Download Direct Interaction between Survivin and Smac/DIABLO Is Essential

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 278, No. 25, Issue of June 20, pp. 23130 –23140, 2003
Printed in U.S.A.
Direct Interaction between Survivin and Smac/DIABLO
Is Essential for the Anti-apoptotic Activity of Survivin during
Taxol-induced Apoptosis*
Received for publication, January 29, 2003, and in revised form, March 19, 2003
Published, JBC Papers in Press, March 26, 2003, DOI 10.1074/jbc.M300957200
Zhiyin Song, Xuebiao Yao, and Mian Wu‡
From the Department of Molecular and Cell Biology, Key Laboratory of Structural Biology, School of Life Sciences,
University of Science and Technology of China, Hefei, Anhui 230027, People’s Republic of China
Survivin is a member of the inhibitor of apoptosis
protein (IAP) family that has been implicated in both
apoptosis inhibition and cell cycle control. However, its
inhibitory mechanism and subcellular localization remain controversial. In this report, we provided evidence
for the first time that Survivin physically interacts with
Smac/DIABLO both in vitro and in vivo. A point mutation (D71R) in the baculovirus IAP repeat motif and a
C-terminal deletion mutant (Surv-BIR) of Survivin fail
to bind to Smac/DIABLO and abrogate its ability to inhibit apoptosis. The N-terminal of mature Smac/DIABLO
is absolutely required for Survivin䡠Smac complex formation. Subcellular distributions of Survivin and Smac/
DIABLO showed that they co-localized within the cytosol during interphase. In addition, Survivin was found
to be incapable of binding to caspase. We also identified
that the co-presence of Smac/DIABLO and XIAP was
required for Survivin to inhibit caspase cleavage in a
cell-free system. In conclusion, our results provide the
first evidence that the interaction between Smac/DIABLO and Survivin is an essential step underling the
inhibition of apoptosis induced by Taxol.
Among the regulators of apoptosis, considerable interest has
been focusing on the inhibitors of apoptosis protein (IAP)1
family, which were first identified as negative regulators in
programmed cell death characterized by the presence of one to
three copies of the baculovirus IAP repeat (BIR) domain (1).
However, anti-apoptotic function in physiological cell death has
not been established for all IAPs, because some members in
this family play essential roles in cell division, rather than
merely acting as the regulators of apoptosis (2). Survivin (16.5
KDa) is a special member of the inhibitor-of-apoptosis proteins
(IAPs) family containing a single BIR and lacking a RING
finger motif. It is mostly expressed in the vast majority of
tumors or in the embryonic development, but not in normal
* This research was supported by the Key Project Fund (KSCX2-201-004), a special grant (to M. W.) from the Chinese Academy of Sciences, the National Natural Science Foundation of China (Grants
30121001 and 90208027), and a 973 grant (Grant 2002CB713700) from
the Ministry of Science and Technology of China. The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed. Tel.: 86-551-3606264; Fax: 86-551-360-6264; E-mail: [email protected].
1
The abbreviations used are: IAP, inhibitor of apoptosis protein; BIR,
baculovirus IAP repeat; GST, glutathione S-transferase; Ab, antibody;
GFP, green fluorescence protein; EGFP, enhanced GFP; BSA, bovine
serum albumin; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; cyto c, cytochrome c; TRAIL, tumor necrosis factor-related
apoptosis-inducing ligand.
adult tissues (3, 4). Survivin is cell cycle-regulated and predominantly expressed in the G2/M phase (5). The degradation of
Survivin is believed to be through the ubiquitin-proteasome
pathway (6). Accumulating evidence supports the idea that
Survivin is a bifunctional protein that acts as a cell division
regulator (7–14) and an apoptosis suppressor (3, 5, 15, 16). The
mechanism of how Survivin inhibits apoptosis remains controversial. Tamm et al. (16) demonstrated that Survivin was able
to bind to the effector caspase-3 and caspase-7 in vitro and
proposed that Survivin may inhibit caspase activity in physiological cell death by the similar mechanism. However, comparison between the x-ray crystallographic structures of Survivin
and that of the XIAP (BIR2)䡠caspase-3 complex fails to reveal
any clues of how Survivin could directly suppress caspase-3
(17). Verdecia et al. (18) and Banks et al. (19) have demonstrated that Survivin is unable to directly bind to caspase-3 in
vitro and does not inhibit caspase-3 activity. Wang and colleagues identified a novel mitochondrial-associated protein
Smac/DIABLO that was able to interact with IAPs (20, 21) and
may help explain the inhibitory function of Survivin.
Smac/DIABLO is a mitochondria protein but is released into
the cytosol in response to some of apoptotic stimuli, including
UVB-irradiation, etoposide, or glucocorticoids (20 –23). After
mitochondria import, the N terminus of precursor Smac/DIABLO is removed by limited proteolysis to generate a mature
form of the molecule (20, 27). Mature Smac/DIABLO was found
to promote caspase activation by binding and neutralizing the
IAPs, including XIAP, CIAP-1, and CIAP-2 (20, 21, 23). Numbers of reports confirmed that mature Smac/DIABLO interacts
with BIR2 and BIR3 of XIAP, and the N terminus of mature
Smac/DIABLO is absolutely required for this interaction (24,
26 –29). However, whether Smac/DIABLO physically interacts
with Survivin has yet to be fully characterized.
In this report, we investigated the effect of interactions between Survivin and Smac/DIABLO on the apoptosis induced by
Taxol (paclitaxel). We have demonstrated that Taxol is able to
trigger apoptosis significantly in HeLa cell, resulting in the
activation of cellular caspases-3, -7, and -9 and release of Smac/
DIABLO and cytochrome c from the mitochondrial. Ectopic
expression of Survivin was able to significantly block the Taxolinduced cell death. Our GST pull-down binding assay and
immunoprecipitation demonstrate for the first time that Survivin directly interacted with Smac/DIABLO but not with
caspases. We also found mutant Survivin with a single amino
acid change at residue Asp-71 (D71R) or truncated Survivin
lacking the C terminus (residues 1–97) failed to bind to Smac/
DIABLO and abrogated its ability to inhibit apoptosis. On the
contrary, these two Survivin mutants displayed some apoptotic
effects. In addition, we have shown that wild type Survivin
does not interact with Smac mutants M-Smac (a methionine
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The Role of Survivin in Apoptosis
was added to the begin of mature Smac) or ⌬74Smac (residues
75–239). We examined subcellular distributions of Survivin
and Smac/DIABLO within living cells at different stages of the
cell cycle and found that Survivin and Smac are co-localized in
the cytosol during interphase, and when cells enter into M
phase Survivin acts as a chromosome passenger protein. In
addition, by using the cell-free system, we were able to demonstrate that both Smac/DIABLO and XIAP are required in order
for inhibition of caspase cleavage by Survivin. Combined, our
data strongly support our proposed hypothetical mode for Survivin in the inhibition of apoptosis. During apoptosis Survivin
binds to Smac, which is released from induced mitochondria.
Therefore, by reducing Smac/DIABLO antagonism to IAPs,
such as XIAP, the free XIAP directly interacts with caspases
and cell death is blocked.
