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A critical role of RICK/RIP2 polyubiquitination in Nod‐induced NF‐κB activation

A critical role of RICK/RIP2 polyubiquitination in Nod‐induced NF‐κB activation The EMBO Journal (2008) 27, 373–383 & 2008 European Molecular Biology Organization All Rights Reserved 0261-4189/08 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-jB activation 1 2 immune diseases (Inohara et al, 2005; Fritz et al, 2006). Two Mizuho Hasegawa , Yukari Fujimoto , 1 3 NLR family members, Nod1 and Nod2, play an essential role Peter C Lucas , Hiroyasu Nakano , 2 1,4 in the recognition of specific bacterial peptidoglycan (PGN)- Koichi Fukase , Gabriel Nu´ n˜ ez 1,5, related molecules (Chamaillard et al, 2003; Inohara et al, and Naohiro Inohara * 2003; Girardin et al, 2003a, b). Nod1 and Nod2 mediate Department of Pathology, The University of Michigan Medical School, transcriptional activation of innate immune genes including Ann Arbor, MI, USA, Department of Chemistry, Graduate School of antimicrobial peptides, proinflammatory cytokines and che- Science, Osaka University, Toyonaka, Osaka, Japan, Department of mokines, that recruit immune cells to the sites of microbial Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan, Comprehensive Cancer Center, The University of stimulation (Chamaillard et al, 2003; Kobayashi et al, 2005; Michigan Medical School, Ann Arbor, MI, USA and Department of Boughan et al, 2006; Marks et al, 2006; Masumoto et al, 2006; Biochemistry 2nd, Interdisciplinary Graduate School of Medicine Voss et al, 2006). Although the precise structure of the and Engineering, University of Yamanashi, Chuou, Yamanashi, Japan bacterial molecules that stimulate NLR signaling remains largely unknown, the core recognition moieties of Nod1 Nod1 and Nod2 are intracellular proteins that are involved and Nod2 have been identified as g-glutamyl-meso-diamino- in host recognition of specific bacterial molecules and are pimelic acid (iE-DAP), and muramyl dipeptide (MDP), re- genetically associated with several inflammatory diseases. spectively (Chamaillard et al, 2003; Girardin et al, 2003a, b; Nod1 and Nod2 stimulation activates NF-jB through Inohara et al, 2003). Previous studies using synthetic and RICK, a caspase-recruitment domain-containing kinase. natural PGN-related molecules containing MDP and/or iE- However, the mechanism by which RICK activates NF-jB DAP revealed that these molecules induce/enhance innate in response to Nod1 and Nod2 stimulation is unknown. and adaptive immune responses (Goto and Aoki, 1987; Fritz Here we show that RICK is conjugated with lysine- et al, 2007). 63-linked polyubiquitin chains at lysine 209 (K209) located The induction of immune response genes through Nod1 in its kinase domain upon Nod1 or Nod2 stimulation and and Nod2 signaling largely depends on NF-kB, a proinflam- by induced oligomerization of RICK. Polyubiquitination of matory transcriptional factor (Inohara et al, 1999, 2000; RICK at K209 was essential for RICK-mediated IKK activa- Ogura et al, 2001; Masumoto et al, 2006). Transcriptional tion and cytokine/chemokine secretion. However, RICK activation of proinflammatory genes is initiated by nuclear polyubiquitination did not require the kinase activity of translocation of NF-kB following its release from the IkB RICK or alter the interaction of RICK with NEMO, a proteins (Karin and Ben-Neriah, 2000; Hoffmann and regulatory subunit of IjB kinase (IKK). Instead, polyubi- Baltimore, 2006). Previous studies demonstrated that RICK quitination of RICK was found to mediate the recruit- (RIP2/CARDIAK/RIPK2) is a critical downstream mediator of ment of TAK1, a kinase that was found to be essential Nod1 and Nod2 signaling (Inohara et al, 1999; Ogura et al, for Nod1-induced signaling. Thus, RICK polyubiquiti- 2001; Chin et al, 2002; Kobayashi et al, 2002; Park et al, nation links TAK1 to IKK complexes, a critical step in 2007). RICK is composed of N-terminal kinase and C-terminal Nod1/Nod2-mediated NF-jB activation. caspase-recruitment domains (CARD) linked via an inter- The EMBO Journal (2008) 27, 373–383. doi:10.1038/ mediate (IM) region (Inohara et al, 1998; McCarthy et al, sj.emboj.7601962; Published online 13 December 2007 1998; Thome et al, 1998). RICK physically associates with Subject Categories: signal transduction; immunology Nod1 and Nod2 through CARD–CARD interaction and acti- Keywords: NLR; Nod1; Nod2; RICK; TAK1 vates IkB kinase (IKK), leading to phosphorylation and degradation of IkBs (Inohara et al, 1999, 2000; Ogura et al, 2001). Functional studies revealed that close proximity of RICK molecules by self-oligomerization of Nod1 and Nod2 is important for NF-kB activation (Inohara et al, 2000). RICK is Introduction known to interact with NEMO (IKKg/IKBKG) and this inter- Members of the nucleotide-binding domain-like receptor action is essential for NF-kB activation in RICK-mediated (NLR) protein family are found in both mammals and plants signaling (Inohara et al, 2000). Furthermore, Nod2 stimula- (Inohara et al, 2005; Fritz et al, 2006). Several mammalian tion with MDP induces NEMO polyubiquitination and NLRs, like plant NLRs, are involved in host resistance against this event is required for optimal NF-kB activation (Abbott microbial pathogens and genetically associated with human et al, 2004). However, the molecular mechanism by which the IKK complex is activated via RICK to induce NF-kB *Corresponding author. Department of Pathology, Comprehensive activation in response to NLR stimulation is unknown. Cancer Center, University of Michigan Medical School, 1500 E. Medical Center Dr., C574 MSRB2, Ann Arbor, MI 48109, USA. Here we show that RICK is conjugated with K63-linked Tel.: þ 1 734 936 3317; Fax: þ 1 734 647 9654; polyubiquitin chains upon Nod1 and Nod2 stimulation. E-mail: [email protected] RICK polyubiquitination was essential for NF-kB activation and chemokine/cytokine secretion in response to Nod1 sti- Received: 26 September 2007; accepted: 23 November 2007; published online: 13 December 2007 mulation. Furthermore, we provide evidence for a bridging &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 2 2008 373 | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al function of RICK ubiquitination by linking TAK1 to the IKK KF1B or MDP, respectively. Immunoblotting analysis revealed complex that is critical for NF-kB activation during Nod that RICK was polyubiquitinated after KF1B and MDP stimu- signaling. lation (Figure 2A). RICK polyubiquitination was detected 1 h after stimulation with Nod1 and Nod2 agonists. The poly- ubiquitination of RICK by Nod1 and Nod2 stimulation was specific in that it was not detected in parental HEK293 cells Results stimulated with TNFa (Figure 2A). The ubiquitination of Nod1 and Nod2 stimulation induce lysine 63-linked RICK is not due to overexpression of proteins because we polyubiquitination of RICK also found significant ubiquitination of expressed RICK In order to determine the mechanism by which Nod1 stimu- whose levels were similar to those of endogenous RICK in lation induces NF-kB activation, we used mouse embryonic MEFs (see below, Figures 5D and 7D). To determine the fibroblasts (MEFs) because we previously found that non- linkage type of polyubiquitin chains on RICK, Nod1- and immune cells are much more sensitive to Nod1-stimulatory Nod2-expressing cells were transfected with RICK-Myc molecules than hematopoietic cells (Hasegawa et al, 2006; and HA-tagged wild-type (WT) or lysine-mutant Ub proteins Masumoto et al, 2006). Stimulation of MEFs with KF1B, a containing single lysine mutations at position 48 (K48R) or 63 synthetic specific Nod1-stimulatory compound, induced se- (K63R). Upon Nod1 and Nod2 stimulation with KF1B and cretion of the chemokine CCL2 (Figure 1A) and phosphoryla- MDP, respectively, RICK was strongly polyubiquitinated with tion of IkBa (Figure 1B). Such responses were dependent on K48R Ub, but much less with K63R Ub (Figure 2A). Thus, the RICK in that the induction of CCL2 production induced by polyubiquitin chains on RICK were linked primarily through KF1B, but not TNFa, was abolished in RICK-deficient MEFs lysine 63 of ubiquitin. (Figure 1A). Previous studies showed that enforced oligomerization of Recently, ubiquitination of RIP, a mediator of TNFa signal- RICK by Nod1 and Nod2 induces NF-kB activation (Inohara ing, was found to be important for IKK activation (Ea et al, et al, 2000). Similarly, oligomerization of RICK-DCARD-Fpk3, 2006; Li et al, 2006). Because our previous studies suggest a RICK fusion protein in which the CARD is replaced with functional and structural similarities between RIP and RICK, three tandem FKBP-related dimerization domains, results in we first hypothesized that Nod1 stimulation might result in NF-kB activation that is dependent on the FKBP-specific ubiquitination of RICK. As shown in Figure 1, RICK from dimerizer AP1510 (Inohara et al, 2000; Figure 2B). Using MEFs stimulated with KF1B co-immunoprecipitated with this approach, we tested if oligomerization of RICK-DCARD- high-molecular-weight ubiquitinated proteins (Figure 1C, Fpk3 results in RICK polyubiquitination in HEK293Tcells that upper panel). To further assess a role of ubiquitination in coexpress HA-Ub. Immunoblotting analysis revealed that Nod1 and Nod2 signaling, we tested if RICK is ubiquitinated RICK-DCARD-Fpk3 was polyubiquitinated in an AP1510-de- upon Nod1 and Nod2 stimulation, using human embryonic pendent manner whereas the control Fpk3 protein was not kidney (HEK) 293 cells constitutively expressing Nod1 and (Figure 2B). These results suggest that induced proximity of Nod2. Nod1- and Nod2-expressing HEK293 cells were trans- RICK (1–435) lacking the CARD domain mimics the inter- fected with plasmids producing Myc-RICK and HA-Ub and action between RICK and Nod proteins and induces RICK stimulated with specific synthetic Nod1 and Nod2 agonists, signaling. The kinase domain of RICK is essential for RICK ubiquitination To determine which region of RICK is required for polyubi- quitination, we constructed RICK-DCARD-Fpk3-mutant pro- teins lacking various regions of RICK and tested their ability to be ubiquitinated in the absence and presence of the dimerizer AP1510 (Figure 3A). Immunoblotting analysis showed that all RICK-DCARD-Fpk3 proteins containing resi- dues 1–292 of RICK were ubiquitinated in an AP1510-depen- dent manner, whereas that encompassing residues 293–435 was not (Figure 3B). These results indicate that the region Figure 1 Nod1 stimulation induces ubiquitination of RICK. (A) composed of amino-acid residues 1–292 located within the MEFs from WT and RICK-deficient mice were stimulated with kinase region of RICK contains the polyubiquitinated site(s). 