Direct Interaction between Survivin and Smac/DIABLOIs Essential for the Anti-apoptotic Activity of Survivin duringTaxol-induced Apoptosis*

Received for publication, January 29, 2003, and in revised form, March 19, 2003Published, 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 apoptosisprotein (IAP) family that has been implicated in bothapoptosis inhibition and cell cycle control. However, itsinhibitory mechanism and subcellular localization re-main controversial. In this report, we provided evidencefor the first time that Survivin physically interacts withSmac/DIABLO both in vitro and in vivo. A point muta-tion (D71R) in the baculovirus IAP repeat motif and aC-terminal deletion mutant (Surv-BIR) of Survivin failto bind to Smac/DIABLO and abrogate its ability to in-hibit apoptosis. The N-terminal of mature Smac/DIABLOis absolutely required for Survivin�Smac complex forma-tion. Subcellular distributions of Survivin and Smac/DIABLO showed that they co-localized within the cy-tosol during interphase. In addition, Survivin was foundto be incapable of binding to caspase. We also identifiedthat the co-presence of Smac/DIABLO and XIAP wasrequired for Survivin to inhibit caspase cleavage in acell-free system. In conclusion, our results provide thefirst evidence that the interaction between Smac/DIA-BLO and Survivin is an essential step underling theinhibition of apoptosis induced by Taxol.

Among the regulators of apoptosis, considerable interest hasbeen focusing on the inhibitors of apoptosis protein (IAP)1

family, which were first identified as negative regulators inprogrammed cell death characterized by the presence of one tothree copies of the baculovirus IAP repeat (BIR) domain (1).However, anti-apoptotic function in physiological cell death hasnot been established for all IAPs, because some members inthis family play essential roles in cell division, rather thanmerely acting as the regulators of apoptosis (2). Survivin (16.5KDa) is a special member of the inhibitor-of-apoptosis proteins(IAPs) family containing a single BIR and lacking a RINGfinger motif. It is mostly expressed in the vast majority oftumors or in the embryonic development, but not in normal

adult tissues (3, 4). Survivin is cell cycle-regulated and predom-inantly expressed in the G2/M phase (5). The degradation ofSurvivin is believed to be through the ubiquitin-proteasomepathway (6). Accumulating evidence supports the idea thatSurvivin is a bifunctional protein that acts as a cell divisionregulator (7–14) and an apoptosis suppressor (3, 5, 15, 16). Themechanism of how Survivin inhibits apoptosis remains contro-versial. Tamm et al. (16) demonstrated that Survivin was ableto bind to the effector caspase-3 and caspase-7 in vitro andproposed that Survivin may inhibit caspase activity in physio-logical cell death by the similar mechanism. However, compar-ison between the x-ray crystallographic structures of Survivinand that of the XIAP (BIR2)�caspase-3 complex fails to revealany clues of how Survivin could directly suppress caspase-3(17). Verdecia et al. (18) and Banks et al. (19) have demon-strated that Survivin is unable to directly bind to caspase-3 invitro and does not inhibit caspase-3 activity. Wang and col-leagues identified a novel mitochondrial-associated proteinSmac/DIABLO that was able to interact with IAPs (20, 21) andmay help explain the inhibitory function of Survivin.

Smac/DIABLO is a mitochondria protein but is released intothe cytosol in response to some of apoptotic stimuli, includingUVB-irradiation, etoposide, or glucocorticoids (20–23). Aftermitochondria import, the N terminus of precursor Smac/DIA-BLO is removed by limited proteolysis to generate a matureform of the molecule (20, 27). Mature Smac/DIABLO was foundto promote caspase activation by binding and neutralizing theIAPs, including XIAP, CIAP-1, and CIAP-2 (20, 21, 23). Num-bers of reports confirmed that mature Smac/DIABLO interactswith BIR2 and BIR3 of XIAP, and the N terminus of matureSmac/DIABLO is absolutely required for this interaction (24,26–29). However, whether Smac/DIABLO physically interactswith Survivin has yet to be fully characterized.

In this report, we investigated the effect of interactions be-tween Survivin and Smac/DIABLO on the apoptosis induced byTaxol (paclitaxel). We have demonstrated that Taxol is able totrigger apoptosis significantly in HeLa cell, resulting in theactivation of cellular caspases-3, -7, and -9 and release of Smac/DIABLO and cytochrome c from the mitochondrial. Ectopicexpression of Survivin was able to significantly block the Taxol-induced cell death. Our GST pull-down binding assay andimmunoprecipitation demonstrate for the first time that Sur-vivin directly interacted with Smac/DIABLO but not withcaspases. We also found mutant Survivin with a single aminoacid change at residue Asp-71 (D71R) or truncated Survivinlacking the C terminus (residues 1–97) failed to bind to Smac/DIABLO and abrogated its ability to inhibit apoptosis. On thecontrary, these two Survivin mutants displayed some apoptoticeffects. In addition, we have shown that wild type Survivindoes not interact with Smac mutants M-Smac (a methionine

* This research was supported by the Key Project Fund (KSCX2-2-01-004), a special grant (to M. W.) from the Chinese Academy of Sci-ences, the National Natural Science Foundation of China (Grants30121001 and 90208027), and a 973 grant (Grant 2002CB713700) fromthe Ministry of Science and Technology of China. The costs of publica-tion of this article were defrayed in part by the payment of pagecharges. 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.: 86-551-360-6264; Fax: 86-551-360-6264; E-mail: wumian88@yahoo.com.

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, bovineserum albumin; PBS, phosphate-buffered saline; FITC, fluorescein iso-thiocyanate; cyto c, cytochrome c; TRAIL, tumor necrosis factor-relatedapoptosis-inducing ligand.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 25, Issue of June 20, pp. 23130–23140, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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was added to the begin of mature Smac) or �74Smac (residues75–239). We examined subcellular distributions of Survivinand Smac/DIABLO within living cells at different stages of thecell cycle and found that Survivin and Smac are co-localized inthe cytosol during interphase, and when cells enter into Mphase Survivin acts as a chromosome passenger protein. Inaddition, by using the cell-free system, we were able to demon-strate that both Smac/DIABLO and XIAP are required in orderfor inhibition of caspase cleavage by Survivin. Combined, ourdata strongly support our proposed hypothetical mode for Sur-vivin in the inhibition of apoptosis. During apoptosis Survivinbinds 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 caspasesand cell death is blocked.

