From the Departments of Molecular Microbiology and
Immunology and §§ Neurology and Pharmacology and
Molecular Sciences, Johns Hopkins Schools of Public Health and
Medicine, Baltimore, Maryland 21205, the ¶ Metabolism Branch, NCI,
National Institutes of Health, Bethesda, Maryland 20892-1578, the
Human Genome Sciences, Inc., Rockville, Maryland 20850, and the
Department of Biochemistry, Merck Research
Laboratories, Rahway, New Jersey 07065
Received for publication, November 10, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although human c-IAP1 and c-IAP2 have been
reported to possess antiapoptotic activity against a variety of stimuli
in several mammalian cell types, we observed that full-length c-IAP1
and c-IAP2 failed to protect cells from apoptosis induced by Bax
overexpression, tumor necrosis factor The IAP (inhibitor of apoptosis) proteins
Op-IAP and Cp-IAP were originally identified in baculoviruses because
they could functionally replace the baculovirus-caspase inhibitor P53
(1, 2). The closest homologues to the baculovirus IAPs are the Drosophila IAPs, D-IAP1 and D-IAP2, and the human IAPs,
c-IAP1 (MIHB), c-IAP2 (MIHC), and XIAP (hILP, MIHA) (3, 4). These molecules all share two or three copies of the BIR (baculovirus IAP
repeat) motif at their N termini and a RING finger at their C termini
and are direct inhibitors of caspases, a family of death-inducing proteases (5-10).
All of these IAP proteins have been shown to inhibit apoptosis in one
or more paradigms. D-IAP1 was consistently retrieved in a genetic
screen for caspase inhibitors (11), and both D-IAP1 and D-IAP2
overexpression inhibits apoptosis in the Drosophila retina
(12). Human c-IAP1 and c-IAP2 are less potent inhibitors of apoptosis
compared with XIAP, correlating with the observation that XIAP is a
more potent caspase inhibitor (~100-fold) than c-IAP1 or c-IAP2
in vitro (3). Both biochemical and structural data support a
model where the individual BIR motifs of XIAP are specific inhibitors
of different caspases. That is, the second BIR motif (BIR-2) and
adjacent sequences of XIAP interact directly with activated caspase-3
and are sufficient to inhibit caspase-3 (13, 14). In addition, BIR-3 of
XIAP binds to and inhibits caspase-9. However, NMR structure analysis
predicts that the mechanism of caspase inhibition by BIR-3 will be
distinct from that of BIR-2 (15), supporting a model where the
individual BIR repeats have independent and nonredundant functions
(14).
The role of the C-terminal RING finger seems to be cell type- and/or
death stimulus-dependent. In some situations the RING is
required for antiapoptotic activity, but in others the RING inhibits
the antiapoptotic function of IAP proteins. Although the RING finger of
Op-IAP is required for protection of insect cells from apoptosis
induced by actinomycin D treatment or baculovirus infection, this
domain is not required for inhibition of Hid-induced apoptosis in the
same insect cell line (16-18). Partial inhibition of cell death
induced by HID as well as Reaper and Grim is conferred by BIR-2 of
Op-IAP (most equivalent to BIR-3 of c-IAP1) plus a critical
carboxyl-proximal flanking sequence. However, BIR-2 plus the RING
finger is a more potent protector than BIR-2 (with proximal sequences)
alone, implying that the RING finger contributes to the antiapoptotic
function of Op-IAP (19).
The RING fingers of a number of proteins including c-IAP1 and XIAP were
recently shown to function as E3 ubiquitin ligases in
proteosome-dependent protein degradation (20). This
activity may predominantly facilitate self-destruction as shown for
c-IAP1 and XIAP, resulting in cell death. Alternatively, the
antiapoptotic function of IAP proteins may be explained by
ubiquitination and degradation of caspases as suggested for the RING
finger of c-IAP2 (21). The RING finger domains of XIAP, D-IAP1, and
D-IAP2 also were shown to interact with signaling factors for the bone
morphogenetic protein kinase receptors and may participate in these
signal transduction pathways as well (22).
Earlier work in mammalian cells had suggested that the RING finger of
full-length c-IAP1 does not interfere with its antiapoptotic activity
in mammalian cells. That is, in a variety of stably or transiently
transfected cells, c-IAP1 and c-IAP2 were reported to inhibit apoptosis
induced by different death stimuli including serum withdrawal,
menadione, staurosporine, caspase-1, Bak, or K+
depolarization (23-26). In contrast to these reports using mammalian cells, full-length human c-IAP1 failed to inhibit Reaper-induced cell
death in a Drosophila eye model unless the RING finger was deleted (12). Thus, in insect cells, the RING finger appears to
negatively regulate the antiapoptotic function of human c-IAP1. Consistent with this finding, expression of the spacer-RING region of
D-IAP1 in transgenic flies resulted in a small eye phenotype caused by
excessive cell death in the eye disc. Furthermore, the RING finger
regions of baculovirus Cp-IAP and Drosophila D-IAP1 can
induce apoptosis in the lepidopteran cell line SF-21 (17).
