From Millennium Pharmaceuticals, Inc., Cambridge,
Massachusetts 02139 and § The Center for Apoptosis
Research and the Department of Microbiology and Immunology, Kimmel
Cancer Institute, Thomas Jefferson University, Philadelphia,
Pennsylvania 19107
Received for publication, March 20, 2001
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ABSTRACT |
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BCL10 belongs to the caspase recruitment
domain (CARD) family of proteins that regulate apoptosis and NF- Protein modules play an important role in the assembly of
signaling proteins into specific signal transduction pathways (1). The
death domain, death effector domain, caspase recruitment domain (CARD),1 and the recently
identified PYRIN (DAPIN/PAAD) domain are protein modules
found in many proteins that regulate apoptosis (2-5). The CARD domain
consists of six or seven antiparallel Besides regulating apoptosis, numerous CARD proteins have been found to
play an important role in signaling pathways that lead to the
activation of NF- Expression Plasmids and Antibodies--
Plasmids expressing
CARD10 with C-terminal FLAG epitopes were constructed using pCMV-Tag 4A
(Stratagene). Constructs encoding epitope-tagged BCL10 were described
previously (19). For mammalian two-hybrid assays, the
pCMV-CARD10-CARD/BD plasmids were constructed by inserting the
CARD domain of CARD10 (residues 1-138) into pCMV-BD (Stratagene). The
panel of CARD domains used for the mammalian two-hybrid screen was
described previously (10, 11). CARD7-CARD/AD contains the CARD
domain (residues 1335-1429) of CARD7(3), CARD8-CARD/AD contains the
CARD domain (residues 318-431) of
CARD82 (accession
number AF322184), CASP5-CARD/AD contains the CARD domain
(residues 1-152) of caspase-5, and CASP13-CARD/AD contains the CARD
(residues 1-113) of caspase-13. The BCL10 antibody was described
previously (25).
Reporter Gene Assays--
For mammalian two-hybrid assays, 293T
cells in 6-well plates (35-mm wells) were transfected with the
following plasmids: 750 ng of pCMV-CARD10/BD, 750 ng of pCMV-AD fused
to individual CARD domains, 250 ng of pFR-Luc firefly reporter
(Stratagene), and 250 ng of pRL-TK renilla reporter (Promega). For
NF- Co-immunoprecipitation Assays--
293T cells transfected with
plasmids were lysed in 50 mM Tris, pH 8.0, 120 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40 buffer
and incubated with indicated antibodies. The immune complexes were precipitated with protein G-Sepharose (Amersham Pharmacia Biotech), washed extensively, and then subjected to SDS polyacrylamide gel electrophoresis and immunoblotted with polyclonal anti-FLAG (Santa Cruz
Biotechnology, Inc.).
In vitro Binding Assays--
In vitro binding assays
between BCL10 and CARD10 proteins were performed as described
previously (26). In brief, BCL10 wt and the L41R mutant were
expressed in DH5- We performed a mammalian two-hybrid analysis and screened
Millennium Pharmaceuticals' collection of CARD domains for a selective interaction with the CARD domain of BCL10. The CARD domain of CARD10
interacted with the CARD of BCL10 resulting in a 340-fold increase in
relative luciferase activity (Fig.
1A). Co-expression of
CARD10-CARD with 22 other CARD domains failed to activate luciferase expression indicating that the CARD of CARD10 interacts selectively with the CARD of BCL10. Human CARD10 is a novel CARD family member of
1032 amino acids with a predicted molecular mass of 116 kDa (Fig.
1B). Amino acid sequence analysis revealed that CARD10 has a
domain structure that is similar to CARD9, CARD11, and CARD14, whose
N-terminal CARD domains also interact selectively with the CARD
activation domain of BCL10 (10, 11; see Figs. 1B and
2A). CARD10 is comprised of at least five putative
functional domains, an N-terminal CARD domain, a central coiled-coil
domain, and a C-terminal tripartite structure consisting of a
PDZ domain, an SH3 domain, and a GUK domain with homology to
guanylate kinase. Although the CARD domain of CARD10 (residues 31-117)
shows significant similarity to those found in other CARD family
members (Fig. 2B), it is most
similar to the CARD domains of CARD11 (58% identity), CARD14 (46%
identity), and CARD9 (46% identity). Adjacent to the N-terminal CARD
domain are coiled-coil structures with extensive regions of heptad
repeats that function in protein oligomerization and activation (27).
