Ubiquitin-specific Peptidase 21 Inhibits Tumor NecrosisFactor �-induced Nuclear Factor �B Activation via Binding toand Deubiquitinating Receptor-interacting Protein 1*□S

Received for publication, July 20, 2009, and in revised form, November 1, 2009 Published, JBC Papers in Press, November 12, 2009, DOI 10.1074/jbc.M109.042689

Gufeng Xu‡1, Xiaojie Tan‡§1, Hongmei Wang‡, Wenjing Sun‡, Yi Shi¶, Susan Burlingame‡, Xue Gu‡�, Guangwen Cao§,Ting Zhang�, Jun Qin¶, and Jianhua Yang‡2

From the ‡Texas Children’s Cancer Center, Department of Pediatrics, Dan L. Duncan Cancer Center, and the ¶Center for MolecularDiscovery, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Department of Molecular and CellularBiology, Baylor College of Medicine, Houston, Texas 77030, the §Department of Epidemiology, Second Military Medical University,800 Xiangyin Road, Shanghai 200433, China, and the �Capital Institute of Pediatrics, Beijing 100020, China

Ubiquitination and deubiquitination of receptor-interactingprotein 1 (RIP1) play an important role in the positive and neg-ative regulation of the tumor necrosis factor � (TNF�)-inducednuclear factor �B (NF-�B) activation. Using a combination offunctional genomic and proteomic approaches, we have identi-fied ubiquitin-specific peptidase 21 (USP21) as a deubiquitinasefor RIP1. USP21 is constitutively associated with RIP1 and deu-biquitinates RIP1 in vitro and in vivo. Notably, knockdown ofUSP21 in HeLa cells enhances TNF�-induced RIP1 ubiquitina-tion, I�B kinase � (IKK�), and NF-�B phosphorylation, inhibi-tor of NF-�B � (I�B�) phosphorylation and ubiquitination, aswell as NF-�B-dependent gene expression. Therefore, ourresults demonstrate that USP21 plays an important role in thedown-regulation of TNF�-induced NF-�B activation throughdeubiquitinating RIP1.

Transcription factor nuclear factor �B (NF-�B)3 plays animportant role in controlling the expression of survival factors,cytokines, and proinflammatory molecules in a broad range ofcellular responses (1–3). NF-�B is sequestered in the cytoplasmby a family of inhibitory proteins called inhibitor of NF-�B(I�B) proteins in inactivated cells. Many intercellular stimuliare capable of triggering the activation of a signal transductionpathway that leads to the degradation of I�B proteins throughthe 26 S proteasome (4–6). Degradation of the I�B proteinsallows NF-�B translocation from cytoplasm to the nucleus andactivates the expression of the target genes (7).

Uponbindingof tumornecrosis factor� (TNF�),TNFreceptor1 (TNFR1) recruits several adaptor proteins, including receptor-interacting protein 1 (RIP1/RIPK1) and TNF receptor-associatedfactor2 (TRAF2), to formacomplex (8,9).ThisTNFR1-associatedcomplex initiates the activation of I�B kinase (IKK), which phos-phorylates I�B protein and activates NF-�B (10–17).

Protein ubiquitination is a crucial regulatory mechanism invarious cellular processes, including cell cycle progression, theDNA damage response, and immune responses (18–20). In theTNF�-induced NF-�B signal transduction pathway, the Lys63-linked polyubiquitination of RIP1 protein mediated by TRAF2E3 ligase is essential for TNF�-induced IKK/NF-�B activation,whereas phosphorylation of the I�B proteins by activated IKKleads to their Lys48-linked polyubiquitination, which labels itfor its degradation by the 26 S proteasome (21).Several deubiquitinating enzymes, including CYLD, A20,

Cezanne, ubiquitin-specific peptidase 15 (USP15), and USP31,have been suggested to be involved in the down-regulation ofTNF�-induced NF-�B activation (22–26). However, it remainsunclear how deubiquitination plays a role in the down-regula-tion of TNF�-induced NF-�B activation.The USPs belong to a subclass of the protein-deubiquitinat-

ing enzyme (DUB) superfamily that are categorized into fivesubclasses based on their ubiquitin-protease domains in thehuman genome and have been shown to be involved in a broadrange of biological activities (27). Even though theUSP subclassof DUB represents the bulk of the deubiquitinating enzymesencoded in the human genome, the roles of USP proteins in theTNF�-induced NF-�B signal transduction pathway have notbeen studied in great detail.In this study, we used a functional genomic approach to iden-

tify the USPs that are involved in TNF�-induced NF-�B activa-tion by screening a library of USPswhose overexpression inhib-its TNF�-induced NF-�B activation. By combining with aproteomic approach, here we present evidence that USP21functions as a RIP1 deubiquitinase that deubiquitinates RIP1and down-regulates TNF�-induced NF-�B activation.