MATERIALS AND METHODS

Oligonucleotides—The sequences of the oligonucleotides used in thisstudy are listed as follows. P1, 5�-CGGGATCCGCATGGGTGCCCCGA-CGTTG-3�; P2, 5�-CGGTCGACCCATCCATGGCAGCCAGCTG-3�; P3,5�-GCAAGCTTATGGCGGCTCTGAAGAGTTG-3�; P4, 5�-GCGGATCC-TCCTCACGCAGGTAGGCCT-3�; P5, 5�-CGGTCGACGTTAATTCTTC-AAACTGCTT-3�; P6, 5�-GCGATTAATATGGCGGTTCCTATTGCAC-3�;P7, 5�-CGGCGGCCGCATCCTCACGCAGGTAGGCCT-3�; P8, 5�-CGG-GATCCGATTAATATGAGGAGAGCAGTGTCTTT-3�; P9, 5�-CGGGAT-CCTGATGGCGGTTCCTATTGCACAG-3�; P10, 5�-GGGAGCCAGATC-GCGACCCCATAGAGGA-3�; P11, 5�-CTATGGGGTCGCGATCTGGC-TCCCAGCC-3�; and P12, 5�-CGGGATCCTCAATCCATGGCAGCCA-GCT-3�.

Reagents and Antibodies—The following antibodies were used in thisstudy: polyclonal antibodies Ab-caspase-7, Ab-caspase-3, Ab-survivin,and Ab-actin (Santa Cruz Biotechnology, Santa Cruz, CA); monoclonalantibodies: 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 (Cal-biochem, La Jolla, CA). Taxol used in our experiments is labeled as GCPgrade. Restriction enzymes were mostly purchased from New EnglandBioLabs. Medium compounds were obtained from Oxid. The majority ofbiochemical reagents were ordered from Sigma. Trypan blue was pur-chased from Invitrogen.

Cell Culture and Transfection—HeLa cells were maintained in Dul-becco’s modified Eagle’s medium containing 10% heat-inactivated fetalbovine serum, 1 � nonessential amino acid, 1 � minimal essentialmedium sodium pyruvate, 100 �g/ml penicillin, 100 �g/ml streptomycin(Invitrogen, Grand Island, NY). Cultured cells were incubated in ahumidified atmosphere containing 5% CO2 at 37 °C. Transfection ofcells with various mammalian expression constructs by LipofectAMINE2000 (Invitrogen) was carried out according to the methods provided bythe manufacturer’s instructions.

PCR-mediated Mutagenesis—To obtain the mutant survivin, wehave employed the PCR-mediated mutagenesis method. Two pairs ofprimers, P1/P10 (complementing the regions from nucleotides 1 to 18and nucleotides 196 to 224, respectively) and P11/P2 (complementingthe regions from nucleotides 200 to 228 and nucleotides 408 to 426,respectively), were used to amplify two Survivin fragments. After gelpurification, the resultant two overlapping PCR fragments were mixedwith equal amounts. This mixture was incubated first at 94 °C for 4min, followed by first PCR (PCR1) of 94 °C denaturing for 1 min, 56 °Cannealing 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 theextension at 72 °C. The PCR1 products were used as a template in asecond PCR (PCR2), primed by oligonucleotides P10 and P11 to carryout another 20 cycles of PCR with the same PCR conditions used inPCR1. A prominent band with an expected size of 0.42 kb was visible on1% agarose gel. A point mutation, GAC (Asp) to CGC (Arg), was furtherverified by DNA sequencing determination.

Plasmids Construction—The full-length Survivin coding sequence(nucleotides 1–426) and its mutant variant with a point mutation atamino acid residue 71 (D71R) were cloned into BamHI/SalI sites ofpGEX-5X-3 (Amersham Biosciences, UK), respectively, to generate re-combinant expression vectors pGEX-5X-3/Survivin and pGEX-5X-3/Surv-D71R, and both fragments were inserted in-frame with the GSTgene. The gene fragment (nucleotides 1–291), containing only the Sur-vivin BIR domain (residues 1–97), was generated by the PCR methodusing primers P1 and P5 (see “Oligonucleotides”) and cloned into

BamHI/SalI sites of pGEX-5X-3 to yield pGEX-5X-3/Surv-BIR. Genefragments coding for wild type Survivin, for mutant Survivin (Surv-D71R) with a point mutation at amino acid residue 71, for the truncatedSurvivin (Surv-BIR) lacking its C-terminal and Smac/DIABLO werealso cloned into pEGFP-C1 plasmid, respectively. In addition, the genefragments (reacted by PCR for primer pairs P12/P1) was used to gen-erated pTRE2/A-Surv (coding for antisense Survivin). The DNA frag-ment coding for mature Smac/DIABLO (residues 56–239) and DNAfragment 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 restrictionenzymes 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-lengthSmac/DIABLO gene fragment (residues1–239) resulted from PCR am-plification with primers P3 and P4 inserted into HindIII/BamHI sites ofthe mammalian expression vector pEGFP-N1 (Clontech, Palo Alto, CA)to fuse to the EGFP gene, and the resultant recombinant plasmid wasdesignated as pEGFP-N1/F-Smac. DNA fragments, coding for mutantSmac/DIABLO with an methionine residue added at the N terminus ofmature Smac/DIABLO (designated as M-Smac) and for mutant Smac/DIABLO lacking 73 amino acid residues at its N terminus (designatedas �73Smac), were reacted by PCR away from Smac/DIABLO cDNAusing primer pairs P9/P4 and P8/P4, respectively, and inserted into theBamHI site of pEGFP-N1 in fusion with EGFP to generate plasmidspEGFP-N1/M-Smac and pEGFP-N1/�73Smac.

Expression and Purification of Fusion Proteins—Overnight culturedEscherichia coli cells DH5� or BL21/DE3 containing the fusion plas-mids were grown at a 1:100 dilution in an Amp-LB medium until theoptical density reached A600 � 0.5. Isopropyl-1-thio-�-D-galactopyrano-side was then added to a final concentration of 0.4 mM. Cells wereinduced for another 3 h before they were harvested and subjected tosonication for 10 shot pulses of 20 s each. The maximum protein releasewas determined by the Bradford assay (Bio-Rad) by comparing theknown concentration of bovine serum albumin (BSA). The GST fusionproteins were purified through the glutathione-SepharoseTM 4B beads(Amersham Biosciences). The solubilized His6-Smac or His6-�74Smacfusion proteins were purified by incubating with chelating SepharoseTM

Fast Flow beads (Amersham Biosciences) according to the manufactur-er’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 per-formed as described by Suzuki et al. (50). Taxol-stimulated HeLa cells(2 � 106) were lysed with the 200-�l lysis buffer as described above. Celllysates or bacterially expressed and purified His6 tag proteins wereincubated with GST or GST fusion protein immobilized on glutathione-Sepharose 4B beads (Amersham Biosciences) for overnight at 4 °C andwashed three times with 500 �l of 1� PBS. The bound proteins wereeluted by elution buffer, and eluted products were subjected to SDS-PAGE for Western blot analysis or Coomassie Blue staining.

Cell Death Assay—The ability of Survivin, Smac/DIABLO, or theirmutants to affect cell viability was assayed by transfecting or HeLacells (2 � 104 cells/well) in 24-well plates with 0.3 �g of mammalianexpression vectors using LipofectAMINE 2000. Twenty hours aftertransfection, cells continued to be incubated with 100 nM Taxol for 36 h,and the viability of the cells was measured with the standard trypanblue exclusion method by counting blue dead cells. Data are expressedas percentages of control and are the means of three independentexperiments.