We found that like the Drosophila system, c-IAP1 and c-IAP2
failed to protect mammalian cells unless the RING finger domain was
deleted. Furthermore, c-IAP1 was cleaved during apoptosis to release
the C-terminal spacer-RING domain that was capable of killing cells in
transient transfections.
Plasmid and Virus Constructs--
The human IAPs were identified
in an expressed sequence tag data base (Human Genome Sciences)
by searching with a BLAST program for homology to baculovirus IAP.
Expressed sequence tag clones were used as probes to identify
full-length cDNAs from the libraries that contained the expressed
sequence tag of interest. These clones were essentially identical to
previously published c-IAP1 and c-IAP2. C-terminal truncations were
generated by insertion of oligonucleotides containing stop codons in
all three reading frames into restriction sites at the codon positions
indicated in Fig. 2. The
CARD1 deletion mutant lacks an internal MunI
restriction fragment. The RING mutant was generated by recombinant
polymerase chain reaction mutating Cys586,
His588, and Cys592 to alanines. Cells, Infections, and Transfections--
Mycoplasma-free BHK
and CHO cells were plated at 1 × 104 or 2.5 × 105 cells/well in a 24- or 6-well dish, respectively, and
infected the following day with recombinant Sindbis viruses at a
multiplicity of 10 plaque forming units/per cell. Cell viability was
determined at ~30 h postinfection by trypan blue exclusion which was
previously shown to accurately reflect the apoptotic death induced by
Sindbis virus (27, 30).
Transfected CHO (10 µl LipofectAMINE; Life Technologies, Inc.), BHK
(10 µl of LipofectAMINE), 293 (10 µl of GenePorter; Gene Therapy
Systems), MCF-7 (10 µl of LipofectAMINE), and Rat-1 (2.5 µl of
LipofectAMINE) cells were fixed and stained with
5-bromo-4-chloro-3-indolyl Immunoblot Analysis--
GST fused to amino acids 87-618 of
c-IAP1 was purified from Escherichia coli, cleaved from GST
with thrombin and used to immunize rabbits (HRP, Inc., Denver,
PA) to generate anti-c-IAP1 antibody. Cell lysates were prepared with
RIPA buffer and a mixture of protease inhibitors at 8-24 h
postinfection with recombinant viruses or 16-24 h post-transfection
and separated by SDS-PAGE. Immunoblot analysis was performed with
anti-c-IAP1 (1:1000 dilution) or anti-HA antibody 12CA5 (1:1000
dilution; Roche Molecular Biochemicals).
In Vitro Cleavage Assay--
[35S]Met-labeled
c-IAP1 protein and derivatives were produced by in vitro
translation (TnT T7 Quick System, Promega). 1.5 µl of each
translation mix was incubated with 10 µl of 293 cell extract prepared
as previously described (31) and 1 mM ATP at 37 °C for
~15 h in the absence or presence of 100 µM caspase
inhibitor DEVD-CHO or zVAD-fmk. Cleavage with recombinant caspase-3
(Merck) was performed as previously described (32).
Zinc Binding Assay--
GST fusion proteins were purified from
E. coli using glutathione-bound resin and dialyzed
extensively against phosphate buffered saline to remove unbound zinc.
Protein samples of known concentration (determined by BCA protein
assay) were subjected to atomic absorption spectroscopy, and the
absorbance at 214 nm (specific for incinerated zinc) was compared with
a standard curve of known ZnSO4 concentrations to determine
the molar ratio of zinc to protein in each sample.
To compare the antiapoptotic activities of IAP family proteins,
human c-IAP1 and c-IAP2 were inserted into the Sindbis virus vector and
tested for their ability to inhibit Sindbis virus-induced cell death.
Sindbis virus triggers classic apoptotic death in many cell types,
providing a quantitative analysis of the function of a variety of cell
death regulators (30). Mammalian XIAP/hILP and baculovirus Op-iap were
previously reported to potently protect cells in this assay (33), and
both the pro- and antiapoptotic functions of several Bcl-2 family
members have been assessed in this manner (27). BHK and N18 murine
neuroblastoma cells were infected with recombinant viruses encoding
human c-IAP1, c-IAP2, or XIAP, and cell viability was assessed by dye
exclusion. Although Bcl-xL and XIAP protected cells from
virus-induced cell death, both c-IAP1 and c-IAP2 failed to protect
cells compared with Sindbis virus vector controls encoding the same
cDNAs in reverse orientation (Fig. 1,
A and B).
treatment or Sindbis
virus infection. However, deletion of the C-terminal RING domains of
c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this
region negatively regulates the antiapoptotic function of the
N-terminal BIR domain. This finding is consistent with the observation
by others that the spacer region and RING domain of c-IAP1 functions as
an E3 ligase, promoting autoubiquitination and degradation of c-IAP1.