EXPERIMENTAL PROCEDURES

Identification of USP21-associated Proteins by MassSpectrometry—HEK-293T cells were transfected with theempty vector control and FLAG-RIP1 vector and lysed. FLAG-

* This work was supported, in whole or in part, by National Institutes of HealthGrant 1R21CA106513-01A2 (to J. Y.), American Cancer Society Grant RSG-06-070-01-TBE (to J. Y.), and National Basic Research Program of ChinaGrant 2007CB511900 (to T. Z.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table 1 and Figs. S1–S3.

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: Texas Children’s Cancer

Center, Dept. of Pediatrics, Dan L. Duncan Cancer Center, Baylor College ofMedicine, One Baylor Plaza, Houston, TX 77030. Tel.: 832-824-4572; Fax:832-825-4732; E-mail: jianhuay@bcm.edu.

3 The abbreviations used are: NF-�B, nuclear factor �B; I�B, inhibitor of NF-�B;IKK, I�B kinase; DTT, dithiothreitol; DUB, protein deubiquitinating enzymesuperfamily; HA, hemagglutinin; IL, interleukin; PMSF, phenylmethylsulfo-nyl fluoride; RIP1, receptor-interacting protein 1; sh, small hairpin; TNF�,tumor necrosis factor �; TNFR1, TNF receptor 1; TRAF, TNFR-associatedfactor; USP, ubiquitin-specific peptidase; WCL, whole cell lysate.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 2, pp. 969 –978, January 8, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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RIP1was immunoprecipitated fromcell lysateswith anti-FLAGantibodies after preclearing with normal mouse IgG. Theimmunoprecipitates were separated on SDS-PAGE and stainedwith Coomassie Blue. Each lane was divided into 12 pieces, andproteins in which were identified with mass spectrometry asdescribed (28).Cell Culture and Transfection—HEK-293T and HeLa cells

were cultured in Dulbecco’s modified Eagle’s medium contain-ing penicillin (100 units/ml), streptomycin (100 mg/ml), andglutamine (2 mM) supplemented with 10% fetal bovine serumand transfected with FuGENE 6 or FuGENE HD according tothe manufacturer’s recommendation.Expression Plasmids, USP cDNA Expression Library, Small

Hairpin RNA Constructs, Small Interfering RNA—Human ormouse USP cDNA clones were purchased from Open Biosys-tems Company (Huntsville, AL). A full-length cDNA sequence

for each USP member containing an open reading frame wassubcloned into pcDNA3.1 expression vector (Invitrogen). TheNF-�B-dependent Firefly luciferase reporter plasmid andpCMV promoter-dependent Renilla luciferase reporter werepurchased from Clontech. Mammalian expression vectors forTRAF2, RIP1,CYLD,A20, andUSP21were constructed by sub-cloning cDNAs encoding the full-length wild type human pro-teins into the pcDNA3.1 vectors with an N-terminal Myc,FLAG, or HA tag. The USP21C221A mutant expression con-struct was generated using the QuikChange site-directedmutagenesis kit (Stratagene). Mammalian expression vectorsfor HA-IKK� and FLAG-TRAF5 were obtained from Dr. PaulChiao and Dr. Bryant Darnay, respectively (M. D. AndersonCancer Center, Houston, TX). Lys48-only and Lys63-only ubiq-uitin with N-terminal HA tags were subcloned into pcDNA3.1expression vector (Invitrogen). A pSUPER-retro vector was

FIGURE 1. USP21 inhibits TNF�-induced NF-�B activation. A, effect of overexpression of members of USP subclass of deubiquitinase on TNF�-induced NF-�Bactivation. NF-�B luciferase reporter and control Renilla luciferase reporter vectors were co-transfected into HEK 293T cells with empty vector or expressionvectors encoding different USPs for 42 h. Cells were then either untreated or treated with TNF� (1 ng/ml) for 6 h. Then the relative luciferase activity wasmeasured and normalized with the Renilla activity. Error bars indicate � S.D. in three independent experiments. B, USP21 deubiquitinase activity is required forits inhibitory effect on TNF�-induced NF-�B activation. NF-�B luciferase reporter and control Renilla luciferase reporter vectors were co-transfected intoHEK-293T cells with empty vector or expression vectors encoding USP21 wild type (WT) or C221A mutant with or without Myc tag for 42 h. Cells were then eitheruntreated or treated with TNF� (1 ng/ml) for 6 h. Then, the relative luciferase activity was measured and normalized with the Renilla activity. Error barsindicate � S.D. in three independent experiments. Myc-USP21 proteins in the cell lysates were immunoblotted (IB) with anti-Myc antibodies, and �-actin is aloading control.

