IL-17-induced NF-�B Activation via CIKS/Act1PHYSIOLOGIC SIGNIFICANCE AND SIGNALING MECHANISMS*□S

Received for publication, November 2, 2010, and in revised form, January 18, 2011 Published, JBC Papers in Press, February 18, 2011, DOI 10.1074/jbc.M110.199547

Søren Ulrik Sønder‡, Sun Saret‡, Wanhu Tang‡, Dan E. Sturdevant§, Stephen F. Porcella§, and Ulrich Siebenlist‡1

From the ‡Laboratory of Immunoregulation, NIAID, National Institutes of Health, Bethesda, Maryland 20852 and the §Genomics Unit,Research Technologies Section, Rocky Mountain Laboratories, NIAID, National Institutes of Health, Hamilton, Montana 59840

Interleukin-17 (IL-17) is essential in host defense againstextracellular bacteria and fungi, especially at mucosal sites, butit also contributes significantly to inflammatory and autoim-mune disease pathologies. Binding of IL-17 to its receptor leadsto recruitment of adaptor protein CIKS/Act1 via heterotypicassociation of their respective SEFIR domains and activation oftranscription factor NF-�B; it is not known whether CIKSand/or NF-�B are required for all gene induction events. Herewe report that CIKS is essential for all IL-17-induced immedi-ate-early genes in primary mouse embryo fibroblasts, whereasNF-�B is profoundly involved. We also identify a novel sub-domain in the N terminus of CIKS that is essential for IL-17-mediated NF-�B activation. This domain is both necessary andsufficient for interaction between CIKS and TRAF6, an adaptorrequired for NF-�B activation. The ability of decoy peptides toblock this interaction may provide a new therapeutic strategyfor intervention in IL-17-driven autoimmune and inflammatorydiseases.

The discovery of the inflammatory T helper cell type-17(Th17), a subset distinct from the classical Th1 and Th2 popu-lations, has revolutionized our understanding of T-cell medi-ated immunity (1–3). Th17 cells are critical in host defenseagainstmany pathogens, in particular extracellular bacteria andfungi.When improperly controlled, however,Th17 responses canalso feature prominently in a number of inflammatory and auto-immunediseases, suchas rheumatoidarthritis, systemic lupusery-thematosus, multiple sclerosis, and psoriasis (reviewed in Refs.4–6). The discovery of the Th17 cell type has also focused atten-tion on its signature cytokine IL-17 (also known as IL-17A). Acritical, althoughbynomeans exclusive biologic corollary of IL-17expression is the recruitment of neutrophils to sites of inflamma-tion (7, 8).IL-17A is amember of a family of cytokines that also includes

IL-17B, -C, -D, -E (also known as IL-25), and -F. IL-17A signalsvia a receptor composed of the IL-17RA and RC chains; thesereceptor chains are members of a family that also includes RB,RD, and RE (reviewed by Ref. 9). All receptor polypeptidesencode a so-called SEFIR domain (similar expression to fibro-blast growth factor genes and IL-17Rs) in their cytoplasmic tails

(10, 11). Such a domain is also present on the adaptor proteinCIKS2 (connection to I�B Kinase and Stress-activated proteinkinases (12); also knownasAct1 (13) orTRAF3IP2). IL-17Aand-F, as well as IL-25 have been shown to signal by recruitment ofCIKS to their cognate receptors, mediated via heterotypicSEFIR domain associations (14–17).A number of downstream effectors can be activated by IL-17,

the best studied member of this cytokine family, including thetranscription factors NF-�B, C/EBP, and AP-1, as well as MAPkinases; in addition, IL-17 can potently stabilize mRNAs,although mechanisms remain to be discovered (reviewed byRef. 9). Mere overexpression of CIKS profoundly activates p50/p65 NF-�B complexes via the classical activation pathway (12,13, 18). By contrast, stimulation of cells with IL-17, which sig-nals via CIKS, activatesNF-�Bquiteweakly (15, 19), calling intoquestion the physiologic significance of NF-�B activation.Indeed, IL-17 can synergize with TNF�, which has beenascribed to the fact that TNF�, unlike IL-17, strongly activatesNF-�B, whereas IL-17 stabilizes some short-lived mRNAsinduced by TNF� (20). This in turn has fostered the notion thatmRNA stabilization may be a primary function of IL-17.Another unsettled question concerning IL-17 signaling iswhether the CIKS adaptor is essential for expression of all tar-get genes, especially since some reports suggest the possibilityof CIKS-independent signaling events (15, 21, 22).Precisely how CIKS transmits signals to its downstream

effectors, including NF-�B, is only beginning to be elucidated.Recently a central domain of CIKS has been reported to func-tion as an E3-ubiqutin ligase, capable of adding Lys63-linkedpolyubiquitin chains to the adaptor protein TRAF6 (23).TRAF6 have previously been found essential for IL-17/CIKS-mediated activation of NF-�B (14, 15, 24). The E3-ubiquitinligase function was also reported necessary for mRNA stabili-zation, but in this case via a TRAF6 independent mechanism(23, 25). Ubiquitination of TRAF6 is secondary to recruitmentto CIKS, and CIKS reportedly encodes two TRAF6 bindingsites; these sites may be redundant because signaling was onlyimpaired when both sites on CIKS were rendered non-func-tional throughmutagenesis (23, 24). Once polyubiquitinated byCIKS, the TRAF6 adaptor may activate NF-�B via mechanismsalready established for signaling by Toll, IL-1, and CD40 recep-tors, i.e. by activation of Tak1 and IKK (9, 26).Here we investigate initial transcriptional responses of IL-17

and itsmolecular signalingmechanismswith the use of primary* This work was supported, in whole or in part, by the National Institutes ofHealth intramural research program of NIAID.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1 and S2.

1 To whom correspondence should be addressed: Bldg. 10, Rm. 11B15A,Bethesda, MD 20892. E-mail: usiebenlist@niaid.nih.gov.

2 The abbreviations used are: CIKS, connection to I�B kinase and stress-acti-vated protein kinases; MEF, mouse embryo fibroblasts; IP, immunoprecipi-tate; AA, amino acid; JNK, c-Jun N-terminal kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 15, pp. 12881–12890, April 15, 2011Printed in the U.S.A.

