From the Department of Pathology and Comprehensive
Cancer Center and the ** Howard Hughes Medical Institute and
Departments of Internal Medicine and Biological Chemistry, The
University of Michigan Medical School, Ann Arbor, Michigan 48109, and the ¶ Unidad de Inmunologia, Departamento de Biologia
Molecular, Universidad de Cantabria, Santander 39011, Spain
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ABSTRACT |
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We have identified and characterized CIPER, a
novel protein containing a caspase recruitment domain (CARD) in its N
terminus and a C-terminal region rich in serine and threonine residues. The CARD of CIPER showed striking similarity to E10, a product of the
equine herpesvirus-2. CIPER formed homodimers via its CARD and interacted with viral E10 but not with several apoptosis regulators containing CARDs including ARC, RAIDD, RICK, caspase-2, caspase-9, or
Apaf-1. Expression of CIPER induced NF- The Rel/NF- Structural and functional analyses of TNFR family members and
interleukin-1/Toll receptors have revealed the existence of common
intracellular mechanisms that are responsible for signal transduction
and biological activities. The interleukin-1 receptor and Toll
signaling pathways utilize the adaptor protein MyD88 to recruit the
serine-threonine kinases IRAK1 and IRAK2 to mediate NF- Components of signaling pathways are frequently connected by the
interaction of proteins that contain homology domains. In the cell
death pathway, three types of homology interaction domains have been
identified including the death domain, the death effector domain, and
the caspase recruitment domain (CARD). The death domain is also present
as a protein module in components of the interleukin-1 receptor/Toll
signaling pathways that mediate NF- Isolation of the CIPER cDNA--
The partial nucleotide
sequence of cDNAs encoding peptides with homology to the CARD
domain of human caspase-2 (U13021) were found in EST data bases of
GenBankTM using the TBLASTN program. The entire nucleotide
sequence of EST clones 703916 and 574273 was determined by dideoxy sequencing.
Northern Blot Analysis and in Situ Hybridization--
The entire
cDNA insert of EST clone 703916 was radiolabeled by random priming
using a commercial kit (Boehringer Mannheim) and applied for analysis
of human poly(A)+ RNA blots from various tissues
(CLONTECH Laboratories) according to the
manufacturer's instructions. For in situ hybridization, slides containing mouse embryo tissues were prepared as described (18).
Each specimen was hybridized with a digoxigenin-labeled antisense RNA
probe synthesized from a full-length mouse CIPER cDNA using an
in vitro transcription kit (Boehringer Mannheim). As a
control, a sense CIPER RNA-labeled probe was synthesized and used for
hybridization as above. Hybridization, development, and mounting of
slides were performed as described (18).
Construction of Expression Plasmids--
The entire cDNA
insert (1.8 kilobase pairs) of EST clone 574273 was cloned into the
EcoRI and NotI sites of pcDNA3 (Invitrogen) to produce pcDNA3-CIPER. The entire open reading frame of CIPER was
inserted into the BamHI and XhoI sites of
pcDNA3-Flag or pcDNA3-Myc (19) to produce C-terminal Flag- or
Myc-tagged CIPER. The E10 gene was amplified by polymerase chain
reaction using equine herpesvirus-2 DNA (a gift from A. Davison, University of Glasgow, UK). Deletion mutants of
pcDNA3-CIPER-N (1-119)-Myc, pcDNA3-CIPER-C (120-233)-Myc, and
pcDNA-E10-Myc were constructed by a two-step polymerase chain reaction method (20). pcDNA3-caspase-8-AU1 pcDNA-Apaf-1-Myc and
pcDNA3- Transfection, Expression, Immunoprecipitation, and
Immunodetection of Tagged Proteins--
5 × 106
human 293 or 293T cells were transfected with expression plasmids by a
calcium phosphate method as described (21). The total amount of
transfected plasmid DNA was adjusted with pcDNA3 plasmid to be the
same within individual experiments. After transfection, cells were
harvested at different times and lysed with 0.2% Nonidet P-40 isotonic
lysis buffer (24). For immunoprecipitation, 1 mg of soluble protein was
incubated with 1 µg/ml of polyclonal anti-Myc antibody (Santa Cruz)
overnight at 4 °C, and tagged proteins were immunoprecipitated with
protein A-Sepharose 4B (Zymed Laboratories Inc.).
