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INTRODUCTION |
Apoptosis is an evolutionarily conserved process of cell
autodestruction that plays a critical role in development, the
maintenance of tissue homeostasis, and the protection against diseases
(1, 2). The central executioner of apoptosis is a family of aspartic acid-specific cysteine proteases known as caspases (3, 4). A major
mammalian apoptosis pathway leading to caspase activation is the
intrinsic/mitochondrial pathway, which is induced by diverse intracellular death stimuli such as developmental cues, cellular stresses, severe DNA damages, and oncogenic transformation (5, 6).
These stimuli lead to the release of several cell death inducers from
the mitochondria, including cytochrome c and Smac (also
known as DIABLO). Cytochrome c promotes the formation of a
cytosolic protein complex that activates an initiator caspase, caspase-9 (7). Smac,1 on the
other hand, promotes caspase-9 activation by neutralizing the
inhibitory effect on caspases of the X-linked
inhibitor of apoptosis (XIAP) (8, 9).
The IAPs comprise a group of conserved proteins crucial for the
regulation of apoptosis (reviewed in Ref. 10). Initially identified as
baculoviral proteins that can functionally substitute the
baculovirus-encoded caspase inhibitor p35, the IAP family now includes
several mammalian cellular proteins (e.g. XIAP/MIHA/h-ILP, cIAP1/HIAP2/hMIHB, cIAP2/HIAP-1/hMIHC, NAIP, ML-IAP, and survivin) and
two Drosophila proteins (DIAP1 and DIAP2/dILP).
Structurally, all IAPs contain one to three baculovirus IAP repeats
(BIRs). IAPs can directly inhibit caspases through their BIR domains
and intervening linker regions. For example, XIAP has been shown to inhibit caspases-3, -7, and -9 through the BIR region (11, 12), whereas
the corresponding region of DIAP1 inhibits Drosophila caspases, including DRICE and DCP-1 (13, 14). Smac counteracts the
inhibitory effect of XIAP by masking the caspase-9-binding site on XIAP
(8, 9, 15, 16). The IAP binding motif of Smac was subsequently found to
be conserved in three Drosophila death inducers, HID, Grim,
and Reaper (17), that are required for Drosophila cell death
(reviewed in Ref. 18). These three proteins induce apoptosis through
the inhibition of DIAP1 (13, 19), likely via a mechanism similar to
that of Smac (17). In addition to XIAP, Smac also interacts with cIAP1
and cIAP2 (8, 9). Unlike XIAP, the inhibitory effect of the cIAPs on
caspases is weak (100-1,000-fold less than that of XIAP) (20). The
mechanism by which cIAP1 and cIAP2 regulate apoptosis remains less understood.
Many IAPs, including XIAP, cIAP1, cIAP2, and DIAP1, also contain a
COOH-terminal RING domain. The RING domains on XIAP and cIAP1, like
those found in many other proteins (21), have been shown to possess E3
ubiquitin ligase activity (22, 23). Protein polyubiquitination, which
typically targets proteins for degradation in the 26 S proteasome,
involves the sequential action of three enzymes: the
ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (Ubc
or E2), and a ubiquitin ligase (E3) (24, 25). The substrate specificity
is mainly determined by E3, which binds to both E2 and the substrate
and facilitates the assembly of a multiubiquitin chain on the
substrate. The E3 activity of XIAP and cIAP1 can mediate their
self-degradation during the apoptosis of thymocytes in response to
certain apoptosis stimuli. cIAP1 also regulates the level of TRAF2, an
intracellular signaling molecule for the tumor necrosis factor receptor
2. The degradation of TRAF2 by cIAP1 appears to sensitize cells
to TNF-mediated apoptosis (26). On the other hand, the RING domain of
IAPs has been shown to inhibit apoptosis in a cell type- and/or death
stimulus-dependent manner (27, 28). The cIAP1 RING domain
can mediate the monoubiquitination of caspases-3 and -7 (23), and the
XIAP RING domain promotes caspase-3 degradation through
polyubiquitination (29). However, it remains unclear whether the E3
activity of IAPs targets proapoptotic proteins other than caspases.
