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the RIP Kinase DomainSynthetic Biology Reveals the Uniqueness of

AbbottSteven M. Chirieleison, Sylvia B. Kertesy and Derek W.

http://www.jimmunol.org/content/196/10/4291doi: 10.4049/jimmunol.1502631April 2016;

2016; 196:4291-4297; Prepublished online 4J Immunol 

Referenceshttp://www.jimmunol.org/content/196/10/4291.full#ref-list-1

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The Journal of Immunology

Synthetic Biology Reveals the Uniqueness of the RIP KinaseDomain

Steven M. Chirieleison, Sylvia B. Kertesy, and Derek W. Abbott

The RIP kinases (RIPKs) play an essential role in inflammatory signaling and inflammatory cell death. However, the function of

their kinase activity has been enigmatic, and only recently has kinase domain activity been shown to be crucial for their signal

transduction capacity. Despite this uncertainty, the RIPKs have been the subject of intense pharmaceutical development with a

number of compounds currently in preclinical testing. In this work, we seek to determine the functional redundancy between

the kinase domains of the four major RIPK family members. We find that although RIPK1, RIPK2, and RIPK4 are similar in

that they can all activate NF-kB and induce NF-kB essential modulator ubiquitination, only RIPK2 is a dual-specificity kinase.

Domain swapping experiments showed that the RIPK4 kinase domain could be converted to a dual-specificity kinase and is

essentially indistinct from RIPK2 in biochemical and molecular activity. Surprisingly, however, replacement of RIPK2’s kinase

domain with RIPK4’s did not complement a nucleotide-binding oligomerization domain 2 signaling or gene expression induction

defect in RIPK22/2 macrophages. These findings suggest that RIPK2’s kinase domain is functionally unique compared with other

RIPK family members and that pharmacologic targeting of RIPK2 can be separated from the other RIPKs. The Journal of

Immunology, 2016, 196: 4291–4297.

The RIP kinases (RIPKs) play an essential role in inflam-matory signaling and cell death (1, 2). RIPK1 is requiredfor TNF-induced NF-kB activation and helps regulate the

switch between TNF-induced apoptosis and necroptosis (1–3),partnering with RIPK3 to induce necroptosis (1, 2, 4). RIPK2 is anessential kinase regulating signaling downstream of the Crohndisease susceptibility protein nucleotide-binding oligomerizationdomain 2 (NOD2) (5, 6). In this role, RIPK2 is part of the proteincomplex that recognizes intracellular bacterial infection and helpstailor the cytokine response to eradicate an offending pathogen (7,8). Although less well studied, RIPK4 is the causative gene inpopliteal pterygium syndrome, a disease characterized by earlylethality with multiple developmental abnormalities (9). Given thecollective influence of the RIPKs on innate immune and inflam-matory signaling, there has been intense interest in manipulatingthese kinases pharmacologically for clinical gain. PharmacologicRIPK1, RIPK2, and RIPK3 inhibitors have all been described and

are in various states of clinical development for disorders as di-verse as sepsis, inflammatory bowel disease, and multiple sclerosis(10–19).Despite this pharmaceutical interest, the function of the RIPKs’