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mouse embryo fibroblasts. We demonstrate that CIKS is abso-lutely essential for all initial IL-17-induced transcription andwe, furthermore, show that classical activation of NF-�B isespecially critical for these responses. We also identify a noveldomain in the N terminus of CIKS that is required for interac-tion with TRAF6 and activation of NF-�B. We discuss thesefindings in terms or their potential to open new avenues fortherapeutic intervention in diseases dependent on IL-17cytokines.

EXPERIMENTAL PROCEDURES

Cell Culture and Reagents—Primary mouse embryo fibro-blast cultures (MEFs) were established from wild-type (WT)and CIKS-deficient (knockout) mice as described previously(16).Micewere bred andhoused inNational Institute ofAllergyand Infectious Diseases facilities, and all experiments weredone with approval of the National Institute of Allergy andInfectious Diseases Animal Care and Use Committee and inaccordance with all relevant institutional guidelines. Immortal-ized NEMO-deficientMEFs were kindly donated by Dr.Mano-lis Pasparakis. Cycloheximide and actinomycin D were pur-chased fromSigma; SB203580, JNK inhibitor II, JAK inhibitor I,and PD98059 were from Calbiochem; and MLN120b waskindly supplied by Millennium Pharmaceuticals. FITC-taggedcell penetrating peptides, TAT wild-type CIKS, GRKKRRQR-RRPPQMNRSIPVEVDESEPYP, and TAT E17A CIKS, GRK-KRRQRRRPPQMNRSIPVAVDESEPYP, were purchased fromAmerican Peptide. MEFs were treated for 30 min with thesepeptides in serum-free medium prior to stimulation. Uptake ofFITC-labeled cell penetrating peptide was confirmed by FACSanalysis. Recombinant IL-17 (100 ng/ml, R&D Systems) and/orTNF� (2 ng/ml, Peprotech) were used for stimulation.RNA Isolation and Real Time PCR (RT-PCR)—RNAwas iso-

lated using the RNeasy RNA isolation kit (Qiagen) accordingto the manufacturer’s instructions. cDNA was synthesizedusing oligo(dT) and SuperScript III (Invitrogen). Expressionof I�b�, Ccl2, c/Ebp�, Zc3h12a, Il-6, Cxc1, and �-actin wasquantified by TaqMan qPCR using primers from AppliedBiosystems. All gene expression results are expressed as2���Ct, where ��Ct � (Ct,target � Ct,�-actin) for stimulatedsamples � (Ct,target � Ct,�-actin) for unstimulated controls.Data are shown as the mean � S.E. For the mRNA stabilityexperiment, expression of Cxcl1 was calculated as 2��Ct,where �Ct � (Ct,Cxcl1 � Ct,�-actin). Expression at 0 h was setto 100% and the remaining samples were normalizedaccordingly.Genechip Experiment—RNAs were extracted and DNA

microarray targets were prepared as described previously (27).Gene expression was measured using the Affymetrix 430 2.0Genechip containing themouse genome and data analysis werecarried out as described previously (28) with the followingmodifications. An analysis of variance was performed using thePartek Genomics Suite (Partek, Inc., St. Louis, MO, version 6.36080414) to obtain multiple test corrected p values using thefalse discovery rate method at the 0.05 significance level com-bined with a fold-change value of 1.5. The data discussed in thisarticle have been deposited in the NCBI Gene Expression

Omnibus (29) and are accessible throughGEO Series accessionnumber GSE24873.Western Blot and Antibodies—Whole cell extracts were iso-

lated, loaded on to 10% SDS-polyacrylamide gel, electrophore-sed, and transferred to PVDF (Millipore) membranes. The fol-lowing antibodies were used: anti-FLAG (Sigma); anti-HA,anti-TRAF6, anti-�-actin, and anti-I�B� (all from Santa Cruz);and anti-NF-�B p65, anti-phospho-Ser536 NF-�B p65, anti-ERK, and anti-phospho-ERK (all from Cell Signaling).Alignment, Plasmids, and Lentivirus—Sequence alignment

was carried out using ClustalW2 (30) using default settings.Full-length human CIKS and CIKS deletion/point mutantswere cloned into a Gateway Entry vector (Invitrogen) and sub-cloned into a lentiviral vector or into pcDNA3.1 HA or FLAGTag destination vectors by Gateway LR recombination usingthe manufacturer’s protocols to generate expression clones. Inthese vectors the standard cytomegalovirus promoter wasreplaced by the PolII promoter to ensure low level constitutiveexpression,with the exception of the vectors used in Fig. 2A andthe IL-17RA vector used in Fig. 4B. The TRAF6 expressionvector has been described previously (31). Plasmid constructswere confirmed by sequencing. Lentivirus preparations usedfor transduction of wild-type and mutant CIKS proteins intoCIKS-deficient primary MEFs were generated with theViraPower Lentiviral Expression System (Invitrogen) followingthe manufacturer’s instructions.Transfection, Luciferase Assay, and Immunoprecipitation—

HeLa cells were transfected using Lipofectamine 2000 (Invitro-gen). Whole cell extracts were isolated 48 h after transfection.Immunoprecipitations (IPs) were carried out using IP kits(Sigma) according to the manufacturer’s instructions and ana-lyzed by Western blotting as described above. For luciferaseassays, HeLa cells were co-transfected with the Ig�B Lucreporter as described previously (18). Luciferase activity wasdetermined 24 h later using the Dual Luciferase assay system(Promega) according to the manufacturer’s instructions andnormalized to an ER Renilla internal control (Promega).