Here we present evidence that cIAP1 and cIAP2 are E3 ubiquitin ligases
for Smac and promote the degradation of this critical death inducer.
The substrate-dependent E3 activity of cIAPs requires an
intact RING finger domain. We also identify putative
ubiquitin-conjugating enzymes (E2) for cIAP1 and -2. In addition, DIAP1
is able to ubiquitinate Grim and HID, two Drosophila
functional homologues of Smac. These results show that cIAPs and DIAP1
can also inhibit apoptosis through the degradation of death inducers.
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EXPERIMENTAL PROCEDURES |
Cell Culture, Antibodies, and Recombinant Proteins--
Human
embryonic kidney 293T and cervix carcinoma HeLa cells were cultured in
complete Dulbecco's modified Eagle's medium with 10% fetal bovine
serum. Rabbit polyclonal antibodies against FLAG and hemagglutinin (HA)
tags (Santa Cruz Biotechnology), anti-FLAG monoclonal antibody M2 and
M2 conjugated on agarose beads (Sigma), and yeast ubiquitin-activating
enzyme (E1), ubiquitin-conjugating enzyme (E2) GST-UbcH5c, and
His6-ubiquitin (Boston Biochem) were purchased from the
indicated sources.
Expression Plasmids--
All mammalian expression constructs
were made in pRK5. The cDNAs for cIAP1, cIAP2, XIAP, caspase-9, and
all E2s were of human origin. The cDNA fragments for the
full-length E2s were obtained from a lymph node or a leukocyte cDNA
library (Clontech) by PCR. All E2s were tagged with
an HA at the NH2 terminus, and the position of the tags for
other constructs is indicated in the figures and/or text. The amino
acid residues present in the following deletion mutant constructs
are indicated in parentheses: cIAP1mut(1-587), cIAP2mut(1-573), cIAP1C(452-618), cIAP2C(441-604),
cIAP2
BIR1(101-587), and DIAP1mut(1-381).
Transfection, Immunoprecipitation, and Western Blotting
Analysis--
Transient transfection was performed using the calcium
phosphate precipitation method. Unless indicated otherwise, cells were transfected with 0.1-10 µg of the indicated plasmids to minimize the
difference in the expression levels of various proteins. Sixteen to
22 h after transfection, cell lysates were prepared in 1% Nonidet P-40 lysis buffer (50 mM HEPES, 150 mM NaCl,
10% glycerol, 2 mM dithiothreitol, 1% Nonidet P-40, 1 mM EDTA) and immunoprecipitated with anti-FLAG monoclonal
antibody M2 beads. The immunoprecipitates and cell lysates were
resolved by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.
In Vitro Ubiquitination Assay--
Mature Smac protein was
obtained by the TNT-coupled transcription/translation
system (Promega) according to the manufacturer's instructions.
FLAG-tagged cIAP1, XIAP, and cIAP2mut (which lacked the first BIR
domain) were expressed in 293T cells and immunoprecipitated from the
cell lysates with the anti-FLAG M2 monoclonal antibody conjugated on
agarose beads. For the experiments shown in Fig. 5, A and
B, the immunoprecipitates were washed twice with washing buffer (25 mM Tris-HCl, pH 7.5, 50 mM NaCl,
0.01% Nonidet P-40, 10% glycerol, 1 mM EDTA) and then
added to a ubiquitination reaction mixture containing 120 ng of yeast
E1, 2.4 µg of UbcH5c, 4 µg of His6-ubiquitin, and 3 µl of radiolabeled mature Smac in 50 µl total volume. After
incubation at 30 °C for 90 min with vigorous shaking, the reaction
was terminated by boiling in the Laemmli SDS-loading buffer. Four
microliters of the reaction mixture was resolved by SDS-PAGE followed
by autoradiography. For the experiment in Fig. 5C, in
vitro translated mature Smac was first incubated with the FLAG
immunoprecipitating beads at 30 °C for 90 min to allow Smac to bind
to the bead-bound E3s. After the ubiquitination reaction, the beads
were washed three times with 1% Nonidet P-40 lysis buffer, resolved on
SDS-PAGE, and analyzed by autoradiography.
