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• W2057842968 abstract "X-linked inhibitor of apoptosis protein (XIAP), the most potent member of the inhibitor of apoptosis protein (IAP) family, plays a crucial role in the regulation of apoptosis. XIAP is structurally characterized by three baculovirus IAP repeat (BIR) domains that mediate binding to and inhibition of caspases and a RING domain that confers ubiquitin ligase activity. The caspase inhibitory activity of XIAP can be eliminated by the second mitochondria-derived activator of caspases (Smac)/direct IAP-binding protein with low pI (DIABLO) during apoptosis. Here we report the identification and characterization of a novel isoform of Smac/DIABLO named Smac3, which is generated by alternative splicing of exon 4. Smac3 contains an NH2-terminal mitochondrial targeting sequence required for mitochondrial targeting of Smac3 and an IAP-binding motif essential for Smac3 binding to XIAP. Smac3 is released from mitochondria into the cytosol in response to apoptotic stimuli, where it interacts with the second and third BIR domains of XIAP. Smac3 disrupts processed caspase-9 binding to XIAP, promotes caspase-3 activation, and potentiates apoptosis. Strikingly, Smac3, but not Smac/DIABLO, accelerates XIAP auto-ubiquitination and destruction. Smac3-stimulated XIAP ubiquitination is contingent upon the physical association of XIAP with Smac3 and an intact RING domain of XIAP. Smac3-accelerated XIAP destabilization is, at least in part, attributed to its ability to enhance XIAP ubiquitination. Our study demonstrates that Smac3 is functionally additive to, but independent of, Smac/DIABLO. X-linked inhibitor of apoptosis protein (XIAP), the most potent member of the inhibitor of apoptosis protein (IAP) family, plays a crucial role in the regulation of apoptosis. XIAP is structurally characterized by three baculovirus IAP repeat (BIR) domains that mediate binding to and inhibition of caspases and a RING domain that confers ubiquitin ligase activity. The caspase inhibitory activity of XIAP can be eliminated by the second mitochondria-derived activator of caspases (Smac)/direct IAP-binding protein with low pI (DIABLO) during apoptosis. Here we report the identification and characterization of a novel isoform of Smac/DIABLO named Smac3, which is generated by alternative splicing of exon 4. Smac3 contains an NH2-terminal mitochondrial targeting sequence required for mitochondrial targeting of Smac3 and an IAP-binding motif essential for Smac3 binding to XIAP. Smac3 is released from mitochondria into the cytosol in response to apoptotic stimuli, where it interacts with the second and third BIR domains of XIAP. Smac3 disrupts processed caspase-9 binding to XIAP, promotes caspase-3 activation, and potentiates apoptosis. Strikingly, Smac3, but not Smac/DIABLO, accelerates XIAP auto-ubiquitination and destruction. Smac3-stimulated XIAP ubiquitination is contingent upon the physical association of XIAP with Smac3 and an intact RING domain of XIAP. Smac3-accelerated XIAP destabilization is, at least in part, attributed to its ability to enhance XIAP ubiquitination. Our study demonstrates that Smac3 is functionally additive to, but independent of, Smac/DIABLO. Apoptosis, programmed cell death, is an evolutionarily conserved and genetically regulated biological process that plays a fundamental role in the development and tissue homeostasis of metazoans (1Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2422) Google Scholar, 2Horvitz H. Cancer Res. 1999; 59: S1701-S1706PubMed Google Scholar, 3Vaux D. Korsmeyer S.J. Cell. 1999; 96: 245-254Abstract Full Text Full Text PDF PubMed Scopus (1356) Google Scholar). Dysregulation of apoptosis has been linked to the pathogenesis of a variety of human diseases (4Thompson C. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6172) Google Scholar).Apoptosis is mainly orchestrated by a family of aspartate-specific cysteine proteases known as caspases. Caspases are synthesized as inactive zymogens that bear three domains: an NH2-terminal prodomain, a large subunit, and a small subunit (5Cryns V. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1160) Google Scholar). Caspases involved in apoptosis are generally divided into two categories, the initiator caspases, which include caspase-2, -8, -9, and -10, and effector caspases, such as caspase-3, -6, and -7. A pro-apoptotic signal culminates in activation of an initiator caspase, which, in turn, activates effector caspases (6Thornberry N. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6133) Google Scholar, 7Shi Y. Mol. Cell. 2002; 9: 459-470Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar). There are two well characterized signal pathways leading to the activation of caspases, the death receptor pathway and the mitochondrial pathway. In the mitochondrial pathway, death signals induce the release of cytochrome c from mitochondria into the cytosol and the assembly of an apoptosome consisting of cytochrome c, adapter protein Apaf-1, and procaspase-9, triggering activation of caspase-9, which activates the effector caspases such as caspase-3, resulting in cleavage of a broad spectrum of cellular targets and leading ultimately to apoptosis (8Green D. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar).Among the most important regulators of caspases are the inhibitor of apoptosis proteins (IAPs) 1The abbreviations used are: IAPinhibitor of apoptosis proteinXIAPX-linked IAPBIRbaculovirus IAP repeatSmacsecond mitochondria-derived activator of caspasesDIABLOdirect IAP-binding protein with low PIMTSmitochondrial targeting sequenceHAhemagglutininGFPgreen fluorescent proteinUbubiquitinFBSfetal bovine serumMEFsmouse embryonic fibroblastsHRPhorseradish peroxidaseECLenhanced chemiluminescencemAbmonoclonal antibodyGSTglutathione S-transferaseMG132carbobenzoxyl-leucinyl-leucinyl-leucinalIBMIAP-binding motifRT-PCRreverse transcriptase-PCRPBSphosphate-buffered salineHEKhuman embryonic kidneySTAstaurosporineE3ubiquitin-protein isopeptide ligaseCDDPcisplatin.1The abbreviations used are: IAPinhibitor of apoptosis proteinXIAPX-linked IAPBIRbaculovirus IAP repeatSmacsecond mitochondria-derived activator of caspasesDIABLOdirect IAP-binding protein with low PIMTSmitochondrial targeting sequenceHAhemagglutininGFPgreen fluorescent proteinUbubiquitinFBSfetal bovine serumMEFsmouse embryonic fibroblastsHRPhorseradish peroxidaseECLenhanced chemiluminescencemAbmonoclonal antibodyGSTglutathione S-transferaseMG132carbobenzoxyl-leucinyl-leucinyl-leucinalIBMIAP-binding motifRT-PCRreverse transcriptase-PCRPBSphosphate-buffered salineHEKhuman embryonic kidneySTAstaurosporineE3ubiquitin-protein isopeptide ligaseCDDPcisplatin. (9Salvesen G. Duckett C. Nat. Rev. Mol. Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1566) Google Scholar, 10Deveraux Q. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2263) Google Scholar). The first IAP was discovered in baculoviruses (11Crook N. Clem R.J. Miller L.K. J. Virol. 1993; 67: 2168-2174Crossref PubMed Google Scholar), and many cellular IAP orthologues have since been found in a number of species, ranging from insects to humans (9Salvesen G. Duckett C. Nat. Rev. Mol. Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1566) Google Scholar, 10Deveraux Q. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2263) Google Scholar). Eight IAPs have been identified in mammalian cells to date (9Salvesen G. Duckett C. Nat. Rev. Mol. Cell Biol. 2002; 3: 401-410Crossref PubMed Scopus (1566) Google Scholar, 10Deveraux Q. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2263) Google Scholar, 12Holcik M. Korneluk R.G. Nat. Rev. Mol. Cell. Biol. 2001; 2: 550-556Crossref PubMed Scopus (233) Google Scholar). The anti-apoptotic activity of IAPs, including XIAP, c-IAP1, c-IAP2, and ML-IAP, has been attributed to their ability to bind to and inhibit caspases (13Deveraux Q. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1708) Google Scholar, 14Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1131) Google Scholar). Of these IAPs, XIAP is the most potent inhibitor of caspases and apoptosis (12Holcik M. Korneluk R.G. Nat. Rev. Mol. Cell. Biol. 2001; 2: 550-556Crossref PubMed Scopus (233) Google Scholar, 13Deveraux Q. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1708) Google Scholar). XIAP is structurally characterized by three tandem repeats of the baculovirus IAP repeat (BIR) domain at its NH2 terminus and a COOH-terminal RING finger domain. The inhibitory activity is mediated through the BIR domains. Different BIR domains have been shown to exhibit distinct functions. The second BIR (BIR2) domain together with the immediately proceeding linker region is responsible for binding to and inhibition of active, processed caspase-3 and -7 (15Takahashi R. Deveraux Q. Tamm I. Welsh K. Assa-Munt N. Salvesen G.S. Reed J.C. J. Biol. Chem. 1998; 273: 7787-7790Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar), whereas the third BIR (BIR3) domain is involved in interacting with and suppressing caspase-9 (16Deveraux Q. Leo E. Stennicke H.R. Welsh K. Salvesen G.S. Reed J.C. EMBO J. 1999; 18: 5242-5251Crossref PubMed Scopus (675) Google Scholar, 17Sun C. Cai M. Meadows R.P. Xu N. Gunasekera A.H. Herrmann J. Wu J.C. Fesik S.W. J. Biol. Chem. 2000; 275: 33777-33781Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Like many RING domain-containing proteins, several IAPs, including XIAP, c-IAP1, and c-IAP2, serve as ubiquitin ligases toward themselves and other target proteins, which are subsequently degraded by the 26S proteasome (18Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (861) Google Scholar, 19Huang H. Joazeiro C.A. Bonfoco E. Kamada S. Leverson J.D. Hunter T. J. Biol. Chem. 2000; 275: 26661-26664Abstract Full Text Full Text PDF PubMed Google Scholar, 20Suzuki Y. Nakabayashi Y. Takahashi R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8662-8667Crossref PubMed Scopus (543) Google Scholar, 21Li X. Yang Y. Ashwell J. Nature. 2002; 416: 345-347Crossref PubMed Scopus (390) Google Scholar). Protein ubiquitination and degradation are subject to tight control (22Pickart C. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2887) Google Scholar). It remains to be determined how the auto-ubiquitination and degradation of IAPs are regulated.The caspase-inhibiting activities of IAPs can be relieved by a mitochondrial protein named Smac (second mitochondria-derived activator of caspases) (23Du C. Fang M. Li Y.-L. L Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2887) Google Scholar), also known as DIABLO (direct IAP-binding protein with low pI) (24Verhagen A. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1959) Google Scholar). Similar to cytochrome c, Smac/DIABLO is encoded by a nuclear gene and is subsequently compartmentalized in mitochondria. Upon receiving pro-apoptotic signals, Smac/DIABLO is released from mitochondria into the cytosol where it interacts with IAPs (XIAP, c-IAP1, c-IAP2, and ML-IAP) and abrogates their caspase inhibitory activity, thereby potentiating apoptosis (23Du C. Fang M. Li Y.