In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of
c-IAP1 including the RING finger. In vitro cleavage of
c-IAP1 with apoptotic cell extracts or with purified recombinant
caspase-3 produced similar fragments. Furthermore, transfection of
cells with the spacer-RING domain alone suppressed the antiapoptotic
function of the N-terminal BIR domain of c-IAP1 and induced apoptosis.
Optimal death-inducing activity of the spacer-RING required both the
spacer region and the zinc-binding RING domain of c-IAP1 but did not
require the caspase recruitment domain located within the spacer
region. To the contrary, deletion of the caspase recruitment domain
increased proapoptotic activity, apparently by stabilizing the
C-terminal fragment.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
BIR-1
initiates at a naturally occurring Met at position 131. IAPs and
derivatives were cloned into the BstEII site of the Sindbis
virus vector (dsTE12Q), and recombinant viruses were generated as
previously reported (27-29). C-terminal c-IAP1 fragments and HA-tagged
fragments of c-IAP1 were generated by polymerase chain reaction and
expressed from a modified pSG5 vector for expression in transfected
cells. All clones were verified by DNA sequencing.
-D-galactopyranoside (X-gal)
22-24 h later unless indicated otherwise. DNA concentrations were held
constant within each experiment. Cell viability was determined by
counting 200-600 live/nonapoptotic blue cells/sample and calculated as
indicated in the legends. MCF7 Fas cells (provided by Vishva Dixit)
were cotransfected with 0.5 µg of green fluorescent protein plasmid
and 2 µg of the plasmid of interest, using 2 µl of Lipofectin.
Medium containing 200 units/ml TNF was added 24 h after
transfection, and cell viability was determined 18 h later by
counting green fluorescent protein-positive cells showing apoptotic
morphology relative to the total number of green fluorescent
protein-positive cells.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (32K):
[in a new window]
Fig. 1.
c-IAP1 and c-IAP2 fail to inhibit apoptosis
compared with XIAP and Bcl-xL. A and
B, BHK (A) and N18 murine neuroblastoma
(B) N18 murine cells were infected with recombinant Sindbis
viruses encoding Bcl-xL, c-IAP1, c-IAP2, XIAP, or their
reverse orientations as controls and cell viability was determined
~30 h postinfection by trypan blue exclusion. Results shown are
representative of three independent experiments. Induction of apoptosis
was confirmed by cell morphology (not shown). C, MCF-7 cells
transfected with a -galactosidase plasmid (0.8 µg) to mark
transfected cells and with plasmids encoding murine Bax (0.8 µg),
human Bak (0.8 µg), human Bcl-xL (2 µg), and human
c-IAP1 (2 µg) separately or in combination were assessed for cell
viability by counting viable/nonapoptotic blue cells. Total DNA
concentrations were held constant with control vector DNA. Means ± S.E. are shown for at least three independent experiments.
It is possible that Sindbis virus triggers a cell death pathway that is impervious to c-IAP1. In an alternate assay, c-IAP1 was tested for the ability to inhibit apoptosis induced by mBax or hBak in transiently transfected MCF-7 cells. Although Bcl-xL protected cells from apoptotic death induced by cotransfected Bax or Bak, c-IAP1 failed to protect in contrast to earlier work in the same assays (Fig. 1C).
To determine whether the RING finger could interfere with antiapoptotic
activity, stop codons were inserted into c-IAP1, c-IAP2, or XIAP coding
sequences within the Sindbis virus vector to generate truncated
proteins lacking the C-terminal RING finger or lacking both the spacer
region and RING (Fig. 2A). CHO
cells were infected with recombinant viruses expressing wild type or
truncated IAPs, and cell viability was determined by trypan blue
exclusion. Removing the spacer-RING or RING domains of XIAP had no
effect on antiapoptotic function. This result with virus-induced
apoptosis is consistent with the observation by others that neither the
RING nor the spacer region of XIAP is required for inhibition of
TNF-induced cell death in MCF7-Fas cells (34) and that the BIR
domain of XIAP is sufficient to inhibit caspases (10). However, the
C-terminal truncations of c-IAP1 conferred a gain of antiapoptotic
activity, indicating that the BIR domain was sufficient to inhibit cell death in this assay and that the antiapoptotic function of the BIR
domain was suppressed by the RING domain (Fig. 2B). Similar results were observed in BHK cells (data not shown). The RING deletion
mutant was also expressed at slightly higher levels, perhaps
contributing to its activity (Fig. 2C), but expression of
the BIR domain alone was problematic and difficult to detect (see
below). Thus, changes in protein expression levels may not fully
account for the gain-of-function by C-terminal truncations. A time
course experiment in CHO cells further demonstrated that deletion of
the C-terminal RING finger of c-IAP1 restores the antiapoptotic
function of the BIR domain against Sindbis virus (Fig.