5 mg/ml KF1B (synthetic Nod1-stimulatory compound), 10 ng/ml To determine the role of RICK polyubiquitination in NF-kB TNFa or left alone. Twenty-four hours post-stimulation, the levels of activation, the ability of RICK fusion proteins to activate CCL2 in culture supernatant were determined by ELISA. The results shown are given as mean7s.d. of triplicate cultures and are NF-kB was tested using a reporter assay. Transfection of representative of three experiments. (B) WT or RICK-deficient HEK293T cells with the different constructs revealed that all MEFs were stimulated with 5 mg/ml KF1B for the indicated times. RICK proteins carrying the region between amino acid 1 and Post-stimulation, cells were lysed and immunoblotted with anti- 319 induced NF-kB activation in an AP1510-dependent man- phospho-IkBa, anti-IkBa or IKKb Ab. (C) MEFs were stimulated with or without KF1B for the indicated times. Post-stimulation, cells ner (Figure 3C). In contrast, enforced oligomerization of were lysed and RICK was immunoprecipitated with rabbit anti-RICK the fusion proteins carrying the region between 1 and 292 Ab. Ubiquitinated (upper panel) and total (lower panel). RICK and that between 293 and 435 did not result in NF-kB proteins in the immunoprecipitates were immunodetected by activation (Figure 3C). These results indicate that both mouse monoclonal anti-RICK and anti-Ub Abs, respectively. Nonspecific signals detected with the Ab are indicated by asterisks. the kinase domain (residues 1–292), which contains the 374 The EMBO Journal VOL 27 NO 2 2008 &2008 European Molecular Biology Organization | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al Figure 2 Nod1 and Nod2 stimulation induce K63-linked polyubiquitination of RICK. (A) HEK293 cells constitutively expressing Nod1 (for KF1B stimulation), Nod2 (for MDP stimulation) and parental HEK293 cells (for TNFa stimulation) were transfected with expression plasmid of Myc-RICK in the presence of expression plasmids of HA-tagged WT, K48R, K63R mutant Ub proteins. Cells were stimulated with 100 ng/ml KF1B, 100 ng/ml MDP (synthetic Nod2-stimulatory compound) or 10 ng/ml TNFa, or left alone (0 h control) for the indicated times. Myc-RICK proteins in transfected cells were immunoprecipitated with rabbit anti-Myc Ab and ubiquitinated RICK proteins were then detected by immunoblotting with anti-HA Ab (upper panels labeled as RICK-Ubn). As a control, RICK proteins in the same samples and Ub proteins in total lysate were detected by immunoblotting with the indicated Abs. (B) Oligomerization-dependent RICK ubiquitination. HEK293Tcells were transfected with control pcDNA3-RICK-Fpk3-Myc () or pcDNA3-RICK-Fpk3-Myc (RICK) in the presence of HA-Ub expression plasmids. Eighteen hours post-transfection, cells were treated with or without 200 nM AP1510. Twenty-four hours post-transfection, proteins were immunoprecipitated and ubiquitinated RICK was analyzed as described in panel A. polyubiquitinated site(s), and the IM region (residues 293– ATP-binding site and results in loss of RICK kinase activity 319) are critical for NF-kB activation. (Inohara et al, 1998), did not impair RICK polyubiquitination Previous studies showed that NF-kB activation mediated (Figure 4B). Thus, the conserved K209, but not the kinase by RICK requires NEMO (Inohara et al, 2000). Therefore, we activity of RICK, is essential for polyubiquitination. To deter- hypothesized that at least one of the regions required for NF- mine if K209 is responsible for polyubiquitination of RICK kB activation interacts with NEMO. To explore this hypoth- during Nod1 signaling, we introduced the K209R mutation esis, we tested the ability of RICK-Fpk3-Myc constructs into a full-length RICK construct and tested the ability of WT carrying different regions of RICK to interact with NEMO and K209R-mutant RICK proteins to be ubiquitinated in by co-immunoprecipitation assay. Consistent with a previous Nod1-expressing HEK293 cells upon Nod1 stimulation with study (Inohara et al, 2000), a RICK truncation mutant KF1B. Consistent with the results presented in Figure 4B, the expressing a region between amino-acid residues 1–319 K209R mutation abolished KF1B-induced polyubiquitination interacted with NEMO (Figure 3D). Significantly, a RICK of RICK (Figure 4C). These results indicate K209 is essential mutant composed of residues 1–292 did not associate with for RICK polyubiquitination in Nod1 signaling. NEMO (Figure 3D). These results indicate that the interaction between RICK and NEMO requires the IM region-spanning residues 293–319 of RICK. Polyubiquitination of RICK is essential for Nod1 and Nod2-mediated NF-jB activation Lysine 209 in the kinase domain of RICK is To test if polyubiquitination at K209 of RICK is required for polyubiquitinated in a signal-dependent manner NF-kB activation, we assessed the ability of lysine-mutant To map the polyubiquitinated lysine residue(s) in the kinase RICK-DCARD-Fpk3 proteins to activate NF-kB after stimula- domain of RICK, we first performed close inspection of the tion with different doses of AP1510. All the point mutant amino-acid sequence of RICK. Alignment of amino-acid RICK proteins were expressed at levels comparable with that sequences from the kinase domain and IM region of RICK of WT protein, except the kinase-dead K47M mutant that was revealed 10 lysine residues that are evolutionarily conserved expressed at significantly lower levels (Figure 5A, inset). in 11 animal species (Supplementary Figure 1). Therefore, we Consistent with its lower expression, the K47M mutant introduced site-directed mutations in the 10 absolutely con- showed reduced levels of NF-kB activation when compared served and seven additional highly conserved lysine residues with WT protein (Figure 5A). However, the K47 mutant did and tested if the mutations affect the ability of RICK-DCARD- show a dose-dependent increase in NF-kB and since K47 is Fpk3 to be ubiquitinated in an AP1510-dependent manner essential for the kinase activity of RICK (Inohara et al, 1998), (Figure 4A). As shown in Figure 4B, the point mutant K209R, we conclude that the kinase activity of RICK is required for but not the remaining 16 lysine mutations, abolished the neither NF-kB activation nor polyubiquitination. Notably, the polyubiquitination of RICK. Notably, the K47M mutation, K209R mutation, but not those of the remaining conserved which replaces the essential lysine residue in the conserved lysines, abolished AP1510-dependent NF-kB activation &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 2 2008 375 | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al Figure 3 The kinase domain of RICK is essential for RICK ubiquitination, but is not sufficient to activate NF-kB. (A) Schematic diagram of the structural domains of RICK and different deletion mutants. The results of ubiquitination and NF-kB activation experiments are summarized on the right. The region, which requires NF-kB activation in the IM domain is indicated by a gray box. (B) HEK293T cells were transfected with pcDNA3-RICK-(1–435)-Fpk3-Myc (DC), RICK-(1–292)-Fpk3-Myc, RICK-(1–319)-Fpk3-Myc, RICK-(1–328)-Fpk3-Myc, RICK-(1–356)-Fpk3-Myc, RICK-(1–390)-Fpk3-Myc RICK-(293–435)-Fpk3-Myc and control vector in the presence of HA-Ub expression plasmid and treated with AP1510 as in Figure 2B. Twenty-four hours post-transfection, proteins were immunoprecipitated and ubiquitinated RICK were analyzed as described in Figure 2. (C) HEK293T cells were transfected with pcDNA3-RICK-(1–435)-Fpk3-Myc (DC), RICK-(1–292)-Fpk3-Myc, RICK-(1–319)-Fpk3-Myc, RICK-(1–328)-Fpk3-Myc, RICK-(1–356)-Fpk3-Myc, RICK-(1–390)-Fpk3-Myc RICK-(293–435)-Fpk3-Myc and control vector in the presence of reporter plasmids. Eight hours post-transfection, cells were treated with medium containing the indicated amounts of AP1510. Twenty-four hours post-transfection, ligand-dependent NF-kB activation was determined with reporter assay. The level of NF-kB-dependent transcription activity in cells transfected with control vector is given as 1. The expression levels of Myc-RICK proteins are shown in an inset. (D) HEK293T cells were transfected with control vector (), pcDNA3-RICK-(1–435)-Fpk3-Myc, RICK-(1–319)-Fpk3-Myc and RICK-(1–292)-Fpk3-Myc in the presence of reporter plasmids, and treated with AP1510 as in Figure 2B. Twenty-four hours post-transfection, RICK proteins were immunoprecipitated with rabbit polyclonal anti-Myc Ab, and then Flag-NEMO (upper panel) and Myc-RICK (middle panel) were immuno- detected with mouse monoclonal anti-Flag and anti-Myc Abs, respectively. As control, Flag-NEMO in total lysate was immunodetected with anti-Flag Ab and shown in the lower panel. Figure 4 Lysine 209 is ubiquitinated in the kinase domain of RICK. (A) Location of lysine residues, which were replaced by arginines in RICK site-directed mutants. (B) HEK293T cells were transfected with control vector (), pcDNA3-RICK-DCARD-Fpk3-Myc (WT) and various lysine- to-arginine mutants in the presence HA-Ub expression plasmid and treated with AP1510 as in Figure 2B. Twenty-four hours post-transfection, proteins were immunoprecipitated and ubiquitinated RICK were immunodetected as described in Figure 1. (C) HEK293 cells constitutively expressing Nod1 were transfected with the expression plasmids of WT and K209R-mutant Myc-RICK in the presence of HA-Ub expression plasmid. Eighteen hours post-transfection, cells were stimulated with or without 100 ng/ml KF1B. Twenty-four hours post-transfection, proteins were immunoprecipitated and ubiquitinated RICK were immunodetected as described in Figure 1. 376 The EMBO Journal VOL 27 NO 2 2008 &2008 European Molecular Biology Organization | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al Figure 5 Polyubiqutination at K209 is essential for RICK-mediated signaling. (A) HEK293T cells were transfected with pcDNA3-RICK-DCARD- Fpk3-Myc (WT) or the mutants in the presence of reporter plasmids. Eight hours post-transfection, cells were treated with medium containing the indicated amount of AP1510. Twenty-four hours post-transfection, ligand-dependent NF-kB activation was determined with reporter assay. The level of NF-kB-dependent transcription activity in cells transfected with control vector is given as 1. Myc-tagged RICK proteins detected with anti-Myc Ab are shown in the inset. (B) RICK-deficient MEFs were infected with retrovirus vectors expressing WT or K209R HA-RICK or control vector and selected by hygromycine. RICK proteins detected with anti-RICK Ab. A nonspecific signal detected with the Ab is indicated by an asterisk. (C) RICK-deficient MEFs were infected with control () or retrovirus vectors expressing WT or K209R HA-RICK. Infected MEFs were selected by hygromycin and stimulated with 5 mg/ml KF1B, 10 ng/ml TNFa or left alone. Twelve hours post-stimulation, secretion levels of CCL2 and CXCL1 were determined by ELISA. The results shown are given as mean7s.d. of triplicate cultures and are representative of three experiments. (D) MEFs stably expressing RICK or RICK K209R were stimulated with 5 mg/ml KF1B for the indicated time. Post-stimulation, cells were lysed, RICK was immunoprecipitated and ubiquitinated RICK was immunodetected by anti-Ub Ab (upper panel). Cell lysate was also immunoblotted with anti-phospho-IkBa, anti-IkBa or IKKb Ab (bottom three panels). A nonspecific signal detected with the Ab is indicated by an asterisk. (E) MEFs stably expressing RICK or RICK K209R were stimulated with 5 mg/ml KF1B or TNFa for indicated times, and then anti- NEMO Ab was used to immunoprecipitate the IKK complex. The IKK assay was carried out by using GST-IkBa and g- P-ATP as substrates (upper). The amount of IKKb in the immunoprecipitates was detected by immunoblotting. (Figure 5A). These results indicate that polyubiquitination of A20 inhibits Nod1- and Nod2-mediated NF-jB activation RICK at K209 is essential for NF-kB activation. and polyubiquitination of RICK To test if K209 polyubiquitination on RICK is important for A20 is a deubiquitinase that removes K63-linked polyubiqui- Nod1 signaling, we assessed the ability of WT and K209R- tin chains and functions as a negative feedback regulator of mutant RICK proteins to complement RICK function in em- inflammatory responses (Lee et al, 2000; Boone et al, 2004; bryonic fibroblasts deficient in RICK. In these experiments, Wertz et al, 2004). Previous studies showed that A20 expres- MEFs from RICK-deficient mice were infected with a retro- sion is induced by NF-kB activation (Krikos et al, 1992) and viral vector producing WT or K209R-mutant RICK proteins. Nod1 stimulation (Masumoto et al, 2006). To test if A20 The expression levels of both WTand K209R RICK proteins in negatively regulates Nod signaling through deubiquitination RICK MEFs were similar to that of endogenous RICK of RICK, we tested the ability of A20 to affect RICK poly- in WT MEFs (Figure 5B). Notably, expression of WT RICK ubiquitination and NF-kB activation in response to Nod1 or conferred responsiveness of MEFs deficient in RICK to KF1B, Nod2 stimulation. To assess the latter, A20 was coexpressed and resulted in secretion of chemokines CCL2 and CXCL1 with Myc-RICK and HA-ubiquitin in HEK293T cells and the (Figure 5C). In contrast, the K209R RICK mutant failed to level of polyubiquitinated RICK was determined by immuno- rescue responsiveness to the Nod1 agonist (Figure 5C). blotting. We found that expression of A20 decreased RICK Immunoblotting showed that WT but not K209R-mutant polyubiquitination (Figure 6A). HEK293T cells express RICK was polyubiquitinated upon Nod1 stimulation and