Nod1, an Apaf-1-like Activator of Caspase-9 and Nuclear Factor-κB*

Ced-4 and Apaf-1 belong to a major class of apoptosis regulators that contain caspase-recruitment (CARD) and nucleotide-binding oligomerization domains. Nod1, a protein with an NH2-terminal CARD-linked to a nucleotide-binding domain and a COOH-terminal segment with multiple leucine-rich repeats, was identified. Nod-1 was found to bind to multiple caspases with long prodomains, but specifically activated caspase-9 and promoted caspase-9-induced apoptosis. As reported for Apaf-1, Nod1 required both the CARD and P-loop for function. Unlike Apaf-1, Nod1 induced activation of nuclear factor-kappa-B (NF-κB) and bound RICK, a CARD-containing kinase that also induces NF-κB activation. Nod1 mutants inhibited NF-κB activity induced by RICK, but not that resulting from tumor necrosis factor-α stimulation. Thus, Nod1 is a leucine-rich repeat-containing Apaf-1-like molecule that can regulate both apoptosis and NF-κB activation pathways.

Recent analyses of Apaf-1-deficient mice suggest a central role of Apaf-1 in apoptosis induced by chemotherapeutic drugs, ultraviolet radiation, and signals associated with neuronal development (15,16). Mutant mice deficient in caspase-9 exhibit abnormalities similar, but not identical, to those observed in mice lacking Apaf-1 (17,18). Unlike C. elegans, mice and humans contain multiple initiator caspases, suggesting that mammalian genomes may contain caspase activators other than Apaf-1. Significantly, Apaf-1 knockout mice lack apparent abnormalities in tissues, such as the thymus, whose appropriate cellular development depends on apoptosis (15,16). The latter observation suggests the existence of additional Apaf-1like molecules or apoptosis pathways that are Apaf-1-independent. In this study, we identified and characterized Nod1, an Apaf-1-like protein that associates with and regulates procaspase-9. Unlike Apaf-1, however, Nod1 contains leucine-rich repeats (LRRs) and induces NF-B activation.

MATERIALS AND METHODS
Isolation of the Nod1 cDNA-The partial nucleotide sequences of cDNAs encoding peptides with homology to a consensus CARD motif (10) were found in the EST data base of Human Genome Sciences using the TBLASTN and HMM programs. Overlapping cDNA clones that encode the 5Ј end of the Nod1 mRNA were isolated from a B-cell human lymphoma cDNA library by the polymerase chain reaction (PCR) using two Nod1-specific primers and two universal primers for the vector. The entire open reading frame was verified by sequence analysis of several Nod1 cDNAs isolated from a primary mammary tumor, normal mammary tissue, and BJAB and 293 cells.
Northern Blot and in Situ Hybridization Analysis-A 2-kilobase pair HindIII fragment of Nod1 cDNA was radiolabeled by random priming using a commercial kit (Boeringer Mannheim) and applied for Northern blot analysis of human poly(A) ϩ RNA blots from various tissues (CLON-TECH) according to the manufacturer's instructions. Slides containing mouse embryo tissues were prepared as described (19). Each specimen was hybridized with a digoxigenin-labeled antisense RNA probe synthesized from a mouse Nod1 cDNA (EST clone 944836) using an in vitro transcription kit (Promega). Hybridization, development, and mounting of slides were performed as described (19).
Transfection, Expression, Immunoprecipitation, Immunodetection of Tagged Proteins, and Laser Scanning Confocal Microscopy-293T cells were co-transfected with pcDNA3-Nod1-Myc or pcDNA3-Nod1-HA and expression plasmids indicated in Fig. 4 as described (21). 24 h posttransfection, proteins co-immunoprecipitated with anti-Myc polyclonal antibody were detected with anti-FLAG or anti-HA monoclonal antibody as described (21). As control, each protein in total lysate was detected by immunoblotting. The binding of these factors was confirmed by reciprocal experiments using immunoprecipitation with anti-HA or anti-FLAG antibody and detection with anti-Myc antibody (21). Localization of HA-tagged Nod1 in transfected 293T cells was analyzed by confocal microscopy as described (35).