MATERIALS AND METHODS
Oligonucleotides—The sequences of the oligonucleotides used in this
study are listed as follows. P1, 5⬘-CGGGATCCGCATGGGTGCCCCGACGTTG-3⬘; P2, 5⬘-CGGTCGACCCATCCATGGCAGCCAGCTG-3⬘; P3,
5⬘-GCAAGCTTATGGCGGCTCTGAAGAGTTG-3⬘; P4, 5⬘-GCGGATCCTCCTCACGCAGGTAGGCCT-3⬘; P5, 5⬘-CGGTCGACGTTAATTCTTCAAACTGCTT-3⬘; P6, 5⬘-GCGATTAATATGGCGGTTCCTATTGCAC-3⬘;
P7, 5⬘-CGGCGGCCGCATCCTCACGCAGGTAGGCCT-3⬘; P8, 5⬘-CGGGATCCGATTAATATGAGGAGAGCAGTGTCTTT-3⬘; P9, 5⬘-CGGGATCCTGATGGCGGTTCCTATTGCACAG-3⬘; P10, 5⬘-GGGAGCCAGATCGCGACCCCATAGAGGA-3⬘; P11, 5⬘-CTATGGGGTCGCGATCTGGCTCCCAGCC-3⬘; and P12, 5⬘-CGGGATCCTCAATCCATGGCAGCCAGCT-3⬘.
Reagents and Antibodies—The following antibodies were used in this
study: polyclonal antibodies Ab-caspase-7, Ab-caspase-3, Ab-survivin,
and Ab-actin (Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal
antibodies: mAb-bcl2 (Oncogene, Manhasset, NY), mAb-GFP (MBL,
Japan), mAb-cytochrome c (R&D Systems, Inc., Minneapolis, MN),
mAb-caspase-9 (Immunotech, France), and mAb-Smac/DIABLO (Calbiochem, La Jolla, CA). Taxol used in our experiments is labeled as GCP
grade. Restriction enzymes were mostly purchased from New England
BioLabs. Medium compounds were obtained from Oxid. The majority of
biochemical reagents were ordered from Sigma. Trypan blue was purchased from Invitrogen.
Cell Culture and Transfection—HeLa cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal
bovine serum, 1 ⫻ nonessential amino acid, 1 ⫻ minimal essential
medium sodium pyruvate, 100 ␮g/ml penicillin, 100 ␮g/ml streptomycin
(Invitrogen, Grand Island, NY). Cultured cells were incubated in a
humidified atmosphere containing 5% CO2 at 37 °C. Transfection of
cells with various mammalian expression constructs by LipofectAMINE
2000 (Invitrogen) was carried out according to the methods provided by
the manufacturer’s instructions.
PCR-mediated Mutagenesis—To obtain the mutant survivin, we
have employed the PCR-mediated mutagenesis method. Two pairs of
primers, P1/P10 (complementing the regions from nucleotides 1 to 18
and nucleotides 196 to 224, respectively) and P11/P2 (complementing
the regions from nucleotides 200 to 228 and nucleotides 408 to 426,
respectively), were used to amplify two Survivin fragments. After gel
purification, the resultant two overlapping PCR fragments were mixed
with equal amounts. This mixture was incubated first at 94 °C for 4
min, followed by first PCR (PCR1) of 94 °C denaturing for 1 min, 56 °C
annealing for 1 min, and 72 °C extension for 1 min. After 10 cycles,
another 8-min extension was added to ensure the completion of all the
extension at 72 °C. The PCR1 products were used as a template in a
second PCR (PCR2), primed by oligonucleotides P10 and P11 to carry
out another 20 cycles of PCR with the same PCR conditions used in
PCR1. A prominent band with an expected size of 0.42 kb was visible on
1% agarose gel. A point mutation, GAC (Asp) to CGC (Arg), was further
verified by DNA sequencing determination.
Plasmids Construction—The full-length Survivin coding sequence
(nucleotides 1– 426) and its mutant variant with a point mutation at
amino acid residue 71 (D71R) were cloned into BamHI/SalI sites of
pGEX-5X-3 (Amersham Biosciences, UK), respectively, to generate recombinant expression vectors pGEX-5X-3/Survivin and pGEX-5X-3/
Surv-D71R, and both fragments were inserted in-frame with the GST
gene. The gene fragment (nucleotides 1–291), containing only the Survivin BIR domain (residues 1–97), was generated by the PCR method
using primers P1 and P5 (see “Oligonucleotides”) and cloned into
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BamHI/SalI sites of pGEX-5X-3 to yield pGEX-5X-3/Surv-BIR. Gene
fragments coding for wild type Survivin, for mutant Survivin (SurvD71R) with a point mutation at amino acid residue 71, for the truncated
Survivin (Surv-BIR) lacking its C-terminal and Smac/DIABLO were
also cloned into pEGFP-C1 plasmid, respectively. In addition, the gene
fragments (reacted by PCR for primer pairs P12/P1) was used to generated pTRE2/A-Surv (coding for antisense Survivin). The DNA fragment coding for mature Smac/DIABLO (residues 56 –239) and DNA
fragment coding for mutant Smac/DIABLO (⌬74Smac, residues75–239)
were generated by PCR techniques using primer pairs P6/P7 and P8/P7,
respectively. The resultant PCR products were digested with restriction
enzymes VspI and NotI and cloned into NdeI/NotI sites of pET-22b
(Novagen, Madison, WI) to generate pET-22b/Smac and pET-22b/
⌬74Smac with an His6 tag fusion at its C terminus. A full-length
Smac/DIABLO gene fragment (residues1–239) resulted from PCR amplification with primers P3 and P4 inserted into HindIII/BamHI sites of
the mammalian expression vector pEGFP-N1 (Clontech, Palo Alto, CA)
to fuse to the EGFP gene, and the resultant recombinant plasmid was
designated as pEGFP-N1/F-Smac. DNA fragments, coding for mutant
Smac/DIABLO with an methionine residue added at the N terminus of
mature Smac/DIABLO (designated as M-Smac) and for mutant Smac/
DIABLO lacking 73 amino acid residues at its N terminus (designated
as ⌬73Smac), were reacted by PCR away from Smac/DIABLO cDNA
using primer pairs P9/P4 and P8/P4, respectively, and inserted into the
BamHI site of pEGFP-N1 in fusion with EGFP to generate plasmids
pEGFP-N1/M-Smac and pEGFP-N1/⌬73Smac.
Expression and Purification of Fusion Proteins—Overnight cultured
Escherichia coli cells DH5␣ or BL21/DE3 containing the fusion plasmids were grown at a 1:100 dilution in an Amp-LB medium until the
optical density reached A600 ⫽ 0.5. Isopropyl-1-thio-␤-D-galactopyranoside was then added to a final concentration of 0.4 mM. Cells were
induced for another 3 h before they were harvested and subjected to
sonication for 10 shot pulses of 20 s each. The maximum protein release
was determined by the Bradford assay (Bio-Rad) by comparing the
known concentration of bovine serum albumin (BSA). The GST fusion
proteins were purified through the glutathione-SepharoseTM 4B beads
(Amersham Biosciences). The solubilized His6-Smac or His6-⌬74Smac
fusion proteins were purified by incubating with chelating SepharoseTM
Fast Flow beads (Amersham Biosciences) according to the manufacturer’s instructions. The ultimate elution products were dialyzed with 1⫻
PBS and were used for in vitro interaction assay.
In Vitro Interaction Assay—An in vitro interaction assay was performed as described by Suzuki et al. (50). Taxol-stimulated HeLa cells
(2 ⫻ 106) were lysed with the 200-␮l lysis buffer as described above. Cell
lysates or bacterially expressed and purified His6 tag proteins were
incubated with GST or GST fusion protein immobilized on glutathioneSepharose 4B beads (Amersham Biosciences) for overnight at 4 °C and
washed three times with 500 ␮l of 1⫻ PBS. The bound proteins were
eluted by elution buffer, and eluted products were subjected to SDSPAGE for Western blot analysis or Coomassie Blue staining.