RESULTS

IL-17 Induces Degradation of I�B� and Phosphorylation ofp65 Dependent on CIKS—To understand the significance ofNF-�B and CIKS in the cellular response to IL-17 we made useof freshly isolated primary mouse embryo fibroblasts. We firstre-examinedwhether and, if so, towhat extent this cytokine canactivate NF-�B in primary, freshly isolated, wild-type or CIKS-deficientMEFs. IL-17 induced the degradation of I�B� in wild-type cells, albeit much less so than TNF�, and only TNF�, notIL-17 was able to do so in CIKS-deficient cells (Fig. 1A). ThusIL-17 was able to liberate NF-�B in a CIKS-dependent mannerin primary cells.The transcriptional activity of the dimeric NF-�B complexes

in the nucleus is also determined by phosphorylation, especiallyof p65/RelA. Various potential phosphorylation sites have beenreported for p65; their occurrence, relative significance, andprecise function are not fully understood and appear to be con-text-dependent (32, 33). Here we show that both IL-17 andTNF� were able to induce phosphorylation of p65 at Ser536 inwild-typeMEFs, whichwas fully dependent onCIKS in the case

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of IL-17 but not TNF�, as expected. No synergistic effect wasobservedwhen stimulatingwith both IL-17 andTNF� (Fig. 1B).The Early Transcriptional Response to IL-17 in Primary

Mouse Embryo Fibroblasts—To further explore the role ofNF-�B and CIKS in the transcriptional response to IL-17 inprimary MEFs, we first performed a genome-wide microarrayanalysis to identify genes that were significantly induced byIL-17 within 2 h (see supplemental Fig. S1 for full results). Afterapplying stringent criteria to the results from 6 independentlyperformed experiments, we identified 9 such genes in primarywild-typeMEFs; by contrast, IL-17 failed to induce any genes inCIKS-deficientMEFs. These results were confirmedwith qPCRanalyses (supplemental Fig. S1C). A set of 9 genes encodes forchemokines Cxcl1, Ccl2, and Ccl7, the cytokines Lif and Il-6,transcription factors I�b�, c/Ebp�, and RelB, and RNA-bindingprotein Zc3hl2a; most of these genes have been identified pre-viously in various screens for IL-17-induced genes (21, 34, 35),

although Zc3h12a has been noted only once in a recent screenof liver cells (36), whereas RelB has never been identified previ-ously. Whereas I�b�, c/Ebp�, and Zc3h12a were induced onlywith IL-17, not TNF�, the remaining genes were induced witheither cytokine and are all known potential targets of NF-�B(37–39). Within the latter group, Cxcl1, Lif, and Il-6 appearedto be synergistically induced by the two cytokines, which maybe due to post-transcriptional regulation (20, 40).Despite the early time point after stimulation chosen in our

analyses, Il-6 and c/Ebp� are not immediate-early genes; theyhave previously been reported to depend on induced expres-sion of I�B�, and their peak of expression occurredwell after 2 h(41, 42) (see also supplemental Fig. S2A). We conclude thatCIKS is essential for expression of all IL-17-induced genes inprimary MEFs within 2 h. The expression profiling study alsoidentified immediate-early targets of IL-17 in primary cells thatcould be investigated further for their dependence on NF-�B.

FIGURE 1. CIKS is essential for IL-17-mediated NF-�B activation, which plays a major role in IL-17-induced immediate-early gene expression.A, wild-type (WT) and CIKS-deficient (CIKS KO) primary MEFs were treated with cycloheximide for 30 min (to prevent the rapid NF-�B-mediated re-synthesis ofI�B�) prior to stimulation for 2 h with IL-17 or TNF� or in the absence of stimulation (Control). Cell lysates were analyzed for I�B� and �-actin as a loading control.B, WT and CIKS knockout MEFs were stimulated as shown and lysates were analyzed for the presence of p-Ser536 p65 and total p65. Blots shown arerepresentative of at least three independent experiments. C, real time PCR analyses of RNAs from primary WT MEFs pre-treated for 30 min with the followingpharmacological inhibitors prior to stimulation with IL-17 for 2 h: 10 �M DMSO (control), 10 �M MLN120b (NF-�B inhibitor), 10 �M SB203580 (p38 inhibitor), 10�M JNK inhibitor II, 10 �M JAK inhibitor I, or 10 �M PD98059 (ERK inhibitor). D, real time PCR analyses of RNAs from WT- and NEMO-deficient MEFs stimulatedwith IL-17 for 2 h. Fold-induction is in reference to unstimulated controls. Data are shown as the mean � S.E. for six independent experiments; *, p � 0.05 pairedStudent’s t test.

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The Role of NF-�B in the Immediate-early TranscriptionalResponse to IL-17—To address what downstream effectorsmaycontribute to immediate-early IL-17-induced gene expression,we pre-treated primary wild-type MEFs with inhibitors forNF-�B, JAK, and MAP kinases p38, JNK, and ERK prior tostimulation with IL-17. Induced expression of immediate-earlygenes Cxcl1, Ccl2 and, to a lesser extent I�b� and Zc3h12a,wasdependent on NF-�B, but was not significantly dependent onp38, JNK, JAK, or ERK (Fig. 1C, see also supplemental Fig. S2B).To verify the role of NF-�B, we also investigated the IL-17-

induced expression of the above genes in NEMO-deficientMEFs, as the classical NF-�B activation pathway is completelyabrogated in these cells. Consistent with the inhibitor studies,Ccl2 and Cxcl1 failed to be induced in the absence of NEMO,whereas induced expression of I�b� and Zc3h12a was signifi-cantly decreased, albeit not abolished (Fig. 1D). We concludethat NF-�B is critically involved in the IL-17-induced expres-sion of immediate-early genes, albeit to a variable degree.A Novel Domain in CIKS Required for Activation of NF-�B—

To identify novel domains within CIKS critical for activation ofNF-�B we generated and initially screened a number of CIKSmutants by assessing their ability to induce expression from anNF-�B-dependent luciferase reporter plasmid upon overex-pression in HeLa cells (18). As expected, deletion of the SEFIRdomain (�SEFIR), which is required for self-association (18), orthe central region (�200–400), which carries the E3 ubiquitin-ligase domain (23), significantly reduced the ability of exoge-nously introduced CIKS to activate NF-�B (Fig. 2A). By con-trast, deletion of one of the two purported TRAF6 binding siteson CIKS (�38–42) had no effect on NF-�B activation (23).Surprisingly, deletion of the N-terminal 50 amino acids