Western Blot Analysis—Cells were washed with 1� PBS and resus-pended with 5 volumes of cold lysis buffer (50 mM Tris-HCl (pH 7.5), 250mM NaCl, 5 mM EDTA, 50 mM NaF, 0.5% Nonidet P-40) supplementedwith protease inhibitor mixture (Roche Applied Science). The cell lysatewas incubated on ice for 30 min and was then centrifuged for 10 min at4 °C. The protein content of the supernatant was determined by usinga BCA-200 protein assay kit (Pierce). Equal amounts of proteins (10–20�g) were loaded onto the gel and separated by SDS-PAGE, and theresolved proteins were transferred to nitrocellulose membrane. Afterblocking 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 withprimary antibody for 1 h at room temperature. The membrane was thenwashed with TBST and probed with horseradish peroxidase-conjugatedsecondary antibody for 1 h. The membrane was washed three times inTBST and developed by ECL using the manufacturer’s protocol. Digi-tonin fractionation of cells into membrane and cytosolic fractions usedfor detection of cytochrome c and Smac/DIABLO was performed asdescribed by Ekert et al. (23).

Immunoprecipitation—Cells were lysed in a Triton X-100-based lysis

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buffer (1% Triton X-100, 10% glycerol, 150 mM NaCl, 20 mM Tris, pH7.5, 2 mM EDTA, protease inhibitor mixture) for 1 h, and the nuclearand cellular debris was cleared by centrifugation. Then the cytosoliclysis was incubated with Smac monoclonal antibody bound to ProteinA/G-Sepharose. After a 4-h incubation at 4 °C, the immunoprecipitateswere washed five times in lysis buffer, and proteins were recoveredby boiling the beads in SDS sample buffer and analyzed using aWestern blot.

Immunofluorescence Confocal Microscopy—HeLa cells were grown insix-well chamber slides, and 24 h later, cells were either left untreatedor 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 cellswere then washed with PBS, fixed with 3.7% paraformaldehyde, per-meabilized 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 temper-ature. The primary antibody was recognized by secondary FITC-conju-gated antibody. Finally, the stained cells were analyzed with a laserscanning confocal microscopy system (Fluoview, Olympus).

RESULTS

Smac/DIABLO Is Released from the Mitochondria Accompa-nied with Cytochrome c upon Induction of Apoptosis by Taxol—Taxol is a natural product with potent anti-tumor activity andhas been approved for the treatment of breast, ovarian, andlung cancers (25, 30–32). We incubated HeLa cells with Taxolat a concentration of 100 nM, which is known to induce apo-ptosis in a number of different cell types. After 24-h incubation,the clear morphological changes characteristic of apoptosiswere observed. Taxol-treated cells became rounded and de-tached from the substratum of the flask, followed by cellularshrinkage and membrane blebbing (data not shown). Caspaseactivation was believed to be the key factor during apoptosis.To evaluate which of the caspases is activated during theprocess of Taxol-induced apoptosis in HeLa cells, we analyzedthe cleavage pattern of caspase-9, caspase-7, and caspase-3 byWestern analysis methods. The results, shown in Fig. 1A, in-dicate that treatment with this drug results in the activation ofcaspase-9, caspase-7, and caspase-3 as evidenced by the ap-pearance of caspase-active forms. One apoptotic signaling path-way leading into caspase activation involves the translocationof cytochrome c into the cytosol from the mitochondrial inter-membrane space (33). To further investigate whether the mi-tochondria are involved in this event, Taxol-treated HeLa cellswere first lysed and divided into membrane pellet and cytosolicfraction. The pellet was then treated with 0.5% bile acid deoxy-cholate and separated by centrifugation into a soluble andmembrane fraction. Cytosolic proteins and membrane proteinssolubilized by the deoxycholate treatment were subjected toWestern blot analysis. As shown in Fig. 1B (panel a), an in-creasing amount of cytochrome c was detected in the cytosolicfractions, whereas a decreasing amount of cytochrome c re-mained in the mitochondrial membrane, and after 48 h, themajority of cytochrome c was detected in the cytosolic fractions.We also observed that Smac/DIABLO was predominantly pres-ent within the membrane fractions prior to treatment withTaxol, and by 48 h post-treatment, there was a significant lossof Smac/DIABLO from the membrane fractions and a concom-itant increase of Smac/DIABLO in the cytosolic fractions (Fig.1B, panel a). In addition, to further elucidate the behavior ofSmac/DIABLO or cyto c during Taxol-induced apoptosis, weexamined its subcellular localization in both untreated cellsand treated cells by immunostaining and confocal microscopy(Fig. 1B, panel b). HeLa cells were stained with MitoTrackerRed to define the location of mitochondria and with anti-Smacor anti-cyto c antibody plus an FITC-conjugated secondaryantibody. In uninduced HeLa cells, the localization of FITC andMitoTracker Red staining gave rise to the yellow color indicat-ing that both Smac/DIABLO and cyto c were localized withinthe mitochondria. However, after induction with Taxol for 24 h,

FIG. 1. Mitochondrial cytochrome c and Smac are releasedduring induction of apoptosis by Taxol. A, HeLa cells weretreated with 100 nM Taxol for the indicated times before being har-vested and lysed in lysis buffer. The processing of caspase-9 (a), -7 (b),and -3 (c) was then analyzed by Western blot probing with theirrespective antibodies. In B: a, HeLa cells were exposed to Taxol (100nM) for the indicated times, and harvested cells were separated intomembrane and cytosolic fractions as described previously (33). Thetwo fractions were assessed for the contents of cytochrome c and Smacby immunoblot. Equal loading of proteins was measured by using aBCA-200 protein assay kit. b, the behaviors of cytochrome c and Smacwere investigated by immunostaining in untreated HeLa cells andcells induced by Taxol for 24 h. Cells in i and ii were untreated; thosein iii and iv were treated.

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both Smac/DIABLO and cyto c were partially released intocytoplasm from mitochondria evidenced by their changed local-ization pattern different from MitoTracker Red staining. Thuswe have demonstrated that Taxol treatment results in therelease of cyto c and Smac/DIABLO, suggesting that the mito-chondria pathway is involved.