Cell Death Assay—The ability of Survivin, Smac/DIABLO, or their
mutants to affect cell viability was assayed by transfecting or HeLa
cells (2 ⫻ 104 cells/well) in 24-well plates with 0.3 ␮g of mammalian
expression vectors using LipofectAMINE 2000. Twenty hours after
transfection, cells continued to be incubated with 100 nM Taxol for 36 h,
and the viability of the cells was measured with the standard trypan
blue exclusion method by counting blue dead cells. Data are expressed
as percentages of control and are the means of three independent
experiments.
Western Blot Analysis—Cells were washed with 1⫻ PBS and resuspended with 5 volumes of cold lysis buffer (50 mM Tris-HCl (pH 7.5), 250
mM NaCl, 5 mM EDTA, 50 mM NaF, 0.5% Nonidet P-40) supplemented
with protease inhibitor mixture (Roche Applied Science). The cell lysate
was incubated on ice for 30 min and was then centrifuged for 10 min at
4 °C. The protein content of the supernatant was determined by using
a BCA-200 protein assay kit (Pierce). Equal amounts of proteins (10 –20
␮g) were loaded onto the gel and separated by SDS-PAGE, and the
resolved proteins were transferred to nitrocellulose membrane. After
blocking with 5% nonfat milk in TBST (20 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 0.1% Tween 20) for overnight at 4 °C, the blot was incubated with
primary antibody for 1 h at room temperature. The membrane was then
washed with TBST and probed with horseradish peroxidase-conjugated
secondary antibody for 1 h. The membrane was washed three times in
TBST and developed by ECL using the manufacturer’s protocol. Digitonin fractionation of cells into membrane and cytosolic fractions used
for detection of cytochrome c and Smac/DIABLO was performed as
described by Ekert et al. (23).
Immunoprecipitation—Cells were lysed in a Triton X-100-based lysis
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The Role of Survivin in Apoptosis
buffer (1% Triton X-100, 10% glycerol, 150 mM NaCl, 20 mM Tris, pH
7.5, 2 mM EDTA, protease inhibitor mixture) for 1 h, and the nuclear
and cellular debris was cleared by centrifugation. Then the cytosolic
lysis was incubated with Smac monoclonal antibody bound to Protein
A/G-Sepharose. After a 4-h incubation at 4 °C, the immunoprecipitates
were washed five times in lysis buffer, and proteins were recovered
by boiling the beads in SDS sample buffer and analyzed using a
Western blot.
Immunofluorescence Confocal Microscopy—HeLa cells were grown in
six-well chamber slides, and 24 h later, cells were either left untreated
or exposed to 100 nM Taxol for another 24-h incubation before 100 nM
MitoTracker Red was added and treated for 60 min at 37 °C. The cells
were then washed with PBS, fixed with 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and blocked for 1 h in 3%
BSA in PBS at 4 °C. Cells were incubated with anti-Smac (1:400 in 3%
BSA) or anti-cyto c antibody (1:400 in 3% BSA) for 1 h at room temperature. The primary antibody was recognized by secondary FITC-conjugated antibody. Finally, the stained cells were analyzed with a laser
scanning confocal microscopy system (Fluoview, Olympus).
RESULTS
Smac/DIABLO Is Released from the Mitochondria Accompanied with Cytochrome c upon Induction of Apoptosis by Taxol—
Taxol is a natural product with potent anti-tumor activity and
has been approved for the treatment of breast, ovarian, and
lung cancers (25, 30 –32). We incubated HeLa cells with Taxol
at a concentration of 100 nM, which is known to induce apoptosis in a number of different cell types. After 24-h incubation,
the clear morphological changes characteristic of apoptosis
were observed. Taxol-treated cells became rounded and detached from the substratum of the flask, followed by cellular
shrinkage and membrane blebbing (data not shown). Caspase
activation was believed to be the key factor during apoptosis.
To evaluate which of the caspases is activated during the
process of Taxol-induced apoptosis in HeLa cells, we analyzed
the cleavage pattern of caspase-9, caspase-7, and caspase-3 by
Western analysis methods. The results, shown in Fig. 1A, indicate that treatment with this drug results in the activation of
caspase-9, caspase-7, and caspase-3 as evidenced by the appearance of caspase-active forms. One apoptotic signaling pathway leading into caspase activation involves the translocation
of cytochrome c into the cytosol from the mitochondrial intermembrane space (33). To further investigate whether the mitochondria are involved in this event, Taxol-treated HeLa cells
were first lysed and divided into membrane pellet and cytosolic
fraction. The pellet was then treated with 0.5% bile acid deoxycholate and separated by centrifugation into a soluble and
membrane fraction. Cytosolic proteins and membrane proteins
solubilized by the deoxycholate treatment were subjected to
Western blot analysis. As shown in Fig. 1B (panel a), an increasing amount of cytochrome c was detected in the cytosolic
fractions, whereas a decreasing amount of cytochrome c remained in the mitochondrial membrane, and after 48 h, the
majority of cytochrome c was detected in the cytosolic fractions.
We also observed that Smac/DIABLO was predominantly present within the membrane fractions prior to treatment with
Taxol, and by 48 h post-treatment, there was a significant loss
of Smac/DIABLO from the membrane fractions and a concomitant increase of Smac/DIABLO in the cytosolic fractions (Fig.
1B, panel a). In addition, to further elucidate the behavior of
Smac/DIABLO or cyto c during Taxol-induced apoptosis, we
examined its subcellular localization in both untreated cells
and treated cells by immunostaining and confocal microscopy
(Fig. 1B, panel b). HeLa cells were stained with MitoTracker
Red to define the location of mitochondria and with anti-Smac
or anti-cyto c antibody plus an FITC-conjugated secondary
antibody. In uninduced HeLa cells, the localization of FITC and
MitoTracker Red staining gave rise to the yellow color indicating that both Smac/DIABLO and cyto c were localized within
the mitochondria. However, after induction with Taxol for 24 h,
FIG. 1. Mitochondrial cytochrome c and Smac are released
during induction of apoptosis by Taxol. A, HeLa cells were
treated with 100 nM Taxol for the indicated times before being harvested and lysed in lysis buffer. The processing of caspase-9 (a), -7 (b),
and -3 (c) was then analyzed by Western blot probing with their
respective antibodies. In B: a, HeLa cells were exposed to Taxol (100
nM) for the indicated times, and harvested cells were separated into
membrane and cytosolic fractions as described previously (33). The
two fractions were assessed for the contents of cytochrome c and Smac
by immunoblot. Equal loading of proteins was measured by using a
BCA-200 protein assay kit. b, the behaviors of cytochrome c and Smac
were investigated by immunostaining in untreated HeLa cells and
cells induced by Taxol for 24 h. Cells in i and ii were untreated; those
in iii and iv were treated.
The Role of Survivin in Apoptosis
FIG. 2. Ectopic overexpression of Smac/DIABLO sensitizes
HeLa cells to Taxol-induced apoptosis. A, schematic diagrams of
Smac and its N-terminal deletion mutants. MTS, mitochondria targeting sequence, the first amino acid residue in each Smac or mutant Smac
is numbered. Smac and the ⌬74Smac were expressed in bacteria as His6
fusion proteins, whereas F-Smac, ⌬73Smac, and M-Smac were expressed in HeLa cells as C-terminal EGFP fusion proteins. B, the
expressed Smac and mutant Smac proteins were verified by Western
analysis using anti-GFP antibody. Lane 1, EGFP; lane 2, F-SmacEGFP; lane 3, Smac-EGFP; lane 4, M-Smac-EGFP; lane 5, ⌬73SmacEGFP. Smac-EGFP was generated by removing the N terminus of
F-Smac-EGFP during Taxol-induced apoptosis. The molecular sizes of
the proteins are indicated at the left. C, HeLa cells were transfected
with EGFP, F-Smac-EGFP, M-Smac-EGFP, or ⌬73Smac-EGFP expression vectors, and 20 h after transfection the cells were treated with 100
nM Taxol for another 36 h. The percentage of cell death was determined
by the trypan blue exclusion method.
both Smac/DIABLO and cyto c were partially released into
cytoplasm from mitochondria evidenced by their changed localization pattern different from MitoTracker Red staining. Thus
we have demonstrated that Taxol treatment results in the
release of cyto c and Smac/DIABLO, suggesting that the mitochondria pathway is involved.