-L. L Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2887) Google Scholar, 24Verhagen A. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1959) Google Scholar, 25Chai J. Du C. Wu J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (701) Google Scholar, 26Srinivasula S. Datta P. Fan X.J. Alnemri Fernandes T. Fernandes-Alnemri T. Huang Z. Alnemri E.S. J. Biol. Chem. 2000; 275: 36152-36157Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 27Adrain C. Creagh E.M. Martin S.J. EMBO J. 2001; 20: 6627-6636Crossref PubMed Scopus (355) Google Scholar, 28Vucic D. Deshayes K. Ackerly H. Pisabarro M. Kadkhodayan S. Fairbrother W. Dixit V. J. Biol. Chem. 2002; 277: 12275-12279Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The first four residues named IAP-binding motif (29Srinivasula S. Hegde R. Saleh A. Datta P. Shiozaki E. Chai J. Lee R. Robbins P. Fernandes-Alnemri T. Shi Y. Alnemri E.S. Nature. 2001; 410: 112-116Crossref PubMed Scopus (852) Google Scholar) of mature Smac/DIABLO play an indispensable role in Smac/DIABLO function through mediating the binding of Smac/DIABLO to both the BIR-2 and -3 domains of XIAP (25Chai J. Du C. Wu J.W. Kyin S. Wang X. Shi Y. Nature. 2000; 406: 855-862Crossref PubMed Scopus (701) Google Scholar). A cytosolic isoform termed Smac-S/-β (26Srinivasula S. Datta P. Fan X.J. Alnemri Fernandes T. Fernandes-Alnemri T. Huang Z. Alnemri E.S. J. Biol. Chem. 2000; 275: 36152-36157Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 30Roberts D. Merrison W. MacFarlane M. Cohen G.M. J. Cell Biol. 2001; 153: 221-228Crossref PubMed Scopus (92) Google Scholar) is proapoptotic despite its inability to bind IAP in cells (30Roberts D. Merrison W. MacFarlane M. Cohen G.M. J. Cell Biol. 2001; 153: 221-228Crossref PubMed Scopus (92) Google Scholar). Thus, the detailed mechanism by which Smac/DIABLO acts to enhance apoptosis remains to be fully elucidated. Besides antagonizing the caspase-inhibiting activity, it remains to be elucidated whether Smac/DIABLO and/or its isoform(s) are involved in the regulation of XIAP ubiquitination and stability.Here we report the identification and characterization of a novel splicing variant of the Smac/DIABLO family designated Smac3. Smac3 mRNA is ubiquitously expressed in human tissues. Smac3 is localized to mitochondria and can be released into the cytosol during apoptosis. Smac3 is able to interact with XIAP. By binding to XIAP, Smac3 disrupts the association between XIAP and processed caspase-9, facilitates the activation of caspase-3, and antagonizes the anti-apoptotic function of XIAP. Most importantly, our results indicate that Smac3, but not Smac/DIABLO, promotes XIAP ubiquitination and destruction. Our study suggests that Smac3 is functionally additive to, but independent of, Smac/DIABLO and Smac-S/-β. Smac3 is the first molecule identified in mammalian cells to participate in the regulation of XIAP stability.EXPERIMENTAL PROCEDURESExpression Constructs—To create full-length Smac3 with a COOH-terminal hemagglutinin (HA) tag in pcDNA3 (Invitrogen), PCR amplification was performed using Pfu polymerase (Stratagene) with the following primers. The forward primer 5′-AAGGGATCCGCCACCATGGCGGCTCTGAAGAGTTGGC-3′ contains a BamHI site (underlined) and a translation initiation codon (in boldface). The reverse primer 5′-ATGCTCGAGTCAAGCGTAATCTGGAACATCGTATGGGTAATCCTCACGCAGGTAGGCCTC-3′ contains an XhoI site (underlined), a translation stop codon (in boldface), and