Apoptosis Assay--
HeLa cells were transfected with 0.5 µg
of Grim-HA together with 0.1 µg of a
-galactosidase reporter
plasmid and 1 µg of DIAP1, DIAP mutant, or control vector. Twenty h
after transfection, cells were fixed in 0.5% glutaraldehyde and
stained with
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal).
The percentage of apoptotic cells was determined by the number of
membrane blebbed cells divided by the total number of blue cells, as
previously described (30).
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RESULTS |
Expression of cIAP1 and cIAP2 Decreased the Steady-state Level of
Smac--
The cell death inducer Smac is synthesized as a precursor,
which becomes the mature form with the cleavage of its
NH2-terminal mitochondrial targeting sequence upon entering
the mitochondria (8). During apoptosis, Smac is released into the
cytosol, where it becomes associated with IAPs. To examine whether IAPs
can target Smac for degradation, we co-expressed in 293T cells the
mature Smac protein together with cIAP1, cIAP2, or XIAP. Interestingly, the amount of Smac protein decreased significantly upon co-expression with either cIAP1 or cIAP2 but not with XIAP (Fig.
1A). The down-regulation of
the mature Smac protein was unlikely due to the inhibition of apoptosis
because XIAP effectively blocked apoptosis (data not shown). In
addition, because the expression levels of XIAP were much higher than
those of cIAP1 and cIAP2 (Fig. 1A), the decrease in Smac
expression was unlikely caused by the squelching effect of the
simultaneous overexpression of multiple proteins. To rule out the
possibility that overexpression of cIAPs led to the generalized
inhibition of protein expression, we examined the effect of cIAPs on
endogenous Smac and found that its expression level was not changed by
cIAP overexpression (Fig. 1B).

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Fig. 1.
cIAP1 and cIAP2 induce down-regulation of
mature Smac. A, cIAPs but not XIAP destabilize cytosolic
mature Smac. HEK293 cells were transfected with Smac-HA and each of the
FLAG-tagged IAPs. The cell extracts from transfected cells were
immunoprecipitated (IP) with the anti-FLAG monoclonal
antibody M2. Cell extracts and anti-FLAG immunoprecipitates were
resolved on SDS-PAGE, followed by an immunoblotting (IB)
analysis with the indicated antibodies. Relative sample loading was
shown by an anti-actin immunoblot (middle panel).
Mr standards (in kDa) are shown on the
left. Smac, mature Smac. B, IAPs do
not affect the level of mitochondrial Smac. 293T cells were transfected
with cIAPs and XIAP. Cell extracts and anti-FLAG immunoprecipitates
were analyzed by immunoblotting with the indicated antibodies.
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Ubiquitination of Smac by cIAP1 and cIAP2--
The cIAP-induced
destabilization of Smac and the low expression levels of cIAPs suggest
that cIAP1 and cIAP2 might target Smac as well as themselves for
degradation via ubiquitination. To compare the self-ubiquitination of
the cIAPs with XIAP, we expressed separately FLAG-tagged cIAP1, cIAP2,
and XIAP in 293T cells together with HA-tagged ubiquitin. Both cIAP1
and cIAP2 were modified with multiple ubiquitins, as indicated by the
appearance of higher molecular weight products (Fig.
2A, top panel). In
contrast, the self-ubiquitination of XIAP was substantially weaker,
consistent with its relatively high expression levels (Fig.
2A).

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Fig. 2.
cIAP1 and cIAP2-mediated Smac ubiquitination.
A, auto-ubiquitination of cIAPs. FLAG-cIAP1, -cIAP2, or
-XIAP were transfected with HA-ubiquitin (HA-Ub) into 293T
cells. Anti-FLAG immunoprecipitates (IP) were analyzed by
immunoblotting (IB) with the indicated antibodies.
