Ubiquitin-Protein Ligase Activity of X-Linked Inhibitor Of

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Ubiquitin-Protein Ligase Activity of X-Linked Inhibitor Of Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death Yasuyuki Suzuki, Yui Nakabayashi, and Ryosuke Takahashi* Laboratory for Motor System Neurodegeneration, RIKEN Brain Science Institute, Wako City, Saitama 351-0198, Japan Edited by Suzanne Cory, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, and approved May 24, 2001 (received for review October 24, 2000) The inhibitor of apoptosis (IAP) family of anti-apoptotic proteins RING finger domain specifically inhibits caspase-9 (11). A regulate programmed cell death and/or apoptosis. One such pro- recent report indicates that only the BIR3 domain of XIAP is tein, X-linked IAP (XIAP), inhibits the activity of the cell death required to inhibit caspase-9 and that the RING finger domain proteases, caspase-3, -7, and -9. In this study, using constitutively is unnecessary for potent caspase-9 inhibition (12). Collectively, active mutants of caspase-3, we found that XIAP promotes the these results suggest that the BIR domains of IAP are essential degradation of active-form caspase-3, but not procaspase-3, in in inhibiting caspases. living cells. The XIAP mutants, which cannot interact with Recent results implicate the RING finger domain in specific caspase-3, had little or no activity of promoting the degradation of ubiquitination events (13–15). Protein ubiquitination begins with caspase-3. RING finger mutants of XIAP also could not promote the the formation of a thiol-ester linkage between the COOH degradation of caspase-3. A proteasome inhibitor suppressed the terminus of ubiquitin and the active site cysteine of the ubiquitin- degradation of caspase-3 by XIAP, suggesting the involvement of activating enzyme (E1). Ubiquitin then is transferred to a a ubiquitin-proteasome pathway in the degradation. An in vitro ubiquitin-conjugating enzyme (Ubc or E2), again through a ubiquitination assay revealed that XIAP acts as a ubiquitin-protein thiol-ester linkage. Ubiquitin-protein ligases (E3s), which are ligase for caspase-3. Caspase-3 was ubiquitinated in the presence primarily responsible for providing specificity to ubiquitin con- of XIAP in living cells. Both the association of XIAP with caspase-3 jugation, interact with E2 and the substrate, facilitating the and the RING finger domain of XIAP were essential for ubiquiti- formation of isopeptide bonds between the COOH terminus of nation. Finally, the RING finger mutants of XIAP were less effective ubiquitin and lysines either on a target protein or on the last than wild-type XIAP at preventing apoptosis induced by overex- ubiquitin of a protein-bound multiubiquitin chain. Multiubiq- pression of either active-form caspase-3 or Fas. These results uitin chains are potent targeting signals for protein degradation demonstrate that the ubiquitin-protein ligase activity of XIAP in proteasomes (16). E3s that are highly homologous to IAP with promotes the degradation of caspase-3, which enhances its anti- respect to their RING finger domains, such as Mdm2 and Siah, apoptotic effect. promote degradation of both themselves and specific substrates, such as p53, and the deleted in colorectal cancer gene product poptosis is a physiological cell suicide program critical to the (DCC), respectively (17–20). Recent reports indicate that XIAP Adevelopment and homeostasis of all animals (1). Abnormal and c-IAP1 promote self-degradation in response to apoptotic inhibition of apoptosis is a hallmark of cancer and autoimmune stimuli (21), and that c-IAP2 promotes in vitro monoubiquiti- disease, whereas excessive cell death has been implicated in nation of caspase-3 and -7 (22). In this study, we investigated neurodegenerative disorders (2). The caspases, a family of whether XIAP promotes degradation of caspase-3 through its E3 intracellular cysteine proteases, are the central executioners of activity and whether its E3 activity affects its anti-apoptotic apoptosis (3). Effector caspases, such as caspase-3 and -7, are effect. activated by initiator caspases, such as caspase-9, through pro- teolytic cleavage (3). Once activated, the effector caspases are Materials and Methods responsible for the proteolytic cleavage of a diverse array of Plasmid Constructions. cDNA encoding reversed-caspase-3 (rev- structural and regulatory proteins, resulting in an apoptotic Casp3), a constitutively active mutant of caspase-3, was gener- phenotype (4). ated by PCR according to Srinivasula et al. (23). Using the Inhibitor of apoptosis proteins (IAPs), originally found in resultant plasmid as a template, PCR was performed with the baculoviruses, have been conserved in a number of species, following primers: revCasp3-forward, GGAATTCAGTGGT- ranging from insects to humans throughout evolution and play a GTTGATGATGACAT; revCasp3-reverse, ATTCTCGAGT- role in regulating apoptosis (5, 6). Several members of the human CAGTCTGTCTCAATGCCAC. The PCR product