Nod2, a Nod1/Apaf-1 Family Member That Is Restricted to Monocytes and Activates NF-κB*

Apaf-1 and Nod1 are members of a protein family, each of which contains a caspase recruitment domain (CARD) linked to a nucleotide-binding domain, which regulate apoptosis and/or NF-κB activation. Nod2, a third member of the family, was identified. Nod2 is composed of two N-terminal CARDs, a nucleotide-binding domain, and multiple C-terminal leucine-rich repeats. Although Nod1 and Apaf-1 were broadly expressed in tissues, the expression of Nod2 was highly restricted to monocytes. Nod2 induced nuclear factor κB (NF-κB) activation, which required IKKγ and was inhibited by dominant negative mutants of IκBα, IKKα, IKKβ, and IKKγ. Nod2 interacted with the serine-threonine kinase RICK via a homophilic CARD-CARD interaction. Furthermore, NF-κB activity induced by Nod2 correlated with its ability to interact with RICK and was specifically inhibited by a truncated mutant form of RICK containing its CARD. The identification of Nod2 defines a subfamily of Apaf-1-like proteins that function through RICK to activate a NF-κB signaling pathway.

Apaf-1 and Nod1 (also called CARD4) are members of a family of intracellular proteins that are composed of an Nterminal caspase recruitment domain (CARD), 1 a centrally located nucleotide-binding domain (NBD), and a C-terminal regulatory domain (1,2). Although Apaf-1 possesses WD40 repeats, Nod1 contains leucine-rich repeats (LRRs) in its C terminus (1,2). The structural and functional similarities between Apaf-1 and Nod1 suggest that these proteins share a common molecular mechanism for activation and effector function. In the case of Apaf-1, the WD-40 repeats act as a recognition domain for mitochondrial damage through binding to cytochrome c, allowing Apaf-1 to oligomerize and interact with procaspase-9 through a CARD-CARD homophilic interaction (3,4). Apaf-1 oligomerization is mediated by the NBD and is thought to induce the proximity and proteolytic activation of procaspase-9 molecules in the apoptosome complex (5,6).
Previous studies showed that Nod1 promotes apoptosis when overexpressed in cells, but unlike Apaf-1, it induces NF-B activation (1,2). NF-B activation induced by Nod1 is mediated by the association of the CARD of Nod1 with the corresponding CARD of RICK (also called RIP2 and CARDIAK), a protein kinase that activates NF-B (1,2,(7)(8)(9). Analyses with wild type (wt) and mutant forms of both Nod1 and RICK have suggested that Nod1 and RICK act in the same pathway of NF-B activation, where RICK functions as a downstream mediator of Nod1 signaling (1,2,10). Nod1 self-associates through its NBD and Nod1 oligomerization promotes proximity of RICK molecules and NF-B activation (10). Nod1 also displays striking similarity to a class of disease resistance (R) proteins found in plants (11,12). Like Nod1, these intracellular R proteins contain N-terminal effector domains linked to a NBD and share with Nod1 the presence of multiple LRRs located C-terminally of the NBD (1,12). After specific recognition of pathogen products, these R proteins mediate a defense response associated with metabolic alterations and localized cell death at the site of pathogen invasion (12). The LRRs of R proteins are highly diverse and appear to be involved in the recognition of a wide array of pathogen components (11,12). The binding partner of the LRRs of Nod1 remains unknown. The structural homology of Nod1 with plant R proteins suggest that other LRR-containing Nod-1-like molecules may exist in the human genome to allow activation of these molecules by different sets of intracellular stimuli. We report here the identification and characterization of Nod2, another LRR-containing protein with structural and functional similarity to Nod1. These studies indicate that Nod2 activates NF-B, but unlike Nod1, this new homologue is primarily expressed in monocytes.