2D).
|
In contrast to c-IAP1, deletion of the C-terminal spacer-RING of c-IAP2 failed to confer antiapoptotic activity on c-IAP2 in the Sindbis virus assay (Fig. 2B). Both full-length and C-terminal truncated c-IAP2 proteins were consistently difficult to detect by immunoblot analysis in Sindbis virus-infected cells, and further analyses of c-IAP2 in this model were abandoned. However, c-IAP2 lacking the spacer-RING region was capable of inhibiting apoptosis induced by TNF treatment of MCF-7 cells, whereas full-length c-IAP2 was inactive in this assay (Fig. 2E). Deletion of the C-terminal spacer-RING domain of c-IAP2 significantly stabilized c-IAP2 in transfected 293 cells, although respectable levels of c-IAP1 still failed to protect (Fig. 2F). Taken together, the RING domains of c-IAP1 and c-IAP2 but not XIAP were capable of suppressing the antiapoptotic functions of their BIR domains in the assays tested here, which may in part be due to protein stabilization.
To further investigate the fate of c-IAP1 protein during apoptosis,
lysates prepared from 293 cells infected with recombinant viruses
encoding wild type or mutant c-IAP1 were immunoblotted with a rabbit
polyclonal antiserum generated against recombinant c-IAP1. The 68-kDa
c-IAP1 expressed from recombinant Sindbis virus comigrated with
endogenous c-IAP1 (compare c-IAP1 lanes with the 68-kDa band in BIR and
CARD lanes in Fig. 3A).
Overexpression increased c-IAP1 protein levels only 3-5-fold over
endogenous levels. In addition to full-length protein, an
immunoreactive polypeptide of 52 kDa and a less stable 35-kDa
polypeptide (open circles in Fig. 3A) were
detected following infection with virus encoding full-length c-IAP1,
suggesting that c-IAP1 may be cleaved during apoptosis. Similar results
were obtained in COS-1 cells (data not shown). The BIR domain alone
(lanes 5-8) was only detected on longer exposures (not
shown). Longer exposures also detected the 52-kDa fragment of
endogenous c-IAP1 in control virus-infected cells, but the less stable
35-kDa fragment was below detection limits (Fig. 3B,
lanes 1 and 2).
|
Deletion mutants of c-IAP1 were analyzed to determine which portion of c-IAP1 is contained in these apparent cleavage fragments. Deletion of most of the CARD motif (amino acids 471-561) within the spacer region produced a c-IAP1 protein that was 10-kDa smaller as expected (open triangle in Fig. 3A). This deletion also shifted the 52-kDa fragment to 41 kDa (solid circle in Fig. 3A) and shifted the smaller 35-kDa fragment to 25 kDa (Fig. 3A, inset). In general, the cleavage fragments increased in abundance with time after infection (Fig. 3, A-C). Therefore, both fragments appear to be cleavage products of c-IAP1, and both contain the CARD motif. Both fragments also contain the C-terminal RING finger. Deletion of the 6-kDa RING reduced the size of the 52-kDa cleavage fragment (Fig. 3B), whereas further deletion of BIR-1 had no effect on the size of the cleavage fragment. Deletion of the RING abolished formation of the 35-kDa fragment, perhaps by destabilizing the polypeptide (Fig. 3C). This possibility is consistent with our difficulty in detecting the spacer region when expressed alone (see Fig. 6 below). Taken together, these data indicate that c-IAP1 is cleaved during apoptosis to yield 52- and 35-kDa fragments that are both derived from the C terminus. The 35-kDa fragment is likely to consist of the spacer and RING domains because the predicted size of this region is 31.3 kDa and expression of an engineered spacer-RING fragment migrates at ~35 kDa on SDS gels (see below). The 52-kDa fragment probably contains BIR-2 through the C terminus based on size estimations. The corresponding N-terminal fragments of c-IAP1 could not be identified among the cleavage fragments and may be degraded.
To determine whether the 52- and 35-kDa fragments of c-IAP1 are
generated by caspase cleavage, in vitro translated
35S-labeled c-IAP1 was treated with 293 cell extracts that
contain activated caspases (31, 35, 36). Cleavage of c-IAP1 by the apoptotic cell extract produced a 35-kDa fragment approximately the
same size as that observed in apoptotic cells. This
cleavage was inhibited by a pan
caspase inhibitor zVAD and a caspase-3 inhibitor DEVD (Fig.
4A) but was not inhibited by the caspase-1 inhibitor YVAD
(data not shown). c-IAP1 was also cleaved to produce a 35-kDa fragment
by purified recombinant caspase-3 that was inhibited by zVAD (Fig.
4A). The larger 52-kDa cleavage fragment observed in
virus-infected cells was not detected in this in vitro assay unless the CARD domain was deleted, perhaps stabilizing the
intermediate cleavage product. Consistent with our observations in
apoptotic cells, 293 cell extracts cleaved the in vitro
translated CARD mutant to the expected 41- and 25-kDa fragments
(Fig. 4B and data not shown). This result confirms that the
in vitro cleavage fragments also contain the C-terminal
region of c-IAP1. Consistent with our results in apoptotic cells (Fig.