B and C, ubiquitination of mature Smac by cIAP1
and cIAP2. Smac-HA (B) or Smac-FLAG (C)
were transfected with FLAG-tagged cIAPs or cIAPmuts (B) or
Myc-tagged cIAPs (C). In this experiment as well as the
following ones, the amount of cIAP plasmids used for transfection was
much higher than that of XIAP to minimize the difference in the levels
of protein expression. The anti-FLAG immunoprecipitates and extracts
were analyzed by Western blotting with the indicated antibodies. *, IgG
light chain. D, ubiquitination of Smac is mediated by the
cIAP RING domains. Smac-HA was co-expressed with cIAP, cIAP1mut, or
cIAP2mut (the latter two mutants were missing the last 31 amino acids
of the corresponding wild-type proteins). The extracts and anti-FLAG IP
were analyzed by immunoblotting. E, cell
type-dependent ubiquitination of Smac by cIAPs. Smac-HA was
expressed with FLAG-cIAP1, -cIAP2, or -XIAP in HeLa cells or
with FLAG-cIAP1 in 293T cells. The cell lysates and the anti-FLAG
immunoprecipitates were analyzed by Western blotting. The middle
panel is a longer exposure of the top panel to show the
ubiquitinated products.
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To determine whether cIAPs and XIAP can ubiquitinate mature Smac, we
expressed cIAPs and XIAP together with mature Smac in 293T cells. The
mature Smac protein that bound to cIAP1 and cIAP2 was markedly
ubiquitinated, showing a ladder pattern characteristic of mono- and
polyubiquitination (Fig. 2B, top panels). The
cIAP1-mediated Smac ubiquitination was so strong that the majority of
mature Smac proteins were modified even without the addition of
exogenous ubiquitin. The cIAP self-ubiquitination and cIAP-mediated
Smac ubiquitination were further confirmed by immunoblotting analysis using an antibody against ubiquitin (Fig. 2C). XIAP,
however, did not induce significant ubiquitination of mature Smac (Fig. 2B).
The RING domains of cIAPs are required for their self-ubiquitination
(22). To test whether these domains are also needed for the
cIAP-mediated ubiquitination of Smac, we deleted the RING domains of
cIAP1 and cIAP2. As shown in Fig. 2D, these RING deletion mutants failed to ubiquitinate mature Smac. Thus, ubiquitination of Smac by IAPs is RING domain-dependent.
To examine whether other cellular factors are involved in the
cIAP-induced Smac ubiquitination, we expressed cIAPs and mature
Smac
in HeLa cells. As a positive control, we also expressed them in 293T
cells. We found that the binding efficiency of mature Smac to cIAPs in
these two different cells was comparable. However, no ubiquitination of
mature Smac was observed in HeLa cells whereas the Smac
ubiquitination was readily detected in 293T cells (Fig. 2E, top two panels). This result supports the
notion that the cIAP-dependent ubiquitination of
mature Smac is regulated by other cellular factors.
Ubiquitination of Smac by cIAPs Requires Their Physical
Association--
The interactions between IAPs and mature Smac are
mediated by the IAP BIR domains and the Smac NH2-terminal
region, particularly a 4-amino acid stretch (AVPI). To confirm the
specificity of cIAP-dependent ubiquitination of Smac, we
generated a mutant cIAP1 construct lacking all the BIR domains and a
mutant mature Smac construct with the AVPI motif deleted. The BIR
deletion mutant of cIAP1 failed to bind to and degrade Smac (Fig.
3A), even though this region
retained E3 activity (22). In addition, the AVPI deletion mutant of
Smac only weakly associated with cIAPs. This mutant was not
ubiquitinated by cIAP1 and showed higher stability in the presence of
cIAP1 compared with the wild-type mature Smac (Fig. 3B).
Thus, cIAP-mediated Smac ubiquitination and degradation requires
specific interactions between cIAPs and Smac.

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Fig. 3.
Ubiquitination of Smac requires its
association with cIAP1. A, the cIAP1 BIR region is essential
for Smac ubiquitination. FLAG-IAP1, -IAP1C, or the vector control were
transfected into 293T cells with Smac-HA. The cell lysates were
incubated with anti-FLAG M2. The extracts and anti-FLAG
immunoprecipitates (IP) were analyzed by immunoblotting
(IB). B, deletion of the AVPI motif of Smac
impairs its ubiquitination by cIAP1. Smac-HA or Smac- AVPI-HA
were transfected into 293T cells with either FLAG-cIAP1 or the control
vector.