MATERIALS AND METHODS
Isolation of the Nod2 cDNA-Nucleotide sequences encoding peptides with homology to Nod1 (GenBank TM accession numbers AC007728 and AQ534686) were found in the public genomic data base using the TBLASTN program. The coding region of human nod2 was obtained by RT-PCR amplification and 5Ј RACE using Nod2-specific oligonucleotide primers cDNA fragments and mRNA from primary mammary tissue as a template. 5Ј RACE was performed using a commercial kit (Roche Molecular Biochemicals). For PCR, three sets of primers were used: 5Ј-ATGTGCTCGCAGGAGGCTTTTCAGGCA-3Ј and 5Ј-CGCCTCACCC-ACCACCAGCACAGTGT-3Ј; 5Ј-CATGGCTGG-ACCCCCGCAGAAGAGCCCA-3Ј and 5Ј-CA-TGCCCGGGTTCATCTG-GCTCATCCGG-3Ј; and 5Ј-GCCATGCCCGGGTTCATCTGGCTCAT-C-3Ј and 5Ј-TGAGTCGAGACATGGGGAAAGCTGCTTC-3Ј. For 5Ј RACE, the initial primer 5Ј-AGCAGCTCGACCAGCTGGCTCCTCTG-* This work was supported in part by Grants CA-64556 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF178930.
§ Supported by funds from Tokushima University. ¶ Supported by a fellowship from the Spanish Ministry of Education and Science.
T-3Ј was used and the product was PCR amplified with the anchored primer and second Nod2-specific primer: 5Ј-GACAGGCCCAAGTAC-CCTTA-TTCCAGA-3Ј. The resulting cDNA fragments were digested with restriction enzymes and ligated to generate an unique cDNA containing the entire open reading frame of Nod2. The cDNA sequence was verified by nucleotide sequencing.
Northern Blot and RT-PCR Analysis of nod2 Expression-A 3.7kilobase fragment containing the entire Nod2 coding region was radiolabeled by random priming using a commercial kit (Roche Molecular Biochemicals) and applied for analysis of human poly(A) ϩ RNA blots from various tissues (CLONTECH Laboratories, Palo Alto, CA) according to the manufacturer's instructions. Peripheral blood leukocytes were obtained from heparinized venous blood from healthy volunteers by Ficoll-Paque (Amersham Pharmacia Biotech) density gradient centrifugation. Granulocytes were separated from red blood cells by brief incubation with hypotonic lysis buffer. The mononuclear cell population was fractionated into lymphocytes and monocytes by adherence to plastic dishes. For RT-PCR analysis, 2 g of total RNA from each cell preparations were used to generate first strand cDNA using a commercially available kit (Life Technologies, Inc.). Nod2 cDNA fragments corresponding to the Nod2 coding region were amplified by PCR using two sets of specific primers: P1, 5Ј-ATGTGCTCGCAGGAGGCTTTTC-AGGCA-3Ј; P2, 5Ј-CGCCTCACCCACCA-CCAGCACAGTGT-3Ј; P3, 5Ј-ATGTGCTCGCAGGAGGCTTTTCAGGCA-3Ј; and P4, 5Ј-CG-CCTCAC-CCACCACCAGCACAGTGT-3Ј. As a control, a cDNA fragment of the human glyceraldehyde-3-phosphate dehydrogenase was amplified using the primers 5Ј-GAGTCAACGGATTTGGTCGTAT-3Ј and 5Ј-AGTCTTCTGGGTGGCAGTGAT-3Ј.
Transfection, Expression, Immunoprecipitation, and Immunodetection of Tagged Proteins-HEK293T cells were cotransfected with pcDNA3-Nod2-HA and various expression plasmids as described (2). To test the interaction between wt RICK and Nod2 mutant proteins, HEK293T cells were cotransfected with pcDNA3-FLAG-RICK and wt or mutant Nod2 expression plasmids. Proteins coimmunoprecipitated with anti-HA antibody were detected with anti-FLAG antibody. To test the interaction between wt Nod2 and RICK mutants, HEK293T cells were cotransfected with pcDNA3-HA-Nod2 and pcDNA3-FLAG-RICK, pcDNA3-FLAG-RICK (1-374), or pcDNA3-FLAG-RICK (374 -540) (2). Proteins coimmunoprecipitated with anti-HA antibody were detected with anti-FLAG antibody. Proteins in total lysate were detected by anti-FLAG and anti-HA monoclonal antibody, respectively.