Ectopic Overexpression of Smac/DIABLO Sensitizes HeLaCells to Taxol-induced Apoptosis—Smac/DIABLO is capable ofpromoting apoptosis induced by various apoptotic stimuli, in-cluding TRAIL, UV irradiation, and etoposide (22, 23). We havedemonstrated that treatment of Taxol could result in the re-lease of Smac/DIABLO from mitochondria (Fig. 1B); it is there-fore rational to propose that released cytosolic Smac/DIABLOmay increase the susceptibility of HeLa cells to Taxol induc-tion. To test this hypothesis, we transiently transfected HeLacells 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

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 thecontrol cells transfected with vector. Mature Smac resultedfrom F-Smac cleavage from mitochondria upon Taxol inductionwas also examined by Western blot (Fig. 2B, lane 3). We thendetermined whether overexpressed Smac/DIABLO promotedapoptosis induced by Taxol. 20 h after transfection, cells weretreated with 100 nM Taxol and incubated for another 36 h thencollected and subjected to trypan blue staining. As shown inFig. 2C, elevated expression of full-length Smac, which wascleaved after mitochondria import into the mature Smac/DIA-BLO, significantly increased Taxol-induced apoptosis, whereastwo Smac-deleted mutants, M-Smac and �73Smac, which werereported to be unable to bind to XIAP (26–29), were less pro-apoptotic than full-length Smac. These results suggest that,although the N terminus of mature Smac/DIABLO is likely toplay a major role in the pro-apoptotic effect on cell death, theN-terminal sequence per se is not essential, because the Smacmutant lacking the first 18 amino acid residues (�73Smac) orthe mutant with Met added in ahead of the first amino acid (A)of mature Smac (M-Smac) are able to partially compromise itspro-apoptotic activity. Combined, these data suggest thatSmac/DIABLO has yet an uncharacterized mechanism under-lying its pro-apoptotic activity and are in accord with previousreports that Smac/DIABLO has a dual role in the caspasecascade (27).

Survivin Physically Interacts with Smac/DIABLO—It hasbeen shown that Smac/DIABLO promotes apoptosis by neutral-izing several IAP family members, particularly XIAP (20, 21).Survivin is a special IAP family member that contains a singleIAP repeat (BIR) and lacks the RING finger motif. Survivin isfound to be up-regulated in most transformed cell lines and innearly all human tumors, but it is rarely present in normaladult tissues (3, 16, 34). Overexpression of Survivin was able toblock cell death induced by various stimuli such as TRAIL andtumor necrosis factor (13, 15), yet the detailed mechanismremains controversial (16–18). Particularly, whether Survivininteracts with Smac/DIABLO in vivo and how they interact toeach other have not been documented. To determine whetherSmac/DIABLO can bind to Survivin, we expressed GFP/Sur-vivin, GFP/XIAP, or vector control in HeLa cells then treatedtransfected cells with 100 nM Taxol for 48 h to generate matureSmac, which is an active form (the first 55 amino acid residuesof full-length Smac are cleaved), for interaction with IAPs.Immunoprecipitation of endogenous Smac demonstrated thatSmac strongly interacts with GFP/XIAP or GFP/Survivin butnot with control GFP protein (Fig. 3C, panel a). This suggeststhat Survivin is able to interact with mature Smac in vivo.However, the direct binding of Survivin to Smac/DIABLO hasnot yet been confirmed, because the co-immunoprecipitationexperiment using cell lysate does not exclude the possibilitythat 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 notrely on proteins present in the eukaryotic cell lysate. We success-fully expressed and purified GST/Survivin fusion protein andSmac/DIABLO with an His6 tag fused at its C terminus frombacteria. The soluble GST/Survivin and mature Smac/DIABLO-His6 were mixed and incubated at 4 °C for overnight, and thismixture was used for interaction assay. As shown in Fig. 3C(panel b), GST/Survivin protein was bound specifically to themature Smac protein as evidenced by appearance of an eluted23-kDa mature Smac band, whereas GST protein alone (control)was unable to bind to Smac. This result indisputably demon-strated that Survivin directly interacts with Smac/DIABLO.

FIG. 2. Ectopic overexpression of Smac/DIABLO sensitizesHeLa cells to Taxol-induced apoptosis. A, schematic diagrams ofSmac and its N-terminal deletion mutants. MTS, mitochondria target-ing sequence, the first amino acid residue in each Smac or mutant Smacis numbered. Smac and the �74Smac were expressed in bacteria as His6fusion proteins, whereas F-Smac, �73Smac, and M-Smac were ex-pressed in HeLa cells as C-terminal EGFP fusion proteins. B, theexpressed Smac and mutant Smac proteins were verified by Westernanalysis using anti-GFP antibody. Lane 1, EGFP; lane 2, F-Smac-EGFP; lane 3, Smac-EGFP; lane 4, M-Smac-EGFP; lane 5, �73Smac-EGFP. Smac-EGFP was generated by removing the N terminus ofF-Smac-EGFP during Taxol-induced apoptosis. The molecular sizes ofthe proteins are indicated at the left. C, HeLa cells were transfectedwith EGFP, F-Smac-EGFP, M-Smac-EGFP, or �73Smac-EGFP expres-sion vectors, and 20 h after transfection the cells were treated with 100nM Taxol for another 36 h. The percentage of cell death was determinedby the trypan blue exclusion method.

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FIG. 3. Survivin physically interacts with Smac. A, homology of the BIR domain between Survivin and other IAP family members wasdetermined using NCBI BLAST software. The highly conserved residues in BIR of IAPs are shaded. B, schematic diagrams of Survivin andSurvivin mutants Surv71 (D71R) and Surv-BIR. N, N-terminal. The coiled-coil structure is in lightly shaded color. The asterisk indicates point

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To investigate which region of Survivin or Smac is responsi-ble for interaction, we generated several mutants to performinteraction assay both in vitro and in vivo. Recently, numbersof reports have shown that XIAP directly interacts with Smac/DIABLO, and this interaction involves the BIR domain of XIAPand the amino terminus of mature Smac (24, 26, 27). BothSurvivin and XIAP are members of the IAP family character-ized by the presence of one or more BIR domains. We therebyreasoned that the pattern of binding of Survivin to Smac/DIABLO might be similar to that of XIAP to Smac/DIABLO. ABLAST program search for comparisons between the BIR do-main of Survivin and BIR domains of other IAPs revealed thatSurvivin BIR domain displays high homology to BIR3 andBIR2 domains of XIAP (Fig. 3A). Based on the crystal struc-tural analysis of complex of Smac/DIABLO�XIAP (BIR3), Fesikand coworkers (35) proposed that Glu-314 in the BIR3 domainof XIAP may directly contact with the N-terminal amine ofmature Smac and that the E314S mutant of BIR3 abolishesalmost all binding to mature Smac. In addition, it was proposedthat D214S of the BIR2 mutant domain also lost all the capa-bility of binding to Smac (35). As shown in Fig. 3A, amino acidsGlu-314 in BIR3 domain or Asp-214 in BIR2 domain of XIAPare equivalent to Asp-71 of Survivin, because these amino acidsare highly conserved. To demonstrate whether the amino acidAsp-71 of Survivin is critical for binding to Smac/DIABLO, wemutated Asp-71 to Arg-71 (Fig. 3B) and performed an interac-tion assay as described above. As shown in Fig. 3D (panel a), noeluted Smac band was detected (lane 6), indicating that theD71R mutant Survivin lost its binding ability to interact withmature Smac, and further suggested that Asp-71 of Survivin isvital for the binding to Smac. We further performed a co-precipitation experiment and confirmed that mature Smacdoes not bind to Surv-D71R in vivo (Fig. 3D, panel b). Both theBIR2 and BIR3 domains of XIAP were shown to directly inter-act with Smac/DIABLO, so we then examined whether theSurvivin BIR domain alone (Fig. 3B) is able to bind to Smac/DIABLO. To our surprise, there was no expected Smac band tobe detected (lane 8) using an in vitro interaction assay (Fig. 3D,panel a), and an immunoprecipitation assay also failed to de-tect the GFP/Surv-BIR protein in the final eluted complex (Fig.3D, panel b), implying that, unlike XIAP, Survivin BIR domainalone is unable to bind to Smac/DIABLO. The discrepancy maybe explained by analogy: although XIAP is able to bind toHtrA2/Omi (a homolog of Smac/DIABLO), Survivin is unable tobind to it (36). In addition, the coiled-coil structure at the Cterminus of Survivin is reported to be essential for inhibition ofapoptosis (5), therefore, it is not unwise to argue that thiscoiled-coil structure, which is missing in the Survivin BIRdomain, may be responsible for the interaction with Smac/DIABLO.

