From the Department of Pathology and Comprehensive
Cancer Center, The University of Michigan Medical School, Ann Arbor,
Michigan 48109, the ** Unidad de Inmunologia, Departamento de Biologia
Molecular, Universidad de Cantabria, Santander 39011, Spain, and
§§ Human Genome Sciences,
Rockville, Maryland 20850
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ABSTRACT |
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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- Apoptosis, or programmed cell death, is a process that is
essential for normal development and homeostasis of multicellular organisms (1-3). Genetic studies in the nematode Caenorhabditis elegans have identified core components of the death machinery, which are conserved in vertebrates, including humans (1-3). One of
these, Ced-4, is required for developmental cell death in the worm (1,
2). Ced-4 physically interacts with Ced-3 and promotes the proteolytic
activation of the immature Ced-3 caspase into enzymatically active
subunits (4-7). Apaf-1, a mammalian homologue of Ced-4, has been
identified (8). Both Apaf-1 and Ced-4 are composed of an
NH2-terminal caspase recruitment domain
(CARD)1 linked to a
nucleotide-binding domain (NBD), also known as the NB-ARC or NOD domain
(3, 8-10). Ced-4 and Apaf-1 self-associate via the NBD, a process that
mediates proximity and activation of immature Ced-3/caspase-9 molecules
(11-13). The COOH-terminal region of Apaf-1 lacks homology with Ced-4
and is composed of 12 WD-40 repeats (8). In response to certain
apoptotic stimuli, cytochrome c is released from the
mitochondria and binds to Apaf-1 (8, 14), and in the presence of dATP
or ATP, Apaf-1 associates with and activates procaspase-9 (8, 14).
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-1-like
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- 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 5' 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)+ RNA blots from various tissues
(CLONTECH) 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).
Construction of Expression Plasmids--
The entire open reading
frame of Nod1 was amplified by PCR and was cloned into pcDNA3-Myc
and pcDNA3-HA (20) to generate pcDNA3-Nod1-Myc and
pcDNA3Nod1-HA. Deletion and site-directed mutants of Nod1
(1-648, 649-953, 126-953, V41Q, K208R) were constructed by a
two-step PCR method. pcDNA3-caspase-1-FLAG,
pcDNA3-caspase-2-Myc, pcDNA3-caspase3-FLAG,
pcDNA3-caspase-3-C163S-FLAG, pcDNA3-caspase-4-HA, pcDNA3-caspase-6-FLAG, pcDNA3-caspase-7-FLAG, pcDNA3-caspase-9-FLAG, pcDNA3-caspase-9-C287S-HA, pcDNA3-FLAG-RICK, pcDNA3-FLAG-RICK (1-374), pcDNA3-FLAG-RICK (374-540), pcDNA3-Apaf-1-Myc,
pcDNA3-Ced4-Myc, pcDNA3-Bcl-10-Myc, pcDNA3-PEA15FLAG,
pcDNA3-FADD-Myc, pcDNA3-DEDD-Myc, pcDNA3-FLAG-TRAF1, pcDNA3-FLAG-TRAF2,
pcDNA3-Myc-TRAF5, MT-FLAG-TRAF6, pcDNA3-FLAG-RIP, pcDNA3-Myc-RIP
(558-671), pcDNA3-FLAG-NIK, pcDNA3-TNFR1-FLAG, pcDNA3-TRAMP,
pcDNA3-FLAG-DR4, pcDNA3-FLAG-A20, pRK7-FLAG-IKK 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 post-transfection, 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).