Ectopic Overexpression of Smac/DIABLO Sensitizes HeLa
Cells to Taxol-induced Apoptosis—Smac/DIABLO is capable of
promoting apoptosis induced by various apoptotic stimuli, including TRAIL, UV irradiation, and etoposide (22, 23). We have
demonstrated that treatment of Taxol could result in the release of Smac/DIABLO from mitochondria (Fig. 1B); it is therefore rational to propose that released cytosolic Smac/DIABLO
may increase the susceptibility of HeLa cells to Taxol induction. To test this hypothesis, we transiently transfected HeLa
cells with plasmids pEGFP-N1/F-Smac, pEGFP-N1/M-Smac,
pEGFP-N1/⌬73Smac, and pEGFP-N1 separately (Fig. 2A).
After 24 h of transfection, cell lysates were immunoblotted with
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anti-GFP antibody. Fig. 2B shows that full-length Smac
(F-Smac) and its deleted mutants (M-Smac and ⌬73Smac)
were overexpressed in the transfected cells but not in the
control cells transfected with vector. Mature Smac resulted
from F-Smac cleavage from mitochondria upon Taxol induction
was also examined by Western blot (Fig. 2B, lane 3). We then
determined whether overexpressed Smac/DIABLO promoted
apoptosis induced by Taxol. 20 h after transfection, cells were
treated with 100 nM Taxol and incubated for another 36 h then
collected and subjected to trypan blue staining. As shown in
Fig. 2C, elevated expression of full-length Smac, which was
cleaved after mitochondria import into the mature Smac/DIABLO, significantly increased Taxol-induced apoptosis, whereas
two Smac-deleted mutants, M-Smac and ⌬73Smac, which were
reported to be unable to bind to XIAP (26 –29), were less proapoptotic than full-length Smac. These results suggest that,
although the N terminus of mature Smac/DIABLO is likely to
play a major role in the pro-apoptotic effect on cell death, the
N-terminal sequence per se is not essential, because the Smac
mutant lacking the first 18 amino acid residues (⌬73Smac) or
the mutant with Met added in ahead of the first amino acid (A)
of mature Smac (M-Smac) are able to partially compromise its
pro-apoptotic activity. Combined, these data suggest that
Smac/DIABLO has yet an uncharacterized mechanism underlying its pro-apoptotic activity and are in accord with previous
reports that Smac/DIABLO has a dual role in the caspase
cascade (27).
Survivin Physically Interacts with Smac/DIABLO—It has
been shown that Smac/DIABLO promotes apoptosis by neutralizing several IAP family members, particularly XIAP (20, 21).
Survivin is a special IAP family member that contains a single
IAP repeat (BIR) and lacks the RING finger motif. Survivin is
found to be up-regulated in most transformed cell lines and in
nearly all human tumors, but it is rarely present in normal
adult tissues (3, 16, 34). Overexpression of Survivin was able to
block cell death induced by various stimuli such as TRAIL and
tumor necrosis factor (13, 15), yet the detailed mechanism
remains controversial (16 –18). Particularly, whether Survivin
interacts with Smac/DIABLO in vivo and how they interact to
each other have not been documented. To determine whether
Smac/DIABLO can bind to Survivin, we expressed GFP/Survivin, GFP/XIAP, or vector control in HeLa cells then treated
transfected cells with 100 nM Taxol for 48 h to generate mature
Smac, which is an active form (the first 55 amino acid residues
of full-length Smac are cleaved), for interaction with IAPs.
Immunoprecipitation of endogenous Smac demonstrated that
Smac strongly interacts with GFP/XIAP or GFP/Survivin but
not with control GFP protein (Fig. 3C, panel a). This suggests
that Survivin is able to interact with mature Smac in vivo.
However, the direct binding of Survivin to Smac/DIABLO has
not yet been confirmed, because the co-immunoprecipitation
experiment using cell lysate does not exclude the possibility
that additional cellular factors may be involved in the binding.
To study the direct interaction between Survivin and Smac/
DIABLO, we performed an in vitro binding assay that did not
rely on proteins present in the eukaryotic cell lysate. We successfully expressed and purified GST/Survivin fusion protein and
Smac/DIABLO with an His6 tag fused at its C terminus from
bacteria. The soluble GST/Survivin and mature Smac/DIABLOHis6 were mixed and incubated at 4 °C for overnight, and this
mixture was used for interaction assay. As shown in Fig. 3C
(panel b), GST/Survivin protein was bound specifically to the
mature Smac protein as evidenced by appearance of an eluted
23-kDa mature Smac band, whereas GST protein alone (control)
was unable to bind to Smac. This result indisputably demonstrated that Survivin directly interacts with Smac/DIABLO.
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The Role of Survivin in Apoptosis
FIG. 3. Survivin physically interacts with Smac. A, homology of the BIR domain between Survivin and other IAP family members was
determined using NCBI BLAST software. The highly conserved residues in BIR of IAPs are shaded. B, schematic diagrams of Survivin and
Survivin mutants Surv71 (D71R) and Surv-BIR. N, N-terminal. The coiled-coil structure is in lightly shaded color. The asterisk indicates point
The Role of Survivin in Apoptosis
To investigate which region of Survivin or Smac is responsible for interaction, we generated several mutants to perform
interaction assay both in vitro and in vivo. Recently, numbers
of reports have shown that XIAP directly interacts with Smac/
DIABLO, and this interaction involves the BIR domain of XIAP
and the amino terminus of mature Smac (24, 26, 27). Both
Survivin and XIAP are members of the IAP family characterized by the presence of one or more BIR domains. We thereby
reasoned that the pattern of binding of Survivin to Smac/
DIABLO might be similar to that of XIAP to Smac/DIABLO. A
BLAST program search for comparisons between the BIR domain of Survivin and BIR domains of other IAPs revealed that
Survivin BIR domain displays high homology to BIR3 and
BIR2 domains of XIAP (Fig. 3A). Based on the crystal structural analysis of complex of Smac/DIABLO䡠XIAP (BIR3), Fesik
and coworkers (35) proposed that Glu-314 in the BIR3 domain
of XIAP may directly contact with the N-terminal amine of
mature Smac and that the E314S mutant of BIR3 abolishes
almost all binding to mature Smac. In addition, it was proposed
that D214S of the BIR2 mutant domain also lost all the capability of binding to Smac (35). As shown in Fig. 3A, amino acids
Glu-314 in BIR3 domain or Asp-214 in BIR2 domain of XIAP
are equivalent to Asp-71 of Survivin, because these amino acids
are highly conserved. To demonstrate whether the amino acid
Asp-71 of Survivin is critical for binding to Smac/DIABLO, we
mutated Asp-71 to Arg-71 (Fig. 3B) and performed an interaction assay as described above. As shown in Fig. 3D (panel a), no
eluted Smac band was detected (lane 6), indicating that the
D71R mutant Survivin lost its binding ability to interact with
mature Smac, and further suggested that Asp-71 of Survivin is
vital for the binding to Smac. We further performed a coprecipitation experiment and confirmed that mature Smac
does not bind to Surv-D71R in vivo (Fig. 3D, panel b). Both the
BIR2 and BIR3 domains of XIAP were shown to directly interact with Smac/DIABLO, so we then examined whether the
Survivin BIR domain alone (Fig. 3B) is able to bind to Smac/
DIABLO. To our surprise, there was no expected Smac band to
be detected (lane 8) using an in vitro interaction assay (Fig. 3D,
panel a), and an immunoprecipitation assay also failed to detect the GFP/Surv-BIR protein in the final eluted complex (Fig.