kinase domains has been enigmatic with few bone fide substratesidentified (1, 2, 20). In no case is this truer than in the case ofRIPK2. Initial Basic Local Alignment Search Tool searches sug-gested that RIPK2 was a serine-threonine kinase, and indeed,RIPK2 was shown to autophosphorylate (6, 21, 22). In these initialdescriptions, which were based largely on overexpression studies,RIPK2’s kinase activity was shown to be dispensable for signalingsuch that although the RIPK2 protein was essential for NOD1/2signaling, its kinase activity was unnecessary (6, 21, 22). Hintsto RIPK2’s kinase function began to emerge when it was shownthat the joint p38 and RIPK2 inhibitor, SB203580, could causedecreased expression of RIPK2, presumably through a loss ofprotein stability (23). Although this work was also supported bythe fact that a genetic knockin of kinase-dead RIPK2 showeddecreased expression, this feature is shared by many kinases inwhich a kinase-dead variant shows decreased expression (24). Infact, additional pharmacologic studies using a more diverse andspecific panel of RIPK2 inhibitors have shown that inhibition ofRIPK2 kinase activity does not have a universal role in RIPK2protein stability (11, 12, 19, 25); thus, the role of the kinaseactivity in RIPK2 protein stability still remains unanswered. Alast mystery surrounding the RIPK family of kinases centers onwhich phosphoacceptor they prefer to phosphorylate. RIPK2 wasinitially misclassified as a serine-threonine kinase when in factit is a dual-specificity kinase, capable of phosphorylating ser-ines, threonines, and tyrosines (11). Despite this advance in theNOD–RIPK2 field, the preferred phosphoacceptors of the otherRIPKs remains unstudied.Structural studies have also recently highlighted the differences

between, and the importance of, the kinase domains of this familyof proteins. RIPK2 contains an extended, deep ATP binding pocket,which allows a pharmacologic manipulation likely not affordedby the other RIPKs (11, 16, 18). Although molecular modelingand crystal structures have shown largely superimposable kinase

Department of Pathology, Case Western Reserve University School of Medicine,Cleveland, OH 44106

ORCIDs: 0000-0002-3997-5652 (S.M.C.); 0000-0003-4387-8094 (D.W.A.).

Received for publication December 18, 2015. Accepted for publication March 6,2016.

This work was supported by National Institutes of Health Grants R01 GM086550 andP01 DK091222 (to D.W.A.). S.M.C. is supported by the Case Western ReserveUniversity National Institutes of Health Medical Scientist Training Program(T32GM007250).

S.M.C. generated the novel lentiviral vector, interpreted results, and edited the man-uscript; S.B.K. provided technical assistance in preparing and performing the exper-imentation; and D.W.A. generated the reagents, performed the experimentation,interpreted the results, and wrote the manuscript.

Address correspondence and reprint requests to Dr. Derek W. Abbott, Department ofPathology, Case Western Reserve University School of Medicine, Room 6531 Wol-stein Research Building, 2103 Cornell Road, Cleveland, OH 44106. E-mail address:dwa4@case.edu

Abbreviations used in this article: CARD, caspase activation recruitment domain; F,forward; HA, hemagglutinin; HygR, hygromycin resistance gene; m, murine; MDP,muramyl dipeptide; NEMO, NF-kB essential modulator; NOD2, nucleotide-bindingoligomerization domain 2; R, reverse; RIPK, RIP kinase; WT, wild-type.

Copyright� 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00

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domains among RIPK1, -2, and -3, there are subtle structuraldifferences among these three kinases, which can help explainpharmacologic specificity (18). Lastly, pharmacologic inhibitorsfor RIPK1, RIPK2, and RIPK3 have been developed that inde-pendently target these three kinases (10–19). Although structuralstudies have elucidated subtle differences among the kinase do-mains in this family of proteins, they provide only a snapshot ofthe protein in the lowest energy state at a single point in time. Incontrast, little functional work has been done to determine po-tential in vivo cellular redundancy among the RIPKs. How specificare the RIPK kinase domains for their cellular function? Can oneRIPK domain substitute for another, and does the signal trans-duction specificity of the RIPKs rely on the kinase domain or theirC-terminal effector domains? In this work, we study these centralquestions in the field and show that RIPK2’s kinase domain isuniquely required for innate immune signaling and NOD2-drivengene expression.