Immunoprecipitated proteins or total lysates were subjected to 12%
SDS-polyacrylamide electrophoresis and immunoblotted with monoclonal
antibody to Flag (Kodak) or polyclonal anti-Myc antibody. MCF7 and HeLa
cells were co-transfected with plasmid constructs and
Apoptosis Assays--
1 × 105 cells were
co-transfected with 0.2 µg of pcDNA3- NF- Identification of CIPER--
To identify potential apoptosis
regulatory genes, we searched public data bases of ESTs for clones with
homology to the CARD of caspase-2/ICH-1 (14). Three human and two mouse
ESTs containing overlapping nucleotide sequences with significant amino
acid homology to the CARD of caspase-2 were identified. Sequence
analysis of human and mouse cDNAs revealed open reading frames that
encoded proteins of 233 amino acids (Fig.
1A). Both human and mouse
proteins exhibited a high level of similarity (91% amino acid
identity; Fig. 1B), suggesting that they represent the human
and mouse orthologues. We have designated these proteins CIPER, after
CED-3/ICH-1 prodomain homologous,
E10-like regulator (see below). Analysis of the
CIPER amino acid sequence revealed that it contained a N-terminal CARD with significant amino acid similarity to the CARD present in the
prodomain of caspase-2/ICH-1, RAIDD, caspase-9, CED-3, CED-4, and
Apaf-1 (Fig. 1C). Significantly, the CARD of CIPER was most homologous (51% identical) to E10, a protein encoded by the equine herpesvirus-2 gene (26), whose function remains unknown
(Fig. 1C). Unlike caspases and other caspase regulators, the
C-terminal region of CIPER had no significant similarity to any other
protein in public data bases. Significantly, however, the C-terminal
region of CIPER is rich in Ser/Thr residues (Fig. 1B).
CIPER Is Expressed in Multiple Adult and Embryonic Tissues--
We
performed Northern blot analysis to determine the distribution of CIPER
RNA transcripts in various human tissues. CIPER was detected in all
human tissues examined including spleen, lymph node, peripheral blood
lymphocytes, heart, brain, placenta lung, skeletal muscle, and
pancreas, as two transcripts of 2.5 and 1.3 kilobases (Fig.
2A). We also evaluated the
distribution of CIPER mRNA at stage 15 of mouse embryonic
development by in situ hybridization. In the mouse embryo,
CIPER mRNA labeling was detected in the glanglionic eminence and
the ventricular zone of the developing brain, olfactory epithelium,
tongue, whisker follicles, salivary gland, heart, lung, liver, and
intestinal epithelia (Fig. 2B, a-c).
CIPER Promotes Apoptosis--
To begin to elucidate the
physiological function of CIPER, expression constructs producing
Flag-tagged CIPER were introduced into 293T, HeLa, and MCF7 cells,
which were subsequently observed for features of apoptosis. In these
cells, expression of CIPER exhibited no or modest apoptotic activity
when compared with control plasmid (Fig.
3A). We tested next whether
CIPER could regulate apoptosis activated by caspase-8, a death
protease coupled to death receptor pathways. Significantly, expression
of CIPER augmented apoptosis induced by caspase-8 in all cell lines
tested (Fig. 3A). Enhancement of caspase-8-mediated
apoptosis induced by CIPER required a catalytic active caspase-8
because CIPER did not augment the level of apoptosis induced by a
mutant caspase-8 protein with a single amino acid change
(Cys377 to Ser) in the conserved active pentapeptide (2).
Furthermore, caspase-8-induced apoptosis potentiated by CIPER was
inhibited by the broad-based caspase inhibitors baculovirus p35 and
zVAD-fmk (2). To further verify these observations, we transfected 293 cells with the CIPER cDNA and control plasmid and isolated stably transfected clones that expressed CIPER protein (see Fig. 5). Consistent with the results in transient transfection assays, CIPER
expression enhanced apoptosis induced by TNFR1 and CD95 stimulation in
stably transfected clones (Fig. 3, B and C). In addition, we also tested the ability of CIPER to modulate apoptosis induced by FADD, TRADD, and caspase-4, -9, and -10. CIPER enhanced apoptosis induced by all these pro-apoptotic proteins (2).