Cell Death Assay--
Transfection, Immunodetection of Mature and Precursor Forms of Caspase-8
and Caspase-9--
Total lysate from 293T cells transfected with 50 ng
of pcDNA3-caspase-9-FLAG or pcDNA3-caspase-8-HA in the presence
of 100 ng of pcDNA3-HA or pcDNA3-Nod1-HA was prepared 24 h
post-transfection and was subjected to 15% SDS-polyacrylamide gel
electrophoresis. Tagged caspase-9 and caspase-8 were detected by
anti-FLAG and anti-HA antibodies, respectively. For mutant analysis,
293T cells was co-transfected with 50 ng of pcDNA3-FLAG or
pcDNA3-caspase-9-FLAG and 100 ng of pcDNA3-Nod1,
pcDNA3-Nod (1-648)-HA, pcDNA3-Nod1(649-953)HA, pcDNA3-Nod1-V41Q, pcDNA3-Nod1-K208R, pcDNA3-Apaf-1-Myc,
pcDNA3-FLAG-TRAF2, or pcDNA3-HA. 24 h post-transfection, the
percentage of apoptotic cells was determined in triplicate cultures as
described above. The proform and mature form of tagged caspase-9 were
detected with anti-FLAG antibody in total lysate from an aliquot of the same cultures. To test the interaction between caspase-9 and Nod1 mutants, 293T cells were co-transfected with
pcDNA3-caspase-9-C287S-Myc and WT or mutant Nod1 expression
plasmids. Proteins co-immunoprecipitated with anti-Myc antibody were
detected with anti-HA antibody. As control, immunoprecipitated
caspase-9 was detected by re-blotting with anti-Myc monoclonal
antibody. Nod1 proteins were immunoprecipitated by anti-HA polyclonal
antibody and were detected by anti-HA monoclonal antibody.
NF- 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 NH2-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, Mg2+ binding site,
Ref. 37) (Figs. 1 and 2B). The
COOH-terminal 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- Nod1 Is Expressed in Multiple Tissues and Exhibits Restricted
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
(GenBankTM 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- 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 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 enhancement (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
loss-of-function of RAIDD and Apaf-1/Ced-4, respectively (13, 43-45).
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 wild-type 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-92. Thus, the CARD is essential for Nod-1 to
bind and activate procaspase-9, as well as to promote apoptosis.
Nod1 Induces NF- Nod1 Acts Upstream of NIK, IKKs, and I- Nod1 Interacts with RICK via an Homophilic CARD
Association--
Next we determined the regions of Nod1 involved in
NF- Mutants Forms of Nod1 Inhibit NF-
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
NH2-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 (11-13). Nod1 can also
self-associate, 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-
Several lines of evidence suggest that Nod1 and RICK act in the same
pathway to activate NF-B) and bound RICK, a
CARD-containing kinase that also induces NF-
B activation. Nod1
mutants inhibited NF-
B activity induced by RICK, but not that
resulting from tumor necrosis factor-
stimulation. Thus, Nod1 is a
leucine-rich repeat-containing Apaf-1-like molecule that can regulate
both apoptosis and NF-
B activation pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
B activation.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
, pRK7-FLAG-IKK
-K44A, RSVMad-3MSS(I-
B
S32A/S36A),
pRK7-FLAG-IKK
, pRK7-FLAG-IKK
-K44A, pcDNA3-FLAG-TRAF2(241-501),
pcDNA3-FLAG-TRAF6(289-522), pcDNA3-TRADD-Myc, pcDNA3-CLARP-HA,
pcDNA3-p35, and pcDNA3-
-gal have been described previously (6,
19-34). pcDNA3-caspase-8-HA, pcDNA3-caspase-8-C377S-HA,
caspase-9-C287S, and FADD-Myc were generated by subcloning from
different tag versions previously described into
BamHI/XhoI into pcDNA3-HA and pcDNA3-Myc.
RAIDD, PEA15, DEDD and Bcl-10/CIPER cDNAs (EST clone numbers 504896, 570509, 490307, and 574273, respectively) were cloned into the BamHI and XhoI sites of pcDNA3-Myc and
pcDNA3-FLAG to COOH-terminal Myc- or FLAG-tag proteins. A mutation of
caspase-4 (C258S) was introduced into pcDNA3-caspase-4-HA (25) by
site-directed PCR methodology. The authenticity of all constructs was
confirmed by sequencing.
