From the Cancer Research UK Cellular and Molecular
Pharmacology Group, School of Biological Sciences, University of
Manchester, Stopford Bldg., Oxford Road, Manchester M13 9PT, United
Kingdom and the ¶ Institut de Recherches Servier, 125 chemin de
Ronde, Croissy sur Seine 78290, France
Received for publication, September 9, 2002, and in revised form, February 12, 2003
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ABSTRACT |
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Bid is instrumental in death
receptor-mediated apoptosis where it is cleaved by caspase 8 at
aspartate 60 and aspartate 75 to generate truncated Bid (tBID)
forms that facilitate release of mitochondrial cytochrome
c. Bid is also cleaved at these sites by caspase 3 that is
activated downstream of cytochrome c release after diverse
apoptotic stimuli. In this context, tBid may amplify the apoptotic
process. Bid is phosphorylated in vitro by casein kinases
that regulate its cleavage by caspase 8 (Desagher, S., Osen-Sand,
A., Montessuit, S., Magnenat, E., Vilbois, F., Hochmann, A., Journot,
L. Antonsson, A., and Martinou, J.-C. (2001) Mol. Cell
8, 601-611). Using a Bid decapeptide substrate, we observed that
phosphorylation at threonine 59 inhibited cleavage by caspase 8. This
was also seen when recombinant Bid (rBid) and Bid isolated from murine
kidney were incubated with casein kinase II. However, there were
differences in the susceptibility of rBid and isolated Bid to cleavage
by caspases 3 and 8. Caspase 8 cleaved rBid to generate two C-terminal
products, p15 and p13 tBid, but produced only p15 tBid from isolated
Bid. Contrary to rBid, isolated Bid was resistant to cleavage by
caspase 3, yet was readily cleaved within the cytosolic milieu. Our
data suggest that one or more distinct cellular mechanisms regulate Bid
cleavage by caspases 8 and 3 in situ.
Proteins of the Bcl-2 family are crucially involved in the control
of apoptosis (1-3). This family includes anti-apoptotic proteins such
as Bcl-2 itself and an increasing number of pro-apoptotic proteins like
Bax and Bid that translocate to mitochondria and facilitate the release
of cytochrome c during the induction of apoptosis (2-7).
The pro-apoptotic members of the Bcl-2 family can be further divided in
multidomain proteins such as Bax and Bak, which share three domains of
sequence homology with Bcl-2, and the so-called "BH3-only" proteins
such as Bid and Bad, which share a single region of sequence homology
with Bcl-2, the BH3 domain (1, 3, 5-10).
A key aspect of BH-3-only pro-apoptotic proteins of the Bcl-2 family is
their conversion from latent to active state in response to apoptotic
stimuli or, more broadly, to cellular stress. Two general mechanisms of
activation are known: (a) enhanced expression due to
transcriptional up-regulation and (b) post-translational modification of the protein by phosphorylation or cleavage. Bad provides the best characterized example of pro-apoptotic regulation via
protein phosphorylation (2, 9, 10): multiple serine residues of Bad are
phosphorylated as a consequence of several survival signaling pathways,
promoting its cytosolic sequestration by 14-3-3 and thus preventing its
interaction with mitochondrial anti-apoptotic proteins such as Bcl-2
and Bcl-xL (1, 9, 10). More recently, the phosphorylation
of Bik (11) and Bid (12) have been reported, suggesting that
phosphorylation may be a common mechanism to control the action of
pro-apoptotic proteins related to Bcl-2.
The current consensus assigns a primary role to proteolytic cleavage in
the activation of Bid during death receptor signaling (3, 5, 6).
Ligation of death receptors such as Fas results in the assembly of an
intracellular death-inducing signaling complex (DISC)1 that activates the
initiator caspase 8 (13). Whereas in type I cells DISC formation is
very efficient, leading to rapid activation of caspase 8 and then of
effector caspases such as caspase 3, in type II cells DISC formation
proceeds slowly, and the activation of effector caspases requires
amplification via the mitochondrial pathway (13, 14). Bid cleavage by
DISC-activated caspase 8 provides the major mechanism of engaging the
mitochondrial pathway during Fas-induced apoptosis (3, 5, 6).
Caspase 8 cleaves human Bid at aspartate 60 to generate 15-kDa tBid
that translocates to the mitochondrial outer membrane. tBid is thought
to promote the oligomerization of Bax or Bak at the mitochondrial
surface to facilitates the release into the cytosol of cytochrome
c and other apoptogenic proteins (3, 5-8, 15). However,
in vitro tBid oligomers are also capable of facilitating
cytochrome c release in the absence of interaction with Bax
or Bak (16).
Whatever the mitochondrial role of tBid, it is noteworthy that
recombinant full-length Bid (rBid) is nearly as potent as tBid in the
cell-free assay of cytochrome c release (15, 16-18).
Furthermore, the action of various cellular kinases, including ERK,
JNK, and protein kinase C can attenuate Fas-induced apoptosis (12,
19-22). These observations suggested that mechanisms in addition to,
or upstream of, caspase cleavage might regulate the mitochondrial action of Bid (12, 18). A recent study has shown that this is
indeed the case (12), because the phosphorylation status of Bid
regulates its susceptibility to caspase 8-mediated cleavage. Specifically, treatment of murine Bid with casein kinases (CKs) affects
its cleavage (12). Bid phosphorylation may thus block cleavage by
caspase 8, producing a safeguard mechanism to prevent the activation of
this potent pro-apoptotic molecule in situations of basal caspase 8 activity (12).
In addition to caspase 8-mediated cleavage at Asp-60 and Asp-75,
rBid can also be cleaved by caspase 3 at both of these sites (6, 23).
Given the greater catalytic capacity of caspase 3 compared with caspase
8 and its activation by wider range of apoptotic stimuli, it seems
critical that Bid should be protected from activation by basal caspase
3 activity in healthy cells. We have thus tested whether endogenous Bid
in primary tissues presents modifications that affect its cleavage by
caspase 8 and/or caspase 3 and provide biochemical evidence for both
common and different mechanisms that regulate Bid cleavage by these two caspases.
Western Blotting--
Western blotting was performed as
previously reported (18) after protein separation with SDS-PAGE gels,
which were routinely run until the dye front migrated out of the gel.
The acrylamide content of the gels was normally set at 15% to achieve
optimum resolution of tBid bands (5, 12, 18) but was reduced to 13%
for enhanced resolution of closely migrating bands of full-length Bid.