NF-B Activation Assays-293 cells were co-transfected with 200 ng of pBIIx-Luc (32) or control plasmid pf-Luc plus each expression plasmid and 100 ng of pcDNA3-␤-gal in triplicate in the presence of 2 g of pcDNA3-p35 to prevent caspase activation and cell death. 24 h posttransfection, cell extract was prepared, and its relative luciferase activity was measured as described (36). To test the interaction between WT RICK and Nod1 mutants, 293T cells were co-transfected with pcDNA3-FLAG-RICK and WT or mutant Nod1 expression plasmids. Proteins co-immunoprecipitated with anti-HA antibody were detected with anti-FLAG antibody. As control, RICK and Nod1 proteins in total lysate were detected by anti-FLAG and anti-HA monoclonal antibody, respectively. To test the interaction between WT Nod1 and RICK mutants, 293T cells were co-transfected with pcDNA3-HA-Nod1 and pcDNA3-FLAG-RICK, pcDNA3-FLAG-RICK (1-374), or pcDNA3-FLAG-RICK (374 -540) (21). Proteins co-immunoprecipitated with anti-HA antibody were detected with anti-FLAG antibody. As control, RICK and Nod1 proteins in total lysate were detected by anti-FLAG and anti-HA monoclonal antibody, respectively.

RESULTS AND DISCUSSION
Identification of Nod1-To identify novel Apaf-1-like molecules, we searched EST data bases for cDNAs encoding proteins with homology to a consensus CARD motif and found a novel EST cDNA that encodes a CARD protein (10). Sequence analysis of multiple cDNAs encoding the same protein revealed an open reading frame of 953 amino acids (Fig. 1). We termed this novel protein Nod1. Data base search and alignment analyses revealed that Nod1 is composed of an NH 2 -terminal CARD fused to a region of 319 amino acids (residues 171-490) with homology to an ATP/GTPase domain that includes consensus nucleotide-binding motifs (Walker A box, P-loop and Walker B box, Mg 2ϩ binding site, Ref. 37) (Figs. 1 and 2B). The COOHterminal region of Nod1 (residues 649 -953) contained 10 LRRs that function as a protein-protein interaction domain in many proteins (38), most significantly in several plant resistance proteins that contain NBDs with significant amino acid homology to those of Apaf-1 and Ced-4 (9). The NBD of Nod1 is most homologous to that of CIITA (Fig. 2B), a transcription activator with LRRs (39). The CARD of Nod1 was most similar to the CARD of RICK, also called RIP2/CARDIAK, a serine-threonine kinase that regulates apoptosis and NF-B activation ( Fig. 2A) (21,40,41). Alignment analysis revealed that each of the 10 LRRs of Nod1 contained a putative ␣ helix and ␤ sheet (Fig.  2C). This LRR alignment of Nod1 is consistent with LRRs in several proteins that form a horseshoe-shaped structure with a parallel ␤ sheet lining the inner circumference of the horseshoe and ␣ helices flanking its outer circumference (38,42).
Nod1 Is Expressed in Multiple Tissues and Exhibits Re-stricted Distribution in Embryonic Tissues-Northern blot analysis showed Nod1 to be expressed as a 4.4-kilobase transcript in various human adult tissues including heart, placenta, lung, skeletal muscle, liver, kidney, spleen, thymus, and ovary (Fig. 3A). We evaluated the expression of Nod1 mRNA in the mouse embryo by in situ hybridization. At stage E15.5 of embryonic development, Nod1 labeling was detected in the liver, thymus, cortical region of the kidney, lung, gut epithelium, and in certain regions of the central nervous system (Fig.  3B, panels a-f). Therefore, expression of Nod1 appears to be regulated differentially in embryonic and adult tissues. In the developing brain, Nod1 labeling was detected in the inferior tectal neuroepithelium of the developing inferior colliculus, germinal layer of the neocortex, olfactory epithelium, and choroid plexus (Fig. 3B, panel a). The expression of Nod1 in the developing brain is different and more restricted to that observed for Apaf-1 (15). Genomic Organization and Chromosomal Localization of the Human Nod1 Gene-Search of the human genomic data base with the Nod1 cDNA mapped the Nod1 locus to chromosome 7 at 7p14-15 within human genomic PAC clone DJ0777O23 and BAC clone GS114I09 (GenBank TM accession numbers AC005154 and AC006027, respectively). Comparison of genomic and cDNA sequences revealed that the Nod1 gene is composed of 14 exons, including 7 coding and 7 noncoding exons. 