NF-B Activation Assays-Rat1 fibroblasts and its derivative 5R cell line (13) as well as HEK293T cells were cotransfected with 12 ng of the reporter construct pBVIx-Luc (10), plus indicated amounts of each expression plasmid and 120 ng of pEF-BOS-␤-gal in triplicate as described (2, 10). HL60 cells were induced to differentiate into monocytic cells by stimulation with 200 ng/ml of phorbol 12-myristate 13-acetate (Sigma) for 36 h prior to transfection. Differentiated adherent cells were transfected with 500 ng of pEF-BOS-␤-gal, 500 ng of pBVIx-Luc, and indicated amount of each plasmids by using Fugene 6 transfection reagent (Roche Molecular Biochemicals). 24 -36 h post-transfection, cell extracts were prepared, and the relative luciferase activity was measured as described (2,10). Results were normalized for transfection efficiency with values obtained with pEF-BOS-␤-gal.

RESULTS AND DISCUSSION
Identification of Nod2-To identify novel Nod1/Apaf-1-like molecules, we searched public genomic data bases for genes encoding proteins with homology to Nod1 (2). We found a genomic sequence in human chromosome 16 (GenBank TM accession number AC007728) that encodes a peptide with significant homology to the NBD of Nod1. Analysis with GeneFinder of the genomic region predicted a gene encoding a novel protein with significant homology to Nod1. To determine the ends of the coding region, we performed 5Ј RACE using an oligonucleotide complementary to sequences encoding the N terminus of the predicted protein and sequenced several expressed sequence tag cDNAs which contain partial sequences of the gene (GenBank TM accession numbers AA775466, AA910520, and AI090427). To amplify the cDNA containing the entire open reading frame, we performed RT-PCR with three sets of primers corresponding to overlapping sequences of the coding region of the gene. The predicted open reading frame encodes a protein of 1040 amino acids. A BLAST search of protein data bases indicated that the protein encoded by the new open reading frame was most homologous to Nod1 (34% amino acid identity). We termed this protein Nod2 given that is has a high level of homology with Nod1 and thus represents a novel member of the Apaf-1/Nod1 superfamily (Fig. 1). Analysis of the nucleotide sequence revealed two potential in-frame translation initiation sites separated by 81 nucleotides. Further analysis revealed that both translation initiation sites can be utilized in cells, although the longer open reading frame is preferentially used (see below). For simplicity, we refer here to the longer open reading frame as Nod2 and the product encoded by the shorter open reading frame as Nod2b. A BLAST search and domain analyses revealed that Nod2 is composed of two Nterminal CARDs (residues 28 -220) fused to a centrally located NBD domain (residues 273-577) containing consensus nucleotide-binding motifs followed by 10 tandem LRRs (residues 744 -1020) ( Figs. 1 and 2). Each of the 10 LRRs of Nod2 contained predicted ␣ helix and ␤ sheet sequences that are consistent with the prototypical horseshoe-shaped structure of LRRs (Ref. 14 and Fig. 2C). To our knowledge, Nod2 is the first protein to contain two CARDs.
Chromosomal Localization and Genomic Organization of the Human Nod2 Gene-We identified two human BAC clones, RP11-327F22 and RP11-401P9, containing the genomic se-quence of human Nod2 (GenBank TM accession numbers AC007728 and AC007608, respectively). These BAC clones mapped to chromosome 16 at q12. Comparison of Nod2 cDNA and genomic sequences revealed that the Nod2 gene contains 12 coding exons. 2 The Expression of Nod2 Is Most Abundant in Monocytes-Northern blot analysis showed Nod2 to be expressed as two ϳ7.0and ϳ5.5-kilobase transcripts in peripheral blood leukocytes with little or no detectable expression in various human tissues (Fig. 3A). This highly restricted pattern of expression is in contrast to that of Nod1 and Apaf-1 that are expressed in virtually all adult tissues although at different levels (1,2,3).