CIPER Induces NF- Point Mutations in Critical Residues of the CARD Abolished the
Ability of CIPER to Activate NF-
To further verify the NF- NF- CIPER Self-associates and Interacts with Viral E10--
The CARD
has been proposed to mediate signaling through homophilic interactions
(14). We therefore tested the ability of CIPER to associate with other
CARD-containing proteins. Expression constructs producing several HA-
and Flag-tagged apoptosis regulatory proteins and Myc-tagged CIPER were
transiently co-transfected in 293T cells. Cell lysates were
immunoprecipitated with anti-Myc Ab, and co-immunoprecipitated proteins
were analyzed by immunoblotting with anti-Flag Ab. The analysis shown
in Fig. 7A revealed that CIPER
co-immunoprecipitated with viral E10, the molecule to which it is most
similar in the CARD motif (Fig. 1C). In addition, the analysis revealed that CIPER can form homodimers (Fig. 7A).
However, neither RAIDD nor Apaf-1, two CARD-containing proteins,
associated with CIPER (Fig. 7A). Furthermore, CIPER failed
to interact with other CARD-containing proteins including caspases-1,
-2, -4, and -9, RICK/RIP2, and ARC.2 In addition, CIPER did
not associate with several molecules that mediate NF-
To determine whether the CARD of CIPER is important for dimerization,
we tested the ability of CIPER proteins with mutations in conserved
residues of the CARD to associate with wt CIPER. Immunoprecipitation
analysis revealed that mutations in critical residues of the CARD,
CIPER(L41Q), CIPER(G79R), and CIPER(L41Q/G79R) abolished the ability of
CIPER to homodimerize, whereas the S231A mutation outside the CARD did
not (Fig. 7B). Moreover, the CIPER-N (1-119) mutant that
contains the CARD dimerized with wt CIPER, whereas the CIPER-C
(120-233) mutant did not (Fig. 7B), indicating that the
CARD mediates CIPER homodimerization. Immunoblotting analysis revealed
that wt and mutant forms of CIPER were expressed at comparable levels,
ruling out that the loss of function was due to insufficient levels of
expression (Fig. 7B). These results indicate that the CARD
is necessary and sufficient for CIPER homodimerization.
We have identified a novel CARD-containing protein, CIPER, that can
activate NF-
The CARD was essential for CIPER function because mutations in residues
that are conserved in the CARD destroyed the ability of CIPER to
promote NF-
The CARD was first described as a peptide module present in the
prodomains of upstream caspases and adaptor molecules such as
CED-4/Apaf-1 and RAIDD that mediates the recruitment of caspases (14,
35). Indeed, RAIDD and Apaf-1 can recruit caspase-2 and caspase-9 via
CARD interactions (15, 16, 36). However, we found that CIPER is unable
to bind CARD-containing caspases and caspase regulators such as RAIDD,
ARC, or Apaf-1. These results suggest that CARDs may mediate
interactions not only between caspase regulators and caspases but also
between cellular transducers of the NF-B activation, which was
inhibited by dominant-negative NIK and a nonphosphorylable I
B-
mutant but not by dominant-negative RIP. Mutational analysis revealed that the N-terminal region of CIPER containing the CARD was
sufficient and necessary for NF-
B-inducing activity. Point mutations
in highly conserved residues in the CARD of CIPER disrupted the ability
of CIPER to activate NF-
B and to form homodimers, indicating that
the CARD is essential for NF-
B activation and dimerization. We
propose that CIPER acts in a NIK-dependent pathway of
NF-
B activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B family of transcriptional factors plays an
important role in immune and inflammatory responses, cell survival, and
stress response by regulating the expression of numerous cellular and
viral genes (1). In most cells, nuclear factor
B
(NF-
B)1 is composed of
homodimers or heterodimers of 50-kDa (p50) and 65-kDa (p65) subunits
that are sequestered in the cytoplasm by a member of the I
B family
of inhibitory proteins (1). In response to certain cellular, bacterial,
and viral stimuli, NF-
B is activated through the phosphorylation and
inactivation of I
B (1). Active NF-
B is released and translocated
to the nucleus and binds to cognate DNA sequences. Signaling through
the tumor necrosis factor receptor (TNFR) family and
interleukin-1/Toll receptors induces NF-
B activation.