low, but significant, levels of endogenous Nod1 and Nod2 resulted in IKK activation (Figure 5D and E). Because control (Viala et al, 2004). Consistent with the latter, treatment of TNFa induced similar levels of CCL2 and CXCL1 secretion HEK293T cells with high amount of lipophilic Nod1- and from cells infected with WT and K209R-mutant RICK con- Nod2-stimulatory molecules KF1B (5 mg/ml) and AcMDP structs, these findings indicate that polyubiquitination of (acetyl-(6-O-stearoyl)-muramyl-Ala-D-Glu-NH )(5 mg/ml) in- RICK at K209 is essential for Nod1 signaling but dispensable duced NF-kB activation (Figure 6B). Using this system, we for that induced by TNFa stimulation. tested the ability of A20 to affect the NF-kB activation &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 2 2008 377 | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al Figure 6 A20 functions as a negative regulator of Nod1 and Nod2 signaling via deubiquitination of RICK. (A) HEK293T cells were transfected with Flag-tagged A20 expression plasmids or control vector () in the presence of pcDNA3-RICK-DCARD-Fpk3-Myc (WT) and HA-Ub expression plasmid. Eighteen hours post-transfection, cells were treated with 200 nM AP1510 or left alone. Twenty-four hours post-transfection, proteins immunoprecipitated with rabbit anti-Myc Ab were subjected to SDS–PAGE and analyzed by western blot analysis using anti-HA, Myc and Flag Abs to detect ubiquitinated RICK proteins, RICK proteins, and A20 protein, respectively. (B) HEK293T cells were transfected with expression plasmids of A20 or control vector in the presence of reporter plasmids. Eight post-transfection, cells were treated with medium containing 1 mg/ml KF1B, 1 mg/ml AcMDP, 10 ng/ml TNF or 10 ng/ml IL-1b. Twenty-four hours post-transfection, ligand-dependent NF-kB activation was determined with reporter assay. The level of NF-kB-dependent transcription activity in the absence of A20 is given as 100%. The results shown are given as mean7s.d. of triplicate cultures and are representative of three experiments. (C) HEK293T cells were transfected with pcDNA3 (), A20 interference RNA (RNAi) plasmid or control RNAi plasmid (Ctli) in the presence of reporter plasmids. Thirty-six hours after transfection, cells were treated with medium containing 1 mg/ml KF1B, 1 mg/ml AcMDP, 10 ng/ml TNFa or IL-1b. Forty-eight hours post- transfection, ligand-dependent NF-kB activation was determined with reporter assay. The level of NF-kB-dependent transcription activity in cells transfected with pcDNA3 is given as 1. Expression of A20 and control IKKb proteins is shown in inset. The results shown are given as mean7s.d. of triplicate cultures and are representative of three experiments. (D) HEK293 cells constitutively expressing Nod1 were transfected with pcDNA3 () or A20 RNAi plasmid. Forty-eight hours after transfection, cells were treated with 100 ng/ml KF1B for 1 h. The cells were lysed and endogenous RICK was immunoprecipitated. Ubiquitinated and total RICK proteins were immunodetected with anti-Ub and anti-RICK Abs, respectively. induced by these Nod1- and Nod2-stimulatory molecules in impaired phosphorylation of IkBa (Figure 7B). These results HEK293T cells, and found that NF-kB activation induced by indicate that TAK1 plays an essential role in Nod1 signaling. KF1B and AcMDP, as well as that induced by TNFa and IL-1b, Because K63-linked polyubiquitination of RIP recruits TAK1 was reduced by A20 overexpression (Figure 6B). We next complex to the TNFR1 complex leading to NF-kB activation determined whether endogenous A20 mediates negative feed- (Kanayama et al, 2004), we tested whether RICK polyubiqui- back of NF-kB activation after Nod1 stimulation. To test this, tination at K209 mediates the recruitment of TAK1 complex to we used A20 small-RNA interference (RNAi) construct, RICK. To determine this, Myc-RICK-DCARD-Fpk3 proteins which suppresses specifically the expression of A20 (Saitoh were coexpressed with TAK1, TAB1 and TAB2. We found et al, 2005; also see inset in Figure 6C). Like that induced by that WT Myc-RICK-DCARD-Fpk3 protein co-immunoprecipi- TNFa and IL-1b,NF-kB activation induced by KF1B and tated with TAK1, TAB1 and TAB2 in the presence but not in AcMDP were enhanced by transfection with the A20 RNAi the absence of the dimerizer AP1510 (Figure 7C). These plasmid but not with control plasmid (Figure 6C). In addi- results suggest that polyubiquitinated RICK is associated tion, the ubiquitination level of endogenous RICK induced with the TAK1 complex. To test if polyubiquitination is by Nod1 stimulation was upregulated by expression of A20 required for recruitment of TAK1 complex to RICK, we tested RNAi (Figure 6D). These findings suggest that A20 is a the ability of K209R-mutant construct that is deficient in negative feedback regulator of Nod1 and Nod2 signaling. signal-dependent polyubiquitination, to interact with the TAK1 complex. The analysis revealed that the K209R mutant of RICK did not associate with TAK1 complex when com- Polyubiquitination of RICK is required for the pared with WT RICK (Figure 7C). These results suggest that recruitment of TAK1 polyubiquitination of RICK at K209 is essential for recruit- Recent studies showed that a TAK1 complex containing ment of the TAK1 complex to RICK. To test if the interaction TAK1, TAB1, TAB2 and/or TAB3 is a general mediator of NF-kB activation induced by various inflammatory stimuli of TAK1 with RICK is sufficient to induce NF-kB activation, (Ishitani et al, 2003; Sato et al, 2005; Shim et al, 2005). we assessed if TAK1 is recruited to the RICK (1–292)-mutant construct that is polyubiquitinated upon stimulation but is Because TAK1 has been suggested to play a role in NF-kB unable to activate NF-kB and interact with NEMO (see activation induced by Nod2 signaling (Chen et al, 2004; Figure 3). Notably, RICK (1–292) mutant, but not RICK Windheim et al, 2007), we hypothesized that TAK1 mediates (293–435), associated with the TAK1 complex after addition Nod1-induced innate immune responses. To test this, we examined if KF1B induces the secretion of CCL2 from of AP1510 (Figure 7C). To verify that this finding is not due to TAK1-deficient MEFs. We found that WT MEFs secreted overexpression of recombinant proteins, we further investi- CCL2 in response to KF1B stimulation but TAK1-deficient gated the interactions of TAK1 and NEMO with WT and K209R RICK proteins, which are expressed at levels compar- MEFs did not (Figure 7A). The impaired response in TAK1 able to that of endogenous RICK in RICK MEFs. As expected, MEFs was not due to the lack of expression of the general WT but not K209R-mutant RICK was polyubiquitinated and NF-kB regulators, IKKb and IkBa, but was associated with 378 The EMBO Journal VOL 27 NO 2 2008 &2008 European Molecular Biology Organization | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al Figure 7 RICK polyubiquitination at K209 is required for the recruitment of TAK1/TAB1/TAB2 complex. (A) WTor TAK1-deficient MEFs were 2þ stimulated with 5 mg/ml KF1B, 10 ng/ml TNFa, 10 ng/ml IL-1b, 100 ng/ml LPS, 1 mg/ml sBLP, 50 ng/ml PMA plus 0.7 mg/ml Ca ionophore A23187 or left alone. Twenty-four hours post-stimulation, secretion levels of CCL2 were determined by ELISA. The results shown are given as mean7s.d. of triplicate cultures and are representative of three experiments. (B) WT or TAK1-deficient MEFs were stimulated with 5 mg/ml KF1B, 10 ng/ml TNFa or 10 ng/ml IL-1b for 1 h. Post-stimulation, cells were lysed and lysates immunoblotted with anti-phospho-IkBa, anti-IkBa or IKKb Ab. (C) HEK293T cells were transfected with expression plasmids of RICK-DCARD-Fpk3-Myc (WT), RICK-K209R-DCARD- Fpk3-Myc (K209R), RICK-(1–292)-Fpk3-Myc, RICK-(293–435)-Fpk3-Myc and control vector () in the presence of expression plasmids of Ub, HA-TAK1, Flag-TAB1 and T7-TAB2 and treated with AP1510 as in Figure 2B. Twenty-four hours post-transfection, proteins immunoprecipitated with rabbit anti-Myc Ab were subjected to SDS–PAGE and analyzed by immunoblotting analysis using anti-HA or, anti-Flag, anti-T7 and anti-Ub Abs. (D) MEFs stably expressing RICK or RICK K209R were stimulated with 5 mg/ml KF1B for 1 h. The cells were lysed, RICK was immunoprecipitated and endogenous Ub, TAK1, NEMO and RICK were immunodetected with anti-Ub, anti-TAK1 Ab, anti-NEMO or anti-RICK antibodies. interacted with TAK1 upon Nod1 stimulation, whereas the interaction between RICK and NEMO was constitutive and was not affected by the mutation at K209 (Figure 7D). Together with the findings described in previous figures, this finding suggests that TAK1 recruitment to K209- polyubiquitinated RICK is essential for IKK activation upon Nod1 stimulation. TRAF2/TRAF5 but not TRAF6 are required for Nod1-mediated NF-jB activation and CCL2 secretion Recent findings suggest that TRAFs are ubiquitin ligases (E3s), which regulate several immune responses including Figure 8 TRAF2/TRAF5, but not TRAF6, are required for Nod1- inflammatory responses mediated by NF-kB. Although mediated NF-kB activation and CCL2 secretion. (A) WT, TRAF6 / / TRAF6 is suggested to be important for Nod2-mediated (TRAF6 KO) or TRAF2 TRAF5 (TRAF2/5 DKO) MEFs were signaling, TRAF6-depeleted cells still respond to MDP, a stimulated with 5 mg/ml KF1B, 10 ng/ml TNFa, 10 ng/ml IL-1b or left alone. After 24 h incubation, the secretion levels of CCL2 were Nod2-stimulatory molecule (Abbott et al, 2007). We also determined by ELISA. The results shown are given as mean7s.d. of found that TRAF6 MEFs respond to KF1B, resulting in triplicate cultures and are representative of three experiments. (B) IkBa phosphorylation (Figure 8B) and induced secretion of WT and mutant MEFs were stimulated with 5 mg/ml KF1B for 1 h or CCL2 (Figure 8A). Thus, TRAF6 appears to be a non-essential left alone. The phosphorylated (upper panel) and whole (middle panel) populations of IkBa and IKKb (lower panel) were immuno- E3 for both Nod1 and Nod2 signaling. Notably, Nod1 stimu- detected with specific Abs. lation did not induce NF-kB activation and IkBa phosphor- ylation in MEFs lacking TRAF2 and TRAF5, which are essential for TNFa-induced NF-kB activation and are able to Discussion complement one another (Tada et al, 2001; Figure 8). These results suggest that TNFa- and Nod1-mediated signaling RICK is an essential and specific mediator of Nod1 and Nod2 share common E3s to activate NF-kB. signaling (Chin et al, 2002; Kobayashi et al, 2002; Park et al, &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 2 2008 379 | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al Figure 9 Model for the role of RICK polyubiquitination in Nod signaling. For detail, see text. 2007). However, the mechanism by which RICK mediates associate with RICK (McCarthy et al, 1998; Thome et al, / / Nod1- and Nod2-mediated immune responses has remained 1998). Here we demonstrated that TRAF2 TRAF5 MEFs poorly defined. Here we found an essential role for RICK lack NF-kB activation upon Nod1 stimulation. However, we polyubiquitination in Nod1- and Nod2-mediated NF-kB acti- do not have convincing evidence that TRAF2 and TRAF5 vation. The K63-linked polyubiquitin chain at K209 in RICK directly ubiquitinate RICK. We could not find consensus was found to be important for the recruitment of the TAK1 sequences of TRAF2- and TRAF5-binding sites in regions complex to RICK and IKKs. Similarly, RIP (RIPK1), a RICK near K209 in RICK. Although we still do not know how the homologue, was shown to play a critical role in TNFa- E3(s) specifically recognize RICK as a substrate, K209 is induced NF-kB activation through its polyubiquitination at highly conserved in RICK but not in other kinases and is K377 located in the IM region between the N-terminal kinase located in the putative flexible loop region (Supplementary and C-terminal death domains (Ea et al, 2006; Li et al, 2006). Figure 1). Further studies are required to determine the Like that of RICK, polyubiquitinations of RIP are both critical mechanism by which TRAFs are involved in Nod/RICK- for recruitment of TAK1 complex. Therefore, our results mediated signaling, and conclusively identify the E3(s), indicate that Nod1/Nod2 and TNFa signaling