(�N50) completely abolished NF-�B activation (Fig. 2B). Wetherefore generated additional mutants to identify sequenceswithin the N terminus that may be required for NF-�B activa-tion. Deletion of amino acids 1–35 (�1–35), 1–15 (�1–15), and10–25 (�10–25) all completely eliminated the ability of over-expressed CIKS to activate NF-�B (numbering is based on thelonger of the two human CIKS isoforms; the shorter isoformstarts at position 10, but is otherwise identical) (Fig. 2B). Dele-tion of amino acids 20–35 (�20–35) resulted in partial impair-ment of CIKS to activate NF-�B, whereas deletion of aminoacids 35–50 (�35–50), which contains a site previously thoughtto bindTRAF6 (23, 24) failed to impair this CIKS function. Thissuggested that a previously unknownN-terminal domain, mostlikely contained between positions 10 and 25, was crucial foractivation of NF-�B.A comparison of CIKS sequences from various species

showed a remarkable conservation of amino acids in the inter-val between positions 10 and 21; there is little or no conserva-tion elsewhere in the larger N-terminal region of CIKS whenmore divergent species are compared (Fig. 2C). To furtherdelimit the critical sequences, we generated four point muta-tions in the domain between positions 10 and 21, replacingSer13, Val16, Glu17, or Asp19 with Ala (S13A, V16A, E17A andD19A); in addition we generated a combined mutant in whichall four positions were replaced (“Quad”). As shown in Fig. 2D,the E17Amutation completely abrogated the ability of CIKS toactivate NF-�B and the D19A mutation partially interfered,

whereas the S13A andV16Amutations had no significant effectin this assay. Consistent with this, the Quad mutation com-pletely blocked CIKS from activating NF-�B as well. We con-clude that theN terminus of CIKS harbors a previously uniden-tified domain likely located between amino acid positions 10and 21 that is required for activation of NF-�B in CIKS-trans-fected cells.TheN-terminal Domain of CIKS Is Critical for IL-17-induced

Target Gene Expression and Activation of NF-�B—To under-stand the importance of this newly identified N-terminaldomain in a physiologic context, we employed a lentivirustransduction strategy to reconstitute CIKS-deficient primaryMEFs with minimal levels of wild-type and mutant CIKS pro-teins (�10–25, E17A, and �N50), or with a GFP control, andthen assayed for responses to stimulation with IL-17. We firsttested for IL-17-induced expression of the immediate-early tar-get test genes investigated above: I�b�, Zc3h12a, Cxcl1, andCcl2. Reconstitution with the �N50, �10–25, and E17A CIKSmutants significantly impaired induction of Cxcl1, Ccl2, andZc3h12a, and to a lesser extent I�b�,when compared with cellsreconstituted with wild-type CIKS (Fig. 3,A and B) (see supple-mental Fig. S2C for Il-6 and c/Ebp�).

To directly evaluate the ability of IL-17 to activate NF-�B inthe primary MEFs reconstituted with the wild-type and CIKSmutants shown above (Fig. 3, A and B), we measured IL-17-induced degradation of I�B�. As shown in Fig. 3C, only CIKS-deficient MEFs reconstituted with wild-type CIKS were able todegrade I�B� in response to IL-17, whereas those reconstitutedwith CIKS mutants that crippled the critical N-terminaldomain failed to respond to IL-17, as did those reconstitutedwithGFP only. Together these data demonstrate that the newlyidentified N-terminal CIKS domain is essential for IL-17-in-duced degradation of I�B� and thus for activation of NF-�B-dependent target genes.The N-terminal Domain Is Not Required for IL-17-induced

mRNA Stabilization or ERK MAPK Activation—The ability ofthe N-terminal mutants to partially restore induced expressionof, in particular, I�b�, albeit at significantly reduced levels com-pared with wild-type CIKS-reconstituted cells, suggests thatcertain IL-17-induced signals do not require the N-terminaldomain. In addition to NF-�B activation, IL-17 has beenreported to induce activation of MAPKs, including ERK (43).Primary MEFs reconstituted with wild-type CIKS restored theability of IL-17 to fully activate ERK, as did the �N50 mutantwhen comparedwith cells lacking CIKS (Fig. 3D). There was nosignificant difference between MEFs reconstituted with CIKSor the �N50 mutant. We also noted an apparent, low level ofERK activation in the absence of CIKS; the origin of this weakresponse is presently unclear. These data do show that theN-terminal domain of CIKS is not necessary for full activationof ERKMAPK, although some (other) portion of CIKS appearsto be required.IL-17 can induce the stabilization of short-lived mRNAs,

which has been well documented for the mRNA of Cxcl1, andwhich appears to be the primary cause for the profound synergybetween IL-17 and TNF� observed for this gene (20). Becausethe �N50 mutant was unable to activate NF-�B, we askedwhether this mutant might nevertheless, still be able to syner-

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gize with TNF�, which potently activates NF-�B by itself. Asshown in Fig. 3, E and F, transduction of primary MEFs with alentivirus capable of expressing either wild-type CIKS or the�N50 mutant fully reconstituted the ability of IL-17 to syner-gize with TNF� in the induction ofCxcl1 and Il-6. As expected,no synergy between IL-17 and TNF� was observed in CIKS-deficientMEFs transducedwith the�SEFIRmutant or with theGFP control. These resultswere confirmedby directly assessingthe IL-17-mediated stabilization of Cxcl1 mRNA under theseconditions. As shown in Fig. 3, G and H, Cxcl1 mRNA wasdegraded significantly faster in CIKS-deficientMEFs comparedwithMEFs reconstituted with either CIKS or the �N50mutantupon addition of IL-17; this degradation was followed in thepresence of actinomycin D, which was added to stop transcrip-tion after the initialmRNAwas induced byTNF�. Therewas nosignificant difference in terms of mRNA stabilization by IL-17between MEFs reconstituted with CIKS or the �N50 mutant.These results show that the N-terminal domain of CIKS,whereas critical for IL-17-mediated NF-�B activation, is notrequired for mRNA stabilization. Together with the ability ofthe mutants to also still activate ERK, we further conclude thatthe N-terminal mutations must not have caused an overallimpairment of CIKS structure and function.The N-terminal Segment of CIKS Is Necessary for TRAF6