Tumor necrosis factor family members bind to their cognate receptors,
including TNFR1, TNFR2, CD95(Fas/APO-1), TRAMP(DR3/WSL-1/AIR/LARD), CD27, CD30, and CD40, and regulate cell
proliferation, apoptosis, and proinflammatory responses (2, 3). Some
members of the TNFR family such as TNFR1, CD95, and TRAMP, so-called
death receptors, contain a "death domain" in their cytosolic C
termini that is responsible for signaling cell death (4, 5).
Ligand-induced trimerization of the TNFR1 and CD95 results in the
recruitment of the death domain adapter molecules TRADD and FADD,
respectively, which are critical for the activation of the apoptotic
response (6-9).
B activation
(10, 11). Activation of NF-
B through TNFR family stimulation is
mediated by TNFR-associated factors (TRAFs) (12). Several TRAFs
directly interact with the cytoplasmic domain of TNFR family members,
whereas these adaptor molecules associate with TNFR1 via the adaptor
molecule TRADD and the Ser/Thr kinase RIP (12). TRAFs and RIP activate
the NF-
B-inducing kinase (NIK), which in turn activates the I
B
kinases (13). The I
B-
and I
B-
kinases phosphorylate I
B
leading to its degradation by the ubiquitin pathway and subsequent
NF-
B activation (13).
B activation, notably the adaptor
protein MyD88 and the IRAK1 and IRAK2 kinases (10, 11). The CARD was
originally identified as a conserved sequence of about 90 amino acids
present in several proteins of the cell death pathway including RAIDD,
CED-3, caspase-1, caspase-2, caspase-9, and CED-4 (14). CARDs have been
proposed to mediate the binding between adaptor molecules and caspases,
such as the RAIDD-caspase-2 interaction, which allows the recruitment
of this caspase to the TNFR1 complex (15). In addition, CED-3 and
caspase-9 are similarly associated with their regulators CED-4 and
Apaf-1, respectively, through CARD interactions (16, 17). To identify novel regulators of intracellular pathways, we searched public data bases for expressed sequence tag (EST) clones with
homology to the CARD of caspase-2. In this study, we report the
identification and characterization of a novel CARD-containing
protein, CIPER, which positively regulates both apoptosis and
NF-
B activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-gal were previously described (19, 21, 22). pcDNA3-p35, pcDNA3-TNFR1, and pcDNA3-Fas were gifts of V. Dixit (University of Michigan, Ann Arbor, MI). pcDNA3-RIP
(559-671)-Myc and pRK7-Myc-NIK(KK429-430AA) were provided by D. V. Goeddel (Tularik, South San Francisco, CA). RSVMad-3MSS has been
previously described (23).
-galactosidase-expressing reporter construct (pcDNA3-
-gal) by
LipofectAMINE (Life Technologies, Inc.) according to the
manufacturer's instructions.
-gal plus each expression
plasmid in triplicate. In some experiments, 20 µM of the
caspase inhibitor zVAD-fmk (Enzyme Systems Products) was added at
8 h after transfection. At 18 h after transfection, apoptotic
cells were scored as described (21). Specific apoptosis was calculated
as the percentage of blue cells with apoptotic morphology in each
experimental condition minus the percentage of blue cells with
apoptotic morphology in pcDNA3 vector-transfected cells. At least
300 cells from three random fields were counted in each experiment, and
the data show the mean and S.D. of triplicate cultures. Data shown are
representative of at least three independent experiments. Statistical
significance was determined by one-way analysis of variance followed by
Student-Neuman-Keuls post-doc comparisons.
B Activation Assays--
1 × 106 293 cells were co-transfected with 0.25 µg of pBIIx-Luc or control
plasmid pf-Luc plus each expression plasmid in triplicate in the
presence of 2 µg of pcDNA3-p35 to prevent cell death. At 24 h after transfection, cell extract was prepared, and its luciferase activity was measured by the luminescence spectrophotometer
Monolight 2010 (Analytical Luminescence Laboratory). 5 × 106 Jurkat-T leukemia cells were transfected with 3 µg of
pcDNA3-CIPER-Flag and 3 µg of HIV-CAT or
B-HIV-CAT using
DEAE-dextran as described previously (23). Cells were harvested after
48 h, and CAT assays were performed as described previously (25).