Primary antibodies for Bid were obtained from R&D Systems (#AF860,
raised against the whole recombinant mouse protein, Abingdon, UK) and from Santa Cruz Biotechnology Inc. (C-20, raised against the C
terminus of Bid, and N-19 raised against the N terminus of Bid, Santa
Cruz Biotechnology, Santa Cruz, CA). Polyclonal antibodies to Bid
provided by colleagues (J. C. Reed and X. Wang) confirmed the
specificity of our blotting. To accurately evaluate the size of the
Bid-reactive bands, various molecular weight markers were used,
including Bio-Rad prestained Precision protein standards (Bio-Rad,
Hemel Hempstead, UK). Quantitative loading of proteins was determined
by re-blots of reference proteins such as actin and India ink staining
of the proteins.
Other Reagents--
Phosphate-buffered saline was obtained from
Oxoid (Basingstoke, UK), and cell culture media was from Invitrogen
(Paisley, UK). DNA restriction and modification enzymes were from Roche Molecular Biochemicals (Mannheim, Germany), fluorescent probes were
from Molecular Probes (Eugene, OR), caspase substrates and inhibitors
were from Pharmingen (Oxford, UK), and Alexis (Nottingham, UK), and
electrophoresis reagents were from Bio-Rad. The Bid-derived peptides,
dodecapeptides corresponding to residues 55-67 (substrates), 55-60,
and 61-67 (products) of human Bid, were custom synthesized and
analyzed by Neosystem (Strasbourg, France) and dissolved in distilled
water. Other reagents were purchased from Sigma (Poole, UK) and were of
high analytical grade.
Cell Culture--
Human T lymphoma CEM C7A and Jurkat T cells
were grown in RPMI 1640 medium supplemented with fetal bovine serum
(10% v/v) and L-glutamine (2 mM) as described
previously (18).
Fractionation and Fas Activation of Primary Tissues--
Fas
treatment and fractionation of mouse kidney and liver were carried out
as described earlier (18). The mitochondrial pellets were resuspended
and stored in assay buffer (20 mM K-HEPES, 0.12 M mannitol, 0.08 M KCl, 1 mM EDTA,
pH 7.4) containing a mixture of protease inhibitors (Sigma). After
centrifugation of the post-nuclear extract at 10,000 × g, the supernatant was taken apart as cytosolic extract
(fraction S10) while the mitochondrial pellet was washed as describe
earlier (18). The supernatant of the first mitochondrial wash, fraction
S3, was kept for further analysis. The protein content of the various
fractions was determined using the Bio-Rad Bradford mini-assay in the
presence of non-ionic detergents to solubilize membrane proteins;
bovine serum albumin was used as a standard.
Isolated Proteins--
rBid (murine) was obtained in purified
and active form from R&D Systems. Recombinant human caspases were
purchased from Pharmingen or Calbiochem (Cambridge, MA). Endogenous Bid
was isolated from cytosolic extracts of mouse kidney by the procedure
described previously (18). Briefly, frozen cytosolic extracts were
thawed and clarified by extensive centrifugation at 12,000 × g, then diluted with assay buffer containing 2 mM DTT and protease inhibitors. Subsequently, the extracts
were heated at 70 °C for 15 min, followed by centrifugation at
4 °C for 40 min at 12,000 × g. The supernatant containing the majority of endogenous Bid was filtered through Sartorius 100-kDa filters (Fisher Scientific, Loughborough, UK) by
extensive centrifugation. The filtrates were further fractionated and
then concentrated by ultrafiltration with 30- and 5-kDa filters (Sartorius or Millipore, Watford, UK) and gel filtration
chromatography. Bid produced by the above process is referred to
throughout as "isolated Bid." Importantly, when this isolation
process was applied to recombinant Bid, it had no effect upon its
phosphorylation by casein kinase II (data not shown). The crude
Bid preparations obtained as in a previous study (18) could be used
without further manipulation, because they did not contain other
proteins of the Bcl-2 family, although in some cases the preparations
of endogenous Bid were further purified by ammonium sulfate
fractionation, gel filtration, and immunoprecipitation.
In Vitro Modification of Bid--
Phosphatase treatment of
cytosolic extracts and Bid preparations was carried out in either 50 mM Tris-Cl buffer, pH 7.5 (for alkaline phosphatase), or 40 mM PIPES-Cl buffer, pH 6.0 (for acid phosphatase (24)),
containing 1 mM DTT, 1 mM EDTA, and 1 mM EGTA. Alkaline phosphatase or potato acid phosphatase
(Sigma) were added at 2-20 units/ml to the protein samples (5 µg/ml
in the case of recombinant Bid) and incubated at either 37 °C for 1 h or, particularly for acid phosphatase, at 30 °C for 2-3 h (24). The suspension was then diluted 5-fold with caspase assay buffer
(20 mM K-HEPES, 5% sucrose, 50 mM NaCl, 5 mM DTT, and 1 mM EDTA, pH 7.4, containing
protease inhibitors) and either incubated with caspases or stopped with
SDS-sample buffer.
To produce caspase cleavage of Bid, recombinant caspase 3 or caspase 8 were suspended at 1-2 µg/ml (or 1000 units/ml for the enzymes
purchased from Calbiochem) in the caspase assay buffer supplemented
with 0.1% v/v of protease inhibitors and mixed with rBid (1-2
µg/ml) or equivalent concentrations of endogenous Bid isolated from
kidney cytosol or other subcellular fractions from mouse kidney (to a
final protein concentration of 0.5-1 mg/ml). The reaction mixture was
incubated at 30 °C for different times and then stopped by addition
of sample buffer and boiling. Samples were frozen and subsequently
evaluated by Western blotting. Treatment of Bid preparations (5 µg/ml, with casein, Calbiochem) was carried out for 2-3 h at
30 °C with kinase II (human recombinant CKII in 50 mM
Tris-Cl buffer, pH 8.0), containing 10 mM
MgCl2, 10 mM Immunoprecipitation of Bid--
For immunoprecipitation of Bid,
cytosolic extracts from mouse kidney were first cleared with protein G
beads (PerBio, Tattenhall, UK) for 1 h, followed by incubation
with 4 µg of Bid antibody (from R&D Systems) overnight on ice. The
antibody-antigen complexes were captured with protein G beads for
1 h and then resuspended in 0.5 ml of Triton buffer (50 mM, Tris-Cl, pH 7.5, containing 50 mM NaF, 10 mM Measurements of Caspase Activity--
The assay of caspase
activity with the model peptide substrates was based upon
reversed-phase HPLC coupled to UV detection. The reaction mixture
contained 100 units of recombinant caspase 8 or 3 suspended in assay
buffer (50 mM K-HEPES, pH. 7.4, containing 100 mM NaCl, 10 mM DTT, 1 mM EDTA, and
0.1% CHAPS) containing 1 mM peptide substrate to a final
volume of 0.1 ml. After incubation at 30 °C for 6 h, the
reaction was stopped by the addition of 20 µl of 20% trichloroacetic
acid. 30 µl of the mixture were then analyzed by HPLC using a
Platinum EPS C18 column (53 × 7 mm, Alltech, Carnforth, UK) and a
Hewlett Packard 1100 apparatus. The column was eluted with a linear
gradient of 0-90% acetonitrile in water/0.1% trifluoroacetic
acid at a flow rate of 2.5 ml/min. Caspase activity of
biological samples was measured with the fluorogenic substrates Ac-DEVD-AFC or Ac-IETD-AFC (where AFC is
7-amido-4-(trifluoromethyl) coumarin), in caspase assay buffer (compare
with Refs. 18 and 25) using a PerkinElmer Life Sciences LS50B
luminescence spectrometer with excitation at 360 nm and emission at 480 nm and disposable Kartell cuvettes.