Binding—To identify specific functions of the newly identifiedN-terminal CIKS domain, we next tested the ability of mutantCIKS to self-associate. The ability to self-associate is thought tobe required for activation of NF-�B and other downstream sig-nals and it requires the SEFIR domain (18). As shown in Fig. 4A,the �N50 mutant was fully capable of associating with a dif-

ferently tagged version of self or wild-type CIKS in co-trans-fected cells; as a negative control, the �SEFIR mutant wasunable to associate with wild-type CIKS or the�N50mutant,as expected.Next we asked whether loss the N-terminal domain might

affect association with the IL-17RA receptor. This receptorand CIKS can associate to some extent in transfected cells,even in the absence of a signal, as has also been noted byothers (14, 15). We therefore ectopically expressed IL-17RAand wild-type and mutant CIKS proteins in HeLa cells andthen immunoprecipitated IL-17RA and immunoblotted forCIKS. All of the N-terminal domain-crippled CIKS mutantsas well as �35–50 were still able to associate with IL17RA,whereas loss of the SEFIR domain abolished this association,as expected (Fig. 4B). Therefore, loss of the N-terminal 50amino acids had no effect on the ability of CIKS to self-associate or to associate with IL-17RA.We then investigated whether loss of the newly identified

N-terminal domain of CIKS impacted association withTRAF6. This adaptor protein had previously been shown tobe required for IL-17/CIKS-mediated NF-�B activation, butnot mRNA stabilization (23, 25). We therefore tested forassociation of endogenous TRAF6 with the IL-17RA recep-tor in the experiment described above, in which IL-17RA andthe various CIKS forms were exogenously expressed (Fig.4B). Although all N-terminal domain-crippled CIKS mu-tants, including E17A, were co-immunoprecipitated withIL-17RA, none facilitated the co-immunoprecipitation ofendogenous TRAF6. By contrast, wild-type CIKS and the�35–50 mutant, which retains the newly identified domain

FIGURE 2. A conserved domain in the N terminus of CIKS is required for NF-�B activation. A, B, and D, HeLa cells were co-transfected with the indicated HA-or FLAG-tagged CIKS constructs, the NF-�B luciferase reporter and a Renilla construct were used for internal control. Results are recorded as fold-induction ofNF-�B activity relative to cells transfected with an empty expression vector. Data are shown as the mean � S.E. for four independent experiments; *, p � 0.05Student’s t test compared with cells transfected with WT CIKS. Lower panels show approximately equal expression of transfected CIKS proteins in cell lysates;�-actin served as the loading control. C, a short N-terminal sequence of CIKS is highly conserved across multiple species. Alignment of CIKS sequences fromHomo sapiens (human), Mus musculus (mouse), Bos taurus (cow), Gallus gallus (chicken), and Xenopus tropicalis (frog). Asterisk denotes identical amino acids;colon denotes conserved substitutions; and dot denotes semi-conserved substitutions.

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(see above), readily facilitated co-immunoprecipitation ofendogenous TRAF6. These data demonstrate that the N-ter-minal CIKS domain comprised of amino acids 10 to 21 isessential for TRAF6 interaction, which explains why thisdomain is also critical for IL-17-induced NF-�B activation.The N-terminal CIKS Domain Is Both Necessary and Suffi-

cient for TRAF6 Interaction andMayProvide aTarget to ImpairSignaling—We next asked whether the N-terminal domain ofCIKS might be sufficient for binding to TRAF6. To this end wegenerated vectors capable of expressing FLAG-tagged proteinsinwhichGSTwas fused to thewild-type or E17AmutantN-ter-minal 50 amino acids or amino acids 10–25 of CIKS. As shownin Fig. 5A, exogenously expressed TRAF6 could be co-immu-noprecipitated with fusions carrying the wild-type N-terminaldomain (1–50 or 10–25); by contrast, TRAF6 failed to be co-immunoprecipitated if these fusions carried the E17A muta-tion. These data clearly indicate that the newly identifiedN-ter-minal domain of CIKS is not only necessary, but can also besufficient to facilitate association with TRAF6.Given the importance of the N-terminal domain for TRAF6

binding and NF-�B activation, we also investigated whetherthis domain could be targeted to interfere with CIKS-mediatedNF-�B activation. To this end we generated fusion peptides

composed of the cell penetrating portion of HIV-TAT and thewild-type N-terminal domain of CIKS (AA 10–25), or a E17Amutant version of this CIKS sequence as a negative control.When MEFs were pre-exposed to these peptides, the cell-pen-etrating peptide carrying the wild-type N-terminal domain sig-nificantly interfered with IL-17-induced expression of Cxcl1 aswell as Zc3h12a, whereas the E17Amutant control peptide didnot (Fig. 5,B, control for uptake of peptides shown inC)). Theseresults raise the possibility that the N-terminal TRAF6 bindingdomain could be a target for therapeutic intervention in IL-17-driven pathogenesis.

DISCUSSION

The present study shows that IL-17-mediated activation ofthe classical NF-�B pathway in primary MEFs is fully depen-dent on the adaptor CIKS; in turn, IL-17-induced NF-�B is crit-ical for immediate-early gene induction, despite the relativelyweak level of activation when compared with that of TNF�.Although IL-17-induced expression of some immediate-earlygenes appears to be almost completely dependent on NF-�B,others are at least partially dependent. The present study alsoshows that CIKS is absolutely required for all IL-17-inducedtranscription at 2 h post-stimulation. These insights emphasize