-galactosidase
staining, and cell death assays of 293T cells were performed as
described (21). 293T cells were transfected with the indicated amount
of pcDNA3-caspase-9-FLAG, pcDNA3-caspase-4-HA, pcDNA3-caspase-8-HA, or
50 ng of pcDNA3-FADD-Myc, 700 ng of pcDNA3-CLARP-HA, 100 ng of
pcDNA3-FLAG-TRAMP, 500 ng of pcDNA3-TNFR1-FLAG or pcDNA3 in the
presence of 100 ng of pcDNA3-HA or pcDNA3-Nod1-HA, and 200 ng of
pcDNA3-
-gal. In all cases, pcDNA3 was added so that the total amount
of DNA was 2 µg. The reproducibility of each experiment was confirmed
by independent, blinded observers.
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 µg of pcDNA3-p35 to prevent caspase activation
and cell death. 24 h post-transfection, 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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).
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Fig. 1.
Deduced amino acid sequence, domain structure
of human nod1. A, amino acid sequence of Nod1.
Sequences homologous to the CARD, NBD, and LRRs are indicated by
reverse highlighting, underlining, and
arrows, respectively. The consensus sequence of the P-loop
(Walker A box) is indicated by boxes. B,
schematic representation of Nod1. Numbers corresponds to
amino acid residues shown in A. The region homologous to the
CARD, NBD, and LRRs are indicated by a black closed box, a
closed shaded box, and hatched boxes,
respectively.
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Fig. 2.
Alignment of human Nod1 and related
proteins. A, alignment of CARDs of Nod1, RICK
(GenBankTM accession number AF027706), ARC
(GenBankTM accession number AF043244), RAIDD
(GenBankTM accession number U79115), caspase-2
(GenBankTM accession number U13021), Ced-3
(GenBankTM accession number L29052), Ced-4
(GenBankTM accession number X69016), caspase-9
(GenBankTM accession number U56390), Apaf-1
(GenBankTM accession number AF013263), and c-IAP-1
(GenBankTM accession number L49431). The residues identical
and similar to those of Nod1 are shown by reverse and
dark highlighting, respectively. The putative helices,
H1 to H6, are shown according to the three-dimensional structure of the
CARD of RAIDD (23). B, alignment of NBDs of Nod1, CIITA
(GenBankTM accession number X74301), Apaf-1
(GenBankTM accession number AF013263), and Ced-4
(GenBankTM accession number X69016). The residues identical
and similar to those of Nod1 are shown by reverse and dark highlights,
respectively. The consensus sequence of the P-loop (residues 280-284,
Walker A box) and the Mg2+ binding site (residues 280-284,
Walker B box) are indicated by boxes. C,
alignment of LRRs in Nod1. The conserved positions with leucine and
other hydrophobic residues are indicated by highlights. The putative
helix and
sheet are shown according to the three-dimensional
structure of the ribonuclease inhibitor (42).
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Fig. 3.
Northern blot and In situ
hybridization analysis of Nod1 expression. A,
Northern blot analysis of nod1 expression in human adult
tissues. B, in situ hybdridization analysis of
nod1 expression in mouse tissues parasagital sections of
mouse embryo at stage E15 were hybridized with a nod1 probe
(panels a-e). High-power magnification of the thymus and
heart (panel b), liver (panel c), lung
(panel d), and liver and kidney (panel e). The
cerebellum (C), choroid plexus (CP), gut
epithelium (GE), germinal layer (GL), heart
(h), inferior colliculus (IC), kidney (K), lung
(Lg), liver (Lv), olfactory epithelium
(OE), and thymus (T) are indicated by
arrows. Notice that the heart and cerebellum are
negative.
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).
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Fig. 4.
Interaction between Nod1 and multiple
regulators of apoptosis and NF- B
activation. 293T cells were co-transfected with Myc- or HA-tagged
Nod1 and the plasmids expressing FLAG-, Myc-, or HA-tagged proteins
indicated in the figure. Proteins were immunoprecipitated with anti-Myc
antibody, and co-immunoprecipitated proteins were detected with
anti-FLAG or anti-HA antibody are shown in the lanes labeled as
IP. Each protein in total lysate is shown in the lanes
labeled as Total. In the case where tested proteins were
Myc-tagged (indicated by boxed panels), proteins were first
immunoprecipitated with anti-Myc antibody, and co-immunoprecipitated
HA-tagged Nod1 was detected by immunoblotting with anti-HA
antibody
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Fig. 5.