a 27-bp sequence encoding an HA tag (in italics). pcDNA3-Smac3/HA was then generated by subcloning the PCR product into the BamHI and XhoI sites of pcDNA3. pcDNA3-Smac3 and pcDNA3-mature Smac3/HA were constructed by the same strategy. The construct pCMV-Smac3/FLAG expressing full-length Smac3 with FLAG tag at its COOH terminus was generated by cloning the PCR product into p3xFLAG-CMV-14 vector (Sigma). cDNA encoding residues 1–53 of Smac3 was generated by PCR and cloned into pEGFP-N3 (Clontech). To create a vector expressing mature Smac3 with a COOH-terminal hexahistidine tag, the sequence encompassing mature Smac3 was generated by PCR and subcloned into the NdeI and XhoI sites of pET-30c(+) (Novagen). To make a construct expressing mature Smac3 with an NH2-terminal hexahistidine tag, the sequence encompassing mature Smac3 was amplified by PCR and subcloned into the BamHI and XhoI sites of pET-30c(+). To construct vectors to produce different XIAP fragments in bacteria, the BIR1 (residues 1–124), BIR2 (residues 123–235), and BIR3 (residues 236–358) cDNAs were generated by PCR and cloned into pGEX4T-1 (Amersham Biosciences). All the constructs were verified by DNA sequencing.pcDNA3-myc-XIAP (31Liston P. Roy N. Tamai K. Lefebvre C. Baird S. Cherton-Horvat G. Farahani R. McLean M. Ikeda J. MacKenzie A. Korneluk R.G. Nature. 1996; 379: 349-353Crossref PubMed Scopus (867) Google Scholar) was a gift from Drs. P. Liston and R. Korneluk. XIAP and XIAP 3xBIR in pEBB-FLAG (32Duckett C. Nava V.E. Gedrich R.W. Clem R.J. Van Dongen J.L. Gilfillan M.C. Shiels H. Hardwick J.M. Thompson C.B. EMBO J. 1996; 15: 2685-2694Crossref PubMed Scopus (517) Google Scholar) were provided by Dr. C. Duckett. The construct expressing XIAP H467A mutant in pEBB-FLAG (18Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (861) Google Scholar) was a gift from Dr. J. Ashwell. Plasmids containing wild-type XIAP or several mutants (H343A, D214S/W310A/E314S, D148A/D214S/H343A, and D148A/D214S/W310A/E314S) with FLAG tag (33Silke J. Hawkins C. Ekert P. Chew J. Day C. Pakusch M. Verhagen A. Vaux D. J. Cell Biol. 2002; 157: 115-124Crossref PubMed Scopus (119) Google Scholar) were obtained from Drs. J. Silke and D. Vaux as generous gifts. pCW7 (expressing Myc-tagged ubiquitin) and pCW8 (expressing Myc-tagged ubiquitin K48R) (34Ward C. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1127) Google Scholar) were gifts from Dr. R. Kopito. HA-tagged ubiquitin expression vector (35Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (846) Google Scholar) was a gift of Dr. D. Bohmann. The cDNAs for Bcl-XL, Apaf-1-XL, and caspase-9 in pcDNA3 (36Hu Y. Benedict M.A. Wu D. Inohara N. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4386-4391Crossref PubMed Scopus (495) Google Scholar) were provided by Dr. G. Nunez. Vector expressing Smac-FLAG (23Du C. Fang M. Li Y.-L. L Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2887) Google Scholar) was obtained from Dr. X. Wang.Site-directed Mutagenesis—Site-directed mutagenesis of Smac3 was conducted to generate an A56M substitution mutation using Quick-Change™ Site-directed Mutagenesis kit (Stratagene) with the mutagenic primers: 5′-GGAGTAACCCTGTGTATGGTTCCTATTGCACAGGCTG-3′ and 5′-CAGCCTGTGCAATAGGAACCATACACAGGGTTACTCC-3′. The mutation was verified by DNA sequencing.Cell Culture and Transfection—Human HeLa cervical carcinoma cells, human embryonic kidney (HEK) 293 cells, and HEK293T cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and antibiotics. Smac+/+ and Smac–/– mouse embryonic fibroblasts (MEFs) (37Okada H. Suh W. Jin J. Woo M. Du C. Elia A. Duncan G. Wakeham A. Itie A. Lowe S. Wang X. Mak T. Mol. Cell. Biol. 2002; 22: 3509-3517Crossref PubMed Scopus (157) Google Scholar) were obtained from Dr. T. Mak and cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS and antibiotics. Human MCF7 breast cancer cells and Jurkat T leukemia cells were grown in RPMI 1640 (Invitrogen) containing 10% FBS and antibiotics. HeLa cells and Smac–/– MEFs were transfected by using LipofectAMINE Plus reagent (Invitrogen). 