Caspase 8-mediated Cleavage of Human Bid Peptides Is Decreased by
Phosphorylation--
Multiple forms of Bid are observed in lysates
from primary tissues or cultured cells (5, 12, 15, 18, 26). Fig. 1A illustrates how multiple protein bands are detected with
a Bid-specific antibody in the 20-30 kDa range of cytosolic extracts from mouse tissues and from Jurkat cells, a T lymphoma line frequently used in apoptosis studies (5, 12). Similar results were obtained with
different antibodies and other cell lines, including human colon
carcinoma and mouse pro-myelocytic lines (data not shown). The presence
of these different forms of Bid (compare with Ref. 18) prompted us to
investigate the possibility that endogenous Bid may be
post-translationally modified, consistent with recent findings in
vitro (12).
Our first approach to investigate post-translational modifications of
Bid was based on sequence analysis. Specifically, we compared the
available amino acid sequences for mammalian Bid and focused our
attention on potential phosphorylation sites by computing the
conservation and structural position of hydroxyl residues such as
serines and threonines (Fig.
1B). Conserved serines and a
single threonine were identified that, once mapped on the NMR-deduced
structure of Bid (27), could be considered potential phosphorylation
sites. Remarkably, three of these potential phosphorylation sites lie
adjacent to known cleavage sites in human Bid. Thr-59 is proximal to
the primary caspase 8 cleavage site at Asp-60 (5, 6, 26). Ser-65
is proximal to a recently identified site of cleavage by lysosomal
proteases (25). Ser-76, is one residue distal to the specific site of
cleavage (Asp-75) by granzyme B (6, 28, 29), a non-caspase protease
present in granules of cytotoxic lymphocytes (28, 29), which also
corresponds to a secondary cleavage site for caspases (6, 8, 23, 30). This peculiar location of potential phosphorylation sites suggested that post-translational modifications of Bid, if present in
vivo, would indeed affect its susceptibility to proteolysis.
Thr-59 and Ser-65 are the only hydroxyl amino acids that are conserved in the loop region containing the primary site of caspase 8 cleavage (Fig. 1B), which is shared by effector caspases such as
caspase 3 (6, 23, 30). In principle, phosphorylation of either Thr-59
or Ser-65 could sterically hinder the proteolytic activity of caspases,
because it would insert a bulky phosphate group adjacent to the
cleavage site in the target sequence of the Bid protein. However, the
effect on the catalytic efficiency of caspases will depend critically
on the protein conformation around the substrate cleavage and the
degree of steric specificity in the active cleft of the proteolytic
enzyme (25, 31).
To verify the effect of phosphorylation of specific residues on the
efficiency of caspase 8 cleavage of Bid, we developed a model system
using recombinant caspases and synthetic dodecapeptide substrates
corresponding to residues 55-67 in the human sequence of Bid (noted by
the bar in Fig. 1B). These peptides could be effectively cleaved by caspase 8 at the position
corresponding to Asp-60 of the Bid
protein as detected by HPLC (Fig. 2). We then used equivalent
peptides having the residues corresponding to Thr-59 and Ser-65
of Bid, which were individually or cumulatively phosphorylated. After
incubation with caspase 8 for 6 h, the cleavage rate of the
peptide with Thr-59 phosphorylated was ~4-fold slower than that with
the parent non-phosphorylated peptide (Fig. 2, A and
B, and see figure legend). Conversely, phosphorylation of the residue corresponding to Ser-65 did not significantly affect the
rate of caspase 8 cleavage of the peptide substrates, whereas the
phosphorylation of both Thr-59 and Ser-65 induced a similar, if not
stronger, resistance to cleavage than that with phosphorylation of
Thr-59 alone (Fig. 2C and legend).
These data indicate that, although phosphorylation does not produce a
complete block of caspase 8 cleavage, it can substantially inhibit the
rate and efficiency of proteolysis of Bid in the region of the primary
cleavage site, in broad agreement with previous data obtained with
murine Bid (12). Our data also confirm that the relative importance of
the phosphorylation sites affecting cleavage may differ between
species, with Thr-59 more critical in human Bid as compared with Ser-61
and Ser-65 in murine Bid (12). Experiments equivalent to those in Fig.
2 were undertaken using caspase 3, but the synthetic peptides proved to
be ineffective substrates for caspase 3 activity regardless of the
presence of phosphorylated residues, consistent with the notion that
Bid protein is a better substrate for caspase 8 than for caspase 3 (6).
rBid and Bid Isolated from Murine Kidney Cytosol Have a Different
Susceptibility to Caspase 8 Cleavage and Phosphorylation--
The
presence of multiple forms of endogenous full-length Bid in primary
tissues and cell lysates (Fig. 1A, compare with Refs. 5, 12,
and 18) suggested that rBid produced in bacteria, which
consistently shows a single band in immunoblots, may provide an
inappropriate model for studying the physiological modifications of the
protein. Hence, we extended our studies to compare the susceptibility
to caspases of rBid and Bid isolated from murine kidney (compare with
Ref. 18). Because available Bid antibodies recognize tBid less
efficiently than the uncleaved protein (5, 12, 18), we optimized
Western blotting conditions for the detection of tBid bands, even if
these conditions led to overexposure of the bands corresponding to
uncleaved Bid. Fig. 3 shows that incubation of rBid with caspase 8 generates two tBid forms, p15 and
p13, that correspond to the C-terminal polypeptides resulting from
cleavage at both the primary and secondary site (Fig. 3, lanes
2, compare with Fig. 1B). In contrast, when caspase 8 was incubated with Bid isolated from murine kidney cytosol, only the p15 form of tBid was produced (Fig. 3, lane 5; see also Fig.