The COILS2 program (28) predicts with a probability of greater than
70% at least four coiled-coil structures in CARD10 (residues 138-206,
210-256, 263-307, and 326-456) that are interrupted by regions with
a lower coiled-coil potential. The PDZ/SH3/GUK tripartite structure
found at the C terminus (Fig. 2, A, C,
D, and E) classifies CARD10 as a novel member of
the MAGUK family of proteins that function to organize signaling
complexes at plasma membranes (29). Northern blot analysis revealed
that CARD10 is expressed as a 4.4-kilobase transcript in a variety of
adult tissues, including heart, kidney, and liver (Fig. 2F). CARD10 was also expressed abundantly in fetal lung, liver, and kidney
tissues, as well as in multiple cancer cell lines, including HeLa S3,
chronic myelogenous leukemia K562 cells, colorectal
adenocarcinoma SW480 cells, and lung carcinoma A549 cells (data not
shown).
B
signaling pathways. Analysis of BCL10-deficient mice has revealed that
BCL10 mediates NF-
B activation by antigen receptors in B and T
cells. We recently identified a subclass of CARD proteins (CARD9,
CARD11, and CARD14) that may function to connect BCL10 to multiple
upstream signaling pathways. We report here that CARD10 is a novel
BCL10 interactor that belongs to the membrane-associated guanylate
kinase family, a class of proteins that function to organize
signaling complexes at plasma membranes. When expressed in cells,
CARD10 binds to BCL10 and signals the activation of NF-
B through its
N-terminal effector CARD domain. We propose that CARD10 functions as a
molecular scaffold for the assembly of a BCL10 signaling complex that
activates NF-
B.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helices and forms highly
specific homophilic interactions between signaling partners. Thus far,
21 human CARD-containing proteins have been identified. Members of this
family include caspases 1, 2, 4, 5, 9, and 13, Apaf-1, CARD4
(Nod1), Nod2, CARD7 (DEFCAP/NAC), c-IAP-1, c-IAP-2, RICK
(RIP2/CARDIAK), ARC, BCL10, RAIDD, ASC, Iceberg, CARD9, CARD11, and
CARD14 (6-13). CARD domains mediate the assembly of family members
into discrete signaling complexes. For example, Apaf-1 and caspase-9
specifically associate with each other through their CARD domains in
the presence of cytochrome c and dATP resulting in caspase
oligomerization and activation (14).
B. The CARD protein RICK assembles into signaling
complexes with its upstream activators CARD4 and Nod2 and activates the
IKK complex through IKK
(13, 15-17). The CARD protein BCL10 (also
known as CLAP/CIPER/cE10/CARMEN) signals the activation of
NF-
B and has been implicated in B cell lymphomas of
mucosa-associated lymphoid tissue (18-23). Targeted disruption of its
gene in mice has implicated a role for BCL10 in NF-
B activation by
antigen receptors in B and T cells (24). We recently identified several
CARD proteins (CARD9, CARD11, and CARD14) that function to transduce
upstream signals to the activation of BCL10 (10, 11). Interestingly,
CARD11 and CARD14 are members of the membrane-associated guanylate
kinase (MAGUK) family that function to assemble signaling complexes at
the plasma membrane. We report here that CARD10 is a novel CARD/MAGUK
family member that signals the activation of NF-
B through BCL10.
CARD10 and other members of the CARD/MAGUK family likely play an
important role in receptor-mediated activation of BCL10 and
NF-
B.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B assays, 293T cells were transfected with the following
plasmids: 900 ng of pNF-
B luciferase reporter (Stratagene), 100 ng
of pRL-TK renilla reporter (Promega), and 1000 ng of indicated
expression plasmids. Cells were harvested 24 h after transfection,
and firefly luciferase activity was determined using the
dual-luciferase reporter assay system (Promega). In addition, renilla
luciferase activity was determined and used to normalize transfection efficiencies.
bacteria as GST fusion proteins, and
equal amounts of protein were