Equal quantities of extract protein were analyzed in each assay.
Statistical significance was determined by one-way analysis of variance
followed by Student-Neuman-Keuls post-doc comparisons.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Deduced amino acid sequence, domain
structure, and alignment of CIPER. A, schematic
representation of CIPER. Numbers correspond to amino acid
residues shown in B. The CARD is indicated by a closed
box. B, amino acid sequences of human and mouse CIPER.
Conserved identical and nonidentical residues are indicated by
asterisks and dots, respectively. CARDs are
indicated by boxes. Hydrophobic and aromatic amino acid
residues are shown by shading: positive and negative charged
residues are shown by dark and light gray
shading, respectively. helix and
strand breakers are shown
by bold letters. The nucleotide sequences of human and mouse
CIPER are available as accession numbers AF057700 and AF057701 in the
GenBankTM data base. C, alignment of CARDs of
CIPER, E10 (U20824), RAIDD (U79115), caspase-2 (U13021), CED-3
(L29052), caspase-9 (U56390), CED-4 (X69016), and Apaf-1(AF013263).
Conserved H1-8
helices are shown according to the
three-dimensional structure of RAIDD (37).
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Fig. 2.
Tissue distribution of CIPER.
A, expression of CIPER in human tissues by
Northern blot analysis. PBL, peripheral blood lymphocytes.
B, expression of CIPER in mouse embryo at E15d.
a, specific labeling is observed in ventricular zone
(VZ), olfactory epithelium (OF), glanglionic
eminence (GE), salivary gland (SG), tongue
(T), whisker follicles (W), fat deposits
(FD), heart (H), lung (LU), and
intestinal epithelium (I). b and c,
high magnification of salivary gland (b) and lung
(c). d, labeling with sense CIPER probe.
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Fig. 3.
Regulation of apoptosis by CIPER.
A, CIPER potentiates apoptosis induced by caspase-8. 293T,
HeLa, and MCF7 cells were transfected with pcDNA3 or
pcDNA3-CIPER-Flag (1 µg) in the presence or absence of
pcDNA3-caspase-8-AU1 (0.2 µg). The percentage of specific
apoptosis was calculated as described under "Experimental
Procedures" in triplicate cultures. B and C,
CIPER enhances apoptosis induced by TNFR1 or CD95 in stably transfected
293 clones. Cell clones stably transfected with CIPER or control
plasmid were transiently transfected with pcDNA3, pcDNA3-TNFR1
(0.5 µg), or pcDNA3-Fas (1 µg) as indicated. Apoptosis was
determined as described under "Experimental Procedures" in
triplicate cultures.
B Activation--
Signaling through the TNFR1
pathway activates both apoptosis and NF-
B activation (3, 27,
28). To test whether CIPER activates NF-
B, CIPER expression plasmids
were co-transfected with the NF-
B reporter plasmid, pBIIx-Luc, or
control plasmid lacking NF-
B binding sites in 293 cells. CIPER
induced NF-
B activation in a dose-dependent manner (Fig.
4A). Induction of NF-
B
activation by CIPER was specific in that transfection of 293 cells with
expression plamids producing ARC and RAIDD, two CARD-containing
proteins did not promote NF-
B
activation.2 Consistent with
its high level of homology, expression of E10, a product of the equine
herpesvirus-2, also induced NF-
B activation (Fig.
4A). To determine the region of CIPER required for NF-
B activation, we engineered two deletion mutants, CIPER-N (1-119) and
CIPER-C (120-233), to express the N-terminal half including the CARD
and C-terminal half that is rich in Ser/Thr amino acids, respectively.