2 Nod1 Interacts with Caspases with Long Prodomains and RICK-The CARD and its structurally related death effector domain (DED) are peptide motifs that mediate interactions between apoptosis-regulatory proteins (3). To identify the binding partners of Nod1, we tested the ability of Nod1 to associate with a panel of CARD and DED-containing proteins in 293T cells (Fig. 4). We also examined whether Nod1 interacts with additional proteins that regulate apoptosis or NF-B signaling, as Nod1 was found to induce activation of NF-B (see below). In these experiments, Nod1 was tagged at the COOH terminus with a Myc-or HA-epitope and transiently expressed in 293T cells. Overexpressed Nod1 was a cytosolic protein as determined by confocal microscopy. 2 Co-immunoprecipitation assays with Nod1 and tagged proteins revealed that Nod1 preferentially interacted with several procaspases containing long prodomains with CARDs or DEDs, including caspase-1, caspase-2, caspase-4, caspase-8, and caspase-9, but not those with short prodomains like caspase-3 or caspase-7 (Fig. 4). In addition, Nod1 interacted with Nod1 itself, with RICK and CLARP, but not with other CARD or DED-containing proteins including RAIDD, Apaf-1, Ced-4, Bcl-10, FADD, PEA15, or DEDD (Fig. 4). In addition, Nod1 failed to associate with multiple regulators of NF-B activation, including several TRAFs, IKK␣, IKK␤, NIK, or A20 (Fig. 4).
Nod1 Enhances Apoptosis Induced by Caspase-9 but Not That Mediated by Caspase-4, Caspase-8, or CLARP-In order to determine the functional relevance of these interactions, we transiently expressed HA-tagged Nod1 in 293T cells and tested the ability of Nod1 to modulate the apoptosis regulatory function of binding proteins in 293T cells. Although Nod1 did not activate apoptosis by itself, it significantly enhanced apoptosis induced by caspase-9, but not that induced by caspase-4, caspase-8, or CLARP (Fig. 5, A and B). Consistent with its inability to modulate caspase-8-mediated apoptosis, Nod1 did not affect apoptosis induced by FADD, CLARP, TRAMP, or TNFR1 (Fig. 5B), which induce apoptosis through caspase-8 activation (3). In addition, expression of Nod1 did not change the apoptosis regulatory function of caspase-1, caspase-2, or 2 N. Inohara and G. Nuñ ez, unpublished data. RICK. 2 These results indicate that the ability of Nod1 to regulate apoptosis is highly specific and limited thus far to that activated by caspase-9. The effect of Nod1 on caspase-9-induced apoptosis was not due to increased expression of caspase-9, as there was no alteration in the levels of this procaspase-9 in cells transfected with Nod1 plasmids compared with control plasmids (Fig. 5C).
Nod1 Promotes the Activation of Procaspase-9 but Not That of Procaspase-8 -Production of enzymatically active caspases including caspase-9 requires proteolytic processing of the immature form of the enzyme (3). To determine if Nod1 enhances caspase-9 processing, 293T cells were transiently expressed in the presence or absence of HA-tagged Nod1. Expression of Nod1 induced the proteolytic activation of procaspase-9, but not that of procaspase-8 (Fig. 5C). Thus, the ability of Nod1 to enhance caspase-9, but not caspase-8-mediated, apoptosis correlates with its ability to induce proteolytic processing of procaspases. By analogy with Nod1, Apaf-1 also binds to several caspases with long prodomains, but only promotes the activation of procaspase-9 (24), suggesting that binding of Nod1 or Apaf-1 alone is not sufficient for activation of target proteins.
