Synthetic Biology Reveals the Uniqueness of the RIP Kinase Domain Steven M. Chirieleison, Sylvia B. Kertesy and Derek W. Abbott This information is current as of September 26, 2021. J Immunol 2016; 196:4291-4297; Prepublished online 4 April 2016; doi: 10.4049/jimmunol.1502631 http://www.jimmunol.org/content/196/10/4291 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Synthetic Biology Reveals the Uniqueness of the RIP Kinase Domain

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 Downloaded from 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.

he RIP kinases (RIPKs) play an essential role in inflam- are in various states of clinical development for disorders as di- http://www.jimmunol.org/ matory signaling and cell death (1, 2). RIPK1 is required verse as sepsis, inflammatory bowel disease, and multiple sclerosis T for TNF-induced NF-kB activation and helps regulate the (10–19). switch between TNF-induced apoptosis and necroptosis (1–3), Despite this pharmaceutical interest, the function of the RIPKs’ partnering with RIPK3 to induce necroptosis (1, 2, 4). RIPK2 is an kinase domains has been enigmatic with few bone fide substrates essential kinase regulating signaling downstream of the Crohn identified (1, 2, 20). In no case is this truer than in the case of disease susceptibility protein nucleotide-binding oligomerization RIPK2. Initial Basic Local Alignment Search Tool searches sug- domain 2 (NOD2) (5, 6). In this role, RIPK2 is part of the protein gested that RIPK2 was a serine-threonine kinase, and indeed, complex that recognizes intracellular bacterial infection and helps RIPK2 was shown to autophosphorylate (6, 21, 22). In these initial tailor the cytokine response to eradicate an offending pathogen (7, descriptions, which were based largely on overexpression studies, by guest on September 26, 2021 8). Although less well studied, RIPK4 is the causative gene in RIPK2’s kinase activity was shown to be dispensable for signaling popliteal pterygium syndrome, a disease characterized by early such that although the RIPK2 protein was essential for NOD1/2 lethality with multiple developmental abnormalities (9). Given the signaling, its kinase activity was unnecessary (6, 21, 22). Hints collective influence of the RIPKs on innate immune and inflam- to RIPK2’s kinase function began to emerge when it was shown matory signaling, there has been intense interest in manipulating that the joint p38 and RIPK2 inhibitor, SB203580, could cause these kinases pharmacologically for clinical gain. Pharmacologic decreased expression of RIPK2, presumably through a loss of RIPK1, RIPK2, and RIPK3 inhibitors have all been described and protein stability (23). Although this work was also supported by the fact that a genetic knockin of kinase-dead RIPK2 showed decreased expression, this feature is shared by many kinases in Department of Pathology, Case Western Reserve University School of Medicine, which a kinase-dead variant shows decreased expression (24). In Cleveland, OH 44106 fact, additional pharmacologic studies using a more diverse and ORCIDs: 0000-0002-3997-5652 (S.M.C.); 0000-0003-4387-8094 (D.W.A.). specific panel of RIPK2 inhibitors have shown that inhibition of Received for publication December 18, 2015. Accepted for publication March 6, RIPK2 kinase activity does not have a universal role in RIPK2 2016. protein stability (11, 12, 19, 25); thus, the role of the kinase This work was supported by National Institutes of Health Grants R01 GM086550 and P01 DK091222 (to D.W.A.). S.M.C. is supported by the Case Western Reserve activity in RIPK2 protein stability still remains unanswered. A University National Institutes of Health Medical Scientist Training Program last mystery surrounding the RIPK family of kinases centers on (T32GM007250). which phosphoacceptor they prefer to phosphorylate. RIPK2 was S.M.C. generated the novel lentiviral vector, interpreted results, and edited the man- initially misclassified as a serine-threonine kinase when in fact uscript; S.B.K. provided technical assistance in preparing and performing the exper- imentation; and D.W.A. generated the reagents, performed the experimentation, it is a dual-specificity kinase, capable of phosphorylating ser- interpreted the results, and wrote the manuscript. ines, threonines, and tyrosines (11). Despite this advance in the Address correspondence and reprint requests to Dr. Derek W. Abbott, Department of NOD–RIPK2 field, the preferred phosphoacceptors of the other Pathology, Case Western Reserve University School of Medicine, Room 6531 Wol- RIPKs remains unstudied. stein Research Building, 2103 Cornell Road, Cleveland, OH 44106. E-mail address: [email protected] Structural studies have