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 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- 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, 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 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 (HA)-ubiquitin, generated as previously described (7, 26). cDNA expres- Despite the homology within the kinase domains, the RIPKs sion constructs for RIPK1 and RIPK3 were obtained from Vishva Dixit show differential molecular abilities (Genentech), and a cDNA expression