3A), the
CARD mutant of c-IAP1 was more stable in the
in vitro translation mix compared with wild type full-length
c-IAP1 (Fig. 4B). Cleavage of the
CARD mutant of c-IAP1
was detectable as early as 1 h after addition of caspase-3, and
densitometry of the uncleaved protein indicated that 72% was cleaved
by 4 h (Fig. 4C).
|
To map the cleavage site responsible for producing the smaller
35/25-kDa fragment, several Asp residues near the beginning of the
spacer region were mutated individually in the CARD mutant of
c-IAP1. Mutation of Asp372 to Ala abolished formation of
the smaller cleavage fragment, whereas mutation of Asp346,
Asp364, and Asp387 had no effect on generation
of this fragment (Fig. 4D and data not shown). Surprisingly,
mutation of Asp372 also impaired generation of the larger
41-kDa fragment. Mutation of Asp372 may impair subsequent
cleavage at a second site, but we cannot formally exclude the
possibility that the larger fragment is a post-translationally modified
form of the smaller fragment.
Because the RING finger squelched the antiapoptotic activity of
full-length c-IAP1 and because a C-terminal fragment of c-IAP1 containing the RING finger is generated by caspases in vitro
and in apoptotic cells, we tested the possibility that the C terminus of c-IAP1 has proapoptotic function. Transfection of BHK cells with a
construct expressing the spacer-RING region of c-IAP1 (amino acids
342-618) induced cell death in a dose-dependent manner
(Fig. 5A). Similar results
were obtained with a fragment containing amino acids 373-618 (data not
shown).
|
To determine which portion of the spacer-RING construct was responsible
for the induction of apoptosis, several mutants were tested in
transfected BHK cells. Deletion of the CARD domain from the spacer-RING
(SR-CARD) enhanced proapoptotic activity and correlated with higher
protein expression levels, again suggesting that the CARD domain
contributes to the instability of the spacer-RING region (Fig.
5B). Similar results were obtained with HA-tagged constructs
in CHO and 293 cells (Fig. 5, D and F) and Rat-1
(not shown), except that the
CARD mutant of HA-spacer-RING was
generally a more potent killer in these cell types, again correlating
with increased protein expression compared with the CARD-containing construct (Fig. 5E). The spacer or RING regions alone lacked
pro-death activity compared with control vector (Fig. 5, B,
D, and F). The RING domain alone was not detected
by the polyclonal anti-c-IAP1 antibody (Fig. 5B) but was
likely to be expressed efficiently based on results with an HA-tagged
version (Fig. 5E). The spacer region alone was consistently
present at lower levels even when detected via an HA tag (Fig. 5,
B and E). Thus, these experiments do not
eliminate the possibility that the spacer region was sufficient for
proapoptotic function. However, mutation of three conserved Cys/His
residues in the RING (RING*) abolished proapoptotic activity of
spacer-RING, verifying that the RING was required for pro-death activity (Fig. 5C). The pro-death activity of spacer-RING
(lacking the CARD domain) in 293 cells was abolished by cotransfection with the caspase inhibitor P35, suggesting that caspases mediate apoptosis induced by the spacer-RING. Higher molecular mass
species of the spacer, RING, and spacer-RING domains were frequently
observed, suggesting that these proteins are post-translationally
modified in cells (plus signs, Figs. 2F and 5,
A-C).
To confirm that the spacer-RING domain of c-IAP1 can interfere with the
antiapoptotic activity of the BIR domain, we asked whether spacer-RING
could impair protection by the BIR domain from Bax-induced cell death.
Viability of cells cotransfected with Bax and the BIR domain confirmed
that BIR suppressed Bax-induced cell death. Furthermore, cotransfection
with spacer-RING abolished the protective activity of BIR in CHO and
293 cells (Fig. 6).
|
To formally demonstrate that the predicted RING domain of c-IAP1 indeed
binds zinc, a GST-RING protein was purified from E. coli and
tested for its ability to liberate zinc during combustion. Consistent
with the fact that RING fingers of other proteins are known to bind 2 mol of zinc, GST-RING bound the same molar ratio of zinc as two BIR
domains (GST-BIR2-3) that each coordinate one zinc atom (Fig.
7). BIR domains each contain a single
zinc finger, confirmed in the recently determined structure of several
BIR domains (13, 15, 37). This result also suggests that the lack of
cell killing activity by the RING finger alone (with or without an
HA-tag) is probably not due to defects in protein folding.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In contrast to XIAP, full-length c-IAP1 and c-IAP2 failed to
protect cells from apoptosis induced by one or more death stimuli including Bax overexpression, virus infection, and TNF treatment. However, deletion of the C-terminal spacer-RING domains of c-IAP1 and
c-IAP2 restored the latent antiapoptotic function of the N-terminal BIR
domains (Fig. 2). This finding suggests that the C terminus of c-IAP1
and c-IAP2 can negatively regulate the antiapoptotic activity of the
BIR domains. Interestingly, deletion of the C-terminal spacer-RING of
c-IAP2 occurs as the result of a chromosome translocation event
commonly found in mucosa-associated lymphoid tissue (MALT) lymphomas
(38). Perhaps this translocation produces a constitutively antiapoptotic c-IAP2 protein. However, the MALT1 gene to
which cIAP-2 is fused in the t(11;18) MALT lymphoma translocations was recently found to encode a paracaspase that is also likely to contribute to disease (39). The fusion effectively deletes the prodomain of the paracaspase in many of these translocation events (40), linking it to the BIR domain of c-IAP2.