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cIAP1, cIAP2, and XIAP Associate with Overlapping but Partially
Distinct Subsets of E2s--
A eukaryotic genome encodes only one, or
at most a few, E1. In contrast, it encodes a dozen or so E2s. To
determine which E2s can associate with cIAP1, cIAP2, and XIAP, each IAP
was expressed in 293T cells with the E2s that have been previously
found to associate with various RING domain E3s. The COOH-terminal
region of cIAP2 was used in these experiments because it had higher
stability. Co-immunoprecipitation assays revealed that cIAP1
preferentially interacted with UbcH7 and its close homologue UbcH8, as
well as with UbcH5C and UbcH5A, whereas cIAP2 associated strongly with all UbcH5 family E2s and weakly with UbcH7, but not with UbcH8 (Fig. 4,
A and B). XIAP, on
the other hand, associated with all E2s tested but with a weak binding
affinity to UbcH5C and UbcH8 (Fig. 4C). It is common for an
E3 to bind to multiple E2s. For example, Parkin, a
RING-containing E3 that is associated with Parkinson's
disease, interacts with several E2s (31). E2 recruitment in
vivo can also be determined by the presence of other regulatory proteins and the availability of E2s. Because XIAP binds to several E2s, the weak ligase activity of XIAP toward Smac was unlikely caused
by a defective E2 binding. In addition, because only the cIAP2
COOH-terminal region that encompasses the RING domain was used for the
binding assay (Fig. 4B), the NH2-terminal BIR
domains are dispensable for E2 recruitment.

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Fig. 4.
cIAP1, cIAP2, and XIAP interact with
overlapping but partially distinct subsets of E2s. FLAG-cIAP1
(A), -cIAP2C (containing the CARD and the RING domains,
B), or -XIAP (C) were transfected into 293T cells
with either the indicated HA-tagged E2 (+) or the control vector ( ).
The cell lysates were incubated with anti-FLAG M2. The
immunoprecipitated proteins and the cell extracts were analyzed by
Western blotting with the indicated antibody.
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In Vitro Reconstitution of Ubiquitination of Smac by
cIAPs--
Having identified putative E2s for cIAP1, cIAP2, and XIAP,
we next examined the ubiquitination of Smac by these IAPs using an
in vitro ubiquitination assay. FLAG-tagged full-length cIAP1 and XIAP and a deletion mutant of cIAP2 lacking the first BIR domain
were expressed in mammalian cells and affinity purified with an
anti-FLAG antibody. The mutant cIAP2 was used because it was expressed
at a higher level than the wild-type proteins yet still retained the
capacity to bind to Smac (data not shown). When the purified cIAP1 or
cIAP2
BIR1 protein was added to a ubiquitination reaction mixture
containing recombinant E1 and E2 (UbcH5C) and in vitro
translated, metabolically labeled mature Smac, ubiquitination of Smac
was evident as shown by the appearance of higher molecular weight
materials (Fig. 5, A and
B, lane 4 versus 1-3). The
ubiquitination was significantly enhanced when recombinant ubiquitin
was also included in the reaction (lane 5 versus
4). In comparison, XIAP ubiquitinated Smac very weakly even though
the amount of XIAP used was much higher than that used with cIAP1 (Fig.
5C, lane 3 versus 2). These results
confirm that both cIAP1 and cIAP2 are strong ubiquitin ligases but
suggest that XIAP is a weak ubiquitin ligase for mature Smac.

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Fig. 5.
In vitro ubiquitination of mature
Smac by cIAPs and XIAP. In vitro translated,
35S-labeled mature Smac was incubated with recombinant E1,
UbcH5c, His6-Ub, and FLAG-cIAP1 (A and
C), -cIAP2 BIR1 (which lacked the first BIR domain,
B), or -XIAP (C). The IAP proteins were expressed
in 293T cells and purified using the anti-FLAG M2 beads. The total
reaction mixture (A and B) or the M2 beads-bound
fraction (C) were resolved on SDS-PAGE and analyzed either
by autoradiography (A, B, and the top
panel of C) or immunoblotting (IB) with an
anti-FLAG antibody (C, bottom panel).