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used to generate shRNA plasmids for USP21. The followingtarget sequences have been selected: 5�-AAGATGGCTCA-TCACACACTC-3� (shUSP21-1), 5�-AACTTAGCCCGTTC-CAAGTCT-3� (shUSP21-2). A scramble sequence 5�-AGCGCGCTTTGTAGGATTCG-3� was used as a negative control,and the selected shRNA sequences against USP21were submit-ted to a BLAST search against the human genome sequence toensure specificity. The shA20 plasmid was obtained from Dr.

Peter Storz (MayoClinic). Syntheticsmall interfering RNAs againsthuman USP21 (target sequence, 5�-GCCGTTCCAAGTCTGTGA-3�)and A20 (target sequence, 5�-CAACGGCTACTGCAACGAA-3�) were purchased from Ambion(Austin, TX).Antibodies and Reagents—Anti-

USP21 antibodies were purchasedfrom Abcam and Santa Cruz Bio-technology. Monoclonal anti-A20and RIP1 antibodies were pur-chased from BD Biosciences. Otherantibodies used in this study includeanti-FLAG M2 monoclonal andanti-actin antibodies (Sigma); anti-I�B� and secondary antibodies con-jugated to horseradish peroxidase(Cell Signaling Technology); anti-IKK� (NEMO); anti-HA and anti-Myc epitope antibodies (Santa CruzBiotechnology). Human recombi-nant Lys48-linked and Lys63-linkedpolyubiquitin wild type chainswere purchased from Boston Bio-chem (Cambridge, MA). Recombi-nant human TNF� was purchasedfrom R & D Systems (Minneapolis,MN).Immunoprecipitation and Im-

munoblotting—Cells were washedwith phosphate-buffered saline andlysed for 30 min at 4 °C in lysisbuffer containing 25mMHEPES, pH7.6, 135mMNaCl, 1% Triton X-100,1 mM DTT, 10 �g/ml aprotinin, 10�g/ml leupeptin, 1 mM benzami-dine, 1mMPMSF. Lysates were thencleared by centrifugation, and pro-teins were immunoprecipitatedwith affinity antibody and proteinA-agarose beads at 4 °C. Immuno-precipitates were washed four timeswith lysis buffer and boiled withsample buffer before being sepa-rated by SDS-PAGE and thentransferred onto nitrocellulosemembranes following standardprocedures. After being probed

with the appropriate antibodies, the appropriate IgG horse-radish peroxidase-conjugated antibodies were used as thesecondary antibodies, followed by detection with the ECLPlus Western blotting system (Amersham Biosciences) andvisualized by autography.Preparation of Cytoplasmic andNuclear Extracts—Cytoplas-

mic extracts were prepared by adding buffer A (10 mM HEPES,pH 7.9, 10mMKCl, 0.1mM EDTA, 0.1mM EGTA, 1mMDTT, 1

FIGURE 2. Identification of RIP1 as a USP21-associated protein. A, identification of FLAG-USP21-asso-ciated proteins by mass spectrometry. HEK-293T cells were transfected with control vectors or expressionvectors encoding the N-terminal FLAG-tagged USP21. FLAG-USP21 in the cell lysates were immunopre-cipitated with anti-FLAG antibodies and subjected to 10% SDS-PAGE. Coomassie blue-stained FLAG-USP21 proteins were identified by MALDI-TOF mass spectrometry. B, co-immunoprecipitation of FLAG-USP21 and endogenous RIP1 proteins. FLAG-USP21 in the cell lysates from the transfected HEK-293T cellswere immunoprecipitated (IP) with anti-FLAG antibodies and immunoblotted with anti-RIP1 antibodies.C, co-immunoprecipitation of FLAG-USP21 and HA-RIP1 proteins. Expression vectors encoding HA-RIP1were co-transfected into HEK 293T cells with control vectors or expression vectors encoding FLAG-USP21wild type (WT) or deubiquitinase-deficient (C221A) mutant, respectively. FLAG-USP21 in the cell lysateswas immunoprecipitated with anti-FLAG antibodies and immunoblotted (IB) with anti-HA antibodies.D, co-immunoprecipitation of endogenous USP21 and RIP1 proteins. Endogenous RIP1 in the HeLa celllysates was immunoprecipitated with anti-RIP1 antibodies and immunoblotted with anti-USP21 antibod-ies. E, co-immunoprecipitation of endogenous USP21 and RIP1 proteins at different time points of TNF�stimulation. Endogenous RIP1 in the HeLa cell lysates was immunoprecipitated with anti-RIP1 antibodiesand immunoblotted with anti-USP21 antibodies with 10 ng/ml TNF� treatment at different time points.IgG H. C., IgG heavy chain; IgG L. C., IgG light chain.