Our results in mammalian cells are consistent with those obtained in a Drosophila eye model where deletion of the RING finger domain of D-IAP1 significantly enhanced its antiapoptotic function against Reaper overexpression and during Drosophila eye development (12). Similarly, deletion of the c-IAP1 RING was required to protect the third instar eye disc from Reaper-induced cell death. However, in some situations the RING finger may be required for protective function. Full-length c-IAP1 was reported to protect human MCF7 cells from Reaper-induced apoptosis (41), and deletion of the RING finger of D-IAP1, D-IAP2, and MIHA (murine XIAP) impaired their ability to inhibit caspase-1-induced apoptosis in mammalian cells (42). Furthermore, the RING of baculovirus Cp-IAP is required for antiapoptotic activity and to inhibit caspase-9 (5). Taking all of these studies together, the RING finger of IAP proteins may positively or negatively modulate the function of IAP proteins, and this modulatory function is presumably dependent in part on cell type-specific factors.
At least two factors appear to contribute to the lack of antiapoptotic activity by full-length c-IAP1 during virus-induced apoptosis, protein instability, and susceptibility to caspase cleavage. Deletion of the RING of c-IAP1 was reported to prevent autoubiquitination and degradation of c-IAP1 (20). Deletion of the RING appeared to stabilize c-IAP1 in virus-infected cells but the observed increase in protein levels may not fully explain the gain of antiapoptotic function. c-IAP1 was also cleaved to 52- and 35-kDa fragments by recombinant caspase-3, by apoptotic cell extracts, and in cells undergoing apoptosis. Furthermore, the 35-kDa spacer-RING fragment was capable of inducing apoptosis and was capable of inhibiting the antiapoptotic function of the BIR domain when expressed on separate plasmids. No function has been attached to the CARD of c-IAPs, and it is not required for direct binding to caspases (7) nor for the proapoptotic function of the spacer-RING fragment. To the contrary, the CARD of c-IAP1 appears to destabilize both the full-length and cleaved fragments of c-IAP1, although the mechanism is not known. Perhaps through dimerization or recruitment to a protein complex the CARD promotes degradation of c-IAP1, or maybe one or more of several Lys residues in the CARD serve as ubiquitination sites. However, both the spacer and RING domains of c-IAP1 appear to be post-translationally modified to produce larger, ~7-8-kDa immunoreactive bands (marked + in Fig. 5). This size shift is consistent with monoubiquitination, although other possibilities remain.
Cleavage of c-IAP1 in apoptotic cells appears to be accomplished by
caspases as the same size fragments were generated with caspase-3
in vitro, and a caspase inhibitor blocked cleavage of c-IAP1
by apoptotic cell extracts. Deletion analyses indicate that the 35-kDa
caspase cleavage product of c-IAP1 contains the spacer-RING domain.
This domain of c-IAP1 induces apoptosis in transfected cells,
consistent with the finding that the analogous region of D-IAP1 induces
cell death when overexpressed in the Drosophila eye (12).
The generation of proapoptotic cleavage products of c-IAP1 during cell
death is reminiscent of the finding that Bcl-2 and Bcl-xL
are also converted from antiapoptotic to proapoptotic factors by
caspase cleavage (32, 43). However, the in vivo function of
the c-IAP1 spacer-RING fragment that is generated during apoptosis is
not known. This fragment of c-IAP1 could potentially decrease cell
survival via an independent function perhaps involving ubiquitination
of cIAP-1, the BIR domain, or possibly heterologous targets (presumably
antiapoptotic factors). The RING domain (without spacer) of cIAP-2 was
found to be sufficient for in vitro E3 ligase activity that
selectively promoted ubiquitination of caspases 3 and 7 but not caspase
1 (21). This finding seems inconsistent with the proapoptotic function
of spacer-RING in cells but may imply a role for the spacer region in
directing the proapoptotic function of spacer-RING inside cells.
Alternatively, spacer-RING could function as a dominant negative
inhibitor of c-IAP1, perhaps preventing degradation of caspases.
Consistent with this idea, deletion of the N-terminal BIR domain of
survivin results in a C-terminal fragment that appears to interfere
with endogenous survivin by competing for microtubule binding (44, 45).
![]() |
ACKNOWLEDGEMENT |
---|
We thank Jeremy Berg for assistance with the zinc binding assay.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Amyotrophic Lateral Sclerosis Association and the National Institutes of Health (to J. M. H.) and an American Cancer Society Fellowship (to R. J. C.).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.
§ These authors contributed equally to this work.