FIGURE 3. The N-terminal domain of CIKS is essential for IL-17-mediated NF-�B activation in CIKS-reconstituted primary MEFs, but not for IL-17-induced mRNA stabilization or ERK activation. A–D, CIKS-deficient primary MEFs were reconstituted via lentiviral transduction with wild-type CIKS, a mutantCIKS (CIKS �N50, CIKS �10 –25, CIKS E17A, or CIKS �SEFIR) or with GFP as a negative control, as indicated. A, real time PCR analyses for the indicated genes withRNAs isolated from cells stimulated for 2 h with IL-17 or left unstimulated. Data are shown as the mean � S.E. for six independent experiments and fold-induction is in reference to unstimulated cells; *, p � 0.05 Student’s t test. B, Western blot analyses of cell lysates from a representative experiment used in A toverify approximately equal expression of the transduced FLAG-tagged CIKS proteins, with �-actin serving as a loading control. C, Western blot analyses of celllysates for I�B�; �-actin served as a loading control. Cells contained approximately equal levels of transduced FLAG-tagged CIKS proteins. D, Western blotanalyses for activated phosphor-ERK, total ERK levels, and reconstituted FLAG-tagged CIKS levels. The experiments shown in C and D are representative of atleast 3 independent experiments. E, RT-PCR analyses for the indicated genes of RNAs isolated from primary reconstituted MEFs 2 h after stimulation with IL-17,TNF�, or IL-17 � TNF�, as indicated. Data are recorded as the mean � S.E. for six independent experiments measuring fold-induction relative to unstimulatedcells; *, p � 0.05 Student’s t test. F, Western blot analyses of cell lysates collected from a representative experiment used in E to show expression levels of thetransduced FLAG-tagged CIKS proteins, with �-actin serving as a loading control. G, real time PCR analysis of Cxcl1 mRNA expression levels. RNA was isolatedfrom primary CIKS-deficient MEFs reconstituted with wild-type or mutant CIKS, as indicated, and treated as follows. Cells were stimulated with TNF� for 1 h,followed by addition of first actinomycin D and 10 min later, IL-17; RNA was isolated at 0, 2, 4, and 6 h after addition of IL-17. Data are recorded as the mean �S.E. for five independent experiments measuring % of remaining Cxcl1 mRNA; *, p � 0.05 Student’s t test compared with CIKS-deficient MEFs transduced withCIKS. H, Western blot analyses of cell lysates collected from a representative experiment used in G to show expression levels of the transduced FLAG-taggedCIKS proteins, with �-actin serving as a loading control.

FIGURE 4. The N-terminal domain of CIKS is essential for interaction with TRAF6, but not for interaction with self or with IL-17RA. A, HeLa cells wereco-transfected with tagged wild-type or �N50 mutant CIKS together and with differently tagged wild-type or mutant CIKS constructs as indicated. Cell lysateswere IP to evaluate association of co-expressed CIKS proteins in immunoblots (IB) as indicated. B, FLAG-tagged wild-type or one of several mutant CIKSconstructs were co-transfected together with HA-tagged IL-17RA. Cell lysates were IP with anti-HA IL-17RA and analyzed with immunoblots for the presenceof co-precipitating CIKS proteins as well as endogenous TRAF6. Analyses shown in A and B are representative of at least three independent experiments.

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the significance not just of CIKS but also of NF-�B in IL-17signaling,whichhas previously not been fully appreciated.Con-cerning the mechanism of NF-�B activation, we demonstratethat a new N-terminal domain of CIKS, likely situated betweenamino acids 10 and 21 (numbering refers to the longer of thetwo human isoforms, with the shorter starting at position 10), isabsolutely necessary for association of CIKS with TRAF6, foractivation of NF-�B and consequently, also for proper IL-17-induced expression of immediate-early target genes in primaryMEFs. Furthermore, this new domain appears to be sufficientfor recognition by TRAF6, as the presence of this domain in afusion with an irrelevant carrier protein is able to bestow theability to attract TRAF6. Finally, the newly identified N-termi-nal domain may represent a potential target for therapeuticintervention in diseases in which IL-17 cytokines drive patho-genesis, as decoy cell-penetrating peptides carrying this domainappear to interfere with IL-17-induced and TRAF6/NF-�B-de-pendent gene expression.CIKS was previously thought to contain two putative

TRAF6 binding sites based on matching a consensus TRAF6binding site (AA numbers 38–42 EEESE and AA numbers333–337 EERPA). Mutations in either site had no effect onCIKS function, but a combined mutation of both sites wasreported to significantly impair TRAF6 binding, TRAF6ubiquitination, NF-�B activation, and JNK phosphorylation(23). It is presently unclear how these two mutationstogether, but not individually, interfered with CIKS func-tion, although it is conceivable that together these mutationssomehow impaired the overall structure and thus function ofCIKS. Importantly, as shown here, deletions of or mutationswithin the interval between AA 10 and 21 (specifically theE17A mutant) completely abolished TRAF6 binding of CIKSas well as TRAF6- and NF-�B-dependent signaling. At thesame time, these deletions and mutations had no effect onother, TRAF6-independent functions of CIKS, including theability of CIKS to self-associate or to associate with thereceptor, IL-17-inducedmRNA stabilization, and ERK phos-phorylation. Furthermore, this newly identified TRAF6

binding site on CIKS, in isolation, was able to bind TRAF6.Therefore, the N-terminal domain between AAs 10 and 21 isnecessary for interaction with TRAF6 and, in consequence,for TRAF6/NF-�B-dependent signaling in response to IL-17and, it can be sufficient for interaction with TRAF6. We thusconclude that the newly identified N-terminal domain is thetrue and functionally critical TRAF6 binding site on CIKS. Itremains theoretically possible that secondary TRAF6 bind-ing sites exist within CIKS; if such sites exist, however, theirengagement with TRAF6 would have to depend on an initialinteraction with the new domain described here.The AA sequence between positions 10 and 21 is well con-

served between species, including frog: a sequence that bearslittle similarity elsewhere in the extended N-terminal region ofmammalian CIKS sequences. The 12-AA long domain includesa sequence that conforms with a loose TRAF6 binding consen-sus reported previously (CIKS 10–21, MNRSIPVEVDES;TRAF6 consensus binding site PXEXX(Ar/Ac) (44–46). Thenewly identified TRAF6 binding domain in CIKS is situated ator very close to the N terminus of CIKS (the short form ofhuman CIKS starts at position 10, the beginning of the newlyidentified domain). This may make this site very accessible forinteraction with TRAF6; indeed, exogenously expressed CIKSreadily interacts with TRAF6, even in the absence of any signals(15).IL-17 has been strongly implicated in disease pathogenesis

in a number of inflammatory and autoimmune diseases, suchas rheumatoid arthritis, multiple sclerosis, psoriasis, as wellas severe asthma among others (4–6). Furthermore, IL-25,another member of the IL-17 family that signals via CIKS,has been implicated in asthma, as it induces a strong Th-2-like innate response that also promotes Th2 adaptiveresponses thought to underlie most cases of asthma (16, 17,47). Thus it might be useful to develop a means to blockIL-17 cytokine signaling in such diseases. To date the prooffor this principal has been established in mouse models forthe diseases such as rheumatoid arthritis (48) and experi-mental autoimmune encephalomyelitis (the mouse model

FIGURE 5. The N-terminal domain of CIKS is sufficient to interact with TRAF6. A, HeLa cells were co-transfected with TRAF6 and FLAG-tagged GST fusionscarrying the wild-type CIKS sequence between amino acid positions 10 and 25 or 1 and 50 or the E17A mutant versions of both of these sequences. Cell lysateswere IP with anti-FLAG analyzed in immunoblots (IB) for TRAF6. Analyses shown are representative of at least three independent experiments. B, real time PCRanalyses for the indicated genes with RNAs from primary WT MEFs treated with cell penetrating peptides containing the wild-type CIKS sequence betweenamino acid positions 10 and 25 or an E17A mutant version prior to stimulation with IL-17 for 1 h. Induction values are relative to stimulated, but otherwiseuntreated WT MEFs, arbitrarily set to 100. Data are shown as the mean � S.E. for three independent experiments; *, p � 0.05 Student’s t test. C, FACS analysisconfirms an equal amount of cell penetrating peptides in the MEFs. Analyses shown are representative of at least three independent experiments.