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mMPMSF, 20mMglycerophosphate, 0.1mMNa3VO4, 10�g/mlaprotinin, and 10�g/ml leupeptin) to cell pellets. The cells werethen suspended and chilled on ice for 15 min followed by add-

ing 25�l of 10%Nonidet P-40 and vortexing vigorously for 10 s.Cytoplasmic extracts were collected after centrifugation at12,000 � g for 5 min. For the nuclear extracts, the nuclear pel-

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lets were washed three times using buffer A, as mentionedbefore, and buffer B (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM

EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 20 mM glycero-phosphate, 1 mM Na3VO4, 10 �g/ml aprotinin, and 10 �g/mlleupeptin) was then added to the nuclear pellets. The resuspen-sion was put on ice for 15 min followed by centrifugation at12,000 � g for 5 min. The supernatants were collected asnuclear extracts.In VitroDeubiquitinationAssay—Wild-type ormutantMyc-

tagged USP21 proteins were immunoprecipitated from thetransfected HEK-293T cell lysates prepared with Nonidet P-40lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM

EDTA, 10% (v/v) glycerol, 0.5% (v/v) Nonidet P-40, 1 mM

PMSF, and 1 mM DTT). The immunoprecipitates werewashed three times with washing buffer and then incubatedwith 1.25 �g of either Lys48-linked or Lys63-linked polyubiq-uitin substrates in 15 �l of 50 mM Tris-HCl buffer, pH 7.2,containing 1 mM DTT for 2 h or with 15 �l of cell lysatesprepared from the FLAG-RIP1-transfected HEK-293T cellsin Nonidet P-40 lysis buffer and incubated at 37 °C for 2 h.Reaction mixtures were spun down, and the supernatantswere analyzed by silver staining and immunoblotting withanti-FLAG (M2) antibodies.Luciferase Reporter Assay—HEK-293T or HeLa cells were

transiently co-transfected with an NF-�B-dependent Fireflyluciferase reporter and effector plasmids along with theRenilla luciferase plasmid. The cells were harvested in pas-sive lysis buffer after 48 h, and luciferase assays were per-formed using the Dual Luciferase Reporter Assay system(Promega). The relative luciferase activity was calculatedthrough dividing the Firefly luciferase activity by the Renillaluciferase activity. Data represent three independent exper-iments performed in duplicate.Establishment of the Stable USP21 Knockdown HeLa Cell

Lines—The pSUPER USP21 shRNA retroviral vectors weretransfected into the HEK-293T cells with retrovirus packingvector Pegpam 3e and RDF vector using FuGENE 6. Viralsupernatants were collected after 36 and 48 h. HeLa cells werethen incubated with virus-containing medium in the presenceof 4 �g/ml Polybrene. Stable cell lines were established after 7days of puromycin (2 �g/ml) selection. Knockdown of theUSP21 was confirmed by real time reverse transcription-PCRand immunoblotting analysis.

RESULTS

USP21 Inhibits TNF�-induced NF-�BActivation—Proteinubiquitination and deubiquitination have been suggested toplay important roles in TNF�-induced NF-�B activation(20). To explore whether any member of deubiquitinases in

the USP subclass is involved in regulation of TNF�-inducedNF-�B activation, we first generated a library of mammalianexpression vectors that encode 30 USP deubiquitinases.Then we used an NF-�B-dependent luciferase reporter assayto assess the effects of overexpression of each USP on TNF�-induced NF-�B luciferase reporter activity. To avoid thepotential inhibitory effect of a tag sequence on deubiquiti-nase enzymatic activity, we did not put any tag into the deu-biquitinase protein coding sequence in this USP expressionlibrary. In this screen, as shown in Fig. 1A, USP21 signifi-cantly inhibited TNF�-induced NF-�B luciferase reporteractivity whereas other USPs had no or less effect. To assesswhether the inhibitory effect of overexpression of theseUSPs on the TNF�-induced NF-�B reporter activity is due totheir deubiquitinase activity, we generated expression vec-tors encoding a USP21 deubiquitinase-deficient mutant bysubstitution of a cysteine residue in the USP active site withan alanine (C221A) and found that only USP21 wild type, butnot deubiquitinase-deficient C221A mutant, abolished theTNF�-induced NF-�B activation in a reporter assay (Fig.1B). This result suggests that USP21 deubiquitinase activityis required for its inhibitory role in the TNF�-inducedNF-�B activation. To test further whether USP21 possessesthe deubiquitinase activity and whether the cysteine to ala-nine mutation in the potential active site abrogates its deu-biquitinase, we immunoprecipitated the Myc-tagged USP21wild type and mutant proteins from the cell lysates of thetransfected HEK-293T cells and performed an in vitro deu-biquitination assay with both Lys48- and Lys63-linked ubiq-uitin substrates. In this assay, we found that USP21 wild typebut not C221A mutant proteins catalyze the deubiquitina-tion of both Lys48- and Lys63-linked ubiquitin (data notshown). Together, our data strongly indicate that USP21 isinvolved in the down-regulation of the TNF�-inducedNF-�B activation through its deubiquitinase activity.USP21 Is Associated with RIP1—To determine the molecu-