3D, panel b), implying that, unlike XIAP, Survivin BIR domain
alone is unable to bind to Smac/DIABLO. The discrepancy may
be explained by analogy: although XIAP is able to bind to
HtrA2/Omi (a homolog of Smac/DIABLO), Survivin is unable to
bind to it (36). In addition, the coiled-coil structure at the C
terminus of Survivin is reported to be essential for inhibition of
apoptosis (5), therefore, it is not unwise to argue that this
coiled-coil structure, which is missing in the Survivin BIR
domain, may be responsible for the interaction with Smac/
DIABLO.
The N-terminal of Smac/DIABLO is essential for binding to
XIAP (26 –29), we thereby checked whether this N terminus is
also crucial for binding to Survivin. We made a series of Smac
mutant constructs as depicted in Fig. 2A. Proteins ⌬74Smac
and mature Smac were expressed and purified from bacteria
E. coli cells, and proteins F-Smac, M-Smac, and ⌬73Smac were
expressed in HeLa cells. Among them, proteins ⌬74Smac, M-
23135
Smac, and mature Smac were used in GST pull-down assay
(see “Material and Methods”). Fig. 3E (panel a) shows that
mutant variant ⌬74Smac is not detected in eluted products
(lane 2), indicating that Survivin does not bind to ⌬74Smac,
which lacks 18 amino acid residues at its N terminus of mature
Smac. A further experiment showed that M-Smac-GFP in cell
lysate was not able to bind to GST/Survivin-coupled beads (Fig.
3E, panel b). Taken together, these data suggest that the interactions between Survivin and Smac/DIABLO involve the N
terminus of mature Smac, and, moreover, we propose that wild
type Survivin may directly contact with Ala-1 of mature Smac,
because M-Smac (with a methionine added to the beginning of
mature Smac) nullifies the ability of mature Smac to interact
with Survivin.
Survivin Is Co-localized in Cytosol with Mature Smac during
Interphase—Survivin not only acts as an inhibitor of apoptosis
but also regulates cell division (5, 14, 15). Skoufias et al. (42)
reported that human Survivin is a kinetochore-associated passenger protein and determined its localization by using an
immunostaining method. However, different immunostaining
protocols or antibodies produce different results because of the
co-existence of Survivin splice variants (46). We thereby constructed a series of plasmids that express GFP, GFP/Survivin,
GFP/Surv-BIR, GFP/Surv-D71R, and GFP/Smac (Fig. 4B) to
study their distributions in living cells. We first transiently
transfected HeLa cells with constructed plasmids; 48 h later,
cells were incubated with DNA-staining dye Hoechst 33342 (2
␮g/ml) for 30 min, and the living cells were then examined by
fluorescence microscope. As shown in Fig. 4A, GFP protein
displayed an evenly diffused localization throughout the whole
cells transfected with pEGFP-C1, and its distribution was
found to be independent of the cell cycle. GFP/Survivin was
firstly found to localize in the cytoplasm during interphase and
then translocate into the central spindle midzone when cell
cycle division entered into anaphase. Finally, GFP/Survivin
was detected in the midbody during telophase (Fig. 4A). These
data clearly demonstrate that Survivin is a bona fide chromosomal passenger protein. Our result is in good agreement with
a previous report by Li et al. (5) in which they demonstrate that
Survivin was able to bind to microtubules during interphase
and to be translocated to the nucleus to regulate cell division at
the G2/M phase. To investigate the subcellular distribution of
Survivin mutants, we expressed GFP fusion proteins GFP/
Surv-D71R and GFP/Surv-BIR in HeLa cells and examined
their distributions by fluorescence microscopy. Fig. 4A shows
the intracellular localization of GFP/Surv-D71R in different
phases of the cell cycle in HeLa cells. During interphase mutant GFP/Surv-D71R was distributed mainly in the cytoplasm
similar to GFP/Survivin. As the cell cycle entered into anaphase, it spread evenly throughout the whole cells and was
unable to localize at the spindle midzone. Interestingly enough,
GFP/Surv-D71R displayed a midbody localization pattern identical to that of wild type GFP/Survivin during the late telophase. We also studied the localization of the GFP/Surv-BIR
mutant and found that its localization was the same as that of
GFP protein, which distributed uniformly throughout the cells
and did not localize to either spindle midzone or the midbody.
mutation of Asp-71 to Arg-71 (D71R). In C: a, lysates were prepared from GFP, GFP/XIAP, or GFP/Survivin stably transfected HeLa cells
(treatment by Taxol for 48 h), respectively. Endogenous mature Smacs were immunoprecipitated (IP) from the lysates and examined for interaction
with GFP, GFP/XIAP, and GFP/Survivin by Western blot (WB) with anti-GFP antibody. b, the interactions between GST/Survivin and mature
Smac were examined by GST pull-down binding assay, and GST was used as mock control. In D: a, results from GST mediated pull-down assay.
Survivin mutants did not interact with mature Smac. Lanes 1, 3, 5, and 7 indicate input Smac, and lanes 2, 4, 6, and 8 indicate the final eluted
complex. b, the interaction of GFP-tagged Survivin or Survivin mutants with mature Smac was examined as in C, panel a. In E: a, the interaction
of GST/Survivin with ⌬74Smac was investigated by in vitro interaction assay. Lane 1 denotes input ⌬74Smac, and lane 2 indicates the final eluted
complex. b, lane 1 is a cytosolic fraction containing transfected expressed, which could not be pulled down by immobilized GST/Survivin (lane 2)
and GST control (lane 3). Anti-GFP antibody was used to deleted M-Smac-EGFP.
23136
The Role of Survivin in Apoptosis
FIG. 4. Survivin is co-localized with Smac/DIABLO during interphase. A, HeLa cells were transfected with plasmids, including
pEGFP-C1, pEGFP-C1/Survivin, pEGFP-C1/Surv-D71R, pEGFP-C1/Surv-BIR, and pEGFP-C1/Smac. 48 h after transfection, cells were incubated
with Hoechst 33342 (2 ␮g/ml) for another 30 min, and the living cells were then analyzed with a fluorescence microscope. GFP or GFP fusion
proteins are represented by the green color, and the blue color was generated by Hoechst 33342 staining. Interphase, anaphase, and telophase are
denoted at the top. B, the expressions of five transfected recombinant plasmids indicated in A were verified by Western blot, and the primary
antibody used was anti-GFP monoclonal antibody.
These results suggest that the ␣-helix region at the C-terminal
of Survivin contributes to its binding to microtubule in interphase and to its proper dynamic redistribution during mitosis.