293T cells were transfected by either LT1 transfection reagent (Mirus) or calcium phosphate precipitation method.Western Blotting—Cells were harvested in Triton X-100-based lysis buffer (20 mm Hepes (pH 7.4), 120 mm NaCl, 5 mm EDTA, 1% Triton X-100, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride (Sigma) and complete protease inhibitor mixture (Roche Applied Science)) for 1 h at 4 °C. The debris was removed by centrifugation at 16,000 × g for 20 min at 4 °C. The soluble fractions were recovered, and proteins were quantified using the Micro BCA™ protein assay reagent kit (Pierce). Cellular proteins were resolved on SDS-PAGE and electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) or a nitrocellulose membrane (Bio-Rad) using a semidry transfer apparatus (Bio-Rad). Following blocking, the membrane was incubated with an appropriate primary antibody and then incubated with a corresponding sheep anti-mouse IgG or donkey anti-rabbit IgG conjugated to horseradish peroxidase (Amersham Biosciences). The blots were developed by ECL or ECL Plus method (Amersham Biosciences). Primary antibodies used in this study included the following: anti-FLAG M2 (Sigma), anti-HA mAb (clone 12CA5, Roche Applied Science), anti-HA polyclonal antibody (Santa Cruz Biotechnology), anti-Myc mAb (clone 9E10, Santa Cruz Biotechnology), anti-β-actin mAb (Sigma), anti-GFP antibody (Santa Cruz Biotechnology and Clontech), antibody specific for ubiquitin (Zymed Laboratories Inc.), rabbit anti-caspase-9 cleavage site 315/316 antibody (BIOSOURCE International Inc.), anti-caspase-9 polyclonal antibody (Cell Signaling Technology), anti-caspase-3 antibody (Upstate Biotechnology, Inc.), anti-XIAP mAb (Transduction Laboratories), anti-cytochrome c mAb (clone 7H8.2C12, Pharmingen), and anti-Smac polyclonal antibody (Upstate Biotechnology, Inc.).Immunoprecipitation—Immunoprecipitation was performed as described previously (38Deshpande R. Woods T.L. Fu J. Zhang T. Stoll S.W. Elder J.T. J. Investig. Dermatol. 2000; 115: 477-485Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The precleared supernatants were incubated with anti-FLAG M2 affinity gel (Sigma), anti-Myc mAb conjugated to agarose beads (Santa Cruz Biotechnology), or anti-HA mAb conjugated to agarose beads (Santa Cruz Biotechnology) at 4 °C for 4 h or overnight with constant agitation. The immunoprecipitated materials were analyzed by Western blotting.Far Western Blotting—Affinity-purified recombinant GST-XIAP fragments were fractionated by SDS-PAGE and transferred onto a PVDF membrane. Proteins were denatured by incubating the membrane for 1 h at ambient temperature in denaturation buffer (10 mm Na2HPO4 (pH 7.4), 150 mm NaCl, 5 mm MgCl2, and 1 mm dithiothreitol) containing 6 m guanidine HCl. Proteins were then renatured by several washes of the membrane using gradual reduction of guanidine HCl to 0.3 m. The membrane was incubated in the same buffer at 4 °C overnight. After blocking, the membrane was incubated with whole cell extract of 293T cells expressing Smac3/HA overnight at 4 °C. Bound proteins were detected by Western blotting with an anti-HA mAb.Immunostaining—HeLa cells were transfected with a construct for Smac3/HA. Twenty four to 48 h