is mediated which ubiquitinates K209 in RICK. through similar molecular events. In summary, we have dissected two molecular signaling Previous studies suggested an important role for the IM events that are critical for Nod signaling, namely recruitment domain located between the kinase domain and the CARD of of TAK1 to polyubiquitinated K209 located in the kinase RICK in Nod1-mediated NF-kB activation (Inohara et al, domain, and recruitment of NEMO, which is mediated by 2000). Here we further characterized the structural require- the IM region of RICK. NEMO interacted with RICK-DCARD- ment of IM and found that the region between 293 and 319, Fpk3 in the absence of AP1510, which is required for poly- near to the N-terminal kinase domain, is important for NF-kB ubiquitination of K209 and concomitant NF-kB activation activation. This is also comparable to the observation that the (Figure 3D). As a result, the interaction between RICK and IM region of RIP is required for TNF signaling (Inohara et al, NEMO through the IM region appears to be independent of 2000). However, oligomerization of RICK-Fpk3 fusion protein the interaction between TAK1 and RICK and is not sufficient carrying the IM region alone did not result in NF-kB activa- to activate NF-kB. Therefore, the association of RICK with tion (Figure 3C), whereas that of the equivalent region of RIP both TAK1 and IKK complexes are required for signal trans- was sufficient to induce oligomerization-dependent NF-kB duction resulting in NF-kB activation. This conclusion leads activation (Inohara et al, 2000). These results suggest that the us to propose the following model (Figure 9): the molecular kinase domain of RICK, but not that of RIP, is essential for events during Nod1 and Nod2 signaling are (1) the recruit- NF-kB activation. Previous studies showed that the K47M ment of RICK to Nod1 and Nod2 upon sensing of their kinase-inactive mutant of RICK still activates NF-kB (Inohara specific microbial stimuli, (2) the close proximity of RICK et al, 2000), suggesting that the kinase domain of RICK induced by the self-oligomerization of Nod1 and Nod2, (3) mediates a critical function, other than phosphorylation, polyubiquitination at K209 in RICK by unknown E3(s), (4) which is required for the induction of NF-kB. Indeed, we recruitment of the TAK1 complex to RICK, (5) further recruit- found here that K63-linked polyubiquitination at K209 within ment of IKK complex through the interaction between the IM the kinase domain of RICK is essential for RICK-mediated region of RICK with NEMO, and finally (6) activation of IKKs signaling. In contrast, the critical polyubiquitination site of by TAK1. In this model, Nod1 and Nod2 play an important RIP at K377 is located the IM region (Ea et al, 2006; Li et al, role as switches to turn on the downstream cascade reaction 2006). These results indicate that RICK and RIP differ in the and RICK functions as a bridging protein to link TAK1 and domain localization of the critical K63-linked polyubiquitina- IKK complexes. Similarly, RIP may function as a bridging tion site, providing a mechanistic explanation for the ob- protein between TAK1 and IKK complexes in TNFa signaling. served differences between the two related kinases in the However, the K63-linked polyubiquitination site K377 is induction of NF-kB. Although the localization of the K63- located in the IM region and, therefore, previous studies linked polyubiquitinated site is different in RICK and RIP, could not separate the two molecular events mediated by polyubiquitination of both kinases is regulated by upstream RIP, namely the recruitment of TAK1 and that of the IKKs. stimuli and required for recruitment of the critical TAK1 Further studies are required to explore the differences and complex to these kinases. While polyubiquitination of RICK similarities between Nod and TNF signaling pathways. is essential for Nod1-mediated NF-kB activation, the E3s that The kinase domain of RICK possesses protein kinase mediate polyubiquitination of RICK remain unknown. Some activity (Inohara et al, 1998; McCarthy et al, 1998; Thome possible candidates include the TRAFs, which are known to et al, 1998) and residues critical for catalytic activity are 380 The EMBO Journal VOL 27 NO 2 2008 &2008 European Molecular Biology Organization | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al Luciferase assay evolutionary conserved. However, previous and current stu- NF-kB activation was determined using 0.510 HEK293T cells dies showed that the residue critical for kinase activity is not transfected with expression plasmids in the presence of reporter essential for NF-kB activation, polyubiquitination of RICK plasmids, NF-kB-dependent pBxIV-luc and control pEF1BOS-b-gal, and interaction of RICK with NEMO (Inohara et al, 2000). as described (Inohara et al, 2000; Kobayashi et al, 2002). Therefore, the kinase activity of RICK might regulate other molecules including ERKs, p38 kinases and caspase-1 that Cytokine/chemokine secretion WT and RICK-deficient MEFs were infected with retroviral vectors have been linked to RICK-mediated signaling (Thome et al, pMSCV-hygro-HA-RICK and pMSCV-hygro-HA-RICK K209R pack- 1998; Navas et al, 1999; Chin et al, 2002; Kobayashi et al, aged in HEK293T cells cotransfected with EcoPack plasmid (a gift 2002). Previous studies suggested that the activation of these from Dr G Nolan, UCSF). Infection was carried out in the presence intracellular molecules is important for secretion of certain of 5 mg/ml polybrene for 3 h and incubation continued for 48 h with fresh medium. Hygromycin-resistant (hygro ) MEFs were selected cytokines upon Nod1 and Nod2 stimulation (Inohara et al, with 0.3 mg/ml hygromycin (Roche) for 5 days. A total of 110 2001; Girardin et al, 2003a, b; Viala et al, 2004). Alternatively, hygro MEFs were cultured in 0.25 ml medium in 48-well plates and the kinase domain of RICK may regulate the turnover rate of stimulated with inflammatory stimuli indicated in figure legends. the RICK protein. Consistent with the latter, we found lower Twenty-four hours post-stimulation, the secretion levels of CCL2 and CXCL1 in the medium were determined by sandwich ELISA kits but significant residual polyubiquitination of RICK indepen- (BD, San Diego, CA). Similarly, WT and TAK1-deficient MEFs were dent from K63-Ub upon Nod1 and Nod2 stimulation stimulated with the indicated inflammatory stimuli and CCL2 and (Figure 2), suggesting that RICK is partially polyubiquitinated CXCL1 secretion levels were determined 24 h post-stimulation. in a K63-Ub-independent manner. Furthermore, K48-linked polyubiquitination of RIP during TNFa signaling has been Immunoprecipitation analyses and immunodetection suggested to regulate proteasomal degradation of RIP (Wertz HEK293 cells were transfected with expression plasmids or siRNA plasmids and lysed with NP-40 buffer as described (Inohara et al, et al, 2004). Therefore, our finding suggests that K48-linked 1999) The proteins were immunoprecipitated with anti-Myc rabbit polyubiquitination of RICK might regulate RICK protein levels polyclonal (A-14; Santa Cruz) or rabbit anti-RICK (H-300; Santa during Nod signaling. The functional relevance of K63-Ub- Cruz) antibody (Ab). Myc-, HA- or Flag-tagged proteins were independent polyubiquitination of RICK and the role of the immunodetected with horseradish peroxidase-conjugated Abs specific for each tag (A-14 and Y-11 from Santa Cruz for Myc and kinase activity in RICK protein degradation warrant further HA tags, respectively, M2 from Sigma-Aldrich for Flag tag). investigation. A total of 110 MEFs were stimulated with 5 mg/ml KF1B for the indicated time. MEFs were lysed with and RICK proteins were immunoprecipitated by rabbit anti-RICK or rabbit anti-HA Ab (Y-11; Santa Cruz). Ubiquitinated and total RICK proteins, TAK1 or NEMO Materials and methods in the immunoprecipitates were immunodetected by mouse monoclonal anti-Ub (Abcam, Cambridge, MA), anti-RICK (Alexis, Ligand compounds, plasmids and culture cells San Diego, CA), anti-TAK1 (C-9; Santa Cruz) and anti-NEMO Abs Synthetic compounds, iE-DAP and KF1B have been described (B-3; Santa Cruz), respectively. Cell lysates were immunoblotted (Masumoto et al, 2006). MDP, AcMDP, sBLP (Pam Cys-OH) TNFa, with anti-phospho-IkBa, anti-IkBa (Cell Signaling) or IKKb Ab (H- IL-1b, PMA and A23187 were obtained from commercial sources. 470; Santa Cruz). AP1510 is a gift from Ariad (Cambridge, MA). pcDNA3-Fpk3-Myc, pcDNA3-RICK-Fpk3-Myc, RICK-(1–292)-Fpk3-Myc, pcDNA3-RICK- (K47M)-Fpk3-Myc, pcDNA3-Myc-RICK, pCGN-HA-Ub, pBxIV-luc, In vitro kinase assay pcDNA3-b-gal and pMX2-puro-HA-Nod2 have been described 6 A total of 210 MEFs stably expressing RICK or RICK K209R were (Inohara et al, 2000, 2003; Nishito et al, 2006). pCMV-SPORT6- stimulated with 5 mg/ml KF1B or 10 ng/ml TNFa for the indicated UBB encoding human ubiquitin B (IMAGE 4943285) was obtained time, and then cells were lysed in Triton lysis buffer (10 mM HEPES, from Open Biosystems (Huntsville, AL). RICK-(1–319)-Fpk3-Myc, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM RICK-(1–328)-Fpk3-Myc, RICK-(1–356)-Fpk3-Myc, RICK-(1–390)- b-glycerophosphate, 10 mM NaF, 300 mMNa VO and 1 mM DTT). 3 4 Fpk3-Myc and RICK-(293–435)-Fpk3-Myc were generated by sub- The cell lysates were immunoprecipitated with mouse anti-NEMO cloning of RICK amplified by PCR using specific primers and Ab (BD Pharmingen). Resulting immunoprecipitates were washed subcloned into the KpnI and XhoI sites of pcDNA-Fpk3-Myc. three times with lysis buffer and once with kinase buffer (20 mM pMSCV-hygro-HA-RICK and pMSCV-puro-Nod1-Flag are generated HEPES pH 7.6, 2 mM MgCl , 2 mM MnCl ,10 mM ATP, 10 mM 2 2 by subcloning of the HA-RICK and Nod1-Flag genes into pMSCV- b-glycerophosphate, 10 mM NaF, 300 mMNa VO and 1 mM DTT). 3 4 hygro and pMSCV-puro, respectively (Clontech). Point mutations of Immunoprecipitated IKK complexes were incubated with GST-IkBa RICK and Ub, in which individual lysine residues were mutated to as substrates in kinase buffer with g- P-ATP at 301C for 30 min. The TM arginines, were constructed using the QuikChange site-directed samples were fractionated on SDS–PAGE followed by autoradio- mutagenesis kit (Stratagene). The fidelity of all constructs was graphy. The amounts of IKKb in the immunoprecipitates were confirmed by sequencing. pcDNA3-HA-TAK1, pcDNA3-T7-TAB1 detected by immunoblotting. and pcDNA3-Flag-TAB2 are gifts from Dr T Koseki (Tohoku University, Sendai, Japan). pcDNA3-A20 (Vincenz and Dixit, 1996) is a gift from Dr C Vincenz (University of Michigan, MI). Supplementary data pU6-A20i and pU6-Ctli, A20-specific and control siRNA plasmids Supplementary data are available at The EMBO Journal Online (Saitoh et al, 2005) are gifts from Dr S Yamaoka (Tokyo Medical and (http://www.embojournal.org). Dental University, Tokyo, Japan). HEK293T, parental 293 cells and MEFs were cultured as described (Inohara et al, 2000). RICK-, TAK1-, TRAF6- and TRAF2/ Acknowledgements TRAF5-deficient and control WT MEFs are described previously (Naito et al, 1999; Tada et al, 2001; Kobayashi et al, 2002; Shim This work was supported by NIH grants R01 GM60421 (to N et al, 2005). TAK1-deficient MEFs were provided by Dr S Ghosh Inohara) and R01 DK067628 (to G Nun˜ez). We are grateful to L (Yale University, New Heaven, CT) and TRAF6-deficient MEFs McAllister-Lucas (University of Michigan) for stimulating discus- by Dr J Inoue (Tokyo University, Tokyo, Japan). HEK293 cell sions; S Qiu, P Kuffer, Y Nishito and S Chen (University of lines constitutively expressing Nod1-Flag and HA-Nod2 with Michigan) for technical assistance and S Yamaoka (Tokyo Medical NF-kB-dependent GFP reporter were generated by transfection and Dental University, Tokyo), T Koseki (Tohoku University, Japan), of pMSCV-puro-Nod1-Flag and pMX2-puro-HA-Nod2, respectively, J Inoue (Tokyo University, Japan), C Vincenz (University and NFkB-eGFP (Wang et al, 2002), followed by antibiotic Michigan), S Ghosh (Yale University) and Ariad Pharmaceuticals selection. for materials. 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A critical role of RICK/RIP2 polyubiquitination in Nod‐induced NF‐κB activation