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for multiple sclerosis) (49): in these models loss of CIKSabolished or significantly ameliorated disease development,respectively, without causing any other obvious problems,although one must assume that beneficial functions of IL-17cytokine signaling were also abolished. We have shown herethat a cell-penetrating peptide carrying the newly identifiedN-terminal domain of CIKS, but not a negative control car-rying the E17A mutation, interfered with IL-17 induced sig-naling, presumably by inhibiting TRAF6 binding. Thus newtherapeutic methods for IL-17 cytokine-driven diseasesbased on inhibiting CIKS functions via interference withTRAF6 binding may be entertained in future studies. Indeedpeptides carrying a hydrophobic signal sequence fused to theTRANCE receptor binding site for TRAF6 have been shownto interfere with TRANCE-L-induced functions in a similarway (44). In this scenario it must be kept in mind, however,that not all CIKS signaling is mediated via TRAF6, thusTRAF6-independent signals would still be preserved, even ifTRAF6 binding was blocked. To what extent TRAF6-depen-dent versus TRAF6-independent signaling contributes todisease pathogenesis by IL-17 or IL-25 remains to be inves-tigated. Furthermore, it is possible that decoy peptides orsmall molecule inhibitors based on the TRAF6 binding siteon CIKS might inhibit all TRAF6-mediated signals, not justIL-17 cytokine-induced signals. Therefore it will be of inter-est to also identify sequences within the SEFIR domain thatare important for association with the receptor and/or withself, as such sequences might provide a more specific targetfor blocking CIKS function with decoy peptides or smallmolecule inhibitors derived thereof.

Acknowledgments—We thank Dr. Manolis Pasparakis for the gift ofNEMO-deficient MEFs, Dr. Kimmo Virtaneva for RNA extractionsandmicroarray target preparations, andDr. Andrea Paun for criticalreading of themanuscript.We are grateful to Dr. Anthony S. Fauci forcontinued support.

REFERENCES1. Dong, C. (2006) Nat. Rev. Immunol. 6, 329–3332. Weaver, C. T., Hatton, R. D., Mangan, P. R., and Harrington, L. E. (2007)

Annu. Rev. Immunol. 25, 821–8523. McGeachy, M. J., and Cua, D. J. (2008) Immunity 28, 445–4534. Annunziato, F., Cosmi, L., Liotta, F., Maggi, E., and Romagnani, S. (2009)

Nat. Rev. Rheumatol. 5, 325–3315. Di Cesare, A., Di Meglio, P., and Nestle, F. O. (2009) J. Invest. Dermatol.

129, 1339–13506. Miossec, P., Korn, T., and Kuchroo, V. K. (2009) N. Engl. J. Med. 361,

888–8987. Korn, T., Bettelli, E., Oukka, M., and Kuchroo, V. K. (2009) Annu. Rev.

Immunol. 27, 485–5178. Xu, S., and Cao, X. (2010) Cell Mol. Immunol. 7, 164–1749. Gaffen, S. L. (2009) Nat. Rev. Immunol. 9, 556–56710. Gaffen, S. L. (2008) Cytokine 43, 402–40711. Novatchkova, M., Leibbrandt, A., Werzowa, J., Neubuser, A., and Eisen-

haber, F. (2003) Trends Biochem. Sci. 28, 226–22912. Leonardi, A., Chariot, A., Claudio, E., Cunningham, K., and Siebenlist, U.

(2000) Proc. Natl. Acad. Sci. U.S.A. 97, 10494–1049913. Li, X., Commane, M., Nie, H., Hua, X., Chatterjee-Kishore, M., Wald, D.,

Haag, M., and Stark, G. R. (2000) Proc. Natl. Acad. Sci. U.S.A. 97,10489–10493

14. Chang, S. H., Park, H., and Dong, C. (2006) J. Biol. Chem. 281,

35603–3560715. Qian, Y., Liu, C., Hartupee, J., Altuntas, C. Z., Gulen, M. F., Jane-Wit, D.,

Xiao, J., Lu, Y., Giltiay, N., Liu, J., Kordula, T., Zhang, Q. W., Vallance, B.,Swaidani, S., Aronica, M., Tuohy, V. K., Hamilton, T., and Li, X. (2007)Nat. Immunol. 8, 247–256

16. Claudio, E., Sønder, S. U., Saret, S., Carvalho, G., Ramalingam, T. R.,Wynn, T. A., Chariot, A., Garcia-Perganeda, A., Leonardi, A., Paun, A.,Chen, A., Ren, N. Y., Wang, H., and Siebenlist, U. (2009) J. Immunol. 182,1617–1630

17. Swaidani, S., Bulek, K., Kang, Z., Liu, C., Lu, Y., Yin, W., Aronica, M., andLi, X. (2009) J. Immunol. 182, 1631–1640

18. Mauro, C., Vito, P.,Mellone, S., Pacifico, F., Chariot, A., Formisano, S., andLeonardi, A. (2003) Biochem. Biophys. Res. Commun. 309, 84–90