Materials and MethodsCell lines, plasmids, transfection, and Western blotting

Transient transfection assays were performed using calcium phosphatetransfection of HEK293 cells (CRL-1573; American Type Culture Col-lection), which were grown in 10% FBS and 1% penicillin/streptomycin,Myc-K399R NF-kB essential modulator (NEMO), and hemagglutinin(HA)-ubiquitin, generated as previously described (7, 26). cDNA expres-sion constructs for RIPK1 and RIPK3 were obtained from Vishva Dixit(Genentech), and a cDNA expression construct for RIPK4 was obtainedfrom Shiv Pillai (Massachusetts General Hospital). The template forRIPK2 was used as described (7). Gibson subcloning technology was usedto insert each of the RIPKs into the NTAP expression construct (Stra-tagene) (27). The NTAP expression construct contains an N-terminalcalmodulin binding domain and a streptavidin-binding domain. Forimmunoprecipitation and pulldown assays, cell lysates were preparedwith a buffer containing 50 mmol Tris (pH 7.4), 150 mmol NaCl, 1%Triton X-100, 1 mmol EDTA, 1 mmol EGTA, 2.5 mmol sodium pyrophos-phate, 1 mmol b-glycerophosphate, 5 mmol iodoacetimide, 5 mmolN-ethylmaleimide, 1 mmol PMSF, 1 mmol sodium orthovanadate, and pro-tease inhibitor mixture. Streptavidin beads (Sigma-Aldrich) were blockedwith 1% BSA and added to the lysate overnight when an RIPK was pre-cipitated. Immunoprecipitates were washed five times in lysis buffer beforeboiling in an equal volume of 23 Laemmli sample buffer. Western blottingwas performed as described previously (7). For NEMO precipitation assaysassessing ubiquitination, lysates were boiled before immunoprecipitationto denature the lysate and allow direct assessment of NEMO ubiquitination.NEMO was precipitated via its N-terminal 3Xmyc tag (Ab 9E10 clone;Santa Cruz Biotechnology). The K399R NEMO construct was used asthis limits background ubiquitination. For signaling experiments, 10 mg/mlL-18 muramyl dipeptide (MDP; Invivogen) was added to the media forthe given amount of time before lysates were generated using the abovebuffer. Protein concentrations were standardized by the Bio-Rad proteinassay (Bio-Rad), and Western blots were performed as described. TheHA Ab (16B12) was obtained from Covance. The phosphotyrosine Ab(p-Tyr-100) was obtained from Cell Signaling Technology, as were thep–IkB kinase, total IkB kinase, inhibitor of k L-chain gene enhancerin B cells, and p-inhibitor of k L-chain gene enhancer in B cells. TheGADPH Ab was obtained from GenScript. The RIPK2 Ab (H-300) wasobtained from Santa Cruz Biotechnologies, recognizes the C terminus ofRIPK2, and is thus able to blot the chimeric constructs.

Viral production and stable cell line generation

Immortalized RIPK22/2 macrophages were obtained from MichelleKelliher (University of Massachusetts Medical School) and grown in 10%FBS and 1% penicillin/streptomycin. Lentiviral Crispr V2 (Addgene) wasused as a Gibson subcloning template to generate the empty lentiviralconstruct outlined in Fig. 3A. Gibson subcloning was then used to generatethe retroviral constructs containing full-length NTAP-tagged RIPK2 or theNTAP-tagged RIPK3/2 and RIPK4/2 chimeric constructs. HEK293 cellswere transfected via calcium phosphate with pMD.2 (Addgene), psPAX(Addgene), and the RIPK lentivirus in a 1:3:4 molar ratio. Two days later,supernatant was harvested, centrifuged at 1200 rpm for 5 min, and filteredthrough a 0.45-mm filter. Polybrene (8 mg/ml) was added to the viral su-pernatant, and this mixture was added to the RIPK22/2 macrophages. Two

days later, cells were selected in 500 mg/ml Hygromycin-Gold (Invivogen).Selection continued for .2 wk. Greater than 10,000 individual colonieswere pooled, and Western blotting showed roughly equal expression levelsof the transduced construct.