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The EMBO Journal (2008) 27, 373–383 & 2008 European Molecular Biology Organization All Rights Reserved 0261-4189/08 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-jB activation 1 2 immune diseases (Inohara et al, 2005; Fritz et al, 2006). Two Mizuho Hasegawa , Yukari Fujimoto , 1 3 NLR family members, Nod1 and Nod2, play an essential role Peter C Lucas , Hiroyasu Nakano , 2 1,4 in the recognition of specific bacterial peptidoglycan (PGN)- Koichi Fukase , Gabriel Nu´ n˜ ez 1,5, related molecules (Chamaillard et al, 2003; Inohara et al, and Naohiro Inohara * 2003; Girardin et al, 2003a, b). Nod1 and Nod2 mediate Department of Pathology, The University of Michigan Medical School, transcriptional activation of innate immune genes including Ann Arbor, MI, USA, Department of Chemistry, Graduate School of antimicrobial peptides, proinflammatory cytokines and che- Science, Osaka University, Toyonaka, Osaka, Japan, Department of mokines, that recruit immune cells to the sites of microbial Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan, Comprehensive Cancer Center, The University of stimulation (Chamaillard et al, 2003; Kobayashi et al, 2005; Michigan Medical School, Ann Arbor, MI, USA and Department of Boughan et al, 2006; Marks et al, 2006; Masumoto et al, 2006; Biochemistry 2nd, Interdisciplinary Graduate School of Medicine Voss et al, 2006). Although the precise structure of the and Engineering, University of Yamanashi, Chuou, Yamanashi, Japan bacterial molecules that stimulate NLR signaling remains largely unknown, the core recognition moieties of Nod1 Nod1 and Nod2 are intracellular proteins that are involved and Nod2 have been identified as g-glutamyl-meso-diamino- in host recognition of specific bacterial molecules and are pimelic acid (iE-DAP), and muramyl dipeptide (MDP), re- genetically associated with several inflammatory diseases. spectively (Chamaillard et al, 2003; Girardin et al, 2003a, b; Nod1 and Nod2 stimulation activates NF-jB through Inohara et al, 2003). Previous studies using synthetic and RICK, a caspase-recruitment domain-containing kinase. natural PGN-related molecules containing MDP and/or iE- However, the mechanism by which RICK activates NF-jB DAP revealed that these molecules induce/enhance innate in response to Nod1 and Nod2 stimulation is unknown. and adaptive immune responses (Goto and Aoki, 1987; Fritz Here we show that RICK is conjugated with lysine- et al, 2007). 63-linked polyubiquitin chains at lysine 209 (K209) located The induction of immune response genes through Nod1 in its kinase domain upon Nod1 or Nod2 stimulation and and Nod2 signaling largely depends on NF-kB, a proinflam- by induced oligomerization of RICK. Polyubiquitination of matory transcriptional factor (Inohara et al, 1999, 2000; RICK at K209 was essential for RICK-mediated IKK activa- Ogura et al, 2001; Masumoto et al, 2006). Transcriptional tion and cytokine/chemokine secretion. However, RICK activation of proinflammatory genes is initiated by nuclear polyubiquitination did not require the kinase activity of translocation of NF-kB following its release from the IkB RICK or alter the interaction of RICK with NEMO, a proteins (Karin and Ben-Neriah, 2000; Hoffmann and regulatory subunit of IjB kinase (IKK). Instead, polyubi- Baltimore, 2006). Previous studies demonstrated that RICK quitination of RICK was found to mediate the recruit- (RIP2/CARDIAK/RIPK2) is a critical downstream mediator of ment of TAK1, a kinase that was found to be essential Nod1 and Nod2 signaling (Inohara et al, 1999; Ogura et al, for Nod1-induced signaling. Thus, RICK polyubiquiti- 2001; Chin et al, 2002; Kobayashi et al, 2002; Park et al, nation links TAK1 to IKK complexes, a critical step in 2007). RICK is composed of N-terminal kinase and C-terminal Nod1/Nod2-mediated NF-jB activation. caspase-recruitment domains (CARD) linked via an inter- The EMBO Journal (2008) 27, 373–383. doi:10.1038/ mediate (IM) region (Inohara et al, 1998; McCarthy et al, sj.emboj.7601962; Published online 13 December 2007 1998; Thome et al, 1998). RICK physically associates with Subject Categories: signal transduction; immunology Nod1 and Nod2 through CARD–CARD interaction and acti- Keywords: NLR; Nod1; Nod2; RICK; TAK1 vates IkB kinase (IKK), leading to phosphorylation and degradation of IkBs (Inohara et al, 1999, 2000; Ogura et al, 2001). Functional studies revealed that close proximity of RICK molecules by self-oligomerization of Nod1 and Nod2 is important for NF-kB activation (Inohara et al, 2000). RICK is Introduction known to interact with NEMO (IKKg/IKBKG) and this inter- Members of the nucleotide-binding domain-like receptor action is essential for NF-kB activation in RICK-mediated (NLR) protein family are found in both mammals and plants signaling (Inohara et al, 2000). Furthermore, Nod2 stimula- (Inohara et al, 2005; Fritz et al, 2006). Several mammalian tion with MDP induces NEMO polyubiquitination and NLRs, like plant NLRs, are involved in host resistance against this event is required for optimal NF-kB activation (Abbott microbial pathogens and genetically associated with human et al, 2004). However, the molecular mechanism by which the IKK complex is activated via RICK to induce NF-kB *Corresponding author. Department of Pathology, Comprehensive activation in response to NLR stimulation is unknown. Cancer Center, University of Michigan Medical School, 1500 E. Medical Center Dr., C574 MSRB2, Ann Arbor, MI 48109, USA. Here we show that RICK is conjugated with K63-linked Tel.: þ 1 734 936 3317; Fax: þ 1 734 647 9654; polyubiquitin chains upon Nod1 and Nod2 stimulation. E-mail: [email protected] RICK polyubiquitination was essential for NF-kB activation and chemokine/cytokine secretion in response to Nod1 sti- Received: 26 September 2007; accepted: 23 November 2007; published online: 13 December 2007 mulation. Furthermore, we provide evidence for a bridging &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 2 2008 373 | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al function of RICK ubiquitination by linking TAK1 to the IKK KF1B or MDP, respectively. Immunoblotting analysis revealed complex that is critical for NF-kB activation during Nod that RICK was polyubiquitinated after KF1B and MDP stimu- signaling. lation (Figure 2A). RICK polyubiquitination was detected 1 h after stimulation with Nod1 and Nod2 agonists. The poly- ubiquitination of RICK by Nod1 and Nod2 stimulation was specific in that it was not detected in parental HEK293 cells Results stimulated with TNFa (Figure 2A). The ubiquitination of Nod1 and Nod2 stimulation induce lysine 63-linked RICK is not due to overexpression of proteins because we polyubiquitination of RICK also found significant ubiquitination of expressed RICK In order to determine the mechanism by which Nod1 stimu- whose levels were similar to those of endogenous RICK in lation induces NF-kB activation, we used mouse embryonic MEFs (see below, Figures 5D and 7D). To determine the fibroblasts (MEFs) because we previously found that non- linkage type of polyubiquitin chains on RICK, Nod1- and immune cells are much more sensitive to Nod1-stimulatory Nod2-expressing cells were transfected with RICK-Myc molecules than hematopoietic cells (Hasegawa et al, 2006; and HA-tagged wild-type (WT) or lysine-mutant Ub proteins Masumoto et al, 2006). Stimulation of MEFs with KF1B, a containing single lysine mutations at position 48 (K48R) or 63 synthetic specific Nod1-stimulatory compound, induced se- (K63R). Upon Nod1 and Nod2 stimulation with KF1B and cretion of the chemokine CCL2 (Figure 1A) and phosphoryla- MDP, respectively, RICK was strongly polyubiquitinated with tion of IkBa (Figure 1B). Such responses were dependent on K48R Ub, but much less with K63R Ub (Figure 2A). Thus, the RICK in that the induction of CCL2 production induced by polyubiquitin chains on RICK were linked primarily through KF1B, but not TNFa, was abolished in RICK-deficient MEFs lysine 63 of ubiquitin. (Figure 1A). Previous studies showed that enforced oligomerization of Recently, ubiquitination of RIP, a mediator of TNFa signal- RICK by Nod1 and Nod2 induces NF-kB activation (Inohara ing, was found to be important for IKK activation (Ea et al, et al, 2000). Similarly, oligomerization of RICK-DCARD-Fpk3, 2006; Li et al, 2006). Because our previous studies suggest a RICK fusion protein in which the CARD is replaced with functional and structural similarities between RIP and RICK, three tandem FKBP-related dimerization domains, results in we first hypothesized that Nod1 stimulation might result in NF-kB activation that is dependent on the FKBP-specific ubiquitination of RICK. As shown in Figure 1, RICK from dimerizer AP1510 (Inohara et al, 2000; Figure 2B). Using MEFs stimulated with KF1B co-immunoprecipitated with this approach, we tested if oligomerization of RICK-DCARD- high-molecular-weight ubiquitinated proteins (Figure 1C, Fpk3 results in RICK polyubiquitination in HEK293Tcells that upper panel). To further assess a role of ubiquitination in coexpress HA-Ub. Immunoblotting analysis revealed that Nod1 and Nod2 signaling, we tested if RICK is ubiquitinated RICK-DCARD-Fpk3 was polyubiquitinated in an AP1510-de- upon Nod1 and Nod2 stimulation, using human embryonic pendent manner whereas the control Fpk3 protein was not kidney (HEK) 293 cells constitutively expressing Nod1 and (Figure 2B). These results suggest that induced proximity of Nod2. Nod1- and Nod2-expressing HEK293 cells were trans- RICK (1–435) lacking the CARD domain mimics the inter- fected with plasmids producing Myc-RICK and HA-Ub and action between RICK and Nod proteins and induces RICK stimulated with specific synthetic Nod1 and Nod2 agonists, signaling. The kinase domain of RICK is essential for RICK ubiquitination To determine which region of RICK is required for polyubi- quitination, we constructed RICK-DCARD-Fpk3-mutant pro- teins lacking various regions of RICK and tested their ability to be ubiquitinated in the absence and presence of the dimerizer AP1510 (Figure 3A). Immunoblotting analysis showed that all RICK-DCARD-Fpk3 proteins containing resi- dues 1–292 of RICK were ubiquitinated in an AP1510-depen- dent manner, whereas that encompassing residues 293–435 was not (Figure 3B). These results indicate that the region Figure 1 Nod1 stimulation induces ubiquitination of RICK. (A) composed of amino-acid residues 1–292 located within the MEFs from WT and RICK-deficient mice were stimulated with kinase region of RICK contains the polyubiquitinated site(s). 