The CARD and NBD Are Essential for Nod1 to Activate Procaspase-9 -We engineered mutant forms of Nod1 to determine the regions of Nod1 that are required for caspase-9 activation. Expression of a mutant containing the CARD and NBD (residues 1-648) retained its ability to enhance caspase-9-induced apoptosis, but the mutant containing the LRRs (residues 649 -953) alone did not (Fig. 6B). This indicates that Nod1 promotes caspase-9 apoptosis through the CARD and/or NBD, as it was reported previously for Apaf-1 and Ced-4 (38 -42). The conserved lysine residue in the P-loop of Ced-4 and Apaf-1 is critical for both caspase activation and apoptosis enhance-ment (13,43,44) as are the conserved residues in the CARD of RAIDD (41). We therefore introduced point mutations in highly conserved residues of the CARD (V41Q) and the P-loop of Nod1 (K208R), the corresponding mutations of which results in lossof-function of RAIDD and Apaf-1/Ced-4, respectively (13,(43)(44)(45). Both V41Q and K208R mutations inhibited the ability of Nod1 to enhance caspase-9-induced apoptosis and caspase-9 maturation (Fig. 6B). Thus, the CARD and NBD appear essential for Nod1 to activate procaspase-9 and to promote apoptosis. These results suggest that Nod1 and Apaf-1 activate procaspase-9 by a similar mechanism, which may involve conformational changes and NBD oligomerization of these caspase activators, bringing several molecules of procaspase-9 into close proximity (3,11,13).
Nod1 Requires Its CARD to Bind Procaspase-9 -Apaf-1 associates with caspase-9 via the CARD (8,11,46). We therefore determined the regions of Nod1 that are required for association with procaspase-9. We transiently co-transfected 293T cells with expression plasmids producing caspase-9 and wildtype or mutant forms of Nod1. Immunoblotting analysis of protein complexes revealed that residues 1-648 of Nod1 containing the CARD and NBD co-immunoprecipitated with caspase-9, but the LRR domain did not (Fig. 6C). The K208R mutant still bound to caspase-9, although its binding was reduced when compared with wild-type Nod1 (Fig. 4C). Nod1 with a mutation in a highly conserved residue of the CARD (V41Q) failed to associate with caspase-9 (Fig. 6C). Another mutant lacking the CARD (residues 126 -953) also failed to interact with and activate procaspase-9 2 . Thus, the CARD is essential for Nod-1 to bind and activate procaspase-9, as well as to promote apoptosis.
Nod1 Induces NF-B Activation and Synergizes with RICK- The results presented in Fig. 4 showed that Nod1 also interacts with RICK, a CARD-containing serine-threonine kinase that promotes apoptosis and NF-B activation (21,40,41). Because RICK induces NF-B activation, we asked if Nod-1 could activate NF-B. To test if Nod1 activates NF-B, a Nod1 expression plasmid was co-transfected into 293T cells with pBIIx-Luc, a luciferase NF-B reported plasmid or pf-Luc control plasmid lacking NF-B binding sites (36). Nod1 induced activation of NF-B in a dose-dependent manner, whereas Apaf-1 did not (Fig. 7A). We also confirmed that Nod1 induced NF-B activation in parental 293 cells and HeLa cells. 2 RICK alone induced NF-B activation as reported (40,41). Importantly, co-expression of RICK and Nod1 resulted in synergistic NF-B activation (Fig. 7A).
Nod1 Acts Upstream of NIK, IKKs, and I-B␣ to Induce NF-B Activation-Several surface receptors and intracellular mediators, such as RIP, TRAF2, and TRAF6, use different transducing molecules to activate a common set of intracellular components that lead to degradation of I-B␣ and release of cytoplasmic NF-B (47,48). Stimulation of these upstream components culminate in the activation of a common set of signaling molecules that include NIK and IKKs, which lead to inactivation of I-B␣ (33,34). We used mutants forms of these signaling components to map the site of Nod1 action in the NF-B activation pathway. The NF-B activity of Nod1 was abolished or greatly inhibited by dominant negative forms of NIK, IKK␣, IKK␤, and I-B␣ (Fig. 7B), but not by mutant forms of TRAF2, TRAF6, or RIP, which can inhibit NF-B activation mediated by surface receptors (30,32,50). To test if Nod-1 activates NF-B independently of its ability to promote caspase-9-induced apoptosis, we used a catalytically inactive mutant of caspase-9 that acts as a dominant negative and inhibits caspase-9-induced apoptosis (46,51). Expression of mutant caspase-9 did not inhibit the activation of NF-B induced by Nod1 (Fig. 7B). These results suggest that Nod1 acts upstream of NIK, IKKs, and I-B␣, but in a different pathway, or downstream of TRAF2, TRAF6, or RIP. Moreover, the result with dominant negative caspase-9 suggests that the NF-Binducing activity of Nod1 does not require caspase-9 activity.