also recently highlighted the differences Abbreviations used in this article: CARD, caspase activation recruitment domain; F, between, and the importance of, the kinase domains of this family forward; HA, hemagglutinin; HygR, hygromycin resistance gene; m, murine; MDP, of proteins. RIPK2 contains an extended, deep ATP binding pocket, muramyl dipeptide; NEMO, NF-kB essential modulator; NOD2, nucleotide-binding which allows a pharmacologic manipulation likely not afforded oligomerization domain 2; R, reverse; RIPK, RIP kinase; WT, wild-type. by the other RIPKs (11, 16, 18). Although molecular modeling Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 and crystal structures have shown largely superimposable kinase www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502631 4292 UNIQUENESS OF THE RIP KINASE DOMAIN domains among RIPK1, -2, and -3, there are subtle structural days later, cells were selected in 500 mg/ml Hygromycin-Gold (Invivogen). differences among these three kinases, which can help explain Selection continued for .2 wk. Greater than 10,000 individual colonies pharmacologic specificity (18). Lastly, pharmacologic inhibitors were pooled, and Western blotting showed roughly equal expression levels of the transduced construct. for RIPK1, RIPK2, and RIPK3 have been developed that inde- pendently target these three kinases (10–19). Although structural RNA isolation and quantitative RT-PCR studies have elucidated subtle differences among the kinase do- The stably transduced RIPK macrophages were treated with 10 mg/ml MDP mains in this family of proteins, they provide only a snapshot of for the indicated time. Cells were then harvested and RNA extracted using the protein in the lowest energy state at a single point in time. In a Qiagen RNeasy kit using the manufacturer’s instructions. RNA was re- contrast, little functional work has been done to determine po- verse transcribed using a Quantitect reverse transcription kit (Qiagen). The tential in vivo cellular redundancy among the RIPKs. How specific following primer pairs were used for amplification: murine (m)CXCL10- forward (F) 59-TCCTTGTCCTCCCTAGCTCA-39 and mCXCL10-reverse are the RIPK kinase domains for their cellular function? Can one (R), 59-ATAACCCCTTGGGAAGATGG-39; mGPR84-F, 59-GGGAACC- RIPK domain substitute for another, and does the signal trans- TCAGTCTCCAT-39 and mGPR84-R, 59-TGCCACGCCCCAGATAATG-39; duction specificity of the RIPKs rely on the kinase domain or their mIRG1-F, 59-GTTTGGGGTCGACCAGACTT-39 and mIRG1-R, 59- 9 9 C-terminal effector domains? In this work, we study these central CAGGTCGAGGCCAGAAAACT-3 ;mIL-6-F,5-GCCTTCTTGGGA- CTGATGCT-39 and mIL-6-R, 59-TGCCATTGCACAACTCTTTTCT-39; questions in the field and show that RIPK2’s kinase domain is and mGAPDH-F, 59-AGGCCGGTGCTGAGTATGTC-39 and mGAPDH-R, uniquely required for innate immune signaling and NOD2-driven 59-TGCCTGCTTCACCACCTTCT-39. SYBR Green was obtained from gene expression. Bio-Rad, and the real-time PCR reactions were carried out using a CFX96 C1000 Real-Time Thermal Cycler from Bio-Rad. RT-PCR data are presented as the mean 6 SEM. RT-PCR experiments were performed Downloaded from Materials and Methods in duplicate and repeated three times. Significance of comparisons shown Cell lines, plasmids, transfection, and Western blotting was assessed by Student two-tailed t test. Significance levels are shown in each graph. Transient transfection assays were performed using calcium phosphate transfection of HEK293 cells (CRL-1573; American Type Culture Col- lection), which were grown in 10% FBS and 1% penicillin/streptomycin, Results Myc-K399R NF-kB essential modulator (NEMO), and hemagglutinin Despite the homology within the kinase domains, the RIPKs (HA)-ubiquitin, generated as previously described (7, 26). cDNA expres- http://www.jimmunol.org/ sion constructs for RIPK1 and RIPK3 were obtained from Vishva Dixit show differential molecular abilities (Genentech), and a cDNA expression construct for RIPK4 was obtained The RIPKs have been classified into a family of kinases based on from Shiv Pillai (Massachusetts General Hospital). The template for RIPK2 was used as described (7). Gibson subcloning technology was used homology within the kinase domains. All of the kinase domains lie to insert each of the RIPKs into the NTAP expression construct (Stra- in the N terminus of the protein, C-terminal to the kinase domain; tagene) (27). The NTAP expression construct contains an N-terminal however, their domain architecture differs significantly. Although calmodulin binding domain and a streptavidin-binding domain. For both RIPK1 and RIPK3 contain RIP homotypic interaction motif immunoprecipitation and pulldown assays, cell lysates