To determine the cells that express Nod2, we fractionated peripheral blood leukocytes into granulocyte, lymphocyte, and monocyte populations and performed RT-PCR analysis with two different sets of oligonucleotide primers complementary to Nod2 coding sequences. The analysis showed that Nod2 was 2 Y. Ogura, N. Inohara, and G. Nú ñ ez, unpublished results. . Hydrophobic residues are shown in reverse highlighting. Negatively and positively charged residues are highlighted in light and dark gray, respectively. Proline and glycine residues (␣␤ breaker) are in bold type. The putative ␣helices, H1 to H5, are shown according to the three-dimensional structure of the CARD of RAIDD (19). B, alignment of NBDs of Nod2, Nod1, Apaf-1, and Ced-4. The residues identical and similar to those of Nod2 are shown by reverse and dark highlighting, respectively. The consensus sequence of the P-loop (Walker A box) and the Mg 2ϩ binding site (Walker B box) are indicated by boxes. The residues identical and similar to those of Nod2 are shown by reverse and dark highlighting, respectively. C, alignment of LRRs of Nod2. The conserved positions with leucine and other hydrophobic residues are indicated by dark and light gray highlighting, respectively. The putative ␣ helix and ␤ sheet are shown according to the three-dimensional structure of the ribonuclease inhibitor (14). expressed primarily in monocytes (Fig. 3B). Because the Nod2 sequence contained two potential in-frame translation initiation sites separated by 81 nucleotides (Fig. 3C), we determined their usage by transfection of a Nod2 construct containing both translation initiation sites into HEK293T cells. Because the difference in size between both predicted Nod2 products is only 27 amino acids, we expressed a C-terminally truncated Nod2 lacking residues 302-1040 to facilitate the identification of the translation initiation sites. As a control, we engineered Nod2 plasmids that express each translation initiation site separately within a canonical Kozak's translation initiation motif. The analysis revealed that both translation initiation sites in the Nod2 open reading frame were used, although the most N-terminal translation initiation codon was more efficient as assessed by immunoblotting of cell extracts with an antibody that recognizes a C-terminal HA tag (Fig. 3D).
Nod2 Activates NF-B-Because Nod2 shows the highest homology to Nod1 and the latter protein activates NF-B, we first tested whether expression of Nod2 activates NF-B by transfection of Nod2 plasmids into HEK293T cells. Transfection of the wt Nod2 cDNA induced potent activation of NF-B, as measured with a reporter luciferase construct (see below). In addition, we tested the Nod2b cDNA and obtained similar results to those observed with Nod2. 2 We generated a panel of Nod2 mutants to determine the regions of Nod2 that are required for NF-B activation (Fig. 4A). Immunoblotting analysis revealed that these mutant constructs were expressed when transiently transfected into HEK293T cells (Fig. 4B). Expression of as little as 3 ng of wt Nod2 induced 18-fold activation of NF-B (Fig. 4C). Expression of a Nod2 mutant form lacking the LRRs resulted in enhanced NF-B activation, whereas mutants expressing the LRRs or the NBD alone were inactive (Fig.  4C). The enhanced activity of the Nod2 mutant lacking the LRRs could not be explained by increased expression of the mutant (Fig. 4A). Consistent with these results, it was shown previously that deletion of the LRRs of Nod1 and WD-40 repeats of Apaf-1 results in enhanced NF-B activation and increased ability to activate procaspase-9, respectively (2,5,6). Deletion of the CARDs of Nod2, either singly or in combination, resulted in total loss of NF-B activity (Fig. 4C). However, expression of both CARDs alone, but not each CARD separately, was sufficient for NF-B activation (Fig. 4C). Thus, both CARDs of Nod2 are necessary and sufficient for NF-B activation, suggesting that the CARDs acts as an effector domain in Nod2 signaling. The conserved lysine residue in the P-loop of Nod1 and Apaf-1 is important for the activities of these proteins (2,10,15). Similarly, replacement of the corresponding lysine for arginine in Nod2 resulted in diminished NF-B activity that was rescued at least in part by deletion of the LRRs (Fig. 4C).
