From the Department of Pathology and Comprehensive
Cancer Center, the University of Michigan Medical School, Ann Arbor,
Michigan 48109 and
Department of Microbiology, Tokyo Medical and
Dental University, School of Medicine, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan
Received for publication, September 4, 2000, and in revised form, November 2, 2000
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ABSTRACT |
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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- Apaf-1 and Nod1 (also called CARD4) are members of a family of
intracellular proteins that are composed of an N-terminal 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- Isolation of the Nod2 cDNA--
Nucleotide sequences
encoding peptides with homology to Nod1 (GenBankTM
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 5' RACE using
Nod2-specific oligonucleotide primers cDNA fragments and mRNA
from primary mammary tissue as a template. 5' RACE was performed using
a commercial kit (Roche Molecular Biochemicals). For PCR, three sets of
primers were used: 5'-ATGTGCTCGCAGGAGGCTTTTCAGGCA-3' and
5'-CGCCTCACCC-ACCACCAGCACAGTGT-3'; 5'-CATGGCTGGACCCCCGCAGAAGAGCCCA-3' and 5'-CA-TGCCCGGGTTCATCTGGCTCATCCGG-3'; and
5'-GCCATGCCCGGGTTCATCTGGCTCATC-3' and
5'-TGAGTCGAGACATGGGGAAAGCTGCTTC-3'. For 5' RACE, the initial primer
5'-AGCAGCTCGACCAGCTGGCTCCTCTGT-3' was used and the product was
PCR amplified with the anchored primer and second Nod2-specific primer: 5'-GACAGGCCCAAGTACCCTTA-TTCCAGA-3'. 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
3.7-kilobase 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)+
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 µg 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, 5'-ATGTGCTCGCAGGAGGCTTTTCAGGCA-3'; P2,
5'-CGCCTCACCCACCA-CCAGCACAGTGT-3'; P3,
5'-ATGTGCTCGCAGGAGGCTTTTCAGGCA-3'; and P4,
5'-CG-CCTCACCCACCACCAGCACAGTGT-3'. As a control, a cDNA fragment of
the human glyceraldehyde-3-phosphate dehydrogenase was amplified using
the primers 5'-GAGTCAACGGATTTGGTCGTAT-3' and
5'-AGTCTTCTGGGTGGCAGTGAT-3'.
Construction of Expression Plasmids--
The Nod2 cDNA
was cloned into pcDNA3-HA and pcDNA3-Fpk3-Myc (10). Deletion
and site-directed mutants of Nod2 (129-1040, 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 (), or
pcDNA3-FLAG-RICK () (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- 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 (GenBankTM 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 5' 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
(GenBankTM 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 N-terminal 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 Chromosomal Localization and Genomic Organization of the Human Nod2
Gene--
We identified two human BAC clones, RP11-327F22 and
RP11-401P9, containing the genomic sequence of human Nod2
(GenBankTM 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 ~7.0- and
~5.5-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 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-
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).
NF- 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- Enforced Oligomerization of Nod2 Induces NF-
We have shown that Nod2 is a member of the Nod1/Apaf-1 family that
activates NF-B 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
B (NF-
B) activation, which required IKK
and was inhibited by dominant negative mutants of I
B
, IKK
, IKK
, and IKK
. Nod2
interacted with the serine-threonine kinase RICK via a homophilic
CARD-CARD interaction. Furthermore, NF-
B 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-
B signaling pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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, 7-9).
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
125-214, 1-125,
1-301, 1-744, 265-1040, 126-301, 265-744, 744-1040, K305R,
1-744K305R) were constructed by a PCR method and cloned into
pcDNA3-HA and pcDNA3-Fpk3-Myc (10). The authenticity of all
constructs was confirmed by sequencing. pcDNA3-FLAG-RICK, pcDNA3-FLAG-RICK (), pcDNA3-FLAG-RICK (),
pcDNA3-Myc-RICK (), pcDNA3-Myc-RIP (),
pRK7-FLAG-IKK
, pRK7-FLAG-IKK
-K44A,
RSVMad-3MSS(I
-B
-S32A/S36A), pRK7-FLAG-IKK
,
pRK7-FLAG-IKK
-K44A, and pcDNA3-FLAG-IKK
() have been
described previously (2, 10).
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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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.