RNA isolation and quantitative RT-PCR

The stably transduced RIPKmacrophages were treated with 10 mg/ml MDPfor the indicated time. Cells were then harvested and RNA extracted usinga Qiagen RNeasy kit using the manufacturer’s instructions. RNA was re-verse transcribed using a Quantitect reverse transcription kit (Qiagen). Thefollowing primer pairs were used for amplification: murine (m)CXCL10-forward (F) 59-TCCTTGTCCTCCCTAGCTCA-39 and mCXCL10-reverse(R), 59-ATAACCCCTTGGGAAGATGG-39; mGPR84-F, 59-GGGAACC-TCAGTCTCCAT-39 and mGPR84-R, 59-TGCCACGCCCCAGATAATG-39;mIRG1-F, 59-GTTTGGGGTCGACCAGACTT-39 and mIRG1-R, 59-CAGGTCGAGGCCAGAAAACT-39; mIL-6-F, 59-GCCTTCTTGGGA-CTGATGCT-39 and mIL-6-R, 59-TGCCATTGCACAACTCTTTTCT-39;and mGAPDH-F, 59-AGGCCGGTGCTGAGTATGTC-39 and mGAPDH-R,59-TGCCTGCTTCACCACCTTCT-39. SYBR Green was obtained fromBio-Rad, and the real-time PCR reactions were carried out using aCFX96 C1000 Real-Time Thermal Cycler from Bio-Rad. RT-PCR dataare presented as the mean 6 SEM. RT-PCR experiments were performedin duplicate and repeated three times. Significance of comparisons shownwas assessed by Student two-tailed t test. Significance levels are shownin each graph.

ResultsDespite the homology within the kinase domains, the RIPKsshow differential molecular abilities

The RIPKs have been classified into a family of kinases based onhomology within the kinase domains. All of the kinase domains liein the N terminus of the protein, C-terminal to the kinase domain;however, their domain architecture differs significantly. Althoughboth RIPK1 and RIPK3 contain RIP homotypic interaction motifdomains to allow for homotypic protein–protein interactions (28),only RIPK1 also contains a death domain (29). RIPK4 containsAnkyrin repeats (30), and RIPK2 contains a caspase activationrecruitment domain (CARD) (21, 22), which allows it to interactwith NOD2 and serve as a sensor of intracellular bacterial expo-sure (Fig. 1A) (6). Given that there is widespread interest in tar-geting this family of kinases pharmacologically for diseases asdiverse as autoinflammation, sepsis, and autoimmunity (10–19),we sought to formally compare the molecular and biochemicalactivities of the RIPKs to determine unique features and functionalredundancy of this kinase family. NF-kΒ luciferase studiesshowed that RIPK1, RIPK2, and RIPK4 could all activate NF-kB,whereas RIPK3 could not (Fig. 1B). Surprisingly, only RIPK2 wasconfirmed as a dual specificity kinase as only RIPK2 couldautophosphorylate on tyrosine (Fig. 1C). Lastly, every RIPK ex-cept RIPK3 could induce the ubiquitination of NEMO, a keyfeature of NF-kB activation (31) (Fig. 1D). These findings suggestthat RIPK1, -2, and -4 share similar molecular abilities to activatethe NF-kB signaling pathway, whereas RIPK3 diverges. RIPK2uniquely autophosphorylates on tyrosine, and under these bio-chemical conditions is the only dual-specificity kinase among thisfamily.

Domain switching reveals that RIPK2’s and RIPK4’s kinasedomains are functionally similar

Given that RIPK2’s tyrosine autophosphorylation is required fordownstream NOD2 signaling (11), we were surprised that theother RIPKs did not show tyrosine autophosphorylation activity.To determine if this activity was unique to RIPK2’s kinase domainor if it required the specific spacial proximity to the substratepresent in RIPK2’s C terminus [in which Y474 is phosphorylated(11)], synthetic biology techniques were used to generate chimericRIPK constructs. In each of these constructs, the C terminus of