The N-terminal of Smac/DIABLO is essential for binding toXIAP (26–29), we thereby checked whether this N terminus isalso crucial for binding to Survivin. We made a series of Smacmutant constructs as depicted in Fig. 2A. Proteins �74Smacand mature Smac were expressed and purified from bacteriaE. coli cells, and proteins F-Smac, M-Smac, and �73Smac wereexpressed in HeLa cells. Among them, proteins �74Smac, M-

Smac, and mature Smac were used in GST pull-down assay(see “Material and Methods”). Fig. 3E (panel a) shows thatmutant 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 matureSmac. A further experiment showed that M-Smac-GFP in celllysate was not able to bind to GST/Survivin-coupled beads (Fig.3E, panel b). Taken together, these data suggest that the in-teractions between Survivin and Smac/DIABLO involve the Nterminus of mature Smac, and, moreover, we propose that wildtype Survivin may directly contact with Ala-1 of mature Smac,because M-Smac (with a methionine added to the beginning ofmature Smac) nullifies the ability of mature Smac to interactwith Survivin.

Survivin Is Co-localized in Cytosol with Mature Smac duringInterphase—Survivin not only acts as an inhibitor of apoptosisbut also regulates cell division (5, 14, 15). Skoufias et al. (42)reported that human Survivin is a kinetochore-associated pas-senger protein and determined its localization by using animmunostaining method. However, different immunostainingprotocols or antibodies produce different results because of theco-existence of Survivin splice variants (46). We thereby con-structed a series of plasmids that express GFP, GFP/Survivin,GFP/Surv-BIR, GFP/Surv-D71R, and GFP/Smac (Fig. 4B) tostudy their distributions in living cells. We first transientlytransfected 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 byfluorescence microscope. As shown in Fig. 4A, GFP proteindisplayed an evenly diffused localization throughout the wholecells transfected with pEGFP-C1, and its distribution wasfound to be independent of the cell cycle. GFP/Survivin wasfirstly found to localize in the cytoplasm during interphase andthen translocate into the central spindle midzone when cellcycle division entered into anaphase. Finally, GFP/Survivinwas detected in the midbody during telophase (Fig. 4A). Thesedata clearly demonstrate that Survivin is a bona fide chromo-somal passenger protein. Our result is in good agreement witha previous report by Li et al. (5) in which they demonstrate thatSurvivin was able to bind to microtubules during interphaseand to be translocated to the nucleus to regulate cell division atthe G2/M phase. To investigate the subcellular distribution ofSurvivin mutants, we expressed GFP fusion proteins GFP/Surv-D71R and GFP/Surv-BIR in HeLa cells and examinedtheir distributions by fluorescence microscopy. Fig. 4A showsthe intracellular localization of GFP/Surv-D71R in differentphases of the cell cycle in HeLa cells. During interphase mu-tant GFP/Surv-D71R was distributed mainly in the cytoplasmsimilar to GFP/Survivin. As the cell cycle entered into ana-phase, it spread evenly throughout the whole cells and wasunable to localize at the spindle midzone. Interestingly enough,GFP/Surv-D71R displayed a midbody localization pattern iden-tical to that of wild type GFP/Survivin during the late telo-phase. We also studied the localization of the GFP/Surv-BIRmutant and found that its localization was the same as that ofGFP protein, which distributed uniformly throughout the cellsand 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 interactionwith GFP, GFP/XIAP, and GFP/Survivin by Western blot (WB) with anti-GFP antibody. b, the interactions between GST/Survivin and matureSmac 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 elutedcomplex. b, the interaction of GFP-tagged Survivin or Survivin mutants with mature Smac was examined as in C, panel a. In E: a, the interactionof GST/Survivin with �74Smac was investigated by in vitro interaction assay. Lane 1 denotes input �74Smac, and lane 2 indicates the final elutedcomplex. 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.

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These results suggest that the �-helix region at the C-terminalof Survivin contributes to its binding to microtubule in inter-phase 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 wasabsolutely required for the association of Survivin with spindlemidzone. It has been reported that Survivin interacts physi-cally with two kinetochore proteins INCENP and Aurora-B,which are known to be localized to both spindle midzone andmidbody and thus to control cell cycle (43). Koufias et al. (42)reported that Survivin mutant Surv-C84A did not localize toeither spindle midzone at anaphase or midbody in telophase. Ifcomplexing with INCENP and Aurora-B was the only mode forSurvivin to localize to the chromosome during M phase, wewould expect that the Survivin mutant Surv-D71R to have thesame localization pattern with that of Survivin mutant Surv-C84A 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, suggestingthat Survivin could utilize an as yet uncharacterized mecha-nism to become a chromosomal passenger protein during mi-

tosis. In addition, we have demonstrated that Smac directlyinteracts with Survivin (Fig. 3C). Therefore, we expected thatSmac/DIABLO should co-localize with Survivin in certainphase of cell cycle. We constructed a plasmid expressing ma-ture GFP/Smac fusion protein in HeLa cells, and 24 h aftertransfection, GFP/Smac was found to locate exclusively in thecytosol and was not exhibited in the nucleus during the inter-phase (Fig. 4A). As the cell cycle enters into anaphase, unlikeGFP/Survivin, GFP/Smac was completely excluded from thespindle midzone and did not associate with midbody in latetelophase (Fig. 4A). Thus Survivin and Smac were co-localizedin the cytoplasm during interphase, but their subsequent dis-tributions are distinctive during mitosis. However, the possi-bility that Survivin interacts with Smac during M phase stillcannot be completely excluded. Taking together, our findingssuggest that Survivin is a chromosomal passenger protein dur-ing mitosis and it co-localizes with mature Smac in cytosolduring interphase when Smac/DIABLO is released frommitochondria.