Enhancement of caspase-9-induced apoptosis
and caspase-9 maturation by Nod1. A, regulation of
caspase-9-, but not caspase-8-, induced cell death by Nod1. 293T cells
were transfected with 50 ng of caspase-9 (panels a and
b) or caspase-8 (panels c and d)
expression plasmids plus -galactosidase plasmid in the presence of
vector control (panels a and c) or Nod1
expression plasmid (panels b and d). 24 h
post-transfection, the transfected cells were fixed and visualized by
staining. Arrowheads show rounded apoptotic cells with
membrane blebbing. B, regulation of caspase-9-induced cell
death by Nod1. 293T cells were transfected with caspase-9, caspase-4,
caspase-8, FADD, CLARP, TRAMP, or TNFR1 expression plasmids plus
-galactosidase plasmid in the presence of vector control or Nod1
expression plasmids. 24 h post-transfection, the percent of
apoptotic cells ± S.D. was calculated in triplicate cultures.
C, induction of caspase-9 maturation by Nod1. 293T cells
were co-transfected with caspase-9 or caspase-8 expression plasmids in
the presence of vector control or Nod1 plasmids. Tagged caspase-9 and
caspase-8 in total lysate were detected by anti-FLAG- and anti-HA
antibodies, respectively. The asterisk and double
asterisks indicate nonspecific proteins cross-reacting with
antibodies and truncated products from HA-Nod1, respectively.
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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 ± 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.
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).
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Fig. 7.
NF- B Activation by
Nod1. A, activation of NF-
B by Nod1. Induction of
NF-
B activation was determined in triplicate cultures of 293T cells
co-transfected with vector control, Nod1, RICK, TRAF2, or Apaf-1
expression plasmid and pBIIx-Luc or pf-Luc in the presence of control
plasmid pcDNA-3-
-gal. B, inhibition of Nod1-induced
NF-
B activation by dominant negative mutant proteins of the NF-
B
pathway. Induction of NF-
B activation was determined in triplicate
culture of 293T cells co-transfected with Nod1 expression plasmid and
I-
B
S32A/S36A, IKK
K44A, IKK
K44A, NIK KK429-430AA, TRAF2
(241-501), TRAF6 (289-522), RIP (558-671), 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).
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-
B-inducing activity of Nod1 does not
require caspase-9 activity.
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 co-transfected with plasmids producing WT or mutant Nod1
and RICK. The analysis showed that the NH2-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.
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-
B-inducing
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 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.
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.
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
anti-apoptotic 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.
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ACKNOWLEDGEMENTS |
---|
We thank E. Alnemri, Y. Choi, V. M. Dixit, C. S. Duckett, D. V. Goeddel, and J. Tschopp for plasmids; M. Clarke and C.-H. Chang for stimulating discussions; M. Benedict for critical review of the manuscript; and Sue O'Shea for help with analysis of mouse embryos.
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FOOTNOTES |
---|
* This work was supported by Grant R01 CA64556 from the National Institutes of Health (to G. N.) and Grant 23/98 from the Fundacion Marques de Valdecilla, Santander, Spain (to J. M).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) AF113925.
§ Supported by a fellowship from the Japan Science and Technology Corporation.
¶ Supported by a fellowship from the University of Michigan-Parke Davis Fellowship program and NCI Fellowship program.
Supported by MSTP Student Training Grant T32GM07863 from the
National Institutes of Health.
Supported by a postdoctoral fellowship from the Fundacion
Marques de Valdecilla.
¶¶ Supported by Research Career Development Award K04 CA64421 from the National Institutes of Health. To whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109. Tel.: 734-764-8514; Fax: 734-647-9654; E-mail: gabriel.nunez{at}umich.edu.
2 N. Inohara and G. Nuñez, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
CARD, caspase-recruitment domain;
NF-B, nuclear factor-
B;
I-
B, inhibitor of NF-
B;
IKK, I-
B kinase;
NBD, nucleotide-binding
domain;
LRRs, leucine-rich repeats;
EST, expressed sequence tag;
PCR, polymerase chain reaction;
HA, hemagglutinin;
WT, wild-type;
DED, death
effector domain.
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REFERENCES |
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