5A below). This suggested that Bid isolated from kidney
cytosol exhibits an inherent resistance to proteolysis at the secondary
cleavage site that is not seen with the recombinant protein.
We investigated whether the Bid peptide phosphorylated at Thr-59 (T59P,
see Fig. 2B) could act as a competitive inhibitor of Bid
cleavage by the two caspases. This peptide hardly affected cleavage of
rBid by caspase 8 (Fig. 3, lanes 2 and 3) but
showed some inhibitory effect on the cleavage of rBid by caspase 3 (not shown) and on that of isolated Bid by caspase 8 (Fig. 3, lanes 5 and 6). These data, while supporting the contention
that the target site in Bid has higher affinity for caspase 8 than for caspase 3 (6), provide additional evidence that phosphorylation may
alter proteolysis of Bid.
To directly test the possibility that isolated Bid is phosphorylated
(and/or modified) at residues that influence its cleavage by caspases
8, isolated Bid and rBid were incubated with ATP and CKII, the kinase
recently reported to phosphorylate murine Bid, especially at Thr-58
(12). In the presence of ATP, CKII produced profound changes in the
mobility of rBid from a single band to a triplet of closely migrating
bands resolved using 13% acrylamide gels (Fig.
4A). There was no apparent
decrease in intensity of any of the Bid triplet bands in the presence
of CKII consistent with a lack of cleavage (compare lanes 3 and 4), although CKII treatment did not abolish the
production of tBid forms by caspase 8 as detected with routine 15%
acrylamide gels optimized for tBid resolution (Fig. 4B).
CKII did not alter much the mobility of isolated Bid (Fig.
4A, lanes 5 and 7) but did reduce the
caspase 8-mediated loss of isolated full-length Bid (Fig.
4A, lanes 6 and 8) as well as the
production of tBid (not shown). These results were consistent with the
concept that endogenous modifications of isolated Bid, presumably
including phosphorylation that could only be partially mimicked by
in vitro treatment with CKII, diminish its susceptibility to
cleavage by caspase 8 (12).
Differential Cleavage of Endogenous and rBid by Caspase 8 and
Caspase 3--
In previous studies, emphasis has been placed on the
possible post-translational regulation of Bid cleavage by caspase 8, an
event considered to be crucial in death receptor-induced apoptosis of
type II cells (3, 7, 8, 12-14, 19, 22). However, Bid can also be
effectively cleaved by caspase 3 and other downstream caspases (6, 23,
26, 30) that can be rapidly engaged in type I cells (13, 14), or
subsequent to activation of the apoptosome by a variety of death
stimuli (3, 23, 30). Given the potential relevance to the feed-forward
amplification of the death cascade via tBid-induced mitochondrial
damage (2, 3, 23, 30), we investigated whether the proteolytic cleavage of Bid displayed different properties with caspase 8 and an effector caspase, caspase 3. A comparable cleavage of rBid was observed with
either caspase 8 or 3, which both produced the p15 and p13 forms of
tBid (Fig. 5A, lanes
2 and 3). However, Bid isolated from murine kidney was
completely resistant to caspase 3 cleavage (Fig. 5A,
lane 6), whereas it was efficiently cleaved by caspase 8 to
produce p15 tBid (Fig. 5A, lane 7).
The unexpected differences in the proteolytic susceptibility of Bid
(Fig. 5A) could derive from factors that exclusively
influence the reactivity of isolated Bid with caspase 3. To test this
possibility, we examined whether our preparations of isolated,
partially purified Bid contained contaminants that could affect the
catalytic efficiency of caspases and in particular alter susceptibility
to caspase 3 cleavage (compare with Ref. 25). As shown in Fig.
5B, isolated Bid had a slight stimulatory effect, if any, on
the enzymatic rate of DEVD-AFC cleavage by caspase 3 under the
same conditions as those used in the experiments of Bid cleavage in
Fig. 5A. Additionally, we verified that the cleavage of rBid
by caspases was not significantly altered by the presence of isolated
Bid preparations (data not shown). We thus excluded the possibility
that the difference in caspase cleavage of isolated Bid (Fig.
5A) could derive from factors present in the isolated Bid
preparation that inhibit caspase 8 or caspase 3.
Subcellular Localization of Bid Affects Its Cleavage by Caspase
3--
We subsequently investigated whether the differential
susceptibility of isolated Bid to caspase 3 cleavage compared with the recombinant protein may derive from post-translation modifications by
enzymes present in cytosol or other subcellular compartments where
native Bid is present. Therefore, we compared the cleavage of
endogenous Bid present within subcellular fractions of primary tissues
after addition of exogenous caspase 3 using rBid as a standard.
Endogenous Bid was much more susceptible to caspase 3 cleavage within
cytosolic fractions such as S10 than in other fractions, including
mitochondria and mitochondrial wash (fraction S3, Fig.
6A). We then isolated Bid from
the same cytosolic fraction, re-assessed its susceptibility to caspase
3 cleavage and found it to be completely resistant (Fig.
6B). In contrast, the same isolated Bid was efficiently
cleaved by caspase 8 (Fig. 3). Consequently, the results suggested that
once removed from its cytosolic environment, Bid remained a good
substrate for the initiator caspase 8 but became resistant to cleavage
by the effector caspase 3 arguing that Bid cleavage by these two
caspases is differentially regulated. The fact that addition of
exogenous caspase 3 could elicit complete cleavage of cytosolic Bid
(Fig. 3) could be interpreted as a result of
proteolysis-dependent cleavage of endogenous caspase 8, a
primary substrate of caspase 3 (31). This possibility was verified by following the activation of caspase 8 activity in cytosolic extracts treated with caspase 3 (data not shown).
Acid Phosphatase Treatment Enhances Caspase Cleavage of Endogenous
Bid--
Our previous data suggest that phosphorylation of isolated
Bid reduces but does not abolish its cleavage by caspase 8. Consistent with data shown in Fig. 4B, incubation of isolated
full-length Bid with CKII attenuated the decreased in full-length
protein, but did not prevent caspase 8-mediated production of tBid
(Fig. 7A, lanes 2 and 4). To investigate the role of Bid phosphorylation on
its cleavability further, we examined the impact of incubating isolated
Bid with phosphatases anticipating that this should enhance Bid
cleavage by caspase 8. After unsuccessful attempts with alkaline phosphatase, we realized that the acid nature of mouse Bid would require treatment with phosphatase working at low pH to obtain effective de-phosphorylation. Indeed, treatment with potato acid phosphatase (PAP as in Ref. 24) significantly enhanced the cleavage by
caspase 8 of isolated Bid with or without incubation with CKII with
barely detectable full-length Bid remaining (Fig. 7, A and B). Fig. 7B compares the effects of CKII and PAP
on isolated Bid cleavage by caspases 8 and 3. Although CK II treatment
prevented the production of tBid cleavage by caspase 3 (Fig.