post-transfection, cells were treated for 12–16 h with 30 μm cisplatin (Sigma), 50 μm etoposide (Sigma), 500 μm H2O2 (Sigma), 1 μm staurosporine (Sigma), and 1 μg/ml anti-Fas antibody (clone CH11, Upstate Biotechnology, Inc.). Cells were fixed in PBS containing 4% formaldehyde for 30 min at room temperature, permeabilized in PBST (PBS containing 0.2% Triton X-100) for 5–10 min at room temperature, and then blocked in PBS with 1% bovine serum albumin. Cells were incubated with an anti-HA polyclonal antibody at 4 °C overnight, followed by incubation with fluorescein isothiocyanate-conjugated anti-rabbit IgG (Molecular Probes) for 45 min. Following extensive washing with PBST, cells were probed with an anti-cytochrome c mAb (clone 6H2.B4, Pharmingen) overnight at 4 °C, followed by incubation with Texas Red-conjugated anti-mouse IgG (Santa Cruz Biotechnology) for 45 min at room temperature. Cells were visualized by a laser scanning confocal microscope system (Leica).Cycloheximide Experiment—Two or three μg of XIAP construct was cotransfected into 293T cells grown onto 60-mm plates with equal amounts of Smac3/FLAG, Smac/FLAG, or empty vector. After 36 h, cells from each transfection were equally split into multiple plates and cultured overnight. Cells were then treated with 30 μg/ml of protein synthesis inhibitor cycloheximide (Sigma) for the indicated time points, when cells were harvested. Cellular extracts were normalized for total protein content and subjected to immunoblotting using antisera recognizing Myc for XIAP and FLAG for Smac3 or Smac. The blot was stripped and re-probed with anti-β-actin as the loading control.Reverse Transcription-PCR—For cloning of Smac3, total RNA was extracted from HEK293 cells using TRIzol Reagent (Invitrogen). Five μg of total RNA was utilized to synthesize the first strand cDNA using the primer specific for Smac with Moloney murine leukemia virus reverse transcriptase (Invitrogen). PCR amplification was performed using Pfu polymerase (Stratagene) under the following conditions: 1 time at 94 °C for 45 s, 30 times (94 °C for 45 s, 65 °C for 45 s, and 72 °C for 90 s), and 1 time 72 °C for 10 min. For analysis of Smac3 expression in human cell lines, RT-PCR was carried out essentially as described previously (39Qi M. Byers P. Hum. Mol. Genet. 1998; 7: 465-469Crossref PubMed Scopus (61) Google Scholar).Subcellular Fractionation—Cytosolic and mitochondrial proteins of HeLa cells were prepared essentially as described previously (27Adrain C. Creagh E.M. Martin S.J. EMBO J. 2001; 20: 6627-6636Crossref PubMed Scopus (355) Google Scholar, 40Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4433) Google Scholar). The cytosolic and mitochondrial proteins were subjected to Western blotting analysis.Expression and Purification of GST Fusion Proteins—Overnight cultures of Escherichia coli DH5α (Invitrogen) transformed with parental or recombinant pGEX4T-1 plasmid were diluted in LB medium containing ampicillin and incubated at 37 °C with shaking to an A600 of 0.6. Isopropyl-d-thiogalactopyranoside (Amersham Biosciences) was then added to a final concentration of 0.1–0.2 mm. After an additional 3–5 h of growth at 30 °C, cells were pelleted at 6,000 × g for 20 min at 4 °C and resuspended in PBS containing 1 mg/ml lysozyme (Amersham Biosciences). After sonication, Triton X-100 was added to a final concentration of 1%, followed by centrifugation at 12,000 × g for 20 min at 4 °C. The GST fusion proteins were adsorbed to Glutathione-Sepharose 4B beads (Amersham Biosciences) and eluted with 10 mm reduced glutathione (Sigma) in 50 mm Tris-HCl (pH 8.0).Expression and Purification of