CIPER-N (1-119), but not CIPER-C (120-233), induced NF-
B
activation (Fig. 4A), indicating that the N-terminal half of
CIPER that includes the CARD is sufficient and essential for NF-
B
activation (Fig. 4A). Immunoblotting analysis revealed that wt and mutant forms of CIPER were expressed at comparable levels, suggesting that the loss of function was not due to insufficient levels
of expression (Fig. 4A). Interestingly, CIPER was detected as a doublet of 28 and 31 kDa (Fig. 4A). Treatment of
lysates with calf intestinal alkaline phosphatase eliminated the 31-kDa protein, suggesting that the upper band represents a phosphorylated form of CIPER (2). Phosphorylation of CIPER was mapped to the C-terminal region that is rich in Ser/Thr residues as determined by
expression analysis of CIPER-N (1-119) and CIPER-C (120-233) mutants
(Fig. 4A). To verify the result that CIPER activates
NF-
B, CIPER was coexpressed with another NF-
B reporter plasmid,
HIV-CAT, in Jurkat T cells. As demonstrated in 293 cells, CIPER
expression also induced NF-
B activation in Jurkat T cells as
measured with HIV-CAT but not with control plasmid with mutated NF-
B
sites (Fig. 4B).
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Fig. 4.
CIPER induces NF- B
activation. A, NF-
B activation by CIPER in 293 cells. 293 cells were transfected with 0, 0.02, 0.05, 0.1, 0.2, and 0.5 µg of pcDNA3-CIPER-Myc (WT), 0.5 µg of
pcDNA3-CIPER-N(1-119)-Myc (N), or
pcDNA3-CIPER-C(120-233)-Myc (C) in the presence of
pBIIx-Luc or control plasmid pf-Luc in indicated lanes. Cell lysates
were prepared, and luciferase activity was measured as described under
"Experimental Procedures." CIPER-Myc proteins in the same lysates
were subjected to 12% SDS-polyacrylamide gel electrophoresis and
immunoblotting with polyclonal anti-Myc Ab (inset).
B, NF-
B activation by CIPER in Jurkat-T cells. Jurkat-T
cells were transiently transfected with pcDNA3 or
pcDNA3-CIPER-Flag (3 µg) plus HIV-CAT and
B-HIV-CAT as
indicated. Cell lysates were prepared, and CAT activity was measured as
described under "Experimental Procedures."
B--
To verify that the CARD of
CIPER is important for NF-
B activation, we introduced mutations in
conserved residues of the CARD by site-directed mutagenesis. The point
mutants CIPER(L41Q) and CIPER(G79R) and double point mutant
CIPER(L41Q/G79R) were engineered because their counterpart residues in
the CARDs of CED-3 and RAIDD are critical for heterodimerization with
CED-4 and caspase-2, respectively (15, 29). In addition, we constructed
another point mutation, CIPER(S231A), outside the CARD as a control
(Fig. 5A). Immunoblotting
experiments showed that these mutants expressed CIPER protein (Fig. 5).
We used transient co-transfection of plasmids expressing CIPER mutants
and a NF-
B reporter plasmid into 293 cells to determine the regions
of CIPER required for NF-
B activation. The analysis revealed that
wt, CIPER-N (1-119), and CIPER(S231A) promoted NF-
B
activation, whereas the CARD point mutants CIPER(L41Q), CIPER(G79R),
CIPER(L41Q/G79R), and CIPER-C (120-233) did not (Fig. 5B).
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Fig. 5.
Point mutations in the CARD abolish the
ability of CIPER to activate NF- B.
A, schematic diagram of wt and mutant CIPER proteins. The
positions of point mutations introduced in CIPER are shown by
arrows. S/T denotes region of CIPER rich in
serine and threonine residues. B, the CARD of CIPER is
essential for NF-
B activation. 293 cells were transiently
transfected with pcDNA3-CIPER-Myc or Myc-tagged CIPER mutant plasmid
(0.5 µg) in the absence (open bar) or presence
(closed bar) of pcDNA-TNFR1 (0.1 µg). C,
expression of CIPER induces NF-
B activation in stably transfected
293 clones. 293 cell clones expressing wt (WT) or mutant
CIPER (L41Q, N, or C) or vector
control were transiently transfected with NF-
B or control reporter
plasmids, and luciferase activity was measured as described under
"Experimental Procedures." CIPER-Myc proteins in cell lysates were
subjected to 12% SDS-polyacrylamide gel electrophoresis and were
detected by immunoblotting with polyclonal anti-Myc Ab
(inset).
B-activating ability of CIPER, we developed
293 cell clones by stable transfection with expression plasmids
producing wt, CIPER-N (1-119), CIPER-C (120-233), and CIPER(L41Q).