5 mg/ml KF1B (synthetic Nod1-stimulatory compound), 10 ng/ml To determine the role of RICK polyubiquitination in NF-kB TNFa or left alone. Twenty-four hours post-stimulation, the levels of activation, the ability of RICK fusion proteins to activate CCL2 in culture supernatant were determined by ELISA. The results shown are given as mean7s.d. of triplicate cultures and are NF-kB was tested using a reporter assay. Transfection of representative of three experiments. (B) WT or RICK-deficient HEK293T cells with the different constructs revealed that all MEFs were stimulated with 5 mg/ml KF1B for the indicated times. RICK proteins carrying the region between amino acid 1 and Post-stimulation, cells were lysed and immunoblotted with anti- 319 induced NF-kB activation in an AP1510-dependent man- phospho-IkBa, anti-IkBa or IKKb Ab. (C) MEFs were stimulated with or without KF1B for the indicated times. Post-stimulation, cells ner (Figure 3C). In contrast, enforced oligomerization of were lysed and RICK was immunoprecipitated with rabbit anti-RICK the fusion proteins carrying the region between 1 and 292 Ab. Ubiquitinated (upper panel) and total (lower panel). RICK and that between 293 and 435 did not result in NF-kB proteins in the immunoprecipitates were immunodetected by activation (Figure 3C). These results indicate that both mouse monoclonal anti-RICK and anti-Ub Abs, respectively. Nonspecific signals detected with the Ab are indicated by asterisks. the kinase domain (residues 1–292), which contains the 374 The EMBO Journal VOL 27 NO 2 2008 &2008 European Molecular Biology Organization | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al Figure 2 Nod1 and Nod2 stimulation induce K63-linked polyubiquitination of RICK. (A) HEK293 cells constitutively expressing Nod1 (for KF1B stimulation), Nod2 (for MDP stimulation) and parental HEK293 cells (for TNFa stimulation) were transfected with expression plasmid of Myc-RICK in the presence of expression plasmids of HA-tagged WT, K48R, K63R mutant Ub proteins. Cells were stimulated with 100 ng/ml KF1B, 100 ng/ml MDP (synthetic Nod2-stimulatory compound) or 10 ng/ml TNFa, or left alone (0 h control) for the indicated times. Myc-RICK proteins in transfected cells were immunoprecipitated with rabbit anti-Myc Ab and ubiquitinated RICK proteins were then detected by immunoblotting with anti-HA Ab (upper panels labeled as RICK-Ubn). As a control, RICK proteins in the same samples and Ub proteins in total lysate were detected by immunoblotting with the indicated Abs. (B) Oligomerization-dependent RICK ubiquitination. HEK293Tcells were transfected with control pcDNA3-RICK-Fpk3-Myc () or pcDNA3-RICK-Fpk3-Myc (RICK) in the presence of HA-Ub expression plasmids. Eighteen hours post-transfection, cells were treated with or without 200 nM AP1510. Twenty-four hours post-transfection, proteins were immunoprecipitated and ubiquitinated RICK was analyzed as described in panel A. polyubiquitinated site(s), and the IM region (residues 293– ATP-binding site and results in loss of RICK kinase activity 319) are critical for NF-kB activation. (Inohara et al, 1998), did not impair RICK polyubiquitination Previous studies showed that NF-kB activation mediated (Figure 4B). Thus, the conserved K209, but not the kinase by RICK requires NEMO (Inohara et al, 2000). Therefore, we activity of RICK, is essential for polyubiquitination. To deter- hypothesized that at least one of the regions required for NF- mine if K209 is responsible for polyubiquitination of RICK kB activation interacts with NEMO. To explore this hypoth- during Nod1 signaling, we introduced the K209R mutation esis, we tested the ability of RICK-Fpk3-Myc constructs into a full-length RICK construct and tested the ability of WT carrying different regions of RICK to interact with NEMO and K209R-mutant RICK proteins to be ubiquitinated in by co-immunoprecipitation assay. Consistent with a previous Nod1-expressing HEK293 cells upon Nod1 stimulation with study (Inohara et al, 2000), a RICK truncation mutant KF1B. Consistent with the results presented in Figure 4B, the expressing a region between amino-acid residues 1–319 K209R mutation abolished KF1B-induced polyubiquitination interacted with NEMO (Figure 3D). Significantly, a RICK of RICK (Figure 4C). These results indicate K209 is essential mutant composed of residues 1–292 did not associate with for RICK polyubiquitination in Nod1 signaling. NEMO (Figure 3D). These results indicate that the interaction between RICK and NEMO requires the IM region-spanning residues 293–319 of RICK. Polyubiquitination of RICK is essential for Nod1 and Nod2-mediated NF-jB activation Lysine 209 in the kinase domain of RICK is To test if polyubiquitination at K209 of RICK is required for polyubiquitinated in a signal-dependent manner NF-kB activation, we assessed the ability of lysine-mutant To map the polyubiquitinated lysine residue(s) in the kinase RICK-DCARD-Fpk3 proteins to activate NF-kB after stimula- domain of RICK, we first performed close inspection of the tion with different doses of AP1510. All the point mutant amino-acid sequence of RICK. Alignment of amino-acid RICK proteins were expressed at levels comparable with that sequences from the kinase domain and IM region of RICK of WT protein, except the kinase-dead K47M mutant that was revealed 10 lysine residues that are evolutionarily conserved expressed at significantly lower levels (Figure 5A, inset). in 11 animal species (Supplementary Figure 1). Therefore, we Consistent with its lower expression, the K47M mutant introduced site-directed mutations in the 10 absolutely con- showed reduced levels of NF-kB activation when compared served and seven additional highly conserved lysine residues with WT protein (Figure 5A). However, the K47 mutant did and tested if the mutations affect the ability of RICK-DCARD- show a dose-dependent increase in NF-kB and since K47 is Fpk3 to be ubiquitinated in an AP1510-dependent manner essential for the kinase activity of RICK (Inohara et al, 1998), (Figure 4A). As shown in Figure 4B, the point mutant K209R, we conclude that the kinase activity of RICK is required for but not the remaining 16 lysine mutations, abolished the neither NF-kB activation nor polyubiquitination. Notably, the polyubiquitination of RICK. Notably, the K47M mutation, K209R mutation, but not those of the remaining conserved which replaces the essential lysine residue in the conserved lysines, abolished AP1510-dependent NF-kB activation &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 2 2008 375 | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al Figure 3 The kinase domain of RICK is essential for RICK ubiquitination, but is not sufficient to activate NF-kB. (A) Schematic diagram of the structural domains of RICK and different deletion mutants. The results of ubiquitination and NF-kB activation experiments are summarized on the right. The region, which requires NF-kB activation in the IM domain is indicated by a gray box. (B) HEK293T cells were transfected with pcDNA3-RICK-(1–435)-Fpk3-Myc (DC), RICK-(1–292)-Fpk3-Myc, RICK-(1–319)-Fpk3-Myc, RICK-(1–328)-Fpk3-Myc, RICK-(1–356)-Fpk3-Myc, RICK-(1–390)-Fpk3-Myc RICK-(293–435)-Fpk3-Myc and control vector in the presence of HA-Ub expression plasmid and treated with AP1510 as in Figure 2B. Twenty-four hours post-transfection, proteins were immunoprecipitated and ubiquitinated RICK were analyzed as described in Figure 2. (C) HEK293T cells were transfected with pcDNA3-RICK-(1–435)-Fpk3-Myc (DC), RICK-(1–292)-Fpk3-Myc, RICK-(1–319)-Fpk3-Myc, RICK-(1–328)-Fpk3-Myc, RICK-(1–356)-Fpk3-Myc, RICK-(1–390)-Fpk3-Myc RICK-(293–435)-Fpk3-Myc and control vector in the presence of reporter plasmids. Eight hours post-transfection, cells were treated with medium containing the indicated amounts of AP1510. Twenty-four hours post-transfection, ligand-dependent NF-kB activation was determined with reporter assay. The level of NF-kB-dependent transcription activity in cells transfected with control vector is given as 1. The expression levels of Myc-RICK proteins are shown in an inset. (D) HEK293T cells were transfected with control vector (), pcDNA3-RICK-(1–435)-Fpk3-Myc, RICK-(1–319)-Fpk3-Myc and RICK-(1–292)-Fpk3-Myc in the presence of reporter plasmids, and treated with AP1510 as in Figure 2B. Twenty-four hours post-transfection, RICK proteins were immunoprecipitated with rabbit polyclonal anti-Myc Ab, and then Flag-NEMO (upper panel) and Myc-RICK (middle panel) were immuno- detected with mouse monoclonal anti-Flag and anti-Myc Abs, respectively. As control, Flag-NEMO in total lysate was immunodetected with anti-Flag Ab and shown in the lower panel. Figure 4 Lysine 209 is ubiquitinated in the kinase domain of RICK. (A) Location of lysine residues, which were replaced by arginines in RICK site-directed mutants. (B) HEK293T cells were transfected with control vector (), pcDNA3-RICK-DCARD-Fpk3-Myc (WT) and various lysine- to-arginine mutants in the presence HA-Ub expression plasmid and treated with AP1510 as in Figure 2B. Twenty-four hours post-transfection, proteins were immunoprecipitated and ubiquitinated RICK were immunodetected as described in Figure 1. (C) HEK293 cells constitutively expressing Nod1 were transfected with the expression plasmids of WT and K209R-mutant Myc-RICK in the presence of HA-Ub expression plasmid. Eighteen hours post-transfection, cells were stimulated with or without 100 ng/ml KF1B. Twenty-four hours post-transfection, proteins were immunoprecipitated and ubiquitinated RICK were immunodetected as described in Figure 1. 376 The EMBO Journal VOL 27 NO 2 2008 &2008 European Molecular Biology Organization | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al Figure 5 Polyubiqutination at K209 is essential for RICK-mediated signaling. (A) HEK293T cells were transfected with pcDNA3-RICK-DCARD- Fpk3-Myc (WT) or the mutants in the presence of reporter plasmids. Eight hours post-transfection, cells were treated with medium containing the indicated amount of AP1510. Twenty-four hours post-transfection, ligand-dependent NF-kB activation was determined with reporter assay. The level of NF-kB-dependent transcription activity in cells transfected with control vector is given as 1. Myc-tagged RICK proteins detected with anti-Myc Ab are shown in the inset. (B) RICK-deficient MEFs were infected with retrovirus vectors expressing WT or K209R HA-RICK or control vector and selected by hygromycine. RICK proteins detected with anti-RICK Ab. A nonspecific signal detected with the Ab is indicated by an asterisk. (C) RICK-deficient MEFs were infected with control () or retrovirus vectors expressing WT or K209R HA-RICK. Infected MEFs were selected by hygromycin and stimulated with 5 mg/ml KF1B, 10 ng/ml TNFa or left alone. Twelve hours post-stimulation, secretion levels of CCL2 and CXCL1 were determined by ELISA. The results shown are given as mean7s.d. of triplicate cultures and are representative of three experiments. (D) MEFs stably expressing RICK or RICK K209R were stimulated with 5 mg/ml KF1B for the indicated time. Post-stimulation, cells were lysed, RICK was immunoprecipitated and ubiquitinated RICK was immunodetected by anti-Ub Ab (upper panel). Cell lysate was also immunoblotted with anti-phospho-IkBa, anti-IkBa or IKKb Ab (bottom three panels). A nonspecific signal detected with the Ab is indicated by an asterisk. (E) MEFs stably expressing RICK or RICK K209R were stimulated with 5 mg/ml KF1B or TNFa for indicated times, and then anti- NEMO Ab was used to immunoprecipitate the IKK complex. The IKK assay was carried out by using GST-IkBa and g- P-ATP as substrates (upper). The amount of IKKb in the immunoprecipitates was detected by immunoblotting. (Figure 5A). These results indicate that polyubiquitination of A20 inhibits Nod1- and Nod2-mediated NF-jB activation RICK at K209 is essential for NF-kB activation. and polyubiquitination of RICK To test if K209 polyubiquitination on RICK is important for A20 is a deubiquitinase that removes K63-linked polyubiqui- Nod1 signaling, we assessed the ability of WT and K209R- tin chains and functions as a negative feedback regulator of mutant RICK proteins to complement RICK function in em- inflammatory responses (Lee et al, 2000; Boone et al, 2004; bryonic fibroblasts deficient in RICK. In these experiments, Wertz et al, 2004). Previous studies showed that A20 expres- MEFs from RICK-deficient mice were infected with a retro- sion is induced by NF-kB activation (Krikos et al, 1992) and viral vector producing WT or K209R-mutant RICK proteins. Nod1 stimulation (Masumoto et al, 2006). To test if A20 The expression levels of both WTand K209R RICK proteins in negatively regulates Nod signaling through deubiquitination RICK MEFs were similar to that of endogenous RICK of RICK, we tested the ability of A20 to affect RICK poly- in WT MEFs (Figure 5B). Notably, expression of WT RICK ubiquitination and NF-kB activation in response to Nod1 or conferred responsiveness of MEFs deficient in RICK to KF1B, Nod2 stimulation. To assess the latter, A20 was coexpressed and resulted in secretion of chemokines CCL2 and CXCL1 with Myc-RICK and HA-ubiquitin in HEK293T cells and the (Figure 5C). In contrast, the K209R RICK mutant failed to level of polyubiquitinated RICK was determined by immuno- rescue responsiveness to the Nod1 agonist (Figure 5C). blotting. We found that expression of A20 decreased RICK Immunoblotting showed that WT but not K209R-mutant polyubiquitination (Figure 6A). HEK293T cells express RICK was polyubiquitinated upon Nod1 stimulation and low, but significant, levels of endogenous Nod1 and Nod2 resulted in IKK activation (Figure 5D and E). Because control (Viala et