were prepared with a buffer containing 50 mmol Tris (pH 7.4), 150 mmol NaCl, 1% domains to allow for homotypic protein–protein interactions (28), Triton X-100, 1 mmol EDTA, 1 mmol EGTA, 2.5 mmol sodium pyrophos- only RIPK1 also contains a death domain (29). RIPK4 contains phate, 1 mmol b-glycerophosphate, 5 mmol iodoacetimide, 5 mmol Ankyrin repeats (30), and RIPK2 contains a caspase activation by guest on September 26, 2021 N-ethylmaleimide, 1 mmol PMSF, 1 mmol sodium orthovanadate, and pro- recruitment domain (CARD) (21, 22), which allows it to interact tease inhibitor mixture. Streptavidin beads (Sigma-Aldrich) were blocked with NOD2 and serve as a sensor of intracellular bacterial expo- with 1% BSA and added to the lysate overnight when an RIPK was pre- cipitated. Immunoprecipitates were washed five times in lysis buffer before sure (Fig. 1A) (6). Given that there is widespread interest in tar- boiling in an equal volume of 23 Laemmli sample buffer. Western blotting geting this family of kinases pharmacologically for diseases as was performed as described previously (7). For NEMO precipitation assays diverse as autoinflammation, sepsis, and autoimmunity (10–19), assessing ubiquitination, lysates were boiled before immunoprecipitation we sought to formally compare the molecular and biochemical to denature the lysate and allow direct assessment of NEMO ubiquitination. NEMO was precipitated via its N-terminal 3Xmyc tag (Ab 9E10 clone; activities of the RIPKs to determine unique features and functional Santa Cruz Biotechnology). The K399R NEMO construct was used as redundancy of this kinase family. NF-kΒ luciferase studies this limits background ubiquitination. For signaling experiments, 10 mg/ml showed that RIPK1, RIPK2, and RIPK4 could all activate NF-kB, L-18 muramyl dipeptide (MDP; Invivogen) was added to the media for whereas RIPK3 could not (Fig. 1B). Surprisingly, only RIPK2 was the given amount of time before lysates were generated using the above confirmed as a dual specificity kinase as only RIPK2 could buffer. Protein concentrations were standardized by the Bio-Rad protein assay (Bio-Rad), and Western blots were performed as described. The autophosphorylate on tyrosine (Fig. 1C). Lastly, every RIPK ex- HA Ab (16B12) was obtained from Covance. The phosphotyrosine Ab cept RIPK3 could induce the ubiquitination of NEMO, a key (p-Tyr-100) was obtained from Cell Signaling Technology, as were the feature of NF-kB activation (31) (Fig. 1D). These findings suggest p–IkB kinase, total IkB kinase, inhibitor of k L-chain gene enhancer that RIPK1, -2, and -4 share similar molecular abilities to activate k in B cells, and p-inhibitor of L-chain gene enhancer in B cells. The k GADPH Ab was obtained from GenScript. The RIPK2 Ab (H-300) was the NF- B signaling pathway, whereas RIPK3 diverges. RIPK2 obtained from Santa Cruz Biotechnologies, recognizes the C terminus of uniquely autophosphorylates on tyrosine, and under these bio- RIPK2, and is thus able to blot the chimeric constructs. chemical conditions is the only dual-specificity kinase among this family. Viral production and stable cell line generation Immortalized RIPK22/2 macrophages were obtained from Michelle Domain switching reveals that RIPK2’s and RIPK4’s kinase Kelliher (University of Massachusetts Medical School) and grown in 10% domains are functionally similar FBS and 1% penicillin/streptomycin. Lentiviral Crispr V2 (Addgene) was Given that RIPK2’s tyrosine autophosphorylation is required for used as a Gibson subcloning template to generate the empty lentiviral construct outlined in Fig. 3A. Gibson subcloning was then used to generate downstream NOD2 signaling (11), we were surprised that the the retroviral constructs containing full-length NTAP-tagged RIPK2 or the other RIPKs did not show tyrosine autophosphorylation activity. NTAP-tagged RIPK3/2 and RIPK4/2 chimeric constructs. HEK293 cells To determine if this activity was unique to RIPK2’s kinase domain were transfected via calcium phosphate with pMD.2 (Addgene), psPAX or if it required the specific spacial proximity to the substrate (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 filtered present in RIPK2’s C terminus [in which Y474 is phosphorylated through a 0.45-mm filter. Polybrene (8 mg/ml) was added to the viral su- (11)], synthetic biology techniques were used to generate chimeric pernatant, and this mixture was added to the RIPK22/2 macrophages. Two RIPK constructs. In each of these constructs, the C terminus of The Journal of Immunology 4293 Downloaded from http://www.jimmunol.org/

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 by guest on September 26, 2021 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.