Immunoblotting analysis of two independently derived clones transfected
with each construct showed that they expressed wt and mutant CIPER
protein (Fig. 5C). In agreement with the transient assay
shown in Fig. 5B, clones expressing wt or mutant CIPER
containing the CARD (residues 1-119) expressed significantly more
NF-
B activating activity (15-70-fold) than clones transfected with
empty vector (Fig. 5C). Significantly, clones expressing the
point mutant (L41Q) or CIPER-C (120-233), a mutant with deletion of
the CARD, did not express significant NF-
B activity (Fig.
5C), confirming that the CARD is essential for the ability
of CIPER to activate NF-
B. We also tested the ability of CIPER
mutants to promote apoptosis. These experiments showed
that CIPER-N (1-119) and CIPER(S231A) promoted TNFR1- and
FAS-mediated apoptosis, but the CARD mutants, CIPER(L41Q),
CIPER(G79R), and CIPER(L41Q/G79R) did not.2
B Activation Induced by CIPER Is Inhibited by Dominant
Repressor Forms of NIK or I
B but Not by Dominant-negative
RIP--
To determine the molecular ordering of CIPER relative to
other components of the tumor necrosis factor signaling pathway, CIPER
was co-expressed with dominant repressor forms of I
B, RIP, and NIK.
The double mutation S32A/S36A of I
B
cannot be phosphorylated or
degraded and therefore blocks the nuclear translocation of NF-
B and
transactivation of NF-
B-responsive genes (30). The RIP (558-671)
and NIK(KK429-430AA) mutants act as dominant inhibitors of the
endogenous kinases whose activities are required for NF-
B activation
(8, 31). NF-
B activation induced by CIPER was blocked by
overexpression of dominant repressor forms of I
B and NIK but not by
dominant-negative RIP (Fig. 6). The RIP
mutant was functional, because it inhibited NF-
B activation induced by TNFR1 stimulation (Fig. 6). These results suggest that CIPER functions upstream of NIK and I
B, and downstream of or in parallel to RIP in a NF-
B activation pathway. These results are compatible with published reports showing that TRAFs and RIP activate NIK, which
in turn activates the I
B kinases (13).
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Fig. 6.
NF- B activation
induced by CIPER is inhibited by dominant repressor forms of NIK or
I
B
but not by
dominant-negative RIP. 293 cells were transiently co-transfected
with pcDNA3, pcDNA3-CIPER-Flag (0.5 µg), or pcDNA3-TNFR1 (0.2 µg) and pcDNA3-RIP(558-671)-Myc (DN-RIP, 0.5 µg),
pRK7-Myc-NIK(KK429-430AA) (DN-NIK, 0.5 µg), or RSVMad-3MSS
(DN-I
B
, 0.5 µg) as indicated. The amount of plasmid DNA was
adjusted with control plasmid so that all samples were transfected with
the same amount of DNA. NF-
B activation was measured with reporter
plasmid in triplicate cultures as described under "Experimental
Procedures."
B activation
including NIK, RICK/RIP2, and TRAF-1, -2, -3, -4, and
-6.2
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Fig. 7.
CIPER associates with viral E10 and forms
homodimers. A, CIPER associates with viral E10. 293T
cells were transiently transfected with pcDNA3-CIPER-Flag (4 µg)
and pcDNA3-RAIDD-Myc (3 µg), pcDNA3-Apaf-1-Myc (9 µg),
pcDNA3-E10-Myc (3 µg), or pcDNA3-CIPER-Myc (3 µg) plasmids
in indicated lanes. CIPER-Myc proteins were immunoprecipitated with
anti-Myc polyclonal Ab, and co-immunoprecipitated proteins were
detected by immunoblotting with anti-Flag (top panel).
CIPER-Flag and Myc-tagged proteins in cell lysates were detected by
polyclonal Ab to Myc (middle panel) and monoclonal Ab to
Flag (bottom panel), respectively. B, CIPER forms
homodimers through its CARD. 293T cells were transiently transfected
with pcDNA3-CIPER-Flag (4 µg) and pcDNA3-CIPER-Myc or
Myc-Tagged CIPER mutant plasmids (4 µg) as indicated. CIPER-Myc
proteins were immunoprecipitated with anti-Myc polyclonal Ab, and
co-immunoprecipitated proteins were detected by immunoblotting with
anti-Flag Ab (top panel). CIPER-Myc and CIPER-Flag proteins
in cell lysates were detected by polyclonal Ab to Myc (middle
panel) and monoclonal Ab to Flag (bottom panel),
respectively. WT, wild type; WB, Western blot;
IP, immunoprecipitation.