7B, lane 3), resistance to caspase 3 cleavage was
abrogated by PAP treatment that promoted the production of p15 tBid
(Fig. 7B, lane 6). Lastly, we examined the effect
of PAP on the cleavage of Bid left within kidney cytosol (Fig.
7C). Here PAP produced p15 and p13 tBid. This suggested that
de-phosphorylation had enhanced the capacity of endogenous cytosolic
proteases to cleave Bid at both the primary and secondary site, as in
the case of the recombinant protein treated with caspase 8. Comparable
results were obtained using extracts of human lymphoma
cells.2
Caspase cleavage is considered fundamental to the action of
pro-apoptotic Bid, especially in the pathways of apoptosis triggered by
death receptor ligation (3-7, 25). However, Bid is also cleaved during
drug-induced apoptosis upstream or downstream of cytochrome
c release, dependent on cell type. In type II cells, Bid is
cleaved by caspase 3 or by caspase 8 acting as an amplifying executioner caspase in drug-induced apoptosis (32). In type I cells
where drug treatment can induce formation of a DISC, Bid is cleaved by
caspase 8 upstream of caspase 3 (33). Moreover, in certain scenarios,
such as in the neuronal cell death that occurs in Huntington's
disease, pro-caspase 8 can be activated independently of death receptor
ligation (34) to promote apoptosis via components of the death receptor
signaling pathway, presumably including the cleavage of Bid. Thus, the
events that regulate the susceptibility of Bid cleavage are important
for apoptotic pathways engaged by a variety of stimuli. Given the
potent action of tBid on mitochondria it would seem mandatory that
constitutive mechanisms prevent tBid formation by basal caspase
activities in cells that are responding appropriately to survival
signals. The intracellular location and caspase cleavage of Bid are
likely to be regulated by converging pathways of cell survival, as
indicated by increasing, although indirect evidence (12, 14, 19-23,
35). In this regard, the ability of the tumor suppressor PTEN
(phosphatase and tensin homolog (3-phosphoinositide phosphatase)) to
sensitize to drug-induced apoptosis via a pathway dependent on the DISC component FADD (Fas-associated death domain-containing protein) provides another interesting link between the interruption of a
survival signaling pathway and the cleavage of Bid to the promotion of
apoptosis (36).
Although it has been claimed that Bid is not phosphorylated in cultured
cells (37), more recently it has been reported that Bid can be
phosphorylated both in vitro and in vivo,
particularly by enzymes of the casein kinase family (12). Given the
limited information on the possible modification of endogenous Bid
within primary tissues, we have focused our work on a comparison of the cleavage of endogenous Bid (either within or isolated from mouse kidney
cytosol) and rBid by caspases 8 and 3.
Our data provide novel evidence that endogenous Bid is phosphorylated
(or otherwise modified) at sites influencing its cleavage by caspases.
First, we demonstrated in a model system of Bid cleavage that
phosphorylation of conserved residues such as Thr-59 (in the human Bid
sequence), which surround the primary site of caspase cleavage (Fig.
1B), substantially diminished the proteolytic capacity of
caspase 8 (Fig. 2). Second, we found that the Bid that was constitutively located (albeit at low levels) in kidney fractions other
than cytosol (but including mitochondrial Bid) was resistant to
proteolytic cleavage by caspase 3 (Fig. 6B). This resistance was intrinsic to the protein in situ, because Bid isolated
from the cytosolic extracts, contrary to rBid, was also resistant to caspase 3 cleavage (Figs. 5 and 6). Third, we found that
dephosphorylation of endogenous Bid with PAP greatly facilitated its
cleavage by caspases 3 and 8 (Fig. 7).
Phosphorylation of residues around the cleavage site has been shown to
prevent proteolysis by caspase 3 in other proteins, for example
I Structural properties intrinsic to the Bid protein may partially
account for differential cleavage by different caspases. Previous
results (compare with Ref. 6) showed that Bid is a better substrate for
caspase 8 than for caspase 3, and our data indicate that this is
particularly the case for the cleavage at the secondary site (Asp-75,
compare with Fig. 5B). The different efficiency of rBid
cleavage by caspase 8 and 3 could simply derive from the fact that the
protein region around the secondary cleavage site in Bid is much less
flexible than that around the primary cleavage site (27) and
proteolytic enzymes like caspases prefer flexible peptides as
substrates (31). Hence, even in the absence of Bid modification, local
structural determinants would affect the reactivity of Asp-75 with
caspase 3 but not with caspase 8. However, caspase 8 is essentially
unable to produce p13 tBid when reacting with endogenous Bid isolated
from kidney cytosol (Figs. 5 and 6). This implies that residues such as
Ser-76, the only conserved hydroxyl amino acid lying around the
secondary cleavage site (Fig. 1B), may be modified in
vivo to prevent proteolysis. Indeed, phosphate labeling was
reported to be associated also with a peptide comprising Ser-76 after
in vitro phosphorylation and subsequent fragmentation of
murine Bid (12).
Post-translational modifications must be present also around the
primary cleavage site of endogenous Bid to explain its strong resistance to caspase 3 cleavage (Figs. 5 and 6). The same
modifications, however, cannot prevent Bid cleavage by caspase 8 (compare with Fig. 3). Given the discussion above and the evidence that
phosphorylation incompletely inhibits caspase 8 cleavage of peptide
substrates (Fig. 2) and rBid (Fig. 4 and results not shown, compare
with Ref. 12), this difference can be rationalized in terms of a superior catalytic efficiency and substrate specificity of caspase 8. It is increasingly evident that the exposed loop preceding the BH3
domain in Bid structure (residues 50-80, compare with Fig.
1B and Ref. 27) is susceptible to proteolysis by a variety of endogenous proteases. Besides caspase 8 (5, 6) and other caspases
(6, 23, 24, 30), Bid can be cleaved by lysosomal proteases (25) and
calpain (40). Hence, the (poly)phosphorylation at residues 59, 62/65,
and possibly also 76, may serve as a general mechanism for reducing the
spontaneous degradation of Bid by these endogenous proteases.
These modifications notwithstanding, endogenous Bid remains highly
susceptible to cleavage by caspase 8, especially when compared with its
resistance to cleavage by caspase 3 (Figs. 5 and 6). Consequently,
phosphorylation (or other post-translational modifications around the
caspase sites) could serve not only to stabilize the Bid protein but
also to ensure its specific susceptibility to proteolysis by caspase 8. Our results (Figs. 6 and 7) lend support to this possibility, although
other modifications, such as myristoylation (37), may also contribute
to maintain a rigorous specificity for Bid cleavage toward activated
caspase 8. In possible contradiction with this, our data also show that
addition of exogenous (fully activated) caspase 3 leads to complete
cleavage of cytosolic Bid (Fig. 6). However, this cleavage may derive
from indirect reactions, including activation of cytosolic caspases
like caspase 8, and caspase-mediated activation of protein phosphatases
(42).