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RIPK2 (immediately downstream of the kinase domain) was heldconstant, whereas the kinase domains were swapped. For example,the RIPK1/2 chimera contained the N-terminal RIPK1 kinasedomain with RIPK2’s C terminus, although the RIPK3/2 chimeracontained RIPK3’s kinase domain with RIPK2’s C terminus(Fig. 2A). We first determined if these chimeric molecules couldmaintain the interaction with NOD2. NOD2 is known to interactwith RIPK2 through RIPK2’s C-terminal CARD domain (6) andthus should interact with the chimeric kinases. Western blottingfollowing coimmunoprecipitation from transfected cells showedthat all three chimeric constructs as well as wild-type (WT)RIPK2 could interact with NOD2, suggesting that the chimericproteins were folding correctly and could still interact withRIPK2’s key signaling partner (Fig. 2B). To then test if kinasedomain swapping could biochemically function, in vitro kinaseassays were performed. Of the RIPKs, RIPK4 could autophos-phorylate on tyrosine only when RIPK2’s C-terminal domain waspresent (Fig. 2C). Neither the RIPK1/2 or RIPK3/2 chimeric ki-nases could autophosphorylate on tyrosine (Fig. 2C). Domainswapping further revealed that the RIPK3/2 and RIPK4/2 chi-meras could induce NEMO ubiquitination, albeit at lower levelsrelative to WT RIPK2 (Fig. 2D). Lastly, ubiquitination of NEMOwas not sufficient for NF-kB activation, as only the RIPK4/2

chimeric protein, and not the RIPK3/2 chimeric protein, couldactivate NF-kB (Fig. 2E). These findings suggest that, like RIPK2,RIPK4 also possesses tyrosine kinase activity, as well as theability to induce NEMO ubiquitination and cause subsequentNF-kB activation. The chimeric RIPK4/2 protein is thereforefunctionally similar to WT RIP2 and gives us an important toolto now dissect the uniqueness of RIPK2’s kinase domain insignaling and gene expression systems. This line of research isespecially important as numerous pharmaceutical companies haveRIPK inhibitors in clinical development (10–19).

RIPK2’s kinase domain is uniquely required for NOD2signaling

To then answer if RIPK2’s kinase domain is uniquely required forNOD2 signaling, we used synthetic biology techniques to developa novel lentiviral expression construct [generated from the lenti-CRISPR V2 construct (32)] and then made use of immortalizedRIPK22 /2 macrophages. This lentiviral expression constructcontains standard lentiviral long terminal repeats; however,the EF-1 promoter drives exogenous mRNA transcription. Ahygromycin resistance gene (HygR) was Gibson cloned in frameto a C-terminal P2A self-cleaving peptide cassette. Finally, NTAP-tagged RIPK2, RIPK3/2, and RIPK4/2 were Gibson cloned in to

FIGURE 1. Comparison of the molecular activities of the RIPKs. (A) Schematic showing the RIPKs’ domain structure. Homology lies within the kinase

domain in the N terminus, whereas the C termini have differing domain architecture. (B) HEK293 cells were transfected with CMV-Renilla, NF-kB–driven

luciferase, and 1.5 mg of the indicated RIPK construct. Transfection efficiency was standardized to Renilla expression, and luciferase activities were

measured. RIPK1, RIPK2, and RIPK4 could activate NF-kB, but RIPK3 could not. (C) HEK293 cells were transfected as indicated, and streptavidin bead

association isolated the individual RIPK. In vitro kinase assays were performed in the presence or absence of ATP. Only RIPK2 was able to autophos-

phorylate on tyrosine. (D) HEK293 cells were transfected with HA-tagged ubiquitin, myc-tagged NEMO, and the indicated RIPK construct. NEMO was

isolated by immunoprecipitation under stringent conditions, and Western blotting was performed. RIPK1, RIPK2, and RIPK4 were all able to cause NEMO

ubiquitination, whereas RIPK3 was not. Each given experiment was performed in at least three biologic replicates with similar results in each. *p , 0.02.

IP, immunoprecipitation; RHIM, RIP homotypic interaction motif.