RIPK2 (immediately downstream of the kinase domain) was held chimeric protein, and not the RIPK3/2 chimeric protein, could constant, whereas the kinase domains were swapped. For example, activate NF-kB (Fig. 2E). These findings suggest that, like RIPK2, the RIPK1/2 chimera contained the N-terminal RIPK1 kinase RIPK4 also possesses tyrosine kinase activity, as well as the domain with RIPK2’s C terminus, although the RIPK3/2 chimera ability to induce NEMO ubiquitination and cause subsequent contained RIPK3’s kinase domain with RIPK2’s C terminus NF-kB activation. The chimeric RIPK4/2 protein is therefore (Fig. 2A). We first determined if these chimeric molecules could functionally similar to WT RIP2 and gives us an important tool maintain the interaction with NOD2. NOD2 is known to interact to now dissect the uniqueness of RIPK2’s kinase domain in with RIPK2 through RIPK2’s C-terminal CARD domain (6) and signaling and gene expression systems. This line of research is thus should interact with the chimeric kinases. Western blotting especially important as numerous pharmaceutical companies have following coimmunoprecipitation from transfected cells showed RIPK inhibitors in clinical development (10–19). that all three chimeric constructs as well as wild-type (WT) RIPK2 could interact with NOD2, suggesting that the chimeric RIPK2’s kinase domain is uniquely required for NOD2 proteins were folding correctly and could still interact with signaling RIPK2’s key signaling partner (Fig. 2B). To then test if kinase To then answer if RIPK2’s kinase domain is uniquely required for domain swapping could biochemically function, in vitro kinase NOD2 signaling, we used synthetic biology techniques to develop assays were performed. Of the RIPKs, RIPK4 could autophos- a novel lentiviral expression construct [generated from the lenti- phorylate on tyrosine only when RIPK2’s C-terminal domain was CRISPR V2 construct (32)] and then made use of immortalized present (Fig. 2C). Neither the RIPK1/2 or RIPK3/2 chimeric ki- RIPK22/2 macrophages. This lentiviral expression construct nases could autophosphorylate on tyrosine (Fig. 2C). Domain contains standard lentiviral long terminal repeats; however, swapping further revealed that the RIPK3/2 and RIPK4/2 chi- the EF-1 promoter drives exogenous mRNA transcription. A meras could induce NEMO ubiquitination, albeit at lower levels hygromycin resistance gene (HygR) was Gibson cloned in frame relative to WT RIPK2 (Fig. 2D). Lastly, ubiquitination of NEMO to a C-terminal P2A self-cleaving peptide cassette. Finally, NTAP- was not sufficient for NF-kB activation, as only the RIPK4/2 tagged RIPK2, RIPK3/2, and RIPK4/2 were Gibson cloned in to 4294 UNIQUENESS OF THE RIP KINASE DOMAIN Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

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. the vector in frame with the P2A cassette. The end result is an colonies were pooled. Western blotting showed that although expression vector that is driven by EF-1 (a promoter insensitive to RIPK4/2 was initially expressed at a slightly lower level (Fig. 3B), NF-kB activity) and generates a single mRNA containing the upon stronger hygromycin selection, levels of the exogenous pro- resistance gene and our gene of interest. Upon translation, the teins normalized (Fig. 3C, 3D). Signaling experiments were single mRNA product is generated as two individual proteins performed. Although RIPK2 expression could rescue NOD2- (schematic shown in Fig. 3A). Although the RIPK4/2 chimeric dependent signaling in the RIPK22/2 macrophages, expression of protein can both tyrosine autophosphorylate and activate NF-kB, empty vector (Fig. 3C), RIPK3/2, or RIPK4/2 could not (Fig. 3D), the RIPK3/2 protein can perform neither of these functions and suggesting that despite the biochemical similarities between was therefore used as an additional negative control. Lentivirus RIPK2 and RIPK4/2, RIPK4’s kinase domain could not replace was produced and used to infect RIPK22/2 macrophages. Postin- RIPK2 in NOD2 signaling. To then further determine the extent of fection, cells were selected in hygromycin for 2 wk before .10,000 the signaling defect in a manner more quantifiable, NOD2-driven The Journal of Immunology 4295 Downloaded from http://www.jimmunol.org/