Survivin Is Not a Direct Inhibitor of Caspase—Numbers ofreports have demonstrated that Survivin can inhibit apoptosis

FIG. 4. Survivin is co-localized with Smac/DIABLO during interphase. A, HeLa cells were transfected with plasmids, includingpEGFP-C1, pEGFP-C1/Survivin, pEGFP-C1/Surv-D71R, pEGFP-C1/Surv-BIR, and pEGFP-C1/Smac. 48 h after transfection, cells were incubatedwith Hoechst 33342 (2 �g/ml) for another 30 min, and the living cells were then analyzed with a fluorescence microscope. GFP or GFP fusionproteins are represented by the green color, and the blue color was generated by Hoechst 33342 staining. Interphase, anaphase, and telophase aredenoted at the top. B, the expressions of five transfected recombinant plasmids indicated in A were verified by Western blot, and the primaryantibody used was anti-GFP monoclonal antibody.

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induced by various stimuli (3, 5, 15). The mechanism wherebySurvivin inhibits apoptosis still remains controversial. Sur-vivin belongs to the IAP family; therefore, some groups re-ported that Survivin blocks cell death through binding to activecaspase and in turn to inhibit caspases activity. Tamm et al.(16) reported that Survivin can bind to the effectors caspase-3and -7 in vitro and inhibit cell death. O’Connor et al. (39)demonstrated that the inhibitory effect of Survivin appearsthrough its binding to caspase-9. However, some other groupsreported contradictory results: they claimed that, in the proc-ess of inhibition of cell death, Survivin does not directly inter-act with caspase-3 and -9 (18, 19, 37). To test whether Survivinphysically interacts with caspases, we performed an in vitrointeraction assay (see “Materials and Methods”). GST/Survivinfusion protein immobilized on glutathione-Sepharose 4B beadswas incubated for 1 h with Taxol-treated HeLa cytosolic ex-tracts, and the beads were then washed and eluted. The finaleluted products were subject to Western analysis, and theresults are shown in Fig. 5. Neither GST/Survivin nor GSTcontrol was found able to pull down caspase-3, -7, and -9 fromTaxol-stimulated cell lysates, whereas both pro-caspase andprocessed caspase were detected in the cell lysate (input) lanes.As a positive control, mature Smac could be pulled down by theGST/Survivin as shown in Fig. 5. These results illustrate thatSurvivin 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 thelinker region between BIR1 and BIR2 within XIAP is respon-sible for the active site-directed inhibition of caspase-3 and -7(40), whereas a single BIR2 domain (residues 157–242) withouta linker region does not bind to caspase-7 (29). Chai et al. (29)have further demonstrated that the BIR domains of XIAP aredispensable for the inhibition of caspase-3 and -7 and that afusion protein between GST and the linker peptide of XIAPtightly binds to and potently inhibits caspase-7 and -3, whereasthe GST-BIR2 (residues 156–240) fusion protein lacking thelinker region does not. Wu et al. (41) also reported that thelinker of XIAP is the major determinant of binding and inhib-itor for the caspases. Unlike XIAP, Survivin does not contain alinker region, and therefore it is not surprising for us to findthat Survivin does not bind to caspase-3 and -7. In addition,results from a comparison of the x-ray crystallographic struc-tures between Survivin and the XIAP (BIR2)�caspase-3 com-plex also support the hypothesis that Survivin does not sup-press caspase-3 (17). These data are in good agreement withour conclusion that Survivin is unable to bind to caspases.

Survivin Blocks Apoptosis May through Antagonizing theActivity of Smac/DIABLO—Both Survivin and XIAP were able

to inhibit apoptosis, however, XIAP was found to be morepotent than Survivin (16). In a co-transfection experiment con-ducted by Tamm et al. (16), although XIAP was shown toalmost completely block cell death induced by Bax or Fas/CD95, cell death could only be partially inhibited by Survivinunder the same conditions. This more complete inhibition ofapoptosis by XIAP was not due to higher levels of XIAP proteincompared with that of Survivin (16) but to its higher activity,because in Bax or Fas/CD95-induced cells, the Survivin proteinlevel was even higher than that of XIAP. All these observa-tions, plus our finding that Survivin is unable to bind tocaspases, suggest that Survivin blocks apoptosis, maybethrough some other bridge of proteins to inhibit caspase activ-ity indirectly. One of the possible bridge protein candidatescould be the Smac/DIABLO based on our new finding thatSurvivin can directly interact with Smac/DIABLO (Fig. 3C).We reasoned that Survivin inhibits cell death, maybe throughprotecting other IAPs such as XIAP from being neutralized bySmac/DIABLO, thus allowing them to maintain their suppres-sion on caspases. If this hypothesis is correct, mutant Survivinincapable of binding to Smac/DIABLO would be expected tolose its inhibitory activity. We have demonstrated previouslythat Survivin mutants Surv-D71R and Surv-BIR had lost theirability to bind to Smac/DIABLO (Fig. 3D); we therefore inves-tigated whether the lack of interaction between Survivin mu-tant and Smac/DIABLO could result in the loss of the inhibi-tory effect of Survivin. We constructed a series of mammalianexpression vectors to express Survivin, Surv-D71R, or Surv-BIR in HeLa cells, and the transfected cells were then treatedwith 100 nM Taxol for 36 h, the cytotoxic effects were measuredusing the trypan blue exclusion method, and protein expressionwas detected by Western blot analysis (Fig. 6A, panel b). In thisassay, although Survivin reduced Taxol-induced cell death by22%, protein Surv-D71R or Surv-BIR was unable to protectcells from apoptosis (Fig. 6A, panel a). This suggests thatabolishment of the inhibitory function for Surv-D71R and Surv-BIR may be due to the loss of their interactions with Smac/DIABLO. It is interesting to note that Survivin mutant Surv-D71R causes more cell death compared with empty vectorcontrol (Fig. 6A, panel a), suggesting Surv-D71R is a noveldominant negative mutant. The conversion of anti-apoptoticwild type Survivin to pro-apoptotic mutant Survivin (Surv-D71R) is not unusual, because Survivin mutations T34A orC84A or Survivin antisense were reported to be pro-apoptotic(39, 5), but the exact mechanism still awaits further investiga-tion. Recently, Silke et al. (38) reported that XIAP mutants thatwere unable to bind to caspase and Smac/DIABLO completelylost the inhibitory function of XIAP, whereas XIAP mutantsthat were unable to bind to caspase but could bind to Smac/DIABLO retained their inhibitory effects. These experimentaldata strongly support our notion that Survivin blocks apoptosismainly through antagonizing the activity of Smac/DIABLO butnot directly interacting with caspases (Fig. 5).