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the vector in frame with the P2A cassette. The end result is anexpression vector that is driven by EF-1 (a promoter insensitive toNF-kB activity) and generates a single mRNA containing theresistance gene and our gene of interest. Upon translation, thesingle mRNA product is generated as two individual proteins(schematic shown in Fig. 3A). Although the RIPK4/2 chimericprotein can both tyrosine autophosphorylate and activate NF-kB,the RIPK3/2 protein can perform neither of these functions andwas therefore used as an additional negative control. Lentiviruswas produced and used to infect RIPK22/2 macrophages. Postin-fection, cells were selected in hygromycin for 2 wk before.10,000

colonies were pooled. Western blotting showed that althoughRIPK4/2 was initially expressed at a slightly lower level (Fig. 3B),upon stronger hygromycin selection, levels of the exogenous pro-teins normalized (Fig. 3C, 3D). Signaling experiments wereperformed. Although RIPK2 expression could rescue NOD2-dependent signaling in the RIPK22/2 macrophages, expression ofempty vector (Fig. 3C), RIPK3/2, or RIPK4/2 could not (Fig. 3D),suggesting that despite the biochemical similarities betweenRIPK2 and RIPK4/2, RIPK4’s kinase domain could not replaceRIPK2 in NOD2 signaling. To then further determine the extent ofthe signaling defect in a manner more quantifiable, NOD2-driven

FIGURE 2. Domain swapping reveals similar molecular activities between RIPK2 and RIPK4. (A) Schematic showing the chimeric constructs generated

and used. The C terminus of the constructs is identical to the C terminus of RIPK2, whereas the kinase domains have been swapped as indicated. (B)

Cotransfection into HEK293s with the indicated constructs followed by immunoprecipitation (IP) and Western blotting shows that all chimeric RIPK

proteins can bind to NOD2. (C) HEK293 cells were transfected as indicated, and streptavidin bead association isolated the individual RIPK. In vitro kinase

assays were performed in the presence or absence of ATP. Although RIPK2 could autophosphorylate on tyrosines, only the RIPK4/2 chimera retained this

ability. (D) HEK293 cells were transfected with HA-tagged ubiquitin, myc-tagged NEMO, and the indicated RIPK chimera. NEMO was isolated by IP

under stringent conditions, and Western blotting was performed. RIPK2, RIPK3/2, and RIPK4/2 were all able to cause NEMO ubiquitination to a certain

degree, whereas RIPK1/2 was not. (E) HEK293 cells were transfected with CMV-Renilla, NF-kB–driven luciferase, and the indicated RIPK construct.

Transfection efficiency was standardized to Renilla expression, and luciferase activities were measured. Of the chimeric constructs, only the RIPK4/2

chimera could activate NF-kB. Each given experiment was performed in at least three biologic replicates with similar results in each. *p , 0.05.

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gene expression was studied. Our laboratory has previously usedNextGen sequencing technologies to identify the NOD2-drivengenes most sensitive to RIPK2’s kinase activity (12, 25). Weused these genes as readouts for gene expression. In all cases, onlyRIPK2 expression could rescue NOD2-driven gene expression.This was true for IL-6 (Fig. 4A), CXCL10 (Fig. 4B), IRG-1(Fig. 4C), and Gpr84 (Fig. 4D). Together, these data suggestthat despite molecular and biochemical similarities betweenRIPK2 and RIPK4’s kinase domains, RIPK2’s kinase domainfunctions uniquely, a key feature if one hopes to pharmaceuticallytarget RIPK2 for clinical gain.

DiscussionDespite their homology and familial grouping, the RIPKs partic-ipate in varied biologic functions. RIPK1 and RIPK3 are importantin dictating the mechanism of cell death in response to a variety ofinnate immune and inflammatory signaling (1, 2, 33). RIPK2 iscritically required for NOD1/2 signaling in response to intracel-lular bacterial exposure (34), and RIPK4 is required for properdevelopment (9). Despite this, recent structural work has shownthat a number of broad-spectrum kinase inhibitors target RIPK1,RIPK2, and RIPK3 with similar potency (16–18). This samestructural work has shown that although there are subtle structural