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 C D cell colonies were pooled. Streptavidin bead isolation and Western blotting showed that the stable cell lines expressed the gene of interest. ( and ) The by guest on September 26, 2021 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. gene expression was studied. Our laboratory has previously used differences that may help direct medicinal chemistry toward NextGen sequencing technologies to identify the NOD2-driven specific inhibitors, these three kinases overlap significantly in a genes most sensitive to RIPK2’s kinase activity (12, 25). We three-dimensional structural context, potentially making such used these genes as readouts for gene expression. In all cases, only efforts futile (18). Additionally, work presented in this manuscript RIPK2 expression could rescue NOD2-driven gene expression. shows that they have overlapping molecular and biochemical This was true for IL-6 (Fig. 4A), CXCL10 (Fig. 4B), IRG-1 activities. RIPK1, RIPK2, and RIPK4 all induce NEMO ubiq- (Fig. 4C), and Gpr84 (Fig. 4D). Together, these data suggest uitination and subsequent NF-kB activation. Despite this, only that despite molecular and biochemical similarities between RIPK2 autophosphorylates on tyrosine and is the only RIPK RIPK2 and RIPK4’s kinase domains, RIPK2’s kinase domain proven to be a dual-specificity kinase. Given the interest in functions uniquely, a key feature if one hopes to pharmaceutically pharmacologically targeting a family of kinases with both similar target RIPK2 for clinical gain. and divergent molecular activities (10–19), it was important to determine the functional redundancy of the kinase domain be- Discussion tween the RIPK family members. To this end, domain-swapping Despite their homology and familial grouping, the RIPKs partic- synthetic biology approaches were used. In this context, the only ipate in varied biologic functions. RIPK1 and RIPK3 are important kinase domain that could replicate RIPK2 kinase domain function in dictating the mechanism of cell death in response to a variety of in an in vitro system was the RIPK4 kinase domain. Substituting innate immune and inflammatory signaling (1, 2, 33). RIPK2 is RIPK4’s kinase domain for RIPK2’s allowed NOD2 binding, critically required for NOD1/2 signaling in response to intracel- tyrosine autophosphorylation, NEMO ubiquitination, and NF-kB lular bacterial exposure (34), and RIPK4 is required for proper activation, all key molecular events in which RIPK2 is required development (9). Despite this, recent structural work has shown downstream of NOD2 activation. Surprisingly, despite the mo- that a number of broad-spectrum kinase inhibitors target RIPK1, lecular similarities between the two kinase domains, the RIPK4 RIPK2, and RIPK3 with similar potency (16–18). This same kinase domain could not substitute for RIPK2 in an endogenous structural work has shown that although there are subtle structural setting. It could not support NOD2-induced signaling in RIPK22/2 4296 UNIQUENESS OF THE RIP KINASE DOMAIN Downloaded from

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- http://www.jimmunol.org/ length RIPK2 allowed NOD2-driven gene expression of IL-6 (A), CXCL10 (B), IRG-1 (C), and Gpr84 (D). Mu, macrophage. macrophages and could not replace RIPK2’s role in driving dependent on its kinase domain but not its kinase activity, and this NOD2-induced gene expression. These findings suggest that scaffolding activity cannot be replaced even by RIPK2’s closest RIPK2’s kinase domain is uniquely required for NOD2 signaling homolog. and cannot be replaced by even its closest homologs, implying Another interesting finding in this study centers on tyrosine that unique pharmacologic targeting of the RIPK family members phosphorylation. RIPK2 is known to be a dual-specificity kinase is readily achievable. (11), but work in this manuscript shows that this feature is not by guest on September 26, 2021 RIPK2’s role in innate immune signaling has largely centered shared by the other RIPK family members. Given this, it is sur- on its scaffolding function. RIPK2 clearly helps nucleate signaling prising that RIPK4 is able to autophosphorylate on tyrosine when complexes to transduce signals from NOD1 and NOD2, and ge- its C-terminal