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Fig. 1.
Deduced amino acid sequence and domain
structure of human Nod2. A, amino acid sequence of
Nod2. CARD1 and CARD2, NBD, and LRRs are indicated by reverse
highlighting, underlining, and arrows,
respectively. The consensus sequence of the P-loop (Walker A box) and
the Mg2+ binding site (Walker B box) are indicated by
boxes. B, domain structure of Nod2. Numbers
corresponding to amino acid residues are shown in A. The
region homologous to the CARDs, NBD, and LRRs are indicated by
black, shaded, and hatched boxes,
respectively.
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Fig. 2.
Alignment of human Nod2 and related
proteins. A, alignment of CARDs of Nod2, Nod1
(GenBankTM accession number AF113925), RICK (AF027706), ARC
(AF043244), RAIDD (U79115), Caspase-2 (U13021), Ced-3 (L29052), Ced-4
(X69016), Caspase-9 (U56390), Apaf-1 (AF013263), and c-IAP-1 (L49431).
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 Mg2+ 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).
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Fig. 3.
Expression analysis of Nod2.
A, Northern blot analysis of nod2 expression in
human tissues. PBL, peripheral blood leukocytes;
kb, kilobases. B, RT-PCR analysis of
nod2 expression in granulocyte, monocyte, and lymphocyte
enriched populations. Two sets of Nod2 oligonucleotide primers (P1-P2
and P3-P4) were used to amplify the nucleotide sequences of the CARDs
and LRRs, respectively. C, nucleotide sequence of the 5'
region of nod2. Two potential in-frame translation
initiation sites separated by 81 nucleotides are indicated by
arrows. D, immunoblotting of nod2 gene
products expressed in HEK293T cells. Cells were transfected with
control plasmid (lane 1), constructs containing both
potential translation initiator sites of Nod2 (lane 2), as a
control the second translation initiation site corresponding to that of
Nod2b (lane 3), or the most N-terminal translation
initiation site (lane 4) in the context of a canonical
translation initiation motif. In all cases, a Nod2 protein lacking
residues 302-1040 and HA-tagged at its C terminus was expressed to
facilitate detection of nod2 gene products. Nod2 proteins
were detected by immunoblotting with anti-HA antibody and indicated by
a and b.
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).
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Fig. 4.
Mutational analysis of Nod2.
A, wt and mutant Nod2 proteins. CARDs, NBD, and LRRs are
indicated by black, shaded, and hatched
boxes, respectively. Numbers represent amino acid
residue in Nod2 protein. B, expression analysis of wt and
mutant Nod2 proteins. HEK293T cells were transfected with control
plasmid ( ) or 5 µg of plasmids producing the indicated HA-tagged
Nod2 proteins. Extracts from equal number of cells were
immunoprecipitated with rabbit anti-HA antibody and immunoblotted with
mouse monoclonal anti-HA antibody. The expected size of CARDs, CARD1,
and LRRs mutant proteins are indicated by black arrowheads.
C, NF-
B activation by Nod2 proteins. Induction of NF-
B
activation was determined from triplicate culture of HEK293T cells
cotransfected with the indicated amount of wt or mutant Nod2 expression
plasmids in the presence of pBVIx-Luc and pEF-BOS-
-gal as described
under "Materials and Methods." Values represent the means ± S.D. of triplicate cultures.
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 I
B kinases (IKKs) resulting in
I
B phosporylation and release of cytoplamic NF-
B (16). To
determine whether Nod2 activates an IKK-dependent pathway,
Nod2 was coexpressed with mutant forms of IKK
, IKK
, and I
B
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 I
B
(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.
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Fig. 5.
Nod2 acts through the IKK complex to activate
NF- B. A, inhibition of Nod2
and TNF
-induced NF-
B activation by dominant negative mutant
proteins of the NF-
B pathway. Induction of NF-
B activation was
determined in triplicate cultures of HEK293T cells transfected with 30 ng of Nod2 plasmid (open bars) or stimulated with 10 ng/ml
of TNF
for 4 h (closed bars) and 70 ng of I-
B
S32A/S36A, IKK
K44A, IKK
K44A, RICK (), or RIP ()
expression plasmid in the presence of pBVIx-Luc and pEF-BOS-
-gal.