was double- IAP family proteins, including X-linked IAP (XIAP), c-IAP1 and c-IAP2, have been shown to be potent direct inhibitors of caspase-3, -7, and -9 (7–9). Among the above IAPs, XIAP is the This paper was submitted directly (Track II) to the PNAS office. most potent inhibitor of caspases and apoptosis (6). The struc- Abbreviations: IAP, inhibitor of apoptosis protein; XIAP, X-linked IAP; BIR, baculovirus IAP ture of XIAP is characterized by three tandem repeats of the repeat; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; rev- Casp3, reversed-caspase-3; HA, hemagglutinin; TUNEL, terminal deoxynucleotidyl trans- baculovirus IAP repeat (BIR) domain at its NH2 terminus and ferase-mediated dUTP nick end labeling. a RING finger domain near its COOH terminus. Deletional *To whom reprint requests should be addressed. E-mail: [email protected]. analysis indicates that the second BIR domain (BIR2) of XIAP The publication costs of this article were defrayed in part by page charge payment. This is sufficient to inhibit caspase-3 and -7 (10), whereas a XIAP article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. fragment encompassing the third BIR domain (BIR3) and the §1734 solely to indicate this fact. 8662–8667 ͉ PNAS ͉ July 17, 2001 ͉ vol. 98 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.161506698 Downloaded by guest on September 27, 2021 digested with EcoRI–XhoI and cloned into the EcoRI–XhoI sites tagged UbcH5B and UbcH7 were induced with 1 mM IPTG at of pcDNA3-Myc-N. To generate pcDNA3-FLAG-XIAP, the 30°C for 3 h, then prepared by using Ni-NTA Agarose (Qiagen, EcoRI–XhoI fragment of the full-length XIAP (residues 1–497) Chatsworth, CA) according to the manufacturer’s instructions. of pcDNA3-Myc-XIAP was cloned into pcDNA3-FLAG-N. The Concentrations of the purified proteins were determined by the RING finger domain-deleted form of XIAP (1–437; Coomassie Plus Protein Assay Reagent Kit (Pierce). XIAP⌬RING) was generated by PCR using the following primers: forward, CGGAATTCATGACTTTTAACAGTTTT- In Vitro Translation of Caspases. Wild-type and mutant caspases GAAGG; reverse, CTCCTCGAGTTAAGTACTAATCTCTT- were translated in vitro in the presence of [35S]methionine in TCTG. All of the point mutants were generated by using a rabbit reticulocyte lysate with a TNT T7 Quick Coupled Tran- QuickChange site-directed mutagenesis kit (Stratagene). Full- scription/Translation System (Promega). length XIAP (residues 1–497) was cloned into the EcoRI–XhoI sites of pGEX4T1 (Amersham Pharmacia). pGEX4T1-XIAP In Vitro Ubiquitination Assay. 35S-methionine-labeled, in vitro- has been described (7). UbcH5B and UbcH7 were cloned by translated revCasp3 was added to GST-XIAP (250 ng) immo- reverse transcription–PCR. Amino-terminal His6-tagged bilized on glutathione-Sepharose beads. The mixture was incu- UbcH5B and UbcH7 constructs were generated by ligating PCR bated on ice for1htoformaGST-XIAP/revCasp3 complex. fragments into the EcoRI–XhoI sites of pET28a (Novagen). After extensive washing, the GST-XIAP/revCasp3 complex was pCGN-hemagglutinin (HA)-ubiquitin was a gift from Shiget- used to perform the ubiquitination assay, which was carried out sugu Hatakeyama, Kyusyu University, Fukuoka, Japan. Proper in ubiquitination buffer (50 mM Tris⅐HCl, pH 7.5/2 mM ATP/5 construction of all of the plasmids was confirmed by DNA mM MgCl2/2 mM DTT) containing yeast E1 (200 ng), E2 (150 ␮ ␮ sequencing. ng), and His6-ubiquitin (10 g), in a total volume of 30 l. The mixture was incubated at 30°C for 90 min with agitation to keep Transfection, Immunoblot, and Immunoprecipitation Analysis. 293T the beads in suspension. After the reaction, the samples were and 293 cells were transfected by using either Lipofectamine Plus resolved on SDS/PAGE using a 5–15% linear gradient gel and (GIBCO/BRL) or FuGENE6 (Roche Diagnostics) according to analyzed by autoradiography. the manufacturer’s instructions. A total of 4–5 ϫ 105 cells were plated in 6-well plates, washed the next day, and then transfected DEVDase Activity. Caspase activities were assayed as described (7) by using a total of 2.5 ␮g of plasmid DNA. After 3-h incubation, with some modification. In brief, caspase activity was measured the medium was replaced with fresh complete medium. At 40–48 at 30°C in 100 ␮l of caspase buffer (20 mM Pipes/100 mM NaCl/1 h after transfection, the cells were washed with PBS and mM EDTA/0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1- recovered by scraping. Precipitated cells were lysed in 50 ␮lof propanesulfonate/10% sucrose/10 mM DTT, pH 7.2) containing lysis buffer [20 mM Hepes, pH 7.4/120 mM NaCl/5 mM 20 ␮g of cell lysate and 100 ␮M of the fluorogenic peptide EDTA/1% Triton X-100/10% glycerol/protease inhibitor (Com- substrate, acetyl-DEVD-MCA (Peptide Institute). Activity was plete, Roche Diagnostics)], with or without 20 ␮M of the measured continuously over the indicated time by the release of proteasome inhibitor MG132 (Peptide Institute, Osaka), at 4°C.
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