We also investigated the ability of Nod2 to induce apoptosis. We found that overexpression of Nod2 did not induce apoptosis by itself but enhanced apoptosis induced by caspase-9 expression. 2 These results are similar to those reported for Nod1 and Apaf-1 (1, 2). forms of IKK␣, IKK␤, and IB that have been shown to act as dominant inhibitors of their corresponding endogenous counterparts and/or the IKK complex (16). In addition, we used a truncated mutant of IKK␥/Nemo (residues 134 -419) that is defective in IKK␣ and IKK␤ binding and acts as an inhibitor of NF-B activation induced by RIP and RICK (10). The NF-B activity induced by Nod2 as well as that induced by TNF␣ stimulation were greatly inhibited by mutant IKK␣, IKK␤, IKK␥, and IB␣ (Fig. 5A). Because RICK has been shown to serve as a downstream target of Nod1 (1, 2, 10), we used a truncated form of RICK containing its CARD (residues 406 -540) that acts as a dominant inhibitor of Nod1 activity (1) to test whether NF-B activation induced by Nod2 is similarly inhibited by this RICK mutant. We found that NF-B activation induced by Nod2 was inhibited by mutant RICK but not by a mutant form of RIP that expresses its death effector domain (Fig. 5A). The inhibition by the CARD of RICK was specific in that it did not interfere with ability of TNF␣ to induce NF-B, an activity that was inhibited by the RIP mutant (Fig. 5A). To verify that Nod2 acts upstream of the IKK complex to activate NF-B, we tested the ability of Nod2 to activate NF-B in parental Rat1 fibroblasts and 5R cells, a Rat1-derivative cell line that is defective in IKK␥, an essential subunit of the IKKs (13). We found that Nod2, as well as Nod1 and TNF␣, induced NF-B activity in parental Rat1 cells but not in IKK␥-deficient 5R cells (Fig. 5B). As a control, expression of IKK␤, which functions downstream of IKK␥, induced NF-B activation in both Rat1 and 5R cell lines (Fig. 5B). These results indicate that Nod2 acts through IKK␥/IKK␣/IKK␤ to activate NF-B.

NF-B Activation Induced by Nod2 Requires IKK␥ and Is Inhibited by Dominant Negative Forms of IKKs and RICK-A main pathway of NF-B activation is mediated by IB kinases
Nod2 Associates with RICK via a Homophilic CARD-CARD Interaction-The CARD motif functions as an effector domain that mediates specific homophilic interaction with downstream CARD-containing molecules (17). Because NF-B activation induced by Nod2 was inhibited by a RICK truncated mutant, we tested whether RICK could act as a direct downstream mediator of Nod2 signaling. To test a physical association between Nod2 and RICK, HEK293T cells were cotransfected with plasmids expressing HA-tagged wt or mutant forms of Nod2 and FLAG-tagged RICK, and cellular extracts were immunoprecipitated with anti-HA antibody. Immunoblotting with anti-FLAG antibody revealed that RICK associated with Nod2 (Fig.  6A). The association was mediated by both CARDs of Nod2, because only Nod2 proteins containing both CARDs were capable of interacting with RICK (Fig. 6, A and B). The association of Nod2 with RICK was specific in that Nod2 did not associate with several CARD-containing proteins including Apaf-1, caspase-1, caspase-4, c-IAP-1, c-IAP2, procaspase-9, Bcl-10, RAIDD, and Ced-4 nor with several molecules that activate NF-B including TRAF-1, TRAF-2, TRAF-5, TRAF-6, RIP, NIK, TRADD, IKK␣, IKK␤, or IKK␥. 2 To determine the region of RICK that associates with Nod2, mutant forms of RICK expressing the CARD (residues 374 -540) or lacking the CARD (residues 1-374) were coexpressed with Nod2, and the cell extracts were immunoprecipitated with anti-FLAG antibody. The analysis showed that only the CARD of RICK coimmunoprecipitated with Nod2 (Fig. 6C). Thus, Nod2 and RICK associate via a homophilic CARD-CARD interaction.