19. Awane,M., Andres, P. G., Li, D. J., and Reinecker, H. C. (1999) J. Immunol.162, 5337–5344

20. Hartupee, J., Liu, C., Novotny, M., Li, X., and Hamilton, T. (2007) J. Im-munol. 179, 4135–4141

21. Huang, F., Kao, C. Y., Wachi, S., Thai, P., Ryu, J., and Wu, R. (2007) J. Im-munol. 179, 6504–6513

22. Shen, F., Li, N., Gade, P., Kalvakolanu, D. V., Weibley, T., Doble, B.,Woodgett, J. R., Wood, T. D., and Gaffen, S. L. (2009) Sci. Signal. 2, ra8

23. Liu, C., Qian, W., Qian, Y., Giltiay, N. V., Lu, Y., Swaidani, S., Misra, S.,Deng, L., Chen, Z. J., and Li, X. (2009) Sci. Signal. 2, ra63

24. Kanamori, M., Kai, C., Hayashizaki, Y., and Suzuki, H. (2002) FEBS Lett.532, 241–246

25. Hartupee, J., Liu, C., Novotny, M., Sun, D., Li, X., and Hamilton, T. A.(2009) J. Immunol. 182, 1660–1666

26. Li, X. (2008) Cytokine 41, 105–11327. Shea, P. R., Virtaneva, K., Kupko, J. J., 3rd, Porcella, S. F., Barry, W. T.,

Wright, F. A., Kobayashi, S. D., Carmody, A., Ireland, R. M., Sturdevant,D. E., Ricklefs, S. M., Babar, I., Johnson, C. A., Graham, M. R., Gardner,D. J., Bailey, J. R., Parnell, M. J., Deleo, F. R., andMusser, J. M. (2010) Proc.Natl. Acad. Sci. U.S.A. 107, 4693–4698

28. Li, M., Lai, Y., Villaruz, A. E., Cha, D. J., Sturdevant, D. E., and Otto, M.(2007) Proc. Natl. Acad. Sci. U.S.A. 104, 9469–9474

29. Edgar, R., Domrachev, M., and Lash, A. E. (2002) Nucleic Acids Res. 30,207–210

30. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan,P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R.,Thompson, J. D., Gibson, T. J., and Higgins, D. G. (2007) Bioinformatics23, 2947–2948

31. Leonardi, A., Ellinger-Ziegelbauer, H., Franzoso, G., Brown, K., andSiebenlist, U. (2000) J. Biol. Chem. 275, 271–278

32. Neumann, M., and Naumann, M. (2007) FASEB J. 21, 2642–265433. Perkins, N. D. (2006) Oncogene 25, 6717–673034. Kao, C. Y., Chen, Y., Thai, P., Wachi, S., Huang, F., Kim, C., Harper, R.W.,

and Wu, R. (2004) J. Immunol. 173, 3482–349135. Shen, F., Ruddy, M. J., Plamondon, P., and Gaffen, S. L. (2005) J. Leukocyte

Biol. 77, 388–39936. Sparna, T., Retey, J., Schmich, K., Albrecht, U., Naumann, K., Gretz, N.,

Fischer, H. P., Bode, J. G., and Merfort, I. (2010) BMC Genomics 11, 22637. Ueda, A., Okuda, K., Ohno, S., Shirai, A., Igarashi, T., Matsunaga, K.,

Fukushima, J., Kawamoto, S., Ishigatsubo, Y., and Okubo, T. (1994) J. Im-munol. 153, 2052–2063

38. Bren, G. D., Solan, N. J., Miyoshi, H., Pennington, K. N., Pobst, L. J., andPaya, C. V. (2001) Oncogene 20, 7722–7733

39. Hoffmann, A., Leung, T. H., and Baltimore, D. (2003) EMBO J. 22,5530–5539

40. Schluns, K. S., Cook, J. E., and Le, P. T. (1997) J. Immunol. 158, 2704–271241. Yamamoto, M., Yamazaki, S., Uematsu, S., Sato, S., Hemmi, H., Hoshino,

K., Kaisho, T., Kuwata, H., Takeuchi, O., Takeshige, K., Saitoh, T.,Yamaoka, S., Yamamoto, N., Yamamoto, S., Muta, T., Takeda, K., andAkira, S. (2004) Nature 430, 218–222

42. Yamazaki, S., Matsuo, S., Muta, T., Yamamoto, M., Akira, S., andTakeshige, K. (2008) J. Biol. Chem. 283, 32404–32411

43. Andoh, A., Shimada,M., Bamba, S., Okuno, T., Araki, Y., Fujiyama, Y., andBamba, T. (2002) Biochim. Biophys. Acta 1591, 69–74

44. Ye, H., Arron, J. R., Lamothe, B., Cirilli, M., Kobayashi, T., Shevde, N. K.,

IL-17-induced NF-�B Activation via CIKS

APRIL 15, 2011 • VOLUME 286 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 12889

by guest on May 24, 2020

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nloaded from

Segal, D., Dzivenu, O. K., Vologodskaia, M., Yim, M., Du, K., Singh, S.,Pike, J. W., Darnay, B. G., Choi, Y., and Wu, H. (2002) Nature 418,443–447

45. Heinemann, S., Biesinger, B., Fleckenstein, B., and Albrecht, J. C. (2006)J. Biol. Chem. 281, 8565–8572

46. Sorrentino, A., Thakur, N., Grimsby, S., Marcusson, A., von Bulow, V.,Schuster, N., Zhang, S., Heldin, C. H., and Landstrom,M. (2008)Nat. Cell

Biol. 10, 1199–120747. Barlow, J. L., and McKenzie, A. N. (2009) Biofactors 35, 178–18248. Pisitkun, P., Claudio, E., Ren, N., Wang, H., and Siebenlist, U. (2010) Ar-

thritis Rheum. 62, 3334–334449. Kang, Z., Altuntas, C. Z., Gulen, M. F., Liu, C., Giltiay, N., Qin, H., Liu, L.,

Qian,W., Ransohoff, R.M., Bergmann, C., Stohlman, S., Tuohy, V. K., andLi, X. (2010) Immunity 32, 414–425

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and Ulrich SiebenlistSøren Ulrik Sønder, Sun Saret, Wanhu Tang, Dan E. Sturdevant, Stephen F. Porcella

SIGNIFICANCE AND SIGNALING MECHANISMSB Activation via CIKS/Act1: PHYSIOLOGICκIL-17-induced NF-

doi: 10.1074/jbc.M110.199547 originally published online February 18, 20112011, 286:12881-12890.J. Biol. Chem. 

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