B. The CARD of CIPER exhibits a high level of similarity
to that of E10, a gene product of the equine herpesvirus-2. Like its cellular homologue, expression of E10 induced the activation of NF-
B. Although the mechanism by which E10 activates NF-
B remains to be elucidated, it is possible that E10 mediates NF-
B activation via its association with CIPER. This model would be compatible with the observation that CIPER self-associates via its
CARD, a process that might be required for NF-
B activation. Alternatively, both CIPER and its viral homologue E10 might activate NF-
B through a common cellular target. The signaling pathway(s) in
which CIPER acts to promote NF-
B activation remains unclear. NF-
B
activation induced by CIPER was blocked by dominant-repressor forms of
I
B or NIK but not by dominant-negative RIP. These results suggest
that CIPER acts downstream of RIP and upstream of NIK and I
B in a
RIP-dependent NF-
B activation pathway. The results are
also compatible with a model in which CIPER activates a NIK-regulated pathway of NF-
B activation distinct from that signaled through RIP.
Several signaling pathways that activate NF-
B including those
induced by CD40, interleukin-1/Toll receptors are RIP-independent (10,
11, 32).
B activation. The essential role of the CARD could
reflect a requirement for CIPER to recruit additional protein(s) via
the CARD. For example, CIPER self-association might mediate
oligomerization of CIPER-binding factor(s), a process perhaps essential
for signaling NF-
B activation. In this model, the CARD of CIPER
might serve as a domain that would bring into close proximity effector
molecules that mediate NF-
B activation. This model would be
reminiscent of the model proposed for Apaf-1 in the cell death pathway,
in which oligomerization through the CARD would bring into proximity
two catalytic domains of caspase-9, thus initiating proteolysis (17).
CIPER did not bind to RICK/RIP2, NIK, and TRAF1, -2, -3, and -6, proteins that can activate NF-
B (12, 32-34). Identification of
CIPER-interacting proteins might well reveal the physiological function
of CIPER.
B activation pathway.
Similarly, another CARD-containing protein, RICK/RIP2, is a Ser/Thr
kinase that interacts with and regulates the tumor necrosis factor
family of receptors by binding to adaptor molecules devoid of caspase
activity (32, 33). Like CIPER, RICK/RIP2 promotes both NF-
B
activation and apoptosis (32, 33). These results suggest a broader role
for CARD domains in cellular signaling events than previously
appreciated. Activation of NF-
B has been shown to provide an
anti-apoptotic signal in certain cells (27). It is likely, therefore,
that the apoptosis regulatory function of CIPER may depend on the
cellular context. While this manuscript was being reviewed, another
group identified a gene, bcl-10, at the breakpoint of the
recurrent t(1;14)(p22;q32) chromosomal translocation of
mucosa-associated lymphoid tissue (MALT lymphoma) (38). The sequence of
CIPER is identical to that of bcl-10.
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ACKNOWLEDGEMENTS |
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We are grateful to M. Benedict, L. del Peso, and Y. Hu for critical review of the manuscript, C. Vincenz and M. Dyer for stimulating discussions, and A. Davison, V. Dixit and D. Goeddel for reagents.
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FOOTNOTES |
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* This work was supported in part by Grant CA-64556 from the National Institutes of Health (to G. N.) and Grant SAF97-0054 from the Spanish Ministry of Education and Science (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) AF057700 and AF057701.
§ Supported by a fellowship from the Japan Science and Technology Corporation.
Supported by a postdoctoral fellowship from Fundacion Marques
de Valdecilla.
Supported by the Commission for the Advancement of Young
Scientists and Scholars of Zurich.
§§ Recipient of Research Career Development Award CA-64421 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 T. Koseki and G. Núñez, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
NF-B, nuclear
factor
B;
Ab, antibody;
CARD, caspase recruitment domain;
EST, expressed sequence tag;
TNFR, tumor necrosis factor receptor;
wt, wild
type;
TRAF, TNFR-associated factor;
NIK, NF-
B-inducing kinase.
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REFERENCES |
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