Nod1 Interacts with RICK via an Homophilic CARD Association-Next we determined the regions of Nod1 involved in NF-B activation. Expression of residues 1-648 of Nod1 induced NF-B, while mutants containing the NBD plus LRRs (residues 126 -953) or LRRs (residues 649 -953) alone did not (Fig. 7C). Both V41Q and K208R mutations inhibited the ability of Nod1 to induce NF-B activation (Fig. 7C). Thus, the CARD and the P-loop of Nod1 are essential for Nod-1 to activate NF-B. To determine the regions of Nod1 and RICK involved in their interaction, 293T cells were transiently cotransfected with plasmids producing WT or mutant Nod1 and RICK. The analysis showed that the NH 2 -terminal 1-648 amino acids, but not the LRRs of Nod1, mediate the interaction with RICK (Fig. 7D). The V41Q point mutant in the CARD, but not the K208R P-loop mutant, abolished the association with RICK (Fig. 7D). In addition, another Nod-1 mutant (residues 126 -953) failed to associate with RICK. 2 This result indicates that the CARD of Nod1 is critical for the Nod1-RICK interaction (Fig. 7D). Reciprocal experiments revealed that the CARD, but not the kinase domain of RICK, was required for the association with Nod1 (Fig. 7E). Thus, these results suggest that the Nod1/RICK interaction is mediated via their corresponding CARDs.
Mutants Forms of Nod1 Inhibit NF-B Activation Induced by RICK, but Not That Resulting from Tumor Necrosis Factor-␣ Stimulation-Expression of Nod1 promotes both procaspase-9 activation and NF-B activation, and the latter may involve the association of Nod1 with RICK. To determine if the NF-Binducing activity of RICK requires Nod1 or caspase-9 activity, we co-transfected 293T cells with plasmids expressing RICK and mutant forms of Nod1 or catalytically inactive caspase-9 or caspase-3 (as a control). Expression of Nod1 mutants lacking the CARD (residues 126 -953) or containing only the LRRs (residues 649 -953) inhibited RICK-mediated NF-B activation, but not that induced by tumor necrosis factor ␣ (TNF␣) stimulation (Fig. 7F). Together with the results shown in Fig.  7B, the analysis suggest that both RICK and Nod1 activate a TNF␣-independent pathway of NF-B activation. In addition, the NF-B-inducing activity of RICK was unaffected by mutant FIG. 6. Mutational analysis of Nod1. A, wild-type (WT) and mutant Nod1 proteins. CARD, NBD, and LRRs are indicated by gray, hatched, and closed boxes, respectively. B, regulation of caspase-9-induced apoptosis and caspase-9 maturation by Nod1 mutants. 293T cells were co-transfected with vector control or plasmids expressing caspase-9 and WT or mutant Nod1 proteins, Apaf-1, TRAF2, or vector control. The results are given as the mean Ϯ S.D. of triplicate cultures. The proform and mature form of tagged caspase-9 were detected with anti-FLAG antibody in total lysate from an additional culture of the same experiment. C, interaction between caspase-9 and mutant Nod1. 293T cells were co-transfected with vector control, WT or mutant HA-tagged Nod1 expression plasmid and Myc-tagged caspase-9 C287S expression plasmid. Co-immunoprecipitated proteins with anti-Myc (upper panel) or anti-HA (middle panel) polyclonal antibody were detected by anti-HA monoclonal antibody. As control, immunoprecipitated caspase-9 was detected by re-blotting with anti-Myc monoclonal antibody (lower panel). The bands labeled as IgH represent immunoglobulin heavy chain.
caspase-9 or caspase-3, but was inhibited by mutant I-B␣ (Fig.  7F). The latter results indicate that caspase-9 activity is not required for Nod1 or RICK to activate NF-B activation and further suggest that both TNF␣ and the RICK/Nod-1 signaling pathways use common downstream components such as NIK, IKKs, and I-B␣ for activation of NF-B.