Ankyrin repeats are replaced by RIPK2’s inter- netic loss of RIPK2 does not allow signaling through the NOD1 mediate and CARDs. Although native RIPK4 cannot auto- and NOD2 receptors (5, 33, 34). The fact that overexpression phosphorylate on tyrosine residues, the RIPK4/2 chimera can of kinase-dead RIPK2 could activate NF-kB suggested that the autophosphorylate on tyrosines, and this activity matches RIPK2’s kinase domain might be dispensable for RIPK2’s major known tyrosine kinase activity. This surprising result suggests that function (6, 21, 22). Despite this, recent work uncovering specific RIPK4’s kinase domain has the intrinsic ability to be a dual- inhibitors of RIPK2 suggested that although initial and acute specificity kinase; however, its ability to phosphorylate on tyro- NF-kB signaling did not require kinase activity, optimal NOD- sine is substrate-restricted rather than kinase activity restricted. stimulated cytokine and gene expression absolutely require it To our knowledge, this substrate-driven dual-specificity kinase (11, 19). This finding is supported by our prior study using activity is unique and has broader implications for the kinase NextGen RNAseq methods showing that a significant subset of field as a whole, suggesting that phosphoacceptor preferences NOD2-induced genes require RIPK2’s kinase activity for optimal can be altered by substrate selection rather than by intrinsic ki- expression (12, 25). A key question that remains centers on the nase structure. scaffolding function of RIPK2’s kinase domain versus its actual Thus, in addition to categorizing and comparing the RIPKs to kinase activity. To answer this question, we used domain- one another in terms of their ability to activate NF-kB and perform swapping experiments in which we replaced RIPK2’s kinase do- NEMO ubiquitination, this study illustrates two key features of the main with its closest structural homologs (RIPK4 and RIPK3). RIPK family. First, RIPK2’s kinase domain is uniquely structured Surprisingly, we found that despite the fact that a RIPK4/2 chi- in such a way as to nucleate signaling complexes independent of mera could largely replace RIPK2’s function in overexpression its kinase activity. For this reason, its closest homologous kinase systems, it could not replace RIPK2’s function in more endog- domain, RIPK4, cannot replace it structurally despite having enous, acute signaling experiments. This finding is surprising similar kinase activity. Secondly, RIPK4 has substrate-restricted because pharmacologic experiments have shown that although dual-specificity kinase activity that can be induced by physically RIPK2’s kinase activity is required for optimal gene expression, it fusing the substrate to its kinase domain. In the context of sub- is largely dispensable for acute NF-kB signaling (19). This ex- sequent pharmacologic targeting of this family, the work suggests periment shows that there must be structural elements of the that not only might a small molecule exclusively target RIPK2, but RIPK2 kinase domain independent of its kinase activity such that also that by exclusively targeting RIPK2, the function of the other RIPK4 could not replace RIPK2’s role in acute signaling. The RIPKs might not be affected. It also suggests that by developing scaffolding function and acute NF-kB signaling of RIPK2 is type III kinase inhibitors for RIPK2 and RIPK4, one might be able The Journal of Immunology 4297 to identify substrate-specific inhibitors and limit substrate phos- 15. Fayaz, S. M., and G. K. Rajanikant. 2015. Ensembling and filtering: an effective and rapid in silico multitarget drug-design strategy to identify RIPK1 and RIPK3 phorylation rather than eliminate all RIPK2 or RIPK4 phosphor- inhibitors. J. Mol. Model. 21: 314. doi:10.1007/s00894-015-2855-2. ylation. 16. Charnley, A. K., M. A. Convery, A. Lakdawala Shah, E. Jones, P. Hardwicke, A. Bridges, M. Ouellette, R. Totoritis, B. Schwartz, B. W. King, et al. 2015. Crystal structures of human RIP2 kinase catalytic domain complexed with ATP- Acknowledgments competitive inhibitors: Foundations for understanding inhibitor selectivity. We thank Drs. George Dubyak, Tsan Xiao, and Parameswaran Ramakrishnan Bioorg. Med. Chem. 23: 7000–7006. 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