** Present address: Div. of Biology, Kansas State University, Manhattan, KS 66506.
¶¶ To whom correspondence should be addressed: Dept. of Molecular Microbiology and Immunology, E5140, Johns Hopkins School of Public Health, 615 N Wolfe St., Baltimore, MD 21205. Tel.: 410-955-2716; Fax: 410-955-0105; E-mail: hardwick@jhu.edu.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M010259200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CARD, caspase
recruitment domain;
BHK, baby hamster kidney;
CHO, Chinese hamster
ovary;
DEVD-CHO, N-acetyl-Asp-Glu-Val-aspartinal;
HA, hemagglutinin;
PAGE, polyacrylamide gel electrophoresis;
TNF, tumor
necrosis factor ;
zVAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone;
GST, glutathione S-tranferase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Crook, N. E., Clem, R. J., and Miller, L. K. (1993) J. Virol. 67, 2168-2174[Abstract] |
2. | Clem, R. J., and Duckett, C. S. (1997) Trends Cell Biol. 7, 337-339[CrossRef] |
3. |
Deveraux, Q. L.,
and Reed, J. C.
(1999)
Genes Dev.
13,
239-252 |
4. | Miller, L. K. (1999) Trends Cell Biol. 9, 323-328[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Huang, Q.,
Deveraux, Q. L.,
Maeda, S.,
Salvsen, G. S.,
Stennicke, H. R.,
Hammock, B. D.,
and Reed, J. C.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1427-1432 |
6. | Kaiser, W. J., Vucic, D., and Miller, L. K. (1998) FEBS Lett. 440, 243-248[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Roy, N.,
Deveraux, Q. L.,
Takahashi, R.,
Salvesen, G. S.,
and Reed, J. C.
(1997)
EMBO J.
16,
6914-6925 |
8. |
Deveraux, Q. L.,
Roy, N.,
Stennicke, H. R.,
Van, A.rsdale, T.,
Zhou, Q.,
Srinivasula, S. M.,
Alnemri, E. S.,
Salvesen, G. S.,
and Reed, J. C.
(1998)
EMBO J.
17,
2215-2223 |
9. | Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. (1997) Nature 388, 300-304[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Takahashi, R.,
Deveraux, Q.,
Tamm, I.,
Welsh, K.,
Assamunt, N.,
Salvesen, G. S.,
and Reed, J. C.
(1998)
J. Biol. Chem.
273,
7787-7790 |
11. |
Hawkins, C. J.,
Wang, S. L.,
and Hay, B. A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2885-2890 |
12. | Hay, B. A., Wassarman, D. A., and Rubin, G. M. (1995) Cell 83, 1253-1262[Medline] [Order article via Infotrieve] |
13. | Sun, C., Cai, M., Gunasekera, A. H., Meadows, R. P., Wang, H., Chen, J., Zhang, H., Wu, W., Xu, N., Ng, S. C., and Fesik, S. W. (1999) Nature 401, 818-812[CrossRef][Medline] [Order article via Infotrieve] |
14. | Deveraux, Q., Leo, E., Stennicke, H., Welsh, K., Salvesen, G., and Reed, J. (1999) EMBO J. 5242-5251 |
15. |
Sun, C.,
Cai, M.,
Meadows, R. P.,
Xu, N.,
Gunasekera, A. H.,
Herrmann, J.,
Wu, J. C.,
and Fesik, S. W.
(2000)
J. Biol. Chem.
275,
33777-33781 |
16. | Clem, R. J., and Miller, L. K. (1994) Mol. Cell. Biol. 14, 5212-5222[Abstract] |
17. | Harvey, A. J., Soliman, H., Kaiser, W. J., and Miller, L. K. (1997) Cell Death Differ. 4, 733-744[CrossRef] |
18. |
Hawkins, C. J.,
Uren, A. G.,
Hacker, G.,
Medcalf, R. L.,
and Vaux, D. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
13786-13790 |
19. |
Vucic, D.,
Kaiser, W. J.,
and Miller, L. K.
(1998)
J. Biol. Chem.
273,
33915-33921 |
20. |
Yang, Y.,
Fang, S.,
Jensen, J. P.,
Weissman, A. M.,
and Ashwell, J. D.
(2000)
Science
288,
874-877 |
21. |
Huang, H.,
Joazeiro, C. A. P.,
Bonfoco, E.,
Kamada, S.,
Leverson, J. D.,
and Hunter, T.
(2000)
J. Biol. Chem.
275,
26661-26664 |
22. |
Yamaguchi, K.,
Nagai, S.,
Ninomiya-Tsuji, J.,
Nishita, M.,
Tamai, K.,
Irie, K.,
Ueno, N.,
Nishida, E.,
Shibuya, H.,
and Matsumoto, K.
(1999)
EMBO J.
18,
179-187 |
23. | Liston, P., Roy, N., Tamai, K., Lefebvre, C., Baird, S., Cherton-Horvat, G., Farahani, R., McLean, M., Ikeda, J.-E., MacKenzie, A., and Korneluk, R. G. (1996) Nature 379, 349-353[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Uren, A. G.,
Pakusch, M.,
Hawkins, C. J.,
Puls, K. L.,
and Vaux, D. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4974-4978 |
25. |
Orth, K.,
and Dixit, V. M.