These studies identify and characterize Nod1, a protein structurally related to Apaf-1. Like Apaf-1, Nod1 binds caspase-9 and promotes apoptosis induced by caspase-9. Both Apaf-1 and Nod1 share NH 2 -terminal CARDs followed by NBDs. The CARDs of both Nod1 and Apaf-1 appear to mediate, at least in part, the association of the proteins with the CARD of procaspase-9 (8,11). The NBD is involved in Apaf-1 and Ced-4 self-association, a process that may result in procaspase aggregation and autoactivation (11)(12)(13). Nod1 can also selfassociate, suggesting that Apaf-1 and Nod1 may share a common mechanism for procaspase-9 activation. Cytochrome c released from damaged mitochondria binds to Apaf-1, presumably via its WD-40 repeat region, and acts as a co-factor required for procaspase-9 activation (8,13,14). Nod1 lacks WD-40 repeat region and contains instead LRRs. The LRR domain is known to be involved in protein-protein interactions and in bacterial lipopolysaccharide binding to Toll-like proteins (38,52). This suggests that different upstream signal molecules regulate the activation of Apaf-1 and Nod1. Apaf-1 exhibits homology to plant R resistance gene products and related proteins, which also contain NBDs linked to LRRs (9). These proteins act as intracellular receptors for products of invading pathogens and signal a plant response against pathogens that include activation of a form of programmed cell death at the site of pathogen invasion (53). The LRR domain of the R gene products is responsible for specificity for a particular pathogen (54). By analogy, the LRRs of Nod1 may bind to upstream activators that regulate its ability to activate procaspase-9 and/or NF-B. The structural similarity between plant R resistance proteins and Ced-4-like molecules suggests that the basic regulation of cell death machinery is evolutionarily conserved in plants, nematodes, and vertebrates. , or caspase-9 C287S expression plasmid in the presence of pBIIx-Luc and pcDNA-3-␤-gal. C, Activation of NF-B by Nod1 mutants. Induction of NF-B activation was determined from triplicate culture of 293T cells co-transfected with WT or mutant Nod1 expression plasmid and reporter plasmids. D, interaction between RICK and mutant Nod1 proteins. 293T cells were co-transfected with vector control, WT, or mutant Nod1 expression plasmid and RICK expression plasmid. The co-immunoprecipitated RICK was detected by anti-FLAG antibody (upper panel). As control, total lysates were blotted with anti-FLAG (middle panel), or anti-HA (lower panel) antibody. E, interaction between Nod1 and mutant RICK proteins. 293T cells were co-transfected with WT (WT) or mutant RICK (N for the kinase domain and C for the CARD) expression plasmid and Nod1 expression plasmid or vector control. The co-immunoprecipitated RICK was detected by anti-FLAG antibody (upper panel). As control, total lysates were blotted with anti-FLAG (middle panel) or anti-HA (lower panel) antibody. F, suppression of RICK-induced, but not TNF␣-induced, NF-B activation by Nod1 mutants. Induction of NF-B activation was determined from triplicate culture of 293T cells co-transfected with WT or mutant Nod1, caspase-9 C287S, caspase-3 C163S, I-B␣ S32A/S36A expression plasmids, and the reporter plasmids in the presence of RICK expression plasmid (closed bars). 293T cells were also transfected with WT or mutant Nod1 expression plasmid in the absence of RICK expression plasmid, and 22 h post-transfection the cells were treated with 10 ng/ml TNF␣ for 120 min (open bars).
Several lines of evidence suggest that Nod1 and RICK act in the same pathway to activate NF-B. First, Nod1 and RICK associate via their corresponding CARDs. Second, NF-B activation induced by Nod1 and RICK is synergistic. Third, mutant forms of Nod1 that are deficient in function inhibit the NF-B activity induced by RICK. Collectively, these results indicate that Nod1 and RICK form a protein complex that induce activation of NF-B and suggest that Nod1 acts downstream of RICK to activate NF-B. In some cells, NF-B mediates antiapoptotic signals (50,55). Thus, the biological response mediated by Nod1 may depend on the cellular context, as is the case after stimulation of surface receptors that signal both NF-B and apoptosis (50,55). These studies were based on overexpression of Nod-1 and target proteins. Analysis of the endogenous proteins under physiological conditions will be important for understanding the function of Nod1 on caspase and NF-B activation.