As described above, we have illustrated that Survivinblocked Taxol-induced apoptosis through its binding to Smac/DIABLO, because disruption of Smac�Survivin complex forma-tion leads to a failure of apoptotic inhibition (Fig. 6A, panel a).To further verify this result, we established a cell-free systemto examine whether Smac/DIABLO is a mediator involving inthe inhibition of apoptosis by Survivin. We purified recombi-nant Smac fusion (with an His6 tag at its C terminus) and GSTfusion proteins GST/Survivin, GST/Surv-D71R, and GST/Sur-vivin-BIR for a cell-free assay system. The effects of Smac/DIABLO or GST/Survivin were evaluated by studying the proc-essing of caspase-9 and caspase-7 in cytosolic extracts fromempty vector-transfected or XIAP-overexpressed HeLa cells

FIG. 5. Survivin is unable to bind to caspase. Taxol-treated (100nM, 36 h) HeLa cells were collected and lysed with lysis buffer, and thecytosolic extracts were then used for GST-mediated pull-down assay asdescribed under “Materials and Methods.” The cytosolic extracts andfinal eluted products were subject to Western blot analysis and probedwith 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 finaleluted 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 pulleddown using the same conditions as those used in GST pull-down forcaspase-9, -7, and -3. Molecular sizes of proteins are indicated atthe left.

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following the addition of cytochrome c and dATP. As shown inFig. 6B (panel a), cell extracts without exogenously added cytoc and dATP left procaspase-9 uncleaved, and only one band of45 kDa corresponding to procaspase-9 could be detected (Fig.6B, panel a, lane 1). When cyto c and dATP were added, twocleaved bands (37 and 35 kDa) of caspase-9 were detected inthe blot (Fig. 6B, panel a, lane 2). We found that overexpressionof XIAP strongly prevents the appearance of p37 form (which isgenerated by caspase-3 cleavage) of caspase-9 but not of thep35 form (which is generated by auto-cleavage of caspase-9)(Fig. 6B, panel a, lane 3), indicating XIAP had blocked theactivation of caspase-9. Addition of GST/Survivin or GST/Surv-D71R or GST/Survivin-BIR alone into a cell-free mixture frommock cytosolic extracts was unable to block the appearance ofthe p37 form of caspase-9 (Fig. 6B, panel a, lanes 4–6), sug-gesting Survivin by itself is unable to inhibit caspase-9. Toexclude the possibility that bacterially expressed Survivin fu-sion proteins may compromise their inhibitory functions, weused the cytosolic extracts from cells in which Survivin, Surv-D71R, or Surv-BIR was overexpressed and obtained the sameresults, indicating a failure to block caspase-9 cleavage bySurvivin alone is not due to Survivin expressed from Esche-richia coli (data not shown). In addition, adding Smac proteininto the cell-free mixture from XIAP-overexpressed cytosolicextracts abrogated the prevention of caspase activation byXIAP (Fig. 6B, panel a, lane 7) and resulted in the appearanceof the p37 form, thus confirming that Smac is able to stimulatecaspase-9 activation by removing the inhibition of XIAP. If theGST-Survivin was added to the mixture containing both XIAPand Smac, the p37 form will not be generated (Fig. 6B, panel a,lane 8). In contrast, when the mutant Survivin GST-SR71 orGST-BIR was added, the p37 form was detected (Fig. 6B, panela, lanes 9 and 10). From these results we have reached twoconclusions. First, Survivin is capable of blocking caspase-9activation in the presence of Smac and XIAP. Second, Survivinmutants, which are unable to bind to Smac, fail to inhibitactivation of caspase-9 even in the presence of XIAP and Smac.The same cell-free system was used to examine the effect ofSurvivin 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 cleav-age of caspase-7 (Fig. 6B, panel b, lanes 3–6), whereas Smacprotein was found to antagonize the inhibition of XIAP andgenerate a p19-active form of caspase-7 (Fig. 6B, panel b, lane7). When XIAP, Smac, and GST/Survivin were mixed alto-gether in the cell-free system, cleavage of caspase-7 wasblocked (Fig. 6B, panel b, lane 8), whereas the simultaneouspresence of XIAP, Smac, and Survivin mutants GST/Surv-D71R or GST/Surv-BIR were unable to block the cleavage ofcaspase-7, resulting in the appearance of p19 (Fig. 6B, panel b,lanes 9 and 10). XIAP is known to block the activities ofcaspase-9 and caspase-7 by its binding to caspases (29, 40).However, the inhibitory activity of XIAP can be eliminated bySmac through its direct interaction with XIAP, thus freeing thecaspase from the XIAP�caspase complex (20, 21). In this cell-free system, we have demonstrated that Survivin alone wasunable to block caspase activation due to its inability to bind to

purified proteins and compounds used in the cell-free experiment werethe following: cyto c (1 �g/ml), dATP (1 mM), Smac (100 nM), andGST/Survivin, GST/Surv-D71R, and GST/Surv-BIR were all 200 nM.“�” represents presence and “�” is absence. C, HeLa cells were trans-fected 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 treatedwith 100 nM Taxol for another 36 h. The percentage of cell death wasdetermined by the trypan blue exclusion method.

FIG. 6. Survivin antagonizes the pro-apoptotic activity ofSmac/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), pEGFP-C1/Surv-D71R (fourth bar), pEGFP-C1/Surv-BIR (fifth bar), or p EGFP-C1/ant-Surv (sixth bar), respectively. 20 h later, cells were incubatedwith Taxol (100 nM) for 36 h, and the percentage of cell death wascalculated as the means of three independent experiments. b, expres-sion of proteins from transfected constructs in A (panel a) was con-firmed by Western analysis. In B: the analysis of dATP/cyto c-depend-ent caspase-9 (a) and caspase-7 (b) processing in cytosolic extracts frommock or stable XIAP-expressed HeLa cells. The purified proteins GST/Survivin and His tag/Smac and two Survivin mutants GST/Surv-D71Rand GST/Surv-BIR were added to cytosolic extracts in different combi-nations indicated above the Western blot. The concentrations of various

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caspase (Fig. 5). In addition, Survivin was able to rescue inhi-bition of XIAP through its binding to Smac, thus freeing XIAPfrom the XIAP�Smac complex. However, Survivin mutantsSurv-D71R and Surv-BIR were not able to rescue the inhibitoryeffect of XIAP, because these two Survivin mutants were un-able to bind to Smac (Fig. 3D). To further verify whether theresults obtained from the cell-free system actually occurred inthe cells in vivo, we co-expressed Survivin and Smac (1:1) inHeLa cells and found that co-overexpression of Survivin andSmac remarkably reduced the cell death induced by Taxolcompared with overexpression of Smac alone, whereas co-over-expression of Survivin mutant (Surv-D71R or Surv-BIR) andSmac (1:1) were unable to decrease the cell death (Fig. 6C).This suggested that Survivin was able to antagonize the pro-apoptotic activity of Smac, in particular the BIR domain orC-terminal domain of Survivin were required for its antago-nism. Taken together, data from the transfection experiment invivo were consistent with that from cell-free system in vitro.Although our data have provided strong evidence that Survivinhad inhibitory function with the help of XIAP and Smac, thepossibility that Survivin involves yet uncharacterized path-ways to prevent cell death cannot be dismissed. Additionally,the competitiveness of Survivin and XIAP for binding to Smacis required for further investigation.