lar mechanism of USP21 function in TNF�-mediated NF-�Bactivation, we immunoprecipitated the FLAG-tagged USP21from the lysate of the transfected HEK-293T cells withFLAG-USP21 expression vector and identified the co-im-munoprecipitated proteins with FLAG-USP21 by mass spec-trometry (Fig. 2A). We found that receptor (TNFRSF)-inter-acting serine-threonine kinase 1 (RIP1/RIPK1) was one ofthe major co-immunoprecipitated proteins with FLAG-USP21. The rest of the identified USP21-associated proteins,with the exception of ubiquitin, which are more abundantthan RIP1, seem more likely to be nonspecific bindingproteins (supplemental Table 1). This result suggests that

FIGURE 3. USP21 acts as a RIP1 deubiquitinase. A, USP21 inhibits RIP1 polyubiquitination. Expression vectors encoding FLAG-RIP1 and HA-ubiquitin wereco-transfected into HEK-293T cells with control vectors or expression vectors encoding Myc-USP21 wild type (WT), C221A mutant, or CYLD, respectively.FLAG-RIP1 proteins in the cell lysates were immunoprecipitated (IP) with anti-FLAG antibodies and immunoblotted (IB) with anti-HA antibodies to detect thepresence of ubiquitinated FLAG-RIP1. B, USP21 deubiquitinates RIP1 in vitro. HEK-293T cells were transfected with expression vectors encoding FLAG-RIP1,Myc-USP21 WT, and C221A mutant, respectively. Cells were lysed in the lysis buffer containing only PMSF as a protease inhibitor. Myc-USP21 proteins in the celllysates were immunoprecipitated with anti-Myc antibodies and co-incubated with FLAG-RIP1 lysates for 1 h in the deubiquitinating buffer before beinganalyzed by immunoblotting with the anti-FLAG antibodies to detect the presence of ubiquitinated FLAG-RIP1. C, USP21 inhibits TRAF2- and RIP1- but notTRAF6-induced NF-�B activation. Expression vector encoding TRAF2, RIP1, or TRAF6 as well as NF-�B luciferase reporter and control Renilla luciferase reportervectors were co-transfected into HEK-293T cells with empty vector or expression vectors encoding USP21 WT or C221A mutant for 48 h. The relative luciferaseactivity was measured and normalized with the Renilla activity. Error bars indicate � S.D. in three independent experiments.

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RIP1 may be a major USP21-bind-ing protein. To validate the resultfrom the above protein identifica-tion analysis by mass spectrome-try, the FLAG-USP21 proteinsfrom the HEK 293T cells trans-fected with FLAG-USP21 wereimmunoprecipitated from celllysates with anti-FLAG antibodyand immunoblotted with anti-RIP1 antibody. In this assay, wefound that RIP1 was co-immuno-precipitated with FLAG-USP21(Fig. 2B). The association betweenUSP21 and RIP1 was also con-firmed by co-immunoprecipita-tion of overexpressed HA-RIP1and FLAG-USP21 in HEK 293Tcells (Fig. 2C) and co-immunopre-cipitation of endogenous RIP1 andUSP21 in HeLa cells with andwithout TNF� stimulation (Fig. 2,D and E). Together, these resultssuggest that USP21 is a RIP1-asso-ciated protein.USP21 Acts as a RIP1 De-

ubiquitinase—TheLys63-linkedubiq-uitination of RIP1 plays an essentialrole in TNF�-induced NF-�B acti-vation (21). Our results suggest thatthe inhibitory effect of USP21 onTNF�-induced NF-�B activationcould be through its associationwith RIP1 and regulation of RIP1ubiquitination. To test this hy-pothesis, FLAG-RIP1 and HA-ubiquitin expression vectors wereco-transfected with vector controlor expression vectors encodingMyc-tagged USP21 wild type ordeubiquitinase-deficient C221Amutant as well as CYLD into theHEK 293T cells. FLAG-RIP1 pro-teins were immunoprecipitatedwith the antibody for FLAG epitopeand immunoblotted with anti-HAantibody to detect the presence ofubiquitinated RIP1. As shown inFig. 3A, USP21 wild type abrogatedthe RIP1 overexpression-inducedubiquitination of RIP1, whereas theUSP21 deubiquitinase-deficient mu-tant and CYLD failed to do so. Fur-thermore, overexpression of Myc-USP21 wild type had no inhibitoryeffect on FLAG-TRAF2 and FLAG-TRAF5 polyubiquitination (supple-mental Fig. S2). To confirm the