Enforced Oligomerization of Nod2 Induces NF-B Activation-Previous studies showed that the NBD of Nod1 and Apaf-1 mediates oligomerization of these molecules, an activity that is critical for NF-B and caspase-9 activation, respectively (5,6,10). In the case of Nod1, its oligomerization appears to promote proximity of RICK and NF-B activation (10). To test a similar role for Nod2, we constructed plasmids to express chimeric proteins in which wt or Nod2 mutants were fused to three tandem repeated dimerization domains of Fpk (Fpk3), which can be oligomerized by the cell-permeable ligand AP1510 (18). Immunoblotting analysis showed that the chimeric Fpk3-Nod2 constructs were expressed when transfected in HEK293T cells (Fig. 7A). Because wt Nod2 alone induces NF-B activation, we expressed suboptimal amounts of the chimeric Fpk3-Nod2 constructs into HEK293T cells. Under these experimental conditions, expression of Nod2-Fpk3 induced NF-B activation in a ligand-dependent manner (Fig. 7B). Consistent with the results shown in Fig. 4C, enforced oligomerization of both CARDs but not each CARD singly induced NF-B activation. Similarly, NF-B activation induced by a Nod2 P-loop mutant lacking the LRRs (K305R⌬LRR), which have reduced ability to induce NF-B activation, was enhanced by enforced oligomerization (Fig. 7B). A Nod2-Fpk3 construct lacking the LRRs induced NF-B activation in the absence and presence of AP1510 (Fig. 7B). The latter result might be explained by our observations that Nod2 lacking the LRRs has enhanced activity to self-associate and induce NF-B (Ref. 10 and Fig. 4C). To demonstrate that Nod2 can function in monocytic cells, we transfected Nod2 constructs into HL60 cells stimulated with phorbol 12-myristate 13-acetate. Similarly to the case of HEK293T cells, transient expression of Nod2 induced NF-B activation in monocytic HL60 cells (Fig. 7C, left panel). Furthermore, enforced oligomerization of wild type Nod2-Fpk3 or CARDs-Fpk3 was sufficient for NF-B activation in monocytic HL60 cells (Fig. 7C, right panel).
We have shown that Nod2 is a member of the Nod1/Apaf-1 family that activates NF-B through interactions with its Nterminal CARDs, as these domains were necessary and sufficient for NF-B activation. Nod2 associated with RICK via a homophilic CARD-CARD interaction. The NF-B-inducing activity of Nod2 correlated with its ability to associate with RICK and was inhibited by a RICK mutant, suggesting that RICK is a direct downstream target of Nod2. Thus, the signaling pathways of both Nod1 and Nod2 appear to utilize RICK as a downstream mediator of NF-B activation. In contrast to Nod1, two tandem CARDs are present in the N terminus of Nod2, and both were required for association with RICK and NF-B activation. To our knowledge, Nod2 is the first molecule to contain two CARDs. The molecular basis underlying the requirement of both CARDs of Nod2 for RICK binding remains unclear. The presence of both CARDs may enhance the affinity for the CARD of RICK. Another possibility is that upon an initial interaction involving a CARD of Nod2 and the CARD of RICK, Nod2 may undergo a conformational change that allows the second CARD to associate with high affinity to RICK. The intermediate region of RICK associates with IKK␥ (10), providing a direct link between Nod1/Nod2 and the IKK complex. Consistent with this model, we have shown that NF-B activation induced by Nod2 as well as that induced by Nod1 required IKK␥ and was inhibited by dominant negative forms of IKK␥, IKK␣, and IKK␤. The functional role for the LRRs of Nod1 and Nod2 remains unclear. The LRR is a repeated protein-protein interaction module that is presumably involved in the activation of Nod1 and Nod2 by upstream signals. In the case of plant NBD/LRR-containing R proteins, their LRRs appear to be important for the recognition of pathogen components, and their N-terminal domains appear to mediate a signaling cascade that regulates gene expression (11,12). Recent results indicate that Nod1 and Nod2 can confer responsiveness to bacterial components, suggesting that these proteins are functional counterparts of plant R proteins (20). Because both Nod1 and Nod2 activate NF-B, their LRRs may act to recognize a different set of intracellular stimuli that mediate Nod1 and Nod2 oligomerization and association with RICK. Consistent with this model, Nod1 and Nod2 conferred a different pattern of response to bacterial products including lipopolysaccharides (20), suggesting that they are activated by different microbial stimuli. Because Nod2 is expressed primarily in monocytes, Nod2 might serve as an intracellular receptor for bacterial lipopolysaccharides and/or other bacterial products transducing signals in the monocyte/macrophage that lead to activation of NF-B and transcription of regulatory genes.