Hexahistidine Fusion Proteins—Overnight cultures of E. coli BL21(DE3) pLysS (Novagen) transformed with mature Smac3 in pET30-c(+) were diluted in LB supplemented with 30 μg/ml kanamycin and incubated at 37 °C with shaking to an A600 of 0.4–0.6. Isopropyl-d-thiogalactopyranoside was then added to a final concentration of 1 mm. After an additional 4 h of growth at 30 °C, cells were collected by centrifugation and resuspended in lysis buffer (20 mm Tris-HCl (pH 8.0), 500 mm NaCl, 5 mm imidazole, 0.1% Nonidet P-40, 1 mg/ml lysozyme). Cells were then sonicated and centrifuged. The supernatant was incubated with nickel-nitrilotriacetic acid-agarose (Qiagen) for 1–2 h at 4 °C. After three washes with wash buffer (20 mm Tris-HCl (pH 8.0), 500 mm NaCl, and 20 mm imidazole), recombinant proteins were eluted in elution buffer (20 mm Tris-HCl (pH 8.0), 500 mm NaCl, and 0.5 m imidazole). Proteins were dialyzed against 10 mm Na2HPO4 (pH 8.0), 50 mm NaCl, and 10 mm KCl.GST Pull-down Assay—About 2–4 μg each of a recombinant IAP fragment was bound to 20 μl of glutathione resin (Amersham Biosciences) that had been pre-blocked in PBS containing 0.5% nonfat milk and 0.05% bovine serum albumin, and incubated with an equal amount of recombinant Smac3 protein at 4 °C overnight in 1 ml of binding buffer (10 mm Na2HPO4 (pH 7.4), 100 mm NaCl, 2 mm dithiothreitol, 2 mm EDTA, 0.1% Nonidet P-40, 5% glycerol, and protease inhibitors). Alternatively, ∼2 μg each of recombinant GST fusion proteins immobilized onto glutathione beads was incubated with 500 μg of cell extract from 293T cells transfected with wild-type or mutant Smac3/FLAG at 4 °C overnight. After extensive washing with wash buffer containing 10 mm Na2HPO4 (pH 7.4), 150 mm NaCl, 2 mm dithiothreitol, and 0.5% Nonidet P-40, the complex was eluted with SDS sample buffer and visualized by SDS-PAGE with Coomassie Blue staining or detected by Western blotting.In Vivo Ubiquitination Assay—In vivo ubiquitination assay was performed as described previously (35Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (846) Google Scholar) with modification. Briefly, 293T cells were transfected with the indicated plasmids in the presence of ubiquitin. Cell lysates were immunoprecipitated with anti-FLAG or anti-Myc antibody and immunoblotted with anti-HA or anti-ubiquitin antibody to detect ubiquitinated proteins.In Vitro Caspase-3 Activation Assay—Cytosolic extracts were prepared from 293T cells essentially as described previously (13Deveraux Q. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1708) Google Scholar, 40Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4433) Google Scholar). For initiating caspase activation, aliquots of cytosolic extracts were incubated with 10 μm bovine heart cytochrome c (Sigma), 1 mm dATP (Amersham Biosciences), and additional 1 mm MgCl2 at 30 °C. The reactions were stopped by adding 5× SDS sample buffer, followed by Western blotting analysis using anti-human caspase-3 antibody to monitor caspase-3 activation.Cell Death Assays—Cell death assays were performed essentially as described previously (36Hu Y. Benedict M.A. Wu D. Inohara N. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4386-4391Crossref PubMed Scopus (495) Google Scholar). Briefly, HeLa cells or 293T cells (1 × 105) were seeded in each well of 12-well plates. After 16 h, cells were tr" @default.
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• W2057842968 title "Smac3, a Novel Smac/DIABLO Splicing Variant, Attenuates the Stability and Apoptosis-inhibiting Activity of X-linked Inhibitor of Apoptosis Protein" @default.
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