In addition, data from localization of mutant GFP/Surv-D71R
(BIR domain was mutated) indicated that the BIR domain was
absolutely required for the association of Survivin with spindle
midzone. It has been reported that Survivin interacts physically with two kinetochore proteins INCENP and Aurora-B,
which are known to be localized to both spindle midzone and
midbody and thus to control cell cycle (43). Koufias et al. (42)
reported that Survivin mutant Surv-C84A did not localize to
either spindle midzone at anaphase or midbody in telophase. If
complexing with INCENP and Aurora-B was the only mode for
Survivin to localize to the chromosome during M phase, we
would expect that the Survivin mutant Surv-D71R to have the
same localization pattern with that of Survivin mutant SurvC84A or wild type Survivin. However, GFP/Surv-D71R protein,
although unable to locate to the spindle midzone at anaphase,
was able to localize to the midbody in telophase, suggesting
that Survivin could utilize an as yet uncharacterized mechanism to become a chromosomal passenger protein during mi-
tosis. In addition, we have demonstrated that Smac directly
interacts with Survivin (Fig. 3C). Therefore, we expected that
Smac/DIABLO should co-localize with Survivin in certain
phase of cell cycle. We constructed a plasmid expressing mature GFP/Smac fusion protein in HeLa cells, and 24 h after
transfection, GFP/Smac was found to locate exclusively in the
cytosol and was not exhibited in the nucleus during the interphase (Fig. 4A). As the cell cycle enters into anaphase, unlike
GFP/Survivin, GFP/Smac was completely excluded from the
spindle midzone and did not associate with midbody in late
telophase (Fig. 4A). Thus Survivin and Smac were co-localized
in the cytoplasm during interphase, but their subsequent distributions are distinctive during mitosis. However, the possibility that Survivin interacts with Smac during M phase still
cannot be completely excluded. Taking together, our findings
suggest that Survivin is a chromosomal passenger protein during mitosis and it co-localizes with mature Smac in cytosol
during interphase when Smac/DIABLO is released from
mitochondria.
Survivin Is Not a Direct Inhibitor of Caspase—Numbers of
reports have demonstrated that Survivin can inhibit apoptosis
The Role of Survivin in Apoptosis
FIG. 5. Survivin is unable to bind to caspase. Taxol-treated (100
nM, 36 h) HeLa cells were collected and lysed with lysis buffer, and the
cytosolic extracts were then used for GST-mediated pull-down assay as
described under “Materials and Methods.” The cytosolic extracts and
final eluted products were subject to Western blot analysis and probed
with antibodies of caspases-9, -7, and -3 and Smac as needed. Lanes 1,
4, 7, and 10 were cytosolic fractions alone; lane 2, 5, 8, and 11 were final
eluted products pulled from immobilized GST/Survivin. Lane 3, 6, 9,
and 12 were eluted complex pulled from the immobilized GST control.
In the left panel (lanes 1–3), Smac was shown to be successfully pulled
down using the same conditions as those used in GST pull-down for
caspase-9, -7, and -3. Molecular sizes of proteins are indicated at
the left.
induced by various stimuli (3, 5, 15). The mechanism whereby
Survivin inhibits apoptosis still remains controversial. Survivin belongs to the IAP family; therefore, some groups reported that Survivin blocks cell death through binding to active
caspase and in turn to inhibit caspases activity. Tamm et al.
(16) reported that Survivin can bind to the effectors caspase-3
and -7 in vitro and inhibit cell death. O’Connor et al. (39)
demonstrated that the inhibitory effect of Survivin appears
through its binding to caspase-9. However, some other groups
reported contradictory results: they claimed that, in the process of inhibition of cell death, Survivin does not directly interact with caspase-3 and -9 (18, 19, 37). To test whether Survivin
physically interacts with caspases, we performed an in vitro
interaction assay (see “Materials and Methods”). GST/Survivin
fusion protein immobilized on glutathione-Sepharose 4B beads
was incubated for 1 h with Taxol-treated HeLa cytosolic extracts, and the beads were then washed and eluted. The final
eluted products were subject to Western analysis, and the
results are shown in Fig. 5. Neither GST/Survivin nor GST
control was found able to pull down caspase-3, -7, and -9 from
Taxol-stimulated cell lysates, whereas both pro-caspase and
processed caspase were detected in the cell lysate (input) lanes.
As a positive control, mature Smac could be pulled down by the
GST/Survivin as shown in Fig. 5. These results illustrate that
Survivin interacts with Smac but not with caspase-3, -7, and -9,
and therefore Survivin is not a direct suppressor of caspases.
Recently, Takahashi and coworkers have pointed out that the
linker region between BIR1 and BIR2 within XIAP is responsible for the active site-directed inhibition of caspase-3 and -7
(40), whereas a single BIR2 domain (residues 157–242) without
a linker region does not bind to caspase-7 (29). Chai et al. (29)
have further demonstrated that the BIR domains of XIAP are
dispensable for the inhibition of caspase-3 and -7 and that a
fusion protein between GST and the linker peptide of XIAP
tightly binds to and potently inhibits caspase-7 and -3, whereas
the GST-BIR2 (residues 156 –240) fusion protein lacking the
linker region does not. Wu et al. (41) also reported that the
linker of XIAP is the major determinant of binding and inhibitor for the caspases. Unlike XIAP, Survivin does not contain a
linker region, and therefore it is not surprising for us to find
that Survivin does not bind to caspase-3 and -7. In addition,
results from a comparison of the x-ray crystallographic structures between Survivin and the XIAP (BIR2)䡠caspase-3 complex also support the hypothesis that Survivin does not suppress caspase-3 (17). These data are in good agreement with
our conclusion that Survivin is unable to bind to caspases.
Survivin Blocks Apoptosis May through Antagonizing the
Activity of Smac/DIABLO—Both Survivin and XIAP were able
23137
to inhibit apoptosis, however, XIAP was found to be more
potent than Survivin (16). In a co-transfection experiment conducted by Tamm et al. (16), although XIAP was shown to
almost completely block cell death induced by Bax or Fas/
CD95, cell death could only be partially inhibited by Survivin
under the same conditions. This more complete inhibition of
apoptosis by XIAP was not due to higher levels of XIAP protein
compared with that of Survivin (16) but to its higher activity,
because in Bax or Fas/CD95-induced cells, the Survivin protein
level was even higher than that of XIAP. All these observations, plus our finding that Survivin is unable to bind to
caspases, suggest that Survivin blocks apoptosis, maybe
through some other bridge of proteins to inhibit caspase activity indirectly. One of the possible bridge protein candidates
could be the Smac/DIABLO based on our new finding that
Survivin can directly interact with Smac/DIABLO (Fig. 3C).
We reasoned that Survivin inhibits cell death, maybe through
protecting other IAPs such as XIAP from being neutralized by
Smac/DIABLO, thus allowing them to maintain their suppression on caspases. If this hypothesis is correct, mutant Survivin
incapable of binding to Smac/DIABLO would be expected to
lose its inhibitory activity. We have demonstrated previously
that Survivin mutants Surv-D71R and Surv-BIR had lost their
ability to bind to Smac/DIABLO (Fig. 3D); we therefore investigated whether the lack of interaction between Survivin mutant and Smac/DIABLO could result in the loss of the inhibitory effect of Survivin. We constructed a series of mammalian
expression vectors to express Survivin, Surv-D71R, or SurvBIR in HeLa cells, and the transfected cells were then treated
with 100 nM Taxol for 36 h, the cytotoxic effects were measured
using the trypan blue exclusion method, and protein expression
was detected by Western blot analysis (Fig. 6A, panel b). In this
assay, although Survivin reduced Taxol-induced cell death by
22%, protein Surv-D71R or Surv-BIR was unable to protect
cells from apoptosis (Fig. 6A, panel a). This suggests that
abolishment of the inhibitory function for Surv-D71R and SurvBIR may be due to the loss of their interactions with Smac/
DIABLO. It is interesting to note that Survivin mutant SurvD71R causes more cell death compared with empty vector
control (Fig. 6A, panel a), suggesting Surv-D71R is a novel
dominant negative mutant. The conversion of anti-apoptotic
wild type Survivin to pro-apoptotic mutant Survivin (SurvD71R) is not unusual, because Survivin mutations T34A or
C84A or Survivin antisense were reported to be pro-apoptotic
(39, 5), but the exact mechanism still awaits further investigation. Recently, Silke et al. (38) reported that XIAP mutants that
were unable to bind to caspase and Smac/DIABLO completely
lost the inhibitory function of XIAP, whereas XIAP mutants
that were unable to bind to caspase but could bind to Smac/
DIABLO retained their inhibitory effects. These experimental
data strongly support our notion that Survivin blocks apoptosis
mainly through antagonizing the activity of Smac/DIABLO but
not directly interacting with caspases (Fig. 5).