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above result further, the immunoprecipitatedMyc-USP21 wildtype and deubiquitinase-deficient mutant proteins from thetransfected HEK 293T cell lysates were co-incubated withFLAG-RIP1-transfected HEK 293T cell lysates in vitro. In this

assay, USP21 wild type but notdeubiquitinase-deficient C221Amutant abrogated the ubiquitina-tion of RIP1 (Fig. 3B).Consistent with this result,

co-transfection of FLAG-TRAF2 orFLAG-RIP1 with Myc-USP21 wildtype but not the deubiquitinase-de-ficient mutant in HEK-293T cellsresulted in a decreased TRAF2 andRIP1-induced NF-�B activitiesin a NF-�B-dependent luciferasereporter assay, whereas co-overex-pression of USP21 failed to inhibitTRAF6-induced NF-�B activation(Fig. 3C). Furthermore, overexpres-sion of USP21 had no inhibitoryeffect on interleukin 1 (IL-1)-in-duced NF-�B activation (supple-mental Fig. S3). These resultsstrongly suggest that USP21 is abona fide RIP1 deubiquitinase andinhibits TNF�- but not IL-1-in-duced NF-�B activation.USP21 Down-regulates TNF�-in-

duced RIP1 Ubiquitination andIKK/NF-�B Activation—TNF� in-duces a rapid RIP1 ubiquitination,which is required for TNF�-medi-ated NF-�B activation (21). Todetermine whether USP21 is in-volved in down-regulating TNF�-induced RIP1 ubiquitination, wegenerated USP21 stable knockdownHeLa cell lines using a retroviraltransduction system (Fig. 4A). Wethen analyzed the effect of USP21knockdown onTNF�-inducedRIP1ubiquitination. In this assay, wefound that TNF� induced a higherlevel of the RIP1 ubiquitination atthe early time points and that thishigher level of RIP1 ubiquitinationwas sustained at the later time pointsof theTNF� stimulation in theUSP21

knockdown cells compared with the control cells (Fig. 4A). Thisresult demonstrates that USP21 is involved in terminating TNF�-induced RIP1 ubiquitination in the cells.

FIGURE 4. Knockdown of USP21 expression enhances TNF�-induced RIP1 polyubiquitination and IKK�/NF-�B activation. A, knockdown of USP21expression enhances TNF�-induced RIP1 polyubiquitination in HeLa cells. USP21 knockdown HeLa cell lines were first generated after transduction of the HeLacells with the retrovirus expressing shRNA against USP21 and selected by puromycin. The knockdown effect of USP21 expression was examined by bothquantitative reverse transcription-PCR and immunoblotting with anti-USP21 antibodies. Then the sh-control and two sh-USP21 HeLa cell lines were eitheruntreated or treated with TNF� (10 ng/ml) for the time points indicated and subsequently immunoblotted with anti-RIP1 antibodies indicated. �-Actin wasdetected as a loading control. B, knockdown of USP21 expression enhances TNF�-induced IKK and NF-�B phosphorylation and I�B� ubiquitination in the cells.The sh-control and two sh-USP21 HeLa cell lines were either untreated or treated with TNF� (10 ng/ml) for the time points indicated and subsequentlyimmunoblotted with the antibodies indicated. �-Actin was detected as a loading control. C, knockdown of USP21 expression enhances TNF�-induced NF-�Bactivation. NF-�B luciferase reporter plasmid and Renilla luciferase plasmid were co-transfected into the sh-control and two sh-USP21 HeLa cell lines for 42 h,and then the cells were either untreated or treated with TNF� (1 ng/ml) for 6 h. The relative luciferase activity was measured and normalized with the Renillaactivity. Error bars indicate � S.D. in three independent experiments.

FIGURE 5. USP21 negatively regulates TNF�-mediated IL-6 gene expression. A, knockdown of USP21expression enhances the TNF�-induced NF-�B-dependent IL-6 gene expression. The sh-control and sh-USP21HeLa cell lines were either untreated or treated with TNF� (10 ng/ml) for the time points indicated. Total RNAsfrom these cells were extracted. IL-6 transcript levels in the sh-control and sh-USP21 cell lines were measuredusing quantitative reverse transcription-PCR normalized to glyceraldehyde-3-phosphate dehydrogenase. Thedata are presented as the average of three independent experiments � S.D. (error bars). B, knockdown ofUSP21 expression enhances TNF�-induced IL-6 production. The sh-control and sh-USP21 HeLa cell lines wereeither untreated or treated with TNF� (2 ng/ml) for 24 h. The supernatants from these cell cultures werecollected and subjected to the human IL-6 enzyme-linked immunosorbent assay analysis according to themanufacturer’s instructions.