(1997)
J. Biol. Chem.
272,
8841-8844 |
26. | Simons, M., Beinroth, S., Gleichmann, M., Liston, P., Korneluk, R. G., MacKenzie, A. E., Bahr, M., Klockgether, T., Robertson, G. S., Weller, M., and Schulz, J. B. (1999) J. Neurochem. 72, 292-301[CrossRef][Medline] [Order article via Infotrieve] |
27. | Cheng, E. H. Y., Levine, B., Boise, L. H., Thompson, C. B., and Hardwick, J. M. (1996) Nature 379, 554-556[CrossRef][Medline] [Order article via Infotrieve] |
28. | Hardwick, J. M., and Levine, B. (2000) Methods Enzymol. 322, 492-508[Medline] [Order article via Infotrieve] |
29. |
Levine, B.,
Goldman, J. E.,
Jiang, H. H.,
Griffin, D. E.,
and Hardwick, J. M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4810-4815 |
30. | Levine, B., Huang, Q., Isaacs, J. T., Reed, J. C., Griffin, D. E., and Hardwick, J. M. (1993) Nature 361, 739-742[CrossRef][Medline] [Order article via Infotrieve] |
31. | Fearnhead, H. O., McCurrach, M. E., O'Neill, J., Zhang, K., Lowe, S. W., and Lazebnik, Y. A. (1997) Genes Dev. 11, 1266-1276[Abstract] |
32. |
Clem, R. J.,
Cheng, E. H. Y.,
Karp, C. L.,
Kirsch, D. G.,
Ueno, K.,
Takahashi, A.,
Kastan, M. B.,
Griffin, D. E.,
Earnshaw, W. C.,
Veliuona, M. A.,
and Hardwick, J. M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
554-559 |
33. | Duckett, C. S., Nava, V. E., Gedrich, R. W., Clem, R. J., Van, Dongen, J. L., Gilfillan, M. C., Shiels, H., Hardwick, J. M., and Thompson, C. B. (1996) EMBO J. 15, 2685-2694[Abstract] |
34. |
Duckett, C. S.,
Li, F.,
Wang, Y.,
Tomaselli, K. J.,
Thompson, C. B.,
and Armstrong, R. C.
(1998)
Mol. Cell Biol.
18,
608-615 |
35. |
Kirsch, D. G.,
Doseff, A.,
Chau, B. N.,
Lin, D.-S.,
de Souza-Pinto, N. C.,
Hansford, R.,
Kastan, M. B.,
Lazebnik, Y. A.,
and Hardwick, J. M.
(1999)
J. Biol. Chem.
274,
21155-21161 |
36. | Chau, B. N., Cheng, E. H. Y., Kerr, D. A., and Hardwick, J. M. (2000) Mol. Cell 6, 31-40[Medline] [Order article via Infotrieve] |
37. | Hinds, M. G., Norton, R. S., Vaux, D. L., and Day, C. L. (1999) Nat. Struct. Biol. 6, 648-651[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Dierlamm, J.,
Baens, M.,
Wlodarska, I.,
Stefanova-Ouzonova, M.,
Hernandez, J. M.,
Hossfeld, D. K.,
De Wolf-Peeters, C.,
Hagemeijer, A.,
Van den Berghe, H.,
and Marynen, P.
(1999)
Blood
93,
3601-3609 |
39. | Uren, A. G., O'Rouke, K., Aravind, L., Pisabarro, M. T., Seshagiri, S., Konin, E. V., and Dixit, V. M. (2000) Mol. Cell 6, 961-967[Medline] [Order article via Infotrieve] |
40. |
Baens, M.,
Maes, B.,
Steyls, A.,
Geboes, K.,
Marynen, P.,
and De Wolf-Peeters, C.
(2000)
Am. J. Pathol.
156,
1433-1439 |
41. |
McCarthy, J. V.,
and Dixit, V. M.
(1998)
J. Biol. Chem.
273,
24009-24015 |
42. | Hawkins, C., Ekert, P., Uren, A., Holmgreen, S., and Vaux, D. (1998) Cell Death Differ. 5, 569-576[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Cheng, E. H. Y.,
Kirsch, D. G.,
Clem, R. J.,
Ravi, R.,
Kastan, M. B.,
Bedi, A.,
Ueno, K.,
and Hardwick, J. M.
(1997)
Science
278,
1966-1968 |
44. | Li, F., Ambrosini, G., Chu, E. Y., Plescia, J., Tognin, S., Marchisi, P. C., and Altieri, D. C. (1998) Nature 396, 580-584[CrossRef][Medline] [Order article via Infotrieve] |
45. | Li, F., Ackermann, E. J., Bennett, C. F., Rothermel, A. L., Plescia, J., Tognin, S., Villa, A., Marchisio, P. C., and Altieri, D. C. (1999) Nat. Cell Biol. 1, 461-466[CrossRef][Medline] [Order article via Infotrieve] |