differences that may help direct medicinal chemistry towardspecific inhibitors, these three kinases overlap significantly in athree-dimensional structural context, potentially making suchefforts futile (18). Additionally, work presented in this manuscriptshows that they have overlapping molecular and biochemicalactivities. RIPK1, RIPK2, and RIPK4 all induce NEMO ubiq-uitination and subsequent NF-kB activation. Despite this, onlyRIPK2 autophosphorylates on tyrosine and is the only RIPKproven to be a dual-specificity kinase. Given the interest inpharmacologically targeting a family of kinases with both similarand divergent molecular activities (10–19), it was important todetermine the functional redundancy of the kinase domain be-tween the RIPK family members. To this end, domain-swappingsynthetic biology approaches were used. In this context, the onlykinase domain that could replicate RIPK2 kinase domain functionin an in vitro system was the RIPK4 kinase domain. SubstitutingRIPK4’s kinase domain for RIPK2’s allowed NOD2 binding,tyrosine autophosphorylation, NEMO ubiquitination, and NF-kBactivation, all key molecular events in which RIPK2 is requireddownstream of NOD2 activation. Surprisingly, despite the mo-lecular similarities between the two kinase domains, the RIPK4kinase domain could not substitute for RIPK2 in an endogenoussetting. It could not support NOD2-induced signaling in RIPK22/2

FIGURE 3. The kinase domain of RIPK2 is uniquely required for NOD2 signaling. (A) Schematic showing novel lentiviral construct designed to express

the RIPK chimeras. HygR is cloned in frame with the self-cleaving peptide, P2A, and the NTAP-tagged RIPK chimera. A single mRNA is generated under

the EF-1 promoter and upon translation; the P2A sequence allows a translational skip such that during translation, two proteins (HygR [HygroR] and the

NTAP-tagged RIPK) are generated from a single mRNA. (B) Immortalized RIPK22/2 macrophages were transduced with lentivirus containing no RIPK

(empty), RIPK2, RIPK3/2, and RIPK4/2. Two days after transduction, cells were selected with hygromycin. After 2 wk of selection, .10,000 individual

cell colonies were pooled. Streptavidin bead isolation and Western blotting showed that the stable cell lines expressed the gene of interest. (C and D) The

indicated RIPK cell line was treated with 10 mg/ml of the NOD2 agonist L-18 MDP for the indicated time period. Lysates were generated, and Western

blotting was performed. Although the empty vector line showed no signaling (consistent with RIPK2 being genetically absent), cells reconstituted with

RIPK2 show a strong signaling response. Neither RIPK3/2 nor RIPK4/2 reconstituted cells showed a NOD2-dependent signaling response. In (D), the final

two lanes are RIPK2 reconstituted such that a positive control is present on those blots. Each given experiment was performed in at least three biologic

replicates with similar results in each. cPPT, central polypurine tract; IKK, IkB kinase; LTR, long terminal repeat; WPRE, woodchuck posttranscriptional

regulatory element.

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macrophages and could not replace RIPK2’s role in drivingNOD2-induced gene expression. These findings suggest thatRIPK2’s kinase domain is uniquely required for NOD2 signalingand cannot be replaced by even its closest homologs, implyingthat unique pharmacologic targeting of the RIPK family membersis readily achievable.RIPK2’s role in innate immune signaling has largely centered