As described above, we have illustrated that Survivin
blocked Taxol-induced apoptosis through its binding to Smac/
DIABLO, because disruption of Smac䡠Survivin complex formation leads to a failure of apoptotic inhibition (Fig. 6A, panel a).
To further verify this result, we established a cell-free system
to examine whether Smac/DIABLO is a mediator involving in
the inhibition of apoptosis by Survivin. We purified recombinant Smac fusion (with an His6 tag at its C terminus) and GST
fusion proteins GST/Survivin, GST/Surv-D71R, and GST/Survivin-BIR for a cell-free assay system. The effects of Smac/
DIABLO or GST/Survivin were evaluated by studying the processing of caspase-9 and caspase-7 in cytosolic extracts from
empty vector-transfected or XIAP-overexpressed HeLa cells
23138
The Role of Survivin in Apoptosis
FIG. 6. Survivin antagonizes the pro-apoptotic activity of
Smac/DIABLO. In A: Survivin mutants fail to inhibit apoptosis. a,
HeLa cells were transfected with plasmid pEGFP-C1 (first bar),
pEGFP-C1/XIAP (second bar), pEGFP-C1/Survivin (third bar), pEGFPC1/Surv-D71R (fourth bar), pEGFP-C1/Surv-BIR (fifth bar), or p EGFPC1/ant-Surv (sixth bar), respectively. 20 h later, cells were incubated
with Taxol (100 nM) for 36 h, and the percentage of cell death was
calculated as the means of three independent experiments. b, expression of proteins from transfected constructs in A (panel a) was confirmed by Western analysis. In B: the analysis of dATP/cyto c-dependent caspase-9 (a) and caspase-7 (b) processing in cytosolic extracts from
mock or stable XIAP-expressed HeLa cells. The purified proteins GST/
Survivin and His tag/Smac and two Survivin mutants GST/Surv-D71R
and GST/Surv-BIR were added to cytosolic extracts in different combinations indicated above the Western blot. The concentrations of various
following the addition of cytochrome c and dATP. As shown in
Fig. 6B (panel a), cell extracts without exogenously added cyto
c and dATP left procaspase-9 uncleaved, and only one band of
45 kDa corresponding to procaspase-9 could be detected (Fig.
6B, panel a, lane 1). When cyto c and dATP were added, two
cleaved bands (37 and 35 kDa) of caspase-9 were detected in
the blot (Fig. 6B, panel a, lane 2). We found that overexpression
of XIAP strongly prevents the appearance of p37 form (which is
generated by caspase-3 cleavage) of caspase-9 but not of the
p35 form (which is generated by auto-cleavage of caspase-9)
(Fig. 6B, panel a, lane 3), indicating XIAP had blocked the
activation of caspase-9. Addition of GST/Survivin or GST/SurvD71R or GST/Survivin-BIR alone into a cell-free mixture from
mock cytosolic extracts was unable to block the appearance of
the p37 form of caspase-9 (Fig. 6B, panel a, lanes 4 – 6), suggesting Survivin by itself is unable to inhibit caspase-9. To
exclude the possibility that bacterially expressed Survivin fusion proteins may compromise their inhibitory functions, we
used the cytosolic extracts from cells in which Survivin, SurvD71R, or Surv-BIR was overexpressed and obtained the same
results, indicating a failure to block caspase-9 cleavage by
Survivin alone is not due to Survivin expressed from Escherichia coli (data not shown). In addition, adding Smac protein
into the cell-free mixture from XIAP-overexpressed cytosolic
extracts abrogated the prevention of caspase activation by
XIAP (Fig. 6B, panel a, lane 7) and resulted in the appearance
of the p37 form, thus confirming that Smac is able to stimulate
caspase-9 activation by removing the inhibition of XIAP. If the
GST-Survivin was added to the mixture containing both XIAP
and Smac, the p37 form will not be generated (Fig. 6B, panel a,
lane 8). In contrast, when the mutant Survivin GST-SR71 or
GST-BIR was added, the p37 form was detected (Fig. 6B, panel
a, lanes 9 and 10). From these results we have reached two
conclusions. First, Survivin is capable of blocking caspase-9
activation in the presence of Smac and XIAP. Second, Survivin
mutants, which are unable to bind to Smac, fail to inhibit
activation of caspase-9 even in the presence of XIAP and Smac.
The same cell-free system was used to examine the effect of
Survivin and Smac by evaluating the processing of caspase-7.
We found that the presence of XIAP, but not of GST/Survivin,
GST/Surv-D71R, or GST/Surv-BIR, was able to inhibit cleavage of caspase-7 (Fig. 6B, panel b, lanes 3– 6), whereas Smac
protein was found to antagonize the inhibition of XIAP and
generate a p19-active form of caspase-7 (Fig. 6B, panel b, lane
7). When XIAP, Smac, and GST/Survivin were mixed altogether in the cell-free system, cleavage of caspase-7 was
blocked (Fig. 6B, panel b, lane 8), whereas the simultaneous
presence of XIAP, Smac, and Survivin mutants GST/SurvD71R or GST/Surv-BIR were unable to block the cleavage of
caspase-7, resulting in the appearance of p19 (Fig. 6B, panel b,
lanes 9 and 10). XIAP is known to block the activities of
caspase-9 and caspase-7 by its binding to caspases (29, 40).
However, the inhibitory activity of XIAP can be eliminated by
Smac through its direct interaction with XIAP, thus freeing the
caspase from the XIAP䡠caspase complex (20, 21). In this cellfree system, we have demonstrated that Survivin alone was
unable to block caspase activation due to its inability to bind to
purified proteins and compounds used in the cell-free experiment were
the following: cyto c (1 ␮g/ml), dATP (1 mM), Smac (100 nM), and
GST/Survivin, GST/Surv-D71R, and GST/Surv-BIR were all 200 nM.
“⫹” represents presence and “⫺” is absence. C, HeLa cells were transfected or co-transfected with plasmids pEGFP-N1, pEGFP-C1/Survivin,
pEGFP-C1/Survivin:pEGFP-N1/F-Smac(1:1), pEGFP-C1/Surv-D71R:
pEGFP-N1/F-Smac (1:1), pEGFP-C1/Surv-BIR:pEGFP-N1/F-Smac (1:
1), or pEGFP-N1/F-Smac, 20 h after transfection the cells were treated
with 100 nM Taxol for another 36 h. The percentage of cell death was
determined by the trypan blue exclusion method.
The Role of Survivin in Apoptosis
caspase (Fig. 5). In addition, Survivin was able to rescue inhibition of XIAP through its binding to Smac, thus freeing XIAP
from the XIAP䡠Smac complex. However, Survivin mutants
Surv-D71R and Surv-BIR were not able to rescue the inhibitory
effect of XIAP, because these two Survivin mutants were unable to bind to Smac (Fig. 3D). To further verify whether the
results obtained from the cell-free system actually occurred in
the cells in vivo, we co-expressed Survivin and Smac (1:1) in
HeLa cells and found that co-overexpression of Survivin and
Smac remarkably reduced the cell death induced by Taxol
compared with overexpression of Smac alone, whereas co-overexpression of Survivin mutant (Surv-D71R or Surv-BIR) and
Smac (1:1) were unable to decrease the cell death (Fig. 6C).