IP, immunoprecipitate.
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DIAP1-dependent Ubiquitination of Grim and
HID--
Drosophila IAP1 (DIAP1) plays a critical role in
apoptosis inhibition. Flies defective with DIAP1 die in the embryonic
stage because of excessive cell death (13). Upon activation of
apoptosis, the Drosophila death inducers Grim, HID, and
Reaper bind to and eliminate the inhibitory effect of DIAP1 on
caspases, via an NH2-terminal motif that is shared with
each other and with the mammalian functional homologue Smac (13, 17,
19). Several recent studies showed that Hid, Grim, and Reaper promote
the autoubiquitination and degradation of DIAP1 (32-35). To determine
whether DIAP1, like cIAPs, can mediate the ubiquitination of the death
inducers, we examined the effect of DIAP1 on Grim and HID
expression levels. Expression of DIAP1 in 293T cells resulted in marked
self-ubiquitination, with the appearance of a ladder of higher
molecular weight materials typical of ubiquitination. This DIAP1
self-ubiquitination depended on an intact RING domain (Fig.
6, A and B,
bottom panel, lane 2 versus
3). DIAP1 also ubiquitinated the co-expressed Grim and HID
(Fig. 6, A and B, top and middle
panels). The expression levels of Grim and HID were increased,
rather than decreased, upon the expression of DIAP1 (Fig. 6,
A and B, middle panel), consistent with a previous report that the BIR domains of DIAP1 could enhance the
stability of Grim/HID likely through a direct protein-protein interaction (36). However, the levels of Grim and HID were even higher
in the presence of the RING deletion mutant of DIAP1, which lost the
ability to ubiquitinate itself and Grim/HID (Fig. 6, A and
B, lane 3 versus 2). To further
confirm that the DIAP1-mediated ubiquitination leads to degradation of
Grim, we deleted the only lysine residue in the Grim protein. The
resulting Grim mutant could no longer be ubiquitinated by DIAP1, and
concurrently the level of Grim protein was markedly increased compared
with the wild-type protein (data not shown). Taken together, these
results show that the E3 activity of DIAP1 can trigger degradation of both Grim and HID.

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Fig. 6.
DIAP1-induced ubiquitination and degradation
of Grim and HID. A and B, ubiquitination of Grim
(A) and HID (B) induced by DIAP1. HA-Grim
(A) or HA-HID (B) were transfected into 293T
cells with the vector control, FLAG-DIAP1, or FLAG-DIAP1mut (which
lacked the COOH-terminal RING domain). Cell extracts were
immunoprecipitated with the anti-FLAG antibody. The extracts and the
FLAG immunoprecipitation samples were analyzed by Western blotting with
the indicated antibody. In the bottom panels, the
immunoblots were exposed to film for a prolonged period of time after
Western hybridization to show the ubiquitinated species. The total
amount of DIAP1 protein in lanes 2 and 3 was
similar based on results from a short exposure time. *, a nonspecific
band. **, IgG heavy chain. C, involvement of the DIAP1 RING
domain in the inhibition of Grim-mediated apoptosis. HeLa cells were
transfected with Grim plus either DIAP1 or DIAP1mut. Top
panel, apoptosis was scored among the transfected cells as
described under "Experimental Procedures." Data (mean ± S.D.)
were obtained from representative experiments performed in duplicate.
Middle and lower panels, expression of DIAP1,
DIAP1mut, and Grim. DIAP1 interacted with both Grim and HID in HeLa
cells, and unlike cIAPs, was able to induce the ubiquitination of
Grim/HID in these cells (data not shown).
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To address the functional significance of the IAP E3 activity in
apoptosis regulation, an attempt was made to compare the apoptosis
inhibition ability of the wild-type and the E3-defective cIAP mutants.