al, 2004). Consistent with the latter, treatment of TNFa induced similar levels of CCL2 and CXCL1 secretion HEK293T cells with high amount of lipophilic Nod1- and from cells infected with WT and K209R-mutant RICK con- Nod2-stimulatory molecules KF1B (5 mg/ml) and AcMDP structs, these findings indicate that polyubiquitination of (acetyl-(6-O-stearoyl)-muramyl-Ala-D-Glu-NH )(5 mg/ml) in- RICK at K209 is essential for Nod1 signaling but dispensable duced NF-kB activation (Figure 6B). Using this system, we for that induced by TNFa stimulation. tested the ability of A20 to affect the NF-kB activation &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 2 2008 377 | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al Figure 6 A20 functions as a negative regulator of Nod1 and Nod2 signaling via deubiquitination of RICK. (A) HEK293T cells were transfected with Flag-tagged A20 expression plasmids or control vector () in the presence of pcDNA3-RICK-DCARD-Fpk3-Myc (WT) and HA-Ub expression plasmid. Eighteen hours post-transfection, cells were treated with 200 nM AP1510 or left alone. Twenty-four hours post-transfection, proteins immunoprecipitated with rabbit anti-Myc Ab were subjected to SDS–PAGE and analyzed by western blot analysis using anti-HA, Myc and Flag Abs to detect ubiquitinated RICK proteins, RICK proteins, and A20 protein, respectively. (B) HEK293T cells were transfected with expression plasmids of A20 or control vector in the presence of reporter plasmids. Eight post-transfection, cells were treated with medium containing 1 mg/ml KF1B, 1 mg/ml AcMDP, 10 ng/ml TNF or 10 ng/ml IL-1b. Twenty-four hours post-transfection, ligand-dependent NF-kB activation was determined with reporter assay. The level of NF-kB-dependent transcription activity in the absence of A20 is given as 100%. The results shown are given as mean7s.d. of triplicate cultures and are representative of three experiments. (C) HEK293T cells were transfected with pcDNA3 (), A20 interference RNA (RNAi) plasmid or control RNAi plasmid (Ctli) in the presence of reporter plasmids. Thirty-six hours after transfection, cells were treated with medium containing 1 mg/ml KF1B, 1 mg/ml AcMDP, 10 ng/ml TNFa or IL-1b. Forty-eight hours post- transfection, ligand-dependent NF-kB activation was determined with reporter assay. The level of NF-kB-dependent transcription activity in cells transfected with pcDNA3 is given as 1. Expression of A20 and control IKKb proteins is shown in inset. The results shown are given as mean7s.d. of triplicate cultures and are representative of three experiments. (D) HEK293 cells constitutively expressing Nod1 were transfected with pcDNA3 () or A20 RNAi plasmid. Forty-eight hours after transfection, cells were treated with 100 ng/ml KF1B for 1 h. The cells were lysed and endogenous RICK was immunoprecipitated. Ubiquitinated and total RICK proteins were immunodetected with anti-Ub and anti-RICK Abs, respectively. induced by these Nod1- and Nod2-stimulatory molecules in impaired phosphorylation of IkBa (Figure 7B). These results HEK293T cells, and found that NF-kB activation induced by indicate that TAK1 plays an essential role in Nod1 signaling. KF1B and AcMDP, as well as that induced by TNFa and IL-1b, Because K63-linked polyubiquitination of RIP recruits TAK1 was reduced by A20 overexpression (Figure 6B). We next complex to the TNFR1 complex leading to NF-kB activation determined whether endogenous A20 mediates negative feed- (Kanayama et al, 2004), we tested whether RICK polyubiqui- back of NF-kB activation after Nod1 stimulation. To test this, tination at K209 mediates the recruitment of TAK1 complex to we used A20 small-RNA interference (RNAi) construct, RICK. To determine this, Myc-RICK-DCARD-Fpk3 proteins which suppresses specifically the expression of A20 (Saitoh were coexpressed with TAK1, TAB1 and TAB2. We found et al, 2005; also see inset in Figure 6C). Like that induced by that WT Myc-RICK-DCARD-Fpk3 protein co-immunoprecipi- TNFa and IL-1b,NF-kB activation induced by KF1B and tated with TAK1, TAB1 and TAB2 in the presence but not in AcMDP were enhanced by transfection with the A20 RNAi the absence of the dimerizer AP1510 (Figure 7C). These plasmid but not with control plasmid (Figure 6C). In addi- results suggest that polyubiquitinated RICK is associated tion, the ubiquitination level of endogenous RICK induced with the TAK1 complex. To test if polyubiquitination is by Nod1 stimulation was upregulated by expression of A20 required for recruitment of TAK1 complex to RICK, we tested RNAi (Figure 6D). These findings suggest that A20 is a the ability of K209R-mutant construct that is deficient in negative feedback regulator of Nod1 and Nod2 signaling. signal-dependent polyubiquitination, to interact with the TAK1 complex. The analysis revealed that the K209R mutant of RICK did not associate with TAK1 complex when com- Polyubiquitination of RICK is required for the pared with WT RICK (Figure 7C). These results suggest that recruitment of TAK1 polyubiquitination of RICK at K209 is essential for recruit- Recent studies showed that a TAK1 complex containing ment of the TAK1 complex to RICK. To test if the interaction TAK1, TAB1, TAB2 and/or TAB3 is a general mediator of NF-kB activation induced by various inflammatory stimuli of TAK1 with RICK is sufficient to induce NF-kB activation, (Ishitani et al, 2003; Sato et al, 2005; Shim et al, 2005). we assessed if TAK1 is recruited to the RICK (1–292)-mutant construct that is polyubiquitinated upon stimulation but is Because TAK1 has been suggested to play a role in NF-kB unable to activate NF-kB and interact with NEMO (see activation induced by Nod2 signaling (Chen et al, 2004; Figure 3). Notably, RICK (1–292) mutant, but not RICK Windheim et al, 2007), we hypothesized that TAK1 mediates (293–435), associated with the TAK1 complex after addition Nod1-induced innate immune responses. To test this, we examined if KF1B induces the secretion of CCL2 from of AP1510 (Figure 7C). To verify that this finding is not due to TAK1-deficient MEFs. We found that WT MEFs secreted overexpression of recombinant proteins, we further investi- CCL2 in response to KF1B stimulation but TAK1-deficient gated the interactions of TAK1 and NEMO with WT and K209R RICK proteins, which are expressed at levels compar- MEFs did not (Figure 7A). The impaired response in TAK1 able to that of endogenous RICK in RICK MEFs. As expected, MEFs was not due to the lack of expression of the general WT but not K209R-mutant RICK was polyubiquitinated and NF-kB regulators, IKKb and IkBa, but was associated with 378 The EMBO Journal VOL 27 NO 2 2008 &2008 European Molecular Biology Organization | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al Figure 7 RICK polyubiquitination at K209 is required for the recruitment of TAK1/TAB1/TAB2 complex. (A) WTor TAK1-deficient MEFs were 2þ stimulated with 5 mg/ml KF1B, 10 ng/ml TNFa, 10 ng/ml IL-1b, 100 ng/ml LPS, 1 mg/ml sBLP, 50 ng/ml PMA plus 0.7 mg/ml Ca ionophore A23187 or left alone. Twenty-four hours post-stimulation, secretion levels of CCL2 were determined by ELISA. The results shown are given as mean7s.d. of triplicate cultures and are representative of three experiments. (B) WT or TAK1-deficient MEFs were stimulated with 5 mg/ml KF1B, 10 ng/ml TNFa or 10 ng/ml IL-1b for 1 h. Post-stimulation, cells were lysed and lysates immunoblotted with anti-phospho-IkBa, anti-IkBa or IKKb Ab. (C) HEK293T cells were transfected with expression plasmids of RICK-DCARD-Fpk3-Myc (WT), RICK-K209R-DCARD- Fpk3-Myc (K209R), RICK-(1–292)-Fpk3-Myc, RICK-(293–435)-Fpk3-Myc and control vector () in the presence of expression plasmids of Ub, HA-TAK1, Flag-TAB1 and T7-TAB2 and treated with AP1510 as in Figure 2B. Twenty-four hours post-transfection, proteins immunoprecipitated with rabbit anti-Myc Ab were subjected to SDS–PAGE and analyzed by immunoblotting analysis using anti-HA or, anti-Flag, anti-T7 and anti-Ub Abs. (D) MEFs stably expressing RICK or RICK K209R were stimulated with 5 mg/ml KF1B for 1 h. The cells were lysed, RICK was immunoprecipitated and endogenous Ub, TAK1, NEMO and RICK were immunodetected with anti-Ub, anti-TAK1 Ab, anti-NEMO or anti-RICK antibodies. interacted with TAK1 upon Nod1 stimulation, whereas the interaction between RICK and NEMO was constitutive and was not affected by the mutation at K209 (Figure 7D). Together with the findings described in previous figures, this finding suggests that TAK1 recruitment to K209- polyubiquitinated RICK is essential for IKK activation upon Nod1 stimulation. TRAF2/TRAF5 but not TRAF6 are required for Nod1-mediated NF-jB activation and CCL2 secretion Recent findings suggest that TRAFs are ubiquitin ligases (E3s), which regulate several immune responses including Figure 8 TRAF2/TRAF5, but not TRAF6, are required for Nod1- inflammatory responses mediated by NF-kB. Although mediated NF-kB activation and CCL2 secretion. (A) WT, TRAF6 / / TRAF6 is suggested to be important for Nod2-mediated (TRAF6 KO) or TRAF2 TRAF5 (TRAF2/5 DKO) MEFs were signaling, TRAF6-depeleted cells still respond to MDP, a stimulated with 5 mg/ml KF1B, 10 ng/ml TNFa, 10 ng/ml IL-1b or left alone. After 24 h incubation, the secretion levels of CCL2 were Nod2-stimulatory molecule (Abbott et al, 2007). We also determined by ELISA. The results shown are given as mean7s.d. of found that TRAF6 MEFs respond to KF1B, resulting in triplicate cultures and are representative of three experiments. (B) IkBa phosphorylation (Figure 8B) and induced secretion of WT and mutant MEFs were stimulated with 5 mg/ml KF1B for 1 h or CCL2 (Figure 8A). Thus, TRAF6 appears to be a non-essential left alone. The phosphorylated (upper panel) and whole (middle panel) populations of IkBa and IKKb (lower panel) were immuno- E3 for both Nod1 and Nod2 signaling. Notably, Nod1 stimu- detected with specific Abs. lation did not induce NF-kB activation and IkBa phosphor- ylation in MEFs lacking TRAF2 and TRAF5, which are essential for TNFa-induced NF-kB activation and are able to Discussion complement one another (Tada et al, 2001; Figure 8). These results suggest that TNFa- and Nod1-mediated signaling RICK is an essential and specific mediator of Nod1 and Nod2 share common E3s to activate NF-kB. signaling (Chin et al, 2002; Kobayashi et al, 2002; Park et al, &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 2 2008 379 | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al Figure 9 Model for the role of RICK polyubiquitination in Nod signaling. For detail, see text. 2007). However, the mechanism by which RICK mediates associate with RICK (McCarthy et al, 1998; Thome et al, / / Nod1- and Nod2-mediated immune responses has remained 1998). Here we demonstrated that TRAF2 TRAF5 MEFs poorly defined. Here we found an essential role for RICK lack NF-kB activation upon Nod1 stimulation. However, we polyubiquitination in Nod1- and Nod2-mediated NF-kB acti- do not have convincing evidence that TRAF2 and TRAF5 vation. The K63-linked polyubiquitin chain at K209 in RICK directly ubiquitinate RICK. We could not find consensus was found to be important for the recruitment of the TAK1 sequences of TRAF2- and TRAF5-binding sites in regions complex to RICK and IKKs. Similarly, RIP (RIPK1), a RICK near K209 in RICK. Although we still do not know how the homologue, was shown to play a critical role in TNFa- E3(s) specifically recognize RICK as a substrate, K209 is induced NF-kB activation through its polyubiquitination at highly conserved in RICK but not in other kinases and is K377 located in the IM region between the N-terminal kinase located in the putative flexible loop region (Supplementary and C-terminal death domains (Ea et al, 2006; Li et al, 2006). Figure 1). Further studies are required to determine the Like that of RICK, polyubiquitinations of RIP are both critical mechanism by which TRAFs are involved in Nod/RICK- for recruitment of TAK1 complex. Therefore, our results mediated signaling, and conclusively identify the E3(s), indicate that Nod1/Nod2 and TNFa signaling is mediated which ubiquitinates K209 in RICK. through similar molecular events. In summary, we have dissected two molecular