This suggested that Survivin was able to antagonize the proapoptotic activity of Smac, in particular the BIR domain or
C-terminal domain of Survivin were required for its antagonism. Taken together, data from the transfection experiment in
vivo were consistent with that from cell-free system in vitro.
Although our data have provided strong evidence that Survivin
had inhibitory function with the help of XIAP and Smac, the
possibility that Survivin involves yet uncharacterized pathways to prevent cell death cannot be dismissed. Additionally,
the competitiveness of Survivin and XIAP for binding to Smac
is required for further investigation.
DISCUSSION
In this study, we have shown that ectopic overexpression of
Smac/DIABLO sensitizes HeLa cells to apoptosis induced by
Taxol. We demonstrated for the first time that Smac/DIABLO
physically interacts with Survivin both in vitro and in vivo.
Mutational analysis revealed that amino acid Asp-71 in Survivin is critical for its binding to Smac/DIABLO, and a single
amino acid change (D71R) converts Survivin from being antiapoptotic to pro-apoptotic. The deletion experiments showed
that the C-terminal coiled-coil domain of Survivin is responsible for the interaction between Survivin and Smac/DIABLO.
Similarly, we found that by removing 18 amino acids (AVPIAQKSEPHSLSSEALM) or adding one amino acid (Met) at its
N terminus Smac/DIABLO abolishes its ability to interact with
Survivin. In addition, Survivin is co-localized in cytosol with
Smac/DIABLO during the interphase in living cells. More importantly, the Survivin䡠Smac complex formation contributes to
Survivin inhibitory function during Taxol-induced apoptosis in
HeLa cells.
Smac/DIABLO, a mitochondria protein that is released to
cytosol in response to a number of apoptotic stimuli, was found
to bind IAPs and prevent them from inhibiting caspases (20 –
23). Previous reports (22–24, 44) have clearly shown that the
BIR domains of IAPs, such as BIR2 or BIR3 of XIAP and BIR
of ML-IAP, are sufficient to interact with Smac/DIABLO. However, in the present study, we found that the single BIR domain
of Survivin failed to bind to Smac/DIABLO (Fig. 3D), indicating
that the way Survivin binds to Smac/DIABLO is different from
XIAP or ML-IAP. Smac/DIABLO and its newly identified homolog HtrA2/Omi were reported to be able to bind to XIAP in the
same manner, but, unlike Smac/DIABLO, HtrA2/Omi was unable to interact with Survivin (36). Our result may shed some
light on explaining why Survivin does not bind to HtrA2/Omi.
Recently, Survivin has attracted growing attention due to its
expression in various tumors and its potential application in
tumor therapy (3, 45). However, its subcellular localization
remains controversial: different immunostaining protocols or
different antibodies used give rise to diverse results because of
the presence of Survivin splice variants (46). To remedy these
discrepancies, we analyzed the localizations of GFP/Survivin in
living HeLa cells by using confocal laser microscopy. GFP/
Survivin was excluded from the interphase nucleus and was
23139
FIG. 7. Hypothetical model of Survivin in the inhibition of
apoptosis. Smac is released from Taxol-induced mitochondria, then
binds to overexpressed Survivin, which reduces neutralizing effect of
Smac on XIAP. XIAP in turn was able to interact with caspases, and cell
death was blocked.
detected in the central spindle midzone at anaphase and localized to the remnants of a mitotic apparatus, midbody at telophase (Fig. 4A). Interestingly enough, Survivin mutant GFP/
Surv-D71R, which is unable to bind to Smac/DIABLO, lost its
ability to localize at the spindle midzone at anaphase. This
observation may explain the possible mechanism for the proapoptotic effect of Surv-D71R, because the other two dominant
negative mutants, T34A and C84A, reported elsewhere displayed the same localization pattern as D71R (42, 47).
Although it has been confirmed that Survivin is able to
inhibit apoptosis induced by various stimuli, the mechanism by
which Survivin inhibits apoptosis has not been conclusively
determined. As with other IAPs, it has been reported that
Survivin physically interacts with initiator or effector caspases
(16, 37, 48). However, some groups (49, 50) demonstrated that
this did not seem to translate into physiologically meaningful
inhibition of caspase activity, and the x-ray crystal structure of
Survivin also fails to reveal the presence of a “hook and sinker”
region that mediates caspase binding in other IAPs (18, 37).
Results from the present study have shown that Survivin inhibits apoptosis mainly through antagonizing the pro-apoptotic
ability of Smac/DIABLO rather than through binding to
caspases. We demonstrated that Survivin does not interact
with caspases in vitro and that its mutants Surv-D71R and
Surv-BIR, which are unable to bind to Smac/DIABLO, had lost
their anti-apoptotic effect (Fig. 6A, panel a). By using a cell-free
system, we have shown that XIAP but not Survivin, Surv-D71R
or Surv-BIR, prevents cyto c induced caspases activation. This
prevention can be blocked by adding Smac/DIABLO which
neutralizes XIAP through the formation of Smac*XIAP complex (20, 21). When additional Survivin was subsequently
added, prevention of caspases activation could be restored.
However, Surv-D71R or Surv-BIR, when added into the cellfree system containing both XIAP and Smac, were unable to
restore this prevention (Fig. 6B). These data suggest that Survivin is able to antagonize the pro-apoptotic effect of Smac/
DIABLO in vitro. We also demonstrated that Survivin blocks
the pro-apoptotic activity of Smac/DIABLO in HeLa cells (Fig.
6C). Recently, it was reported that XIAP mutants, which had
lost their ability to inhibit caspase-9 and caspase-3 yet maintained their ability to interact with Smac/DIABLO, were able
to inhibit cell death and that this inhibition could be explained
by showing that endogenous XIAP is sufficient to block apoptosis provided it is not antagonized by an IAP antagonist such
as Smac/DIABLO (20, 21). In the present study, we have demonstrated that, although Survivin is unable to inhibit caspases,
it possesses the ability to bind to Smac/DIABLO, thereby
allowing endogenous IAPs such as XIAP to block caspases
without being antagonized. Taken together, we provided a
working model for the inhibition of apoptosis by Survivin. During apoptosis Survivin binds to Smac/DIABLO released from
induced mitochondria, thus reducing antagonism of Smac/
23140
The Role of Survivin in Apoptosis
DIABLO to XIAP, the free XIAP from Smac䡠XIAP complex
directly interacts with caspases, and cell death is blocked (Fig.
7). This mode suggests that the anti-apoptotic effect of Survivin
may be mitochondria-dependent, because mature Smac is released only from mitochondria. This view is strongly consistent
with ideas proposed by some other groups; they pointed to a
more selective role of Survivin in antagonizing mitochondrialdependent apoptosis, because Survivin blocks mitochondrialinduced but not death-receptor-induced apoptosis in transgenic
animals (53). Recent genetic data showed the heterozygous
Survivin mice (surv ⫺/surv⫹) were more sensitive to mitochondrial-dependent cell death (52). Furthermore, cell death following loss/interference of Survivin showed the characteristics of
mitochondrial-dependent apoptosis (39, 45, 51). In addition, it
is worth noting that Survivin mutant Surv-D71R constructed
by our group represents a novel dominant negative mutant,
because overexpression of this mutant not only abolishes the
inhibitory effect of Survivin but also promotes Taxol-induced
apoptosis. This new finding may have clinical usefulness in
designing novel therapeutic drugs.
Acknowledgments—We thank Yi Wang and Shixin Liu for their
excellent technical help.
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