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TNF� induces a strong NF-�B activation through the phos-phorylation and activation of IKK (29). To evaluate the role ofUSP21 in TNF�-induced IKK/NF-�B activation, we analyzedthe effect of USP21 knockdown on TNF�-induced IKK phos-phorylation, I�B� phosphorylation, ubiquitination, and degra-dation, as well as NF-�B RelA subunit phosphorylation atSer536. As shown in Fig. 4B, TNF� induced an increased level ofIKK phosphorylation at the early time points of stimulation, anincreased level of I�B� phosphorylation and ubiquitination, aswell as an increased level of RelA phosphorylation in theUSP21knockdown cells compared with the control cells. We alsofound that TNF� induced a higher level of c-Jun N-terminalkinase phosphorylation at the earlier time points of stimulationin the USP21 knockdown cells compared with the control cells(Fig. 4B).Consistent with the above results, TNF� also induced a high-

er level of the NF-�B-dependent luciferase reporter activity inthe USP21 knockdown cells compared with the control cells(Fig. 4C). Taken together, these results suggest that USP21inhibits TNF�-induced IKK/NF-�B activation by suppressingRIP1 ubiquitination.USP21 Inhibits TNF�-induced NF-�B-dependent IL-6 Gene

Expression—NF-�B activation is necessary for TNF�-inducedIL-6 expression (30–32). To determine the role of USP21 in theregulation of TNF�-induced IL-6 gene expression, weextracted total RNA from the control and USP21 knockdownHeLa cell lines treated with TNF� for the time points indicatedand performed quantitative reverse transcription-PCR toexamine the TNF�-induced IL-6 expression levels in the cells.As shown in Fig. 5A, TNF� induced a higher level of IL-6expression in USP21 knockdown cells compared with the con-trol cells. Consistently, TNF� also induced a higher level of IL-6protein in the cell medium from USP21 knockdown cells com-pared with the control cells in ELISA (Fig. 5B). These results

suggest that USP21 down-regulates TNF�-induced geneexpression by inhibiting TNF�-induced RIP1 ubiquitination.

DISCUSSION

Lys63-linked RIP1 ubiquitination is an essential step inTNF�-induced IKK/NF-�B activation (20, 33). FollowingTNF� stimulation, RIP1 is promptly ubiquitinated at Lys377 byTRAF2 E3 ligase within 5 min. RIP1 will be quickly deubiquiti-nated at the later time points of stimulation, suggesting thattight control of RIP1 ubiquitination is critical for normalTNF�-induced cellular responses. However, the mechanism ofRIP1 deubiquitination following TNF� stimulation to attenu-ate TNF�-induced RIP1 ubiquitination and downstream IKK-NF-�B activation remains to be clearly defined. In this study, weidentify USP21 as a major deubiquitinase of RIP1 in the TNF�-induced NF-�B activation. By using a combination of func-tional genomic screening and proteomic approaches, we dem-onstrate thatUSP21 is critical tomodulateTNF�-inducedRIP1ubiquitination as well as downstream IKK/NF-�B activationthrough constitutive association with RIP1 and suppression ofLys63-linked RIP1 ubiquitination. Our studies suggest thatUSP21 serves as another level of critical yin-yang regulatorycontrol to maintain a delicate balance in TNF�-inducedinflammatory responses by targeting RIP1.Our study here suggests that functional genomic screening

strategy is a powerful tool to identify the new genes involved inthe regulation of TNF�-inducedNF-�B activation.However, inthis type of screening, we cannot guarantee that USPs are over-expressed at the same level. Therefore, this screening servesonly as an initial step to identify the genes whose overexpres-sion significantly inhibits TNF�-induced NF-�B activation.

Interestingly, we found that both A20 and USP21 efficientlyinhibited RIP1 overexpression-induced ubiquitination, andboth FLAG-tagged USP21 and A20 co-immunoprecipitated

FIGURE 6. Working model for the role of USP21 in the negative regulation of TNF�-induced RIP1 polyubiquitination and NF-�B activation. TNF�induces RIP1 polyubiquitination. USP21 is constitutively associated with RIP1 and acts as a RIP1 deubiquitinase to inhibit TNF�-induced RIP1 polyubiquitina-tion and RIP1-mediated NF-�B activation. USP21 plays a critical role in the down-regulation of the NF-�B activation at the early phase of TNF� stimulation,probably followed by acting together at the later time points of stimulation with A20 ubiquitin (Ub)-editing complex, including TAXBP1/Itch/RNF11 proteinsto terminate NF-�B activation.