Paradoxically, overexpression of cIAPs enhanced apoptosis in 293T
cells. Although an anti-apoptotic function of cIAPs has been previously
demonstrated (37, 38), overexpression of cIAPs in some cells has also
been reported to enhance apoptosis, unless the RING domain was deleted
(26, 39). This proapoptotic activity of cIAPs could be because of the
existence of anti-apoptotic cIAP1 substrates, such as TRAF-2 (26). The
E3 activity of cIAP thus plays a complex role in the regulation of
apoptosis. Overexpression of DIAP1, on the other hand, was not
cytotoxic in either 293T or HeLa cells. Rather, DIAP1 potently
inhibited Grim-induced apoptosis in these cells in a transient
transfection apoptosis assay (Fig. 6C, top panel,
and data not shown). In comparison, the E3 defective RING mutant of
DIAP1 was substantially less effective in blocking Grim-induced
apoptosis (Fig. 6C). Therefore, the DIAP1 E3 activity contributes to its apoptosis inhibitory function.
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DISCUSSION |
The IAPs family is a major group of apoptosis regulators. IAPs
have been shown to directly block caspase activity. In this study we
found that cIAP1 and cIAP2 can also target the mitochondrial cell death
inducer Smac for degradation via the COOH-terminal RING domain-mediated
ubiquitination. Similarly, Drosophila DIAP1 possesses E3
activity that can mediate the ubiquitination and degradation of the
Drosophila death inducers Grim and HID. These results
suggest a novel and evolutionarily conserved mechanism for the
anti-apoptotic function of IAPs.
Among the three mammalian IAPs that interact with Smac, cIAP1 and cIAP2
have strong E3 ligase activity toward Smac. In contrast, the
substrate-dependent ligase activity of XIAP is weak, even though it can tightly bind to Smac. A recent report showed that Smac is
rapidly degraded upon release from the mitochondria during death
receptor-mediated apoptosis and that the Smac degradation is dependent
on the proteasome pathway (40). The authors also detected XIAP-mediated
ubiquitination of Smac in vitro. However, by directly
comparing cIAPs and XIAP both in vivo and in
vitro, we are able to show the marked difference in their E2
activities toward Smac. XIAP has been shown to be a much more potent
inhibitor of caspases than cIAPs (20). Therefore, it is likely that
cIAPs and XIAP may inhibit apoptosis through different mechanisms.
Whereas XIAP may directly bind to and inhibit caspases, cIAP1 and cIAP2 may degrade death inducers such as Smac to prevent it from relieving the inhibitory effect of XIAP on caspases.
It has been shown that IAPs bind to Smac through their BIR domains. We
have found in this study that cIAPs can also interact with several E2s.
Thus, cIAPs may function as single molecule E3s that interact with both
the substrate and E2 (Fig.
7A), and these interactions
may be regulated by other cellular factors, as suggested by the lack of
cIAP-mediated ubiquitination in HeLa cells. In contrast, in the SCF
(Skp1, Cullin, F-box) ubiquitin ligase complex, which plays an
important role in cell cycle regulation, the RING-containing E3 (Roc1)
associates with the substrate through several intermediary proteins and
the substrate specificity is determined mainly by the F-box-containing
protein (Fig. 7B) (24, 25). Given that both Smac and
Grim/HID interact with the BIR region of IAPs, this region might
determine the substrate specificity of the IAPs, and it would be
interesting to identify additional IAP substrates that associate with
this region. The inhibition of IAPs by the death inducer Smac/Grim/HID
and the ubiquitination of death inducers by IAPs indicate that there
are dynamic interactions between these two groups of anti- and
proapoptotic proteins. These interactions, like those between the anti-
and proapoptotic members of the Bcl-2 family proteins, may greatly
influence the lives and deaths of cells.

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Fig. 7.
The IAP E3 ubiquitin ligase versus
the SCF E3. IAPs function as single molecule E3 ubiquitin
ligases. IAPs bind to substrates (e.g. Smac/Grim/HID)
through the BIR region and to E2 through the RING domain. In the SCF E3
complex, multiple proteins are responsible for bringing substrates and
the E2 into close proximity. The RING finger protein ROC1 recruits E2
whereas F-box proteins determine the substrate specificity.
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