signaling Previous studies suggested an important role for the IM events that are critical for Nod signaling, namely recruitment domain located between the kinase domain and the CARD of of TAK1 to polyubiquitinated K209 located in the kinase RICK in Nod1-mediated NF-kB activation (Inohara et al, domain, and recruitment of NEMO, which is mediated by 2000). Here we further characterized the structural require- the IM region of RICK. NEMO interacted with RICK-DCARD- ment of IM and found that the region between 293 and 319, Fpk3 in the absence of AP1510, which is required for poly- near to the N-terminal kinase domain, is important for NF-kB ubiquitination of K209 and concomitant NF-kB activation activation. This is also comparable to the observation that the (Figure 3D). As a result, the interaction between RICK and IM region of RIP is required for TNF signaling (Inohara et al, NEMO through the IM region appears to be independent of 2000). However, oligomerization of RICK-Fpk3 fusion protein the interaction between TAK1 and RICK and is not sufficient carrying the IM region alone did not result in NF-kB activa- to activate NF-kB. Therefore, the association of RICK with tion (Figure 3C), whereas that of the equivalent region of RIP both TAK1 and IKK complexes are required for signal trans- was sufficient to induce oligomerization-dependent NF-kB duction resulting in NF-kB activation. This conclusion leads activation (Inohara et al, 2000). These results suggest that the us to propose the following model (Figure 9): the molecular kinase domain of RICK, but not that of RIP, is essential for events during Nod1 and Nod2 signaling are (1) the recruit- NF-kB activation. Previous studies showed that the K47M ment of RICK to Nod1 and Nod2 upon sensing of their kinase-inactive mutant of RICK still activates NF-kB (Inohara specific microbial stimuli, (2) the close proximity of RICK et al, 2000), suggesting that the kinase domain of RICK induced by the self-oligomerization of Nod1 and Nod2, (3) mediates a critical function, other than phosphorylation, polyubiquitination at K209 in RICK by unknown E3(s), (4) which is required for the induction of NF-kB. Indeed, we recruitment of the TAK1 complex to RICK, (5) further recruit- found here that K63-linked polyubiquitination at K209 within ment of IKK complex through the interaction between the IM the kinase domain of RICK is essential for RICK-mediated region of RICK with NEMO, and finally (6) activation of IKKs signaling. In contrast, the critical polyubiquitination site of by TAK1. In this model, Nod1 and Nod2 play an important RIP at K377 is located the IM region (Ea et al, 2006; Li et al, role as switches to turn on the downstream cascade reaction 2006). These results indicate that RICK and RIP differ in the and RICK functions as a bridging protein to link TAK1 and domain localization of the critical K63-linked polyubiquitina- IKK complexes. Similarly, RIP may function as a bridging tion site, providing a mechanistic explanation for the ob- protein between TAK1 and IKK complexes in TNFa signaling. served differences between the two related kinases in the However, the K63-linked polyubiquitination site K377 is induction of NF-kB. Although the localization of the K63- located in the IM region and, therefore, previous studies linked polyubiquitinated site is different in RICK and RIP, could not separate the two molecular events mediated by polyubiquitination of both kinases is regulated by upstream RIP, namely the recruitment of TAK1 and that of the IKKs. stimuli and required for recruitment of the critical TAK1 Further studies are required to explore the differences and complex to these kinases. While polyubiquitination of RICK similarities between Nod and TNF signaling pathways. is essential for Nod1-mediated NF-kB activation, the E3s that The kinase domain of RICK possesses protein kinase mediate polyubiquitination of RICK remain unknown. Some activity (Inohara et al, 1998; McCarthy et al, 1998; Thome possible candidates include the TRAFs, which are known to et al, 1998) and residues critical for catalytic activity are 380 The EMBO Journal VOL 27 NO 2 2008 &2008 European Molecular Biology Organization | | Role of RICK ubiquitination in Nod signaling M Hasegawa et al Luciferase assay evolutionary conserved. However, previous and current stu- NF-kB activation was determined using 0.510 HEK293T cells dies showed that the residue critical for kinase activity is not transfected with expression plasmids in the presence of reporter essential for NF-kB activation, polyubiquitination of RICK plasmids, NF-kB-dependent pBxIV-luc and control pEF1BOS-b-gal, and interaction of RICK with NEMO (Inohara et al, 2000). as described (Inohara et al, 2000; Kobayashi et al, 2002). Therefore, the kinase activity of RICK might regulate other molecules including ERKs, p38 kinases and caspase-1 that Cytokine/chemokine secretion WT and RICK-deficient MEFs were infected with retroviral vectors have been linked to RICK-mediated signaling (Thome et al, pMSCV-hygro-HA-RICK and pMSCV-hygro-HA-RICK K209R pack- 1998; Navas et al, 1999; Chin et al, 2002; Kobayashi et al, aged in HEK293T cells cotransfected with EcoPack plasmid (a gift 2002). Previous studies suggested that the activation of these from Dr G Nolan, UCSF). Infection was carried out in the presence intracellular molecules is important for secretion of certain of 5 mg/ml polybrene for 3 h and incubation continued for 48 h with fresh medium. Hygromycin-resistant (hygro ) MEFs were selected cytokines upon Nod1 and Nod2 stimulation (Inohara et al, with 0.3 mg/ml hygromycin (Roche) for 5 days. A total of 110 2001; Girardin et al, 2003a, b; Viala et al, 2004). Alternatively, hygro MEFs were cultured in 0.25 ml medium in 48-well plates and the kinase domain of RICK may regulate the turnover rate of stimulated with inflammatory stimuli indicated in figure legends. the RICK protein. Consistent with the latter, we found lower Twenty-four hours post-stimulation, the secretion levels of CCL2 and CXCL1 in the medium were determined by sandwich ELISA kits but significant residual polyubiquitination of RICK indepen- (BD, San Diego, CA). Similarly, WT and TAK1-deficient MEFs were dent from K63-Ub upon Nod1 and Nod2 stimulation stimulated with the indicated inflammatory stimuli and CCL2 and (Figure 2), suggesting that RICK is partially polyubiquitinated CXCL1 secretion levels were determined 24 h post-stimulation. in a K63-Ub-independent manner. Furthermore, K48-linked polyubiquitination of RIP during TNFa signaling has been Immunoprecipitation analyses and immunodetection suggested to regulate proteasomal degradation of RIP (Wertz HEK293 cells were transfected with expression plasmids or siRNA plasmids and lysed with NP-40 buffer as described (Inohara et al, et al, 2004). Therefore, our finding suggests that K48-linked 1999) The proteins were immunoprecipitated with anti-Myc rabbit polyubiquitination of RICK might regulate RICK protein levels polyclonal (A-14; Santa Cruz) or rabbit anti-RICK (H-300; Santa during Nod signaling. The functional relevance of K63-Ub- Cruz) antibody (Ab). Myc-, HA- or Flag-tagged proteins were independent polyubiquitination of RICK and the role of the immunodetected with horseradish peroxidase-conjugated Abs specific for each tag (A-14 and Y-11 from Santa Cruz for Myc and kinase activity in RICK protein degradation warrant further HA tags, respectively, M2 from Sigma-Aldrich for Flag tag). investigation. A total of 110 MEFs were stimulated with 5 mg/ml KF1B for the indicated time. MEFs were lysed with and RICK proteins were immunoprecipitated by rabbit anti-RICK or rabbit anti-HA Ab (Y-11; Santa Cruz). Ubiquitinated and total RICK proteins, TAK1 or NEMO Materials and methods in the immunoprecipitates were immunodetected by mouse monoclonal anti-Ub (Abcam, Cambridge, MA), anti-RICK (Alexis, Ligand compounds, plasmids and culture cells San Diego, CA), anti-TAK1 (C-9; Santa Cruz) and anti-NEMO Abs Synthetic compounds, iE-DAP and KF1B have been described (B-3; Santa Cruz), respectively. Cell lysates were immunoblotted (Masumoto et al, 2006). MDP, AcMDP, sBLP (Pam Cys-OH) TNFa, with anti-phospho-IkBa, anti-IkBa (Cell Signaling) or IKKb Ab (H- IL-1b, PMA and A23187 were obtained from commercial sources. 470; Santa Cruz). AP1510 is a gift from Ariad (Cambridge, MA). pcDNA3-Fpk3-Myc, pcDNA3-RICK-Fpk3-Myc, RICK-(1–292)-Fpk3-Myc, pcDNA3-RICK- (K47M)-Fpk3-Myc, pcDNA3-Myc-RICK, pCGN-HA-Ub, pBxIV-luc, In vitro kinase assay pcDNA3-b-gal and pMX2-puro-HA-Nod2 have been described 6 A total of 210 MEFs stably expressing RICK or RICK K209R were (Inohara et al, 2000, 2003; Nishito et al, 2006). pCMV-SPORT6- stimulated with 5 mg/ml KF1B or 10 ng/ml TNFa for the indicated UBB encoding human ubiquitin B (IMAGE 4943285) was obtained time, and then cells were lysed in Triton lysis buffer (10 mM HEPES, from Open Biosystems (Huntsville, AL). RICK-(1–319)-Fpk3-Myc, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM RICK-(1–328)-Fpk3-Myc, RICK-(1–356)-Fpk3-Myc, RICK-(1–390)- b-glycerophosphate, 10 mM NaF, 300 mMNa VO and 1 mM DTT). 3 4 Fpk3-Myc and RICK-(293–435)-Fpk3-Myc were generated by sub- The cell lysates were immunoprecipitated with mouse anti-NEMO cloning of RICK amplified by PCR using specific primers and Ab (BD Pharmingen). Resulting immunoprecipitates were washed subcloned into the KpnI and XhoI sites of pcDNA-Fpk3-Myc. three times with lysis buffer and once with kinase buffer (20 mM pMSCV-hygro-HA-RICK and pMSCV-puro-Nod1-Flag are generated HEPES pH 7.6, 2 mM MgCl , 2 mM MnCl ,10 mM ATP, 10 mM 2 2 by subcloning of the HA-RICK and Nod1-Flag genes into pMSCV- b-glycerophosphate, 10 mM NaF, 300 mMNa VO and 1 mM DTT). 3 4 hygro and pMSCV-puro, respectively (Clontech). Point mutations of Immunoprecipitated IKK complexes were incubated with GST-IkBa RICK and Ub, in which individual lysine residues were mutated to as substrates in kinase buffer with g- P-ATP at 301C for 30 min. The TM arginines, were constructed using the QuikChange site-directed samples were fractionated on SDS–PAGE followed by autoradio- mutagenesis kit (Stratagene). The fidelity of all constructs was graphy. The amounts of IKKb in the immunoprecipitates were confirmed by sequencing. pcDNA3-HA-TAK1, pcDNA3-T7-TAB1 detected by immunoblotting. and pcDNA3-Flag-TAB2 are gifts from Dr T Koseki (Tohoku University, Sendai, Japan). pcDNA3-A20 (Vincenz and Dixit, 1996) is a gift from Dr C Vincenz (University of Michigan, MI). Supplementary data pU6-A20i and pU6-Ctli, A20-specific and control siRNA plasmids Supplementary data are available at The EMBO Journal Online (Saitoh et al, 2005) are gifts from Dr S Yamaoka (Tokyo Medical and (http://www.embojournal.org). Dental University, Tokyo, Japan). HEK293T, parental 293 cells and MEFs were cultured as described (Inohara et al, 2000). RICK-, TAK1-, TRAF6- and TRAF2/ Acknowledgements TRAF5-deficient and control WT MEFs are described previously (Naito et al, 1999; Tada et al, 2001; Kobayashi et al, 2002; Shim This work was supported by NIH grants R01 GM60421 (to N et al, 2005). TAK1-deficient MEFs were provided by Dr S Ghosh Inohara) and R01 DK067628 (to G Nun˜ez). We are grateful to L (Yale University, New Heaven, CT) and TRAF6-deficient MEFs McAllister-Lucas (University of Michigan) for stimulating discus- by Dr J Inoue (Tokyo University, Tokyo, Japan). HEK293 cell sions; S Qiu, P Kuffer, Y Nishito and S Chen (University of lines constitutively expressing Nod1-Flag and HA-Nod2 with Michigan) for technical assistance and S Yamaoka (Tokyo Medical NF-kB-dependent GFP reporter were generated by transfection and Dental University, Tokyo), T Koseki (Tohoku University, Japan), of pMSCV-puro-Nod1-Flag and pMX2-puro-HA-Nod2, respectively, J Inoue (Tokyo University, Japan), C Vincenz (University and NFkB-eGFP (Wang et al, 2002), followed by antibiotic Michigan), S Ghosh (Yale University) and Ariad Pharmaceuticals selection. for materials. 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Journal

The EMBO Journal – Springer Journals

Published: Jan 23, 2008

Keywords: NLR; Nod1; Nod2; RICK; TAK1

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