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with endogenous RIP1, whereas USP21 pulled down moreendogenous RIP1 and was amore efficient RIP1 deubiquitinasein our in vitrodeubiquitination assay conditions comparedwithA20 (supplemental Fig. S1, A, B, and C). Furthermore, A20expression and binding to RIP1 can only be detected after 30min of TNF� stimulation in HeLa cells, whereas USP21 is con-stitutively expressed and bound to RIP1 (supplemental Fig.S1D). Transient knockdown of USP21 but not A20 expressionenhanced TNF�-induced RIP1 polyubiquitination in HeLacells within 15 min of stimulation (supplemental Fig. S1E).Notably, TNF�-induced RIP1 polyubiquitination peaks at 5min and rapidly goes down to the basal level within 30min (Fig.2E). However, A20 cannot be detected in HeLa cells within 30min of TNF� stimulation (supplemental Fig. S1D), suggestingthat the polyubiquitinated RIP1 induced by TNF� is deubiq-uitinated by a DUB in the absence of A20 in the cells. Interest-ingly, knockdown of both USP21 and A20 further enhancedTNF�-induced NF-�B activation compared with single knock-downofUSP21 orA20 (supplemental Fig. S1F). Together, theseresults strongly suggest that USP21 is mainly responsible forRIP1 deubiquitination at the early time points of TNF� stimu-lation, whereas A20 is induced at later time points of stimula-tion and plays an important “gatekeeper” role in the termina-tion of persistent NF-�B activation in HeLa cells.The ubiquitin-editing enzyme A20 (also called TNFAIP3)

has previously been shown to be a potent inhibitor of NF-�Bsignaling that consists of an N-terminal deubiquitinatingdomain of the OTU family and a C-terminal ubiquitin ligasedomain and targets RIP1 by cleaving Lys63-linked polyubiq-uitin chains and promoting Lys48-linked polyubiquitinationthat triggers proteasome-mediated RIP1 degradation (22,34). Furthermore, A20 has been suggested to be a centralgatekeeper in inflammation and immunity (35, 36). In con-trast, as a member of the USP subclass, USP21 only containsa DUB domain (27). Recently, A20 has been shown to form aubiquitin-editing complex with TAXBP1/Itch/RNF11 pro-teins to modulate the TNF�-induced RIP1 ubiquitination(37–39). Future studies are needed to determine whetherUSP21 needs other scaffolding molecules to mediate itsfunction to target RIP1 in TNF�-mediated NF-�B activation.In addition, Cezanne has been reported to be involved in themodulation of RIP1 ubiquitination process through bindingto RIP1 in a TNF�-inducible manner (23). Future studiesshould be conducted to determine how USP21 cooperateswith A20 and Cezanne to modulate the TNF�-induced RIP1deubiquitination process.Previous reports demonstrate that USP21 can be a NEDD-8

protease (40) and a functional deubiquitinase of histoneH2A inthe regulation of transcriptional initiation (41). These resultssuggest that USP21 may target proteins other than RIP1.In conclusion, our results provide direct evidence thatUSP21

interacts with RIP1 physically and acts as a RIP1 deubiquitinasein the TNF�-mediated NF-�B activation. In view of the datapresented here and in previous reports, we propose a workingmodel (Fig. 6) in which TNF� induces RIP1 ubiquitination andNF-�B activation as well as A20 expression. Lys63-linked RIP1would be rapidly deubiquitinated by USP21 at the early timepoints of stimulation, followed lately by cooperative action of

the A20 ubiquitin-editing complex, including TAXBP1/Itch/RNF11, to deuibquitinate Lys63-linked RIP1 completely andubiquitinate RIP1 in the form of Lys48-linked ubiquitin to pro-mote RIP1 degradation and terminate the persistent TNF�-mediated IKK/NF-�B activation.

Acknowledgments—We thank the proteomics core of the DiabetesEndocrinology Research Center (DERC) supported by National Insti-tutes of Health Grant DK079638 for protein identification. We thankDr. Paul Chiao, Dr. Bryant Darnay, and Dr. Peter Storz for providingIKK�, FLAG-TRAF5, and shA20 constructs, respectively.

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Gu, Guangwen Cao, Ting Zhang, Jun Qin and Jianhua YangGufeng Xu, Xiaojie Tan, Hongmei Wang, Wenjing Sun, Yi Shi, Susan Burlingame, Xue

Protein 1B Activation via Binding to and Deubiquitinating Receptor-interactingκFactor

-induced NuclearαUbiquitin-specific Peptidase 21 Inhibits Tumor Necrosis Factor

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