Results are presented as percentages of values obtained with Nod2 and
control plasmid. In the experiment shown, Nod2 and TNF
induced
58 ± 8-fold and 14 ± 1-fold activation of NF-
B,
respectively. Values represent the means ± S.D. of triplicate
cultures. B, induction of NF-
B in parental Rat-1 and
derivative 5R cells. Induction of NF-
B activation was determined
from triplicate cultures of 1 × 105 HEK293T cells
cotransfected with the indicated plasmids and pBVIx-Luc in the presence
of control plasmid pEF-BOS-
-gal. Values represent the means ± S.D. of triplicate cultures.
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.
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Fig. 6.
Interaction of Nod2 with RICK.
A and B, interaction between wt and mutant Nod2
with RICK. HEK293T cells were cotransfected with wt or mutant Nod2 and
RICK expression plasmid. The coimmunoprecipitated RICK was detected by
immunoblotting with anti-FLAG antibody (top panel). Nod2
immunoprecipitates are shown in bottom panel. Total lysates
were blotted with anti-FLAG antibody (middle panel).
C, interaction between Nod2 and wt and mutant RICK. HEK293T
cells were cotransfected with wt Nod2 and wt or mutant RICK- CARD
(residues 1-374) or RICK-CARD (residues 374-540) expression plasmid.
The coimmunoprecipitated (IP) Nod2 was detected by
immunoblotting with anti-HA antibody (top panel). Total
lysates were blotted with anti-FLAG (middle panel) or
anti-HA (bottom panel) antibody. A background band is shown
with an asterisk.
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).
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Fig. 7.
Enforced oligomerization of Nod2 induces
NF- B activation. A, expression
analysis of wt and mutant Fpk3-Nod2 chimeric proteins. HEK293T cells
were transfected with of control plasmid (
) or plasmids producing the
indicated Myc-tagged Fpk3-Nod2 proteins. Extracts from equal number of
cells was immunoprecipitated and immunoblotted with rabbit anti-Myc
antibody. B, enforced oligomerization of Nod2 induces
NF-
B activation. 2 × 105 HEK293T cells were
transfected with 1 ng of the indicated plasmids in the presence of
pBVIx-luc and pEF-BOS-
-gal. 8 h post-transfection, cells were
treated with 500 nM AP1510 (black bars) or left
untreated (white bars). 24 h post-transfection, the
B-dependent transcription was determined as described in
the Methods. Values represent the means ± S.D. of triplicate
cultures. C, overexpression or enforced oligomerization of
Nod2 activate NF-
B in differentiated HL60 cells. 2 × 105 HL60 cells were stimulated with 200 ng/ml of phorbol
12-myristate 13-acetate for 36 h to induce monocytic
differentiation. Differentiated cells were transfected with 500 ng
(left panel) or 50 ng (right panel) of the
indicated plasmids with pBVIx-luc and pEF-BOS-
-gal. In the
right panel, cells express Fpk3-Nod2 chimeric proteins or
control plasmid, were treated with 500 nM AP1510
(black bars) or left untreated for control (white
bars). 36 h post-transfection, the
B-dependent
transcription was determined as described under "Materials and
Methods." Values represent the means ± S.D. of triplicate
cultures.
B through interactions with its N-terminal 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.
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ACKNOWLEDGEMENTS |
---|
We thank Peter Lucas for critical review of the manuscript. We thank Steve Ethier for mammary cells, Victor Rivera (Ariad Pharmaceuticals) for Fpk plasmids and dimerization agent AP1510, and David Goeddel and Marshall Horwitz for plasmids.
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FOOTNOTES |
---|
* 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. The 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 GenBankTM/EMBL 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.
** To whom correspondence should be addressed. Tel.: 734-764-8509; Fax: 734-647-9654; E-mail: bclx@umich.edu.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M008072200
2 Y. Ogura, N. Inohara, and G. Núñez, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
CARD, caspase
recruitment domain;
HA, hemagglutinin;
IB, inhibitor of NF-
B;
IKK, I
B kinase;
LRRs, leucine-rich repeats;
NBD, nucleotide-binding
domain;
NF-
B, nuclear factor
B;
TNF
tumor necrosis factor
, wt, wild type;
RT, reverse transcriptase;
PCR, polymerase chain
reaction;
RACE, rapid amplification of cDNA ends.
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