In conclusion, our study strengthens the contention
that post-translational modifications alter Bid susceptibility to
caspase cleavage and consequently its pro-apoptotic activation (12, 43). Further studies will determine the biochemical effects of Bid
phosphorylation in its reactivity with membrane lipids (18, 44) that
may be crucial in its biological action in mitochondria.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, 2 mM DTT, and, when indicated, 0.2 mM ATP (compare with Ref. 12). Samples where then diluted 5-fold with caspase
assay buffer and either treated with caspases as above or directly
dissolved in sample buffer.
-glycerophosphate, 10 mM sodium
pyrophosphate, 1 mM EDTA, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM
NaVO4, 0.2% v/v Triton X-100, and protease inhibitors).
The protein G beads were then pelleted by centrifugation, washed three times with Triton buffer, resuspended in sample buffer, and then boiled. After centrifugation to separate the beads, the supernatant was
analyzed by Western blotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, immunoblotting of Bid shows multiple
bands in various cellular contexts. Recombinant mouse Bid (5 ng,
lane 1) was used as a reference for the immunoblotting of
endogenous Bid in cytosolic extracts (fraction S10, 20 µg of protein)
of mouse kidney (lane 2), mouse liver (lane 3),
and human cultured Jurkat T lymphoma cells (lane 4). The
results were obtained with a mixture of the C-20 and R&D antibodies to
Bid, as reported previously (18). The presence of multiple bands was
detected also with other Bid-specific antibodies (not shown, and Refs.
12, 18, and 26). The data are representative of three independent
repeat experiments. B, alignment and comparison of sequences
of mammalian Bid. The sequence of human Bid (6) was used as a template
for aligning the protein or DNA-deduced sequences of Bid available from
current releases of GenBankTM. They include the complete
sequence of mouse and rat Bid, as well as that of bovine Bid
reconstructed from multiple overlapping expressed sequence tags. The
last 55 residues toward the C terminus are not shown because they do
not present conserved serines. Amino acids that are conserved in all
sequences are in boldface, and the arrows
indicate the caspase cleavage sites. The horizontal black
bar delineates the region corresponding to the dodecapeptides used
in our simplified model system for Bid cleavage.
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Fig. 2.
Simplified model system for Bid
cleavage. Synthetic dodecapeptides corresponding to amino acid
residues 55-67 of human Bid were used as substrates for caspase 8 as
described under "Experimental Procedures." The products of the
cleavage reactions were subsequently separated by HPLC, and their
elution was detected by the normalized absorbance at 210 nm
(A210). The thick arrows indicate the
cleavage product corresponding to residues Asp-55 through Asp-60, which
showed a slightly different migration when Thr-59 was phosphorylated.
A, chromatogram of unmodified peptide 55-67 after 6-h
incubation with caspase 8, the estimated rate of cleavage was 0.87 µM/h per enzyme unit. B, chromatogram of the
peptide having Thr-59 phosphorylated after 6-h incubation with caspase
8, the estimated rate of cleavage was 0.25 µM/h per
enzyme unit. C, chromatogram of the peptide having both
Thr-59 and Ser-65 phosphorylated, the estimated rate of cleavage was
0.2 µM/h per enzyme unit compared with the rate of 1 µM/h per enzyme unit obtained with the parent peptide
having only Ser-65 phosphorylated (not shown). The data are
representative of three independent repeat experiments.
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Fig. 3.
Caspase 8 cleavage of recombinant and
isolated Bid. Recombinant caspase 8 (1000 units/ml, Calbiochem)
was incubated for 2 h with 2 µg/ml rBid (lanes 1-3)
and an equivalent concentration of Bid isolated from mouse kidney
cytosol (lanes 4-6). Other conditions were as described for
Fig. 1, and the data shown are representative of three independent
repeat experiments. When indicated (lanes 3 and
6), the incubation contained 0.2 mM of the
synthetic dodecapeptide having Thr-59 phosphorylated (compare with Fig.
2). The data shown are representative of three independent repeat
experiments.
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Fig. 4.
Effect of casein kinase II (CKII)
on Bid immunodetection and cleavage by caspase 8. A,
equivalent concentrations of murine rBid (lanes 1-4) and
Bid isolated from kidney cytosol (lanes 5-8) were incubated
for 3 h in the absence (lanes 1, 2,
5, and 6) and presence (lanes 3,
4, 7, and 8) of 0.8 unit/ml of
recombinant CKII and 0.2 mM ATP. Subsequently, the samples
were diluted 5-fold in caspase assay buffer and incubated with
recombinant caspase 8 for 1 h as described for Fig. 3. To enhance
the resolution of the blot of full-length Bid, the protein was
separated with a 13% acrylamide gel and transferred onto a low
porosity polyvinylidene difluoride membrane (Applied Biosystems) using
a 10 mM sodium CAPS buffer, pH 11, instead of the usual
Tris-glycine buffer. B, Western blotting was conducted under
the routine conditions implemented for tBid resolution (compare with
Fig. 3) with recombinant Bid treated with CKII as in A,
either in the presence or absence of ATP (required for protein
phosphorylation (12)). The data are representative of three independent
repeat experiments.
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Fig. 5.
Differential cleavage of Bid by caspase 8 and
caspase 3. A, tBid products resulting from caspase
cleavage of either rBid or Bid isolated from mouse kidney were
separated with enhanced resolution of the SDS-PAGE and related to the
migration of recombinant tBid (lane 7) and precision
molecular weight standards. Conditions were the same as those described
for Fig. 3. B, effect of the isolated Bid preparation
(diluted to the same final concentration as in the experiment of Bid
cleavage, compare with A and B) on the kinetics
of recombinant caspase 3 (0.1 µg/ml) cleavage of the fluorogenic
substrate Ac-DEVD-AFC (50 µM). Data show the progressive
increase of fluorescent product and are representative of triplicate
repeat experiments. The solid lane is the control trace,
whereas the dashed line is the trace obtained in the
presence of isolated Bid. The dotted line shows the
inhibitory effect of 20 µM Asp-Glu-Val-Asp-chloromethyl
ketone (DEVD-CHO).
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Fig. 6.