DISCUSSION

In this study, we have shown that ectopic overexpression ofSmac/DIABLO sensitizes HeLa cells to apoptosis induced byTaxol. We demonstrated for the first time that Smac/DIABLOphysically interacts with Survivin both in vitro and in vivo.Mutational analysis revealed that amino acid Asp-71 in Sur-vivin is critical for its binding to Smac/DIABLO, and a singleamino acid change (D71R) converts Survivin from being anti-apoptotic to pro-apoptotic. The deletion experiments showedthat the C-terminal coiled-coil domain of Survivin is responsi-ble for the interaction between Survivin and Smac/DIABLO.Similarly, we found that by removing 18 amino acids (AVPI-AQKSEPHSLSSEALM) or adding one amino acid (Met) at itsN terminus Smac/DIABLO abolishes its ability to interact withSurvivin. In addition, Survivin is co-localized in cytosol withSmac/DIABLO during the interphase in living cells. More im-portantly, the Survivin�Smac complex formation contributes toSurvivin inhibitory function during Taxol-induced apoptosis inHeLa cells.

Smac/DIABLO, a mitochondria protein that is released tocytosol in response to a number of apoptotic stimuli, was foundto bind IAPs and prevent them from inhibiting caspases (20–23). Previous reports (22–24, 44) have clearly shown that theBIR domains of IAPs, such as BIR2 or BIR3 of XIAP and BIRof ML-IAP, are sufficient to interact with Smac/DIABLO. How-ever, in the present study, we found that the single BIR domainof Survivin failed to bind to Smac/DIABLO (Fig. 3D), indicatingthat the way Survivin binds to Smac/DIABLO is different fromXIAP or ML-IAP. Smac/DIABLO and its newly identified hom-olog HtrA2/Omi were reported to be able to bind to XIAP in thesame manner, but, unlike Smac/DIABLO, HtrA2/Omi was un-able to interact with Survivin (36). Our result may shed somelight on explaining why Survivin does not bind to HtrA2/Omi.

Recently, Survivin has attracted growing attention due to itsexpression in various tumors and its potential application intumor therapy (3, 45). However, its subcellular localizationremains controversial: different immunostaining protocols ordifferent antibodies used give rise to diverse results because ofthe presence of Survivin splice variants (46). To remedy thesediscrepancies, we analyzed the localizations of GFP/Survivin inliving HeLa cells by using confocal laser microscopy. GFP/Survivin was excluded from the interphase nucleus and was

detected in the central spindle midzone at anaphase and local-ized to the remnants of a mitotic apparatus, midbody at telo-phase (Fig. 4A). Interestingly enough, Survivin mutant GFP/Surv-D71R, which is unable to bind to Smac/DIABLO, lost itsability to localize at the spindle midzone at anaphase. Thisobservation may explain the possible mechanism for the pro-apoptotic effect of Surv-D71R, because the other two dominantnegative mutants, T34A and C84A, reported elsewhere dis-played the same localization pattern as D71R (42, 47).

Although it has been confirmed that Survivin is able toinhibit apoptosis induced by various stimuli, the mechanism bywhich Survivin inhibits apoptosis has not been conclusivelydetermined. As with other IAPs, it has been reported thatSurvivin physically interacts with initiator or effector caspases(16, 37, 48). However, some groups (49, 50) demonstrated thatthis did not seem to translate into physiologically meaningfulinhibition of caspase activity, and the x-ray crystal structure ofSurvivin 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 in-hibits apoptosis mainly through antagonizing the pro-apoptoticability of Smac/DIABLO rather than through binding tocaspases. We demonstrated that Survivin does not interactwith caspases in vitro and that its mutants Surv-D71R andSurv-BIR, which are unable to bind to Smac/DIABLO, had losttheir anti-apoptotic effect (Fig. 6A, panel a). By using a cell-freesystem, we have shown that XIAP but not Survivin, Surv-D71Ror Surv-BIR, prevents cyto c induced caspases activation. Thisprevention can be blocked by adding Smac/DIABLO whichneutralizes XIAP through the formation of Smac*XIAP com-plex (20, 21). When additional Survivin was subsequentlyadded, prevention of caspases activation could be restored.However, Surv-D71R or Surv-BIR, when added into the cell-free system containing both XIAP and Smac, were unable torestore this prevention (Fig. 6B). These data suggest that Sur-vivin is able to antagonize the pro-apoptotic effect of Smac/DIABLO in vitro. We also demonstrated that Survivin blocksthe pro-apoptotic activity of Smac/DIABLO in HeLa cells (Fig.6C). Recently, it was reported that XIAP mutants, which hadlost their ability to inhibit caspase-9 and caspase-3 yet main-tained their ability to interact with Smac/DIABLO, were ableto inhibit cell death and that this inhibition could be explainedby showing that endogenous XIAP is sufficient to block apo-ptosis provided it is not antagonized by an IAP antagonist suchas Smac/DIABLO (20, 21). In the present study, we have dem-onstrated that, although Survivin is unable to inhibit caspases,it possesses the ability to bind to Smac/DIABLO, therebyallowing endogenous IAPs such as XIAP to block caspaseswithout being antagonized. Taken together, we provided aworking model for the inhibition of apoptosis by Survivin. Dur-ing apoptosis Survivin binds to Smac/DIABLO released frominduced mitochondria, thus reducing antagonism of Smac/

FIG. 7. Hypothetical model of Survivin in the inhibition ofapoptosis. Smac is released from Taxol-induced mitochondria, thenbinds to overexpressed Survivin, which reduces neutralizing effect ofSmac on XIAP. XIAP in turn was able to interact with caspases, and celldeath was blocked.

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DIABLO to XIAP, the free XIAP from Smac�XIAP complexdirectly interacts with caspases, and cell death is blocked (Fig.7). This mode suggests that the anti-apoptotic effect of Survivinmay be mitochondria-dependent, because mature Smac is re-leased only from mitochondria. This view is strongly consistentwith ideas proposed by some other groups; they pointed to amore selective role of Survivin in antagonizing mitochondrial-dependent apoptosis, because Survivin blocks mitochondrial-induced but not death-receptor-induced apoptosis in transgenicanimals (53). Recent genetic data showed the heterozygousSurvivin mice (surv �/surv�) were more sensitive to mitochon-drial-dependent cell death (52). Furthermore, cell death follow-ing loss/interference of Survivin showed the characteristics ofmitochondrial-dependent apoptosis (39, 45, 51). In addition, itis worth noting that Survivin mutant Surv-D71R constructedby our group represents a novel dominant negative mutant,because overexpression of this mutant not only abolishes theinhibitory effect of Survivin but also promotes Taxol-inducedapoptosis. This new finding may have clinical usefulness indesigning novel therapeutic drugs.

Acknowledgments—We thank Yi Wang and Shixin Liu for theirexcellent technical help.

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Zhiyin Song, Xuebiao Yao and Mian WuAnti-apoptotic Activity of Survivin during Taxol-induced Apoptosis

Direct Interaction between Survivin and Smac/DIABLO Is Essential for the

doi: 10.1074/jbc.M300957200 originally published online March 26, 20032003, 278:23130-23140.J. Biol. Chem. 

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