on its scaffolding function. RIPK2 clearly helps nucleate signalingcomplexes to transduce signals from NOD1 and NOD2, and ge-netic loss of RIPK2 does not allow signaling through the NOD1and NOD2 receptors (5, 33, 34). The fact that overexpressionof kinase-dead RIPK2 could activate NF-kB suggested that thekinase domain might be dispensable for RIPK2’s major knownfunction (6, 21, 22). Despite this, recent work uncovering specificinhibitors of RIPK2 suggested that although initial and acuteNF-kB signaling did not require kinase activity, optimal NOD-stimulated cytokine and gene expression absolutely require it(11, 19). This finding is supported by our prior study usingNextGen RNAseq methods showing that a significant subset ofNOD2-induced genes require RIPK2’s kinase activity for optimalexpression (12, 25). A key question that remains centers on thescaffolding function of RIPK2’s kinase domain versus its actualkinase activity. To answer this question, we used domain-swapping experiments in which we replaced RIPK2’s kinase do-main with its closest structural homologs (RIPK4 and RIPK3).Surprisingly, we found that despite the fact that a RIPK4/2 chi-mera could largely replace RIPK2’s function in overexpressionsystems, it could not replace RIPK2’s function in more endog-enous, acute signaling experiments. This finding is surprisingbecause pharmacologic experiments have shown that althoughRIPK2’s kinase activity is required for optimal gene expression, itis largely dispensable for acute NF-kB signaling (19). This ex-periment shows that there must be structural elements of theRIPK2 kinase domain independent of its kinase activity such thatRIPK4 could not replace RIPK2’s role in acute signaling. Thescaffolding function and acute NF-kB signaling of RIPK2 is

dependent on its kinase domain but not its kinase activity, and thisscaffolding activity cannot be replaced even by RIPK2’s closesthomolog.Another interesting finding in this study centers on tyrosine

phosphorylation. RIPK2 is known to be a dual-specificity kinase(11), but work in this manuscript shows that this feature is notshared by the other RIPK family members. Given this, it is sur-prising that RIPK4 is able to autophosphorylate on tyrosine whenits C-terminal Ankyrin repeats are replaced by RIPK2’s inter-mediate and CARDs. Although native RIPK4 cannot auto-phosphorylate on tyrosine residues, the RIPK4/2 chimera canautophosphorylate on tyrosines, and this activity matches RIPK2’styrosine kinase activity. This surprising result suggests thatRIPK4’s kinase domain has the intrinsic ability to be a dual-specificity kinase; however, its ability to phosphorylate on tyro-sine is substrate-restricted rather than kinase activity restricted.To our knowledge, this substrate-driven dual-specificity kinaseactivity is unique and has broader implications for the kinasefield as a whole, suggesting that phosphoacceptor preferencescan be altered by substrate selection rather than by intrinsic ki-nase structure.Thus, in addition to categorizing and comparing the RIPKs to

one another in terms of their ability to activate NF-kB and performNEMO ubiquitination, this study illustrates two key features of theRIPK family. First, RIPK2’s kinase domain is uniquely structuredin such a way as to nucleate signaling complexes independent ofits kinase activity. For this reason, its closest homologous kinasedomain, RIPK4, cannot replace it structurally despite havingsimilar kinase activity. Secondly, RIPK4 has substrate-restricteddual-specificity kinase activity that can be induced by physicallyfusing the substrate to its kinase domain. In the context of sub-sequent pharmacologic targeting of this family, the work suggeststhat not only might a small molecule exclusively target RIPK2, butalso that by exclusively targeting RIPK2, the function of the otherRIPKs might not be affected. It also suggests that by developingtype III kinase inhibitors for RIPK2 and RIPK4, one might be able

FIGURE 4. The kinase domain of RIPK2 is required for NOD2-driven gene expression. (A–D) The RIPK-reconstituted cells were treated with 10 mg/ml

MDP for 2.5 or 5 h. Quantitative RT-PCR was performed using expression of GADPH as an RNA quantification control. Only cells reconstituted with full-

length RIPK2 allowed NOD2-driven gene expression of IL-6 (A), CXCL10 (B), IRG-1 (C), and Gpr84 (D). Mu, macrophage.

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to identify substrate-specific inhibitors and limit substrate phos-phorylation rather than eliminate all RIPK2 or RIPK4 phosphor-ylation.

AcknowledgmentsWe thank Drs. George Dubyak, Tsan Xiao, and Parameswaran Ramakrishnan

(CaseWestern Reserve University School ofMedicine) for helpful comments

and critiques on the manuscript. Constructs and reagents were obtained from

Vishva Dixit (Genentech), Michelle Kelliher (University of Massachusetts

Medical School), and Shiv Pillai (Massachusetts General Hospital).

DisclosuresThe authors have no financial conflicts of interest.

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