexts. Here, we provide evidence for a
stepwise series of caspase activation events occurring in
response to cytochrome c. Caspase-9 appears to be the
first caspase to become activated in this context, almost certainly due to clustering of this protease via Apaf-1.
Clustering of caspase-9 results in partial activation of this
protease in an autocatalytic manner (Pan et al., 1998a;
Srinivasula et al., 1998
). Caspase-9 then initiates processing of caspase-3 as well as caspase-7. The activation of
caspase-3 in this context appears to occur in a partly autocatalytic manner since the caspase-3 inhibitor, DEVD-CHO, arrested maturation of caspase-3 at an incompletely processed intermediate stage. The pattern of caspase-3
breakdown suggests that caspase-9 attacks this molecule
between the large and small subunits and that caspase-3
subsequently removes its own prodomain by autocatalysis.
Activated caspase-3 in turn activates caspases-2 and -6 and
also appears to be capable of acting in a feedback loop
on caspase-9 to ensure complete activation of the latter.
Somewhat surprisingly, caspase-6 was found to be required for the activation of caspases-8 and -10 in this context (Fig. 10).
Clearly, further work is necessary to determine whether
the sequence of cytochrome c-inducible caspase activation
events that take place in cell extracts also takes place in intact cells. However, recent gene targeting studies provide
support for our model (Hakem et al., 1998; Yoshida et al.,
1998
). CASP-9 null embryonic stem (ES) cells and embryonic fibroblasts were found to be resistant to multiple
proapoptotic stimuli, but not to cytotoxic T lymphocyte or
TNF-mediated killing, arguing that caspase-9 is required
for forms of apoptosis that are thought to be routed along
the mitochondrial pathway (Hakem et al., 1998
). As further evidence of this, CASP-9
/
ES cells were found to be
resistant to UV-induced death, although cytochrome c release still took place (Hakem et al., 1998
). Moreover, UV-induced caspase-3 and caspase-8 processing was impaired
in CASP-9
/
ES cells as was dexamethasone-induced
processing of caspases-2, -7, and -8 in CASP-9
/
thymocytes (Hakem et al., 1998
), supporting our observations that these caspases are activated downstream of caspase-9
in the cytochrome c pathway. Similar observations have
also been made with respect to etoposide-induced caspase-2
and caspase-8 activation in thymocytes from APAF1
/
mice (Yoshida et al., 1998
). Once again, these observations
lend support to our observations that caspases-2 and -8 are
activated downstream of cytochrome c/Apaf-1.
We have confirmed the observation of Wang and colleagues (Li et al., 1997) that Apaf-1 directly binds to
caspase-9 and have extended this observation to show that
caspases-1, -2, -3, -6, -7, -8, and -10 do not bind to this molecule. These observations are at odds with recent findings
that suggest that Apaf-1 can also form complexes with
caspases-4 and -8 (Hu et al., 1998
). However, in the latter
study no direct interaction between these caspases was
demonstrated since coimmunoprecipitation from cell lysates was the criteria used to determine interaction. Thus, binding could have been mediated by an adaptor protein.
Using a Gal4-based yeast two-hybrid system we have confirmed the interaction between the CED-3-homologous region of Apaf-1 and caspase-9 but again failed to detect direct binding of Apaf-1 to caspase-8 (Harte, M.T., C. Adrain,
and S.J. Martin, unpublished data). In addition, the observation that removal of caspase-9 from cell extracts abolished all caspase activating activity of cytochrome c suggests
that this caspase is indispensable for this pathway, irrespective of the ability of Apaf-1 to complex with other caspases.
Although it is generally believed that multiple caspases
participate in the signaling and destruction phases of apoptosis, it is still unclear whether there is significant functional redundancy within this family of proteases. The
observations that caspase-3, caspase-8, and caspase-9 knockout mice die in utero or soon after birth would argue
against redundancy, at least in certain tissues (Kuida et al.,
1996, 1998
; Hakem et al., 1998
; Varfolomeev et al., 1998
).
In addition, in vitro studies that have used dominant negative forms of caspase-9, as well as data available from
CASP-9 null mice, suggest that this caspase occupies a critical position in a major subset of cell death pathways since
apoptosis was abrogated in the absence of this caspase in a
number of contexts (Hakem et al., 1998
; Kuida et al., 1998
;
Pan et al., 1998b
; Srinivasula et al., 1998
).
It is also commonly believed that caspases with long
prodomains are upstream or signaling caspases whereas
those with short prodomains are effector or executioner
caspases. For example, studies on caspase-8 suggest that
this protease is the most proximal caspase to become activated upon ligation of the CD95 (Fas/Apo-1) molecule
since this caspase is directly recruited into the CD95 signaling complex upon receptor aggregation (Kischkel et al., 1995; Boldin et al., 1996
; Fernandes-Alnemri et al., 1996
;
Muzio et al., 1996
). However, recent studies have suggested that caspase-8 is not always activated early in the
context of CD95 signaling (Scaffidi et al., 1998
). This has
led to the suggestion that two distinct cellular types exist
with respect to CD95 signaling: type I, cells that activate
caspase-8 early (within seconds) of CD95 receptor aggregation and type II, cells that activate caspase-8 late and in
a mitochondrial-dependent fashion (Scaffidi et al., 1998
).
In this study we also observed caspase-8 activation late
in the cytochrome c-inducible caspase cascade. It is possible that caspase-8 activation is merely a bystander event in
this model of apoptosis, since the caspase substrates examined (fodrin, PARP, U1snRNP) were almost completely
cleaved within 60 min of addition of cytochrome c to the
extracts (Fig. 1 B) and nuclear destruction was largely
complete by 90 min (Fig. 1 A). In contrast, only a small
portion of the [35S]methionine-labeled caspase-8 that was
added to the extracts had become processed by 60 min under similar conditions (Fig. 5). However, as previously discussed, caspase-8 activation was also found to be impaired
in cells from CASP-9/
as well as APAF-1
/
mice in certain contexts, suggesting that this caspase may be activated
downstream in some situations (Hakem et al., 1998
; Yoshida et al., 1998
).
In summary, our observations suggests that a branched cascade of caspase activation events, with at least one feedback loop, is initiated distal to entry of cytochrome c into the cytosol. Further studies are required to establish whether the sequence of caspase activation events we report is conserved between different cell types and in response to divergent death-promoting stimuli.
![]() |
Footnotes |
---|
Address correspondence to Dr. Seamus J. Martin, Molecular Cell Biology Laboratory, Dept. of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland. Tel.: 353-1-708-3856. Fax: 353-1-708 3845. E-mail: sjmartin{at}ailm.may.ie
Received for publication 28 August 1998 and in revised form 21 December 1998.
We thank Drs. Vishva Dixit and Xiaodong Wang for provision of cDNAs for caspase-3 and Apaf-1, respectively.
This work was supported by a Wellcome Trust Senior Fellowship in Biomedical Science (047580) to S.J. Martin. M.T. Harte is a Higher Education Authority of Ireland postdoctoral Fellow.
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Abbreviations used in this paper |
---|
CEB, cell extract buffer; ES, embryonic stem.
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