Bid cleavage by caspases differs in different
subcellular fractions. A, subcellular fractions
obtained from homogenates of murine kidney were treated at the same
final concentration of 1 mg/ml with recombinant caspase 3 (1 µg/ml,
from Pharmingen) for different times as described under "Experimental
Procedures." Lanes 3-5 contained a cytosolic extract
(fraction S10 consisting of the 10,000 × g supernatant
(18)), whereas lanes 6-8 contained the mitochondrial wash,
obtained after 10,000 × g centrifugation of crude
mitochondria (fraction S3). Lanes 9-11 contained isolated
kidney mitochondria (mitos). rBid (1 µg/ml, lanes
1 and 2) was treated under the same condition. Protein
samples were blotted with the R&D antibody. B, recombinant
caspase 3 was incubated for 1 h with S10 fraction of murine kidney
(lanes 1 and 2), rBid (at a concentration about
3-fold lower than endogenous Bid, lanes 3 and 4),
and Bid isolated from the same cytosolic S10 fraction (lanes
5 and 6). Samples were incubated for 1 h in the
presence or absence of 20 µM Asp-Glu-Val-Asp-chloromethyl
ketone to inhibit caspase activity. Note the absence of detectable tBid
in isolated Bid (lane 6) versus rBid (lane
4) despite the much higher concentration. Data shown are
representative of four replicate experiments.
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Fig. 7.
Phosphatase treatment of endogenous Bid
facilitates its cleavage by caspases. A, Bid isolated
from murine kidney was untreated (lanes 1 and 2)
or treated with CKII as in Fig. 4 (lanes 3-5) and with
potato acid phosphatase (PAP, lane 6) as described under
"Experimental Procedures." At the end of the incubation, samples
were diluted 5-fold with caspase assay buffer and incubated for 1 h with caspase 8 as described in Fig. 6. B, Bid isolated
from murine kidney was treated with CKII (lanes 1-3) or
with PAP (lanes 4-6). At the end of the incubation, samples
were diluted 5-fold with caspase assay buffer and incubated for 1 h with either caspase 8 or caspase 3. C, samples of murine
kidney cytosol were treated with PAP for 1 h at 37 °C and
probed directly with the R&D antibody. Equal protein loading was
confirmed by subsequent reblots for actin (bottom panel) and
with India ink staining (not shown). Comparable results were
obtained with other antibodies. Data shown are representative of three
independent repeat experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-
(38, 39). In this case, phosphorylation confers specific
protection toward proteolytic events that are associated with the
execution phase of caspase-mediated death (38, 39). Protection from
caspase cleavage would affect both tBid action following the processing
of pro-caspase 8 upstream of mitochondria and tBid involvement in the
amplification of caspase cascades downstream of mitochondria. Our
findings clearly indicate that physiological mechanisms are in place to
allow efficient cleavage of Bid by upstream caspase 8 but not
simultaneously by downstream caspases like caspase 3. What produces
this differential specificity in Bid cleavage?
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ACKNOWLEDGEMENT |
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We thank E. Beaulieu for assistance.
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FOOTNOTES |
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* 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.
§ Funded by an Alliance Grant from Institut de la Recherche Servier (Paris).
Supported by the Lister Insitute of Preventive Medicine. To
whom correspondence should be addressed. Tel.: 44-161-275-5447; Fax:
44-161-275-5600; E-mail: cdive@man.ac.uk.
Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M209208200
2 P. Masdehors, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: DISC, death-inducing signaling complex; DTT, dithiothreitol; tBid, truncated Bid; rBid, recombinant Bid; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; CK, casein kinase; PIPES, 1,4-piperazinediethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAP, potato acid phosphatase; HPLC, high-performance liquid chromatography; AFC, 7-amido-4-(trifluoromethyl) coumarin.
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---|
1. |
Adams, J. M.,
and Cory, S.
(1998)
Science
281,
1322-1326 |
2. | Martinou, J. C., and Green, D. R. (2001) Nat. Rev. Mol. Cell Biol. 2, 63-67[CrossRef][Medline] [Order article via Infotrieve] |
3. | Green, D. R. (2000) Cell 102, 1-4[Medline] [Order article via Infotrieve] |
4. |
Wolter, K. G.,
His, Y. T.,
Smith, C. L.,
Nechustan, A.,
Xi, X. G.,
and Youle, R. J.
(1997)
J. Cell Biol.
139,
1281-1292 |
5. | Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. (1998) Cell 94, 481-490[Medline] [Order article via Infotrieve] |
6. | Li, H., Zhu, H., Xu, C., and Yuan, J. (1998) Cell 94, 491-501[Medline] [Order article via Infotrieve] |
7. |
Wei, M. C.,
Lindsen, T.,
Mootha, V. K.,
Eiler, S.,
Gross, A.,
Ashiya, A.,
Thompson, C. B.,
and Korsmeyer, S. J.
(2000)
Genes Dev.
14,
2060-2071 |
8. |
Wei, M. C.,
Zong, W. X.,
Cheng, E. H.,
Lindsten, T.,
Panoutsakopoulou, V.,
Ross, A. J.,
Roth, K. A.,
MacGregor, G. R.,
Thompson, C. B.,
and Korsmeyer, S. J.
(2001)
Science
292,
624-626 |
9. |
del Peso, L.,
Gonzalez-Garcia, M.,
Page, C.,
Herrera, R.,
and Nunez, G.
(1997)
Science
278,
687-689 |
10. | Lizcano, J. M., Morrice, N., and Cohen, P. (2000) Biochem. J. 349, 547-557[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Verma, S.,
Zhao, L.,
and Chinnadurai, G.
(2001)
J. Biol. Chem.
276,
4671-4676 |
12. | Desagher, S., Osen-Sand, A., Montessuit, S., Magnenat, E., Vilbois, F., Hochmann, A., Journot, L., Antonsson, A., and Martinou, J.-C. (2001) Mol. Cell 8, 601-611[Medline] [Order article via Infotrieve] |
13. |
Scaffidi, C.,
Fulda, S.,
Srinivasan, A.,
Friesen, C.,
Li, F.,
Tomaselli, K. J.,
Debatin, K.,
Krammer, P. H.,
and Peter, M. E.
(1998)
EMBO J.
17,
1675-1687 |
14. |
Scaffidi, C.,
Schmitz, I.,
Zha, J.,
Korsmeyer, S. J.,
Krammer, P. H.,
and Peter, M. E.
(1999)
J. Biol. Chem.
274,
22532-22538 |
15. |
Eskes, R.,
Desangher, S.,
Antonsson, B.,
and Martinou, J.-C.
(2000)
Mol. Cell. Biol.
20,
929-935 |
16. |
Grinberg, M.,
Sarig, R.,
Zaltsman, Y.,
Frumkin, D.,
Grammatikakis, N.,
Reuveny, E.,
and Gross, A.
(2002)
J. Biol. Chem.
277,
12237-12245 |
17. |
Kluck, R.,
Degli Esposti, M.,
Perkins, G.,
Renken, C.,
Kuwana, T.,
Bossy-Wetzel, E.,
Goldberg, M.,
Allen, T.,
Barber, M. J.,
Green, D. R.,
and Newmeyer, D. D.
(1999)
J. Cell Biol.
147,
809-822 |
18. |
Degli Esposti, M.,
Erler, J. M.,
Hickman, J. A.,
and Dive, C.
(2001)
Mol. Cell. Biol.
21,
7268-7276 |
19. |
Holmström, T. H.,
Schmitz, I.,
Söderström, T. S.,
Poukkula, M.,
Johnson, V. L.,
Chow, S. C.,
Krammer, P. H.,
and Eriksson, J. E.
(2000)
EMBO J.
19,
5418-5428 |
20. | Xu, K., and Thornalley, P. J. (2001) Br. J. Cancer 84, 670-673[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Ruiz-Ruiz, C.,
Robledo, G.,
Font, J.,
Izquierdo, M.,
and Lopez-Rivas, A.
(1999)
J. Immunol.
163,
4737-4746 |
22. | Zhuang, S., Demirs, J. T., and Kochevar, I. E. (2001) Oncogene 20, 6764-6776[CrossRef][Medline] [Order article via Infotrieve] |
23. | Slee, E. A., Keogh, S. A., and Martin, S. J. (2000) Cell Death Differ. 7, 556-565[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Wilson, M.,
Burt, A. R.,
Milligan, G.,
and Anderson, N. G.
(1996)
J. Biol. Chem.
271,
8537-8540 |
25. |
Stoka, V.,
Turk, B.,
Schendel, S. L.,
Kim, T.-H.,
Cirman, T.,
Snipas, S. J.,
Ellerby, L. M.,
Bredesen, D.,
Freeze, H.,
Abrahamson, M.,
Brömme, D.,
Krajewski, S.,
Reed, J. C.,
Yin, X. M.,
Turk, V.,
and Salvesen, G. S.
(2001)
J. Biol. Chem.
276,
3149-3157 |
26. |
Gross, A.,
Yin, X.,
Wang, A.,
Wei, M. C.,
Jockel, J.,
Milliman, C.,
Erdjument-Bromage, H.,
Tempst, P.,
and Korsmeyer, S. J.
(1999)
J. Biol. Chem.
274,
1156-1163 |
27. | McDonnell, J. M., Fushman, D., Milliman, C., Korsmeyer, S. J., and Cowburn, D. (1999) Cell 96, 625-634[Medline] [Order article via Infotrieve] |
28. |
Barry, M.,
Heibein, H. A.,
Pinkowski, M. J.,
Lee, S. F.,
Moyer, R. W.,
Green, D. R.,
and Bleackley, R. C.
(2000)
Mol. Cell. Biol.
20,
3781-3794 |
29. |
Alimonti, J. B.,
Shi, L.,
Baijal, P. K.,
and Greenberg, A. H.
(2001)
J. Biol. Chem.
276,
6974-6982 |
30. |
Bossy-Wetzel, E.,
and Green, D. R.
(1999)
J. Biol. Chem.
274,
17484-17490 |
31. | Stennicke, H. R., and Salvesen, G. S. (1999) Cell Death Differ. 6, 1054-1059[CrossRef][Medline] [Order article via Infotrieve] |
32. | Engels, I. H., Stepczynska, A., Stroh, C., Lauber, K., Berg, C., Schwenzer, R., Wajant, H., Janicke, R. U., Porter, A. G., Belka, C., Gregor, M., Schulze-Osthoff, K., and Wesselborg, S. (2000) Oncogene 19, 4563-4573[CrossRef][Medline] [Order article via Infotrieve] |
33. | Fulda, S., Meyer, E., Freisen, C., Susin, S. A., Kroemer, G., and Debatin, K. M. (2001) Oncogene 20, 1063-1075[CrossRef][Medline] [Order article via Infotrieve] |
34. | Gervais, F. G., Singaraja, R., Xanthoudakis, S., Gutekunst, C. A., Leavitt, B. R., Metzler, M., Hackam, A. S., Tam, J., Vaillancourt, J. P., Houtzager, V., Rasper, D. M., Roy, S., Hayden, M. R., and Nicholson, D. W. (2002) Nat. Cell Biol. 4, 95-105[CrossRef][Medline] [Order article via Infotrieve] |
35. | Fulda, S., Kufer, M. U., Meyer, E., van Valen, F., Dockhorn-Dworniczak, B., and Debatin, K. M. (2001) Oncogene 20, 5865-5877[CrossRef][Medline] [Order article via Infotrieve] |
36. | Yuan, X. J., and Whang, Y. E. (2002) Oncogene 21, 319-327[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Zha, J. S.,
Weiler, K.,
Wei, M. C.,
and Korsmeyer, S. J.
(2000)
Science
290,
1761-1765 |
38. |
Barkett, M.,
Xue, D.,
Horvitz, H. R.,
and Gilmore, T. D.
(1997)
J. Biol. Chem.
272,
29419-29422 |
39. | Taylor, J. A., Bren, G. D., Pennington, K. N., Trushin, S. A., Asin, S., and Paya, C. V. (1999) J. Mol. Biol. 290, 839-850[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Chen, M.,
He, H.,
Zhan, S.,
Krajewski, S.,
Reed, J. C.,
and Gottlieb, R. A.
(2001)
J. Biol. Chem.
276,
30724-30728 |
41. |
Plesnila, N.,
Zinkel, S.,
Le, D. A.,
Amin-Hanjani, S.,
Wu, Y.,
Qiu, J.,
Chiarugi, A.,
Thomas, S. S.,
Kohane, D. S.,
Korsmeyer, S. J.,
and Moskowitz, M. A.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
15318-15323 |
42. |
Santoro, M. F.,
Annand, R. R.,
Robertson, M. M.,
Peng, Y.-W.,
Brady, M. J.,
Mankovich, J. A.,
Hackett, M. C.,
Ghayur, T.,
Walter, G.,
Wong, W. W.,
and Giegel, D. A.
(1998)
J. Biol. Chem.
273,
13119-13128 |
43. |
Ravi, R.,
and Bedi, A.
(2002)
Cancer Res.
62,
4180-4185 |
44. | Lutter, M., Fang, M., Luo, X., Nishijima, M., Xie, X., and Wang, X. (2000) Nat. Cell Biol. 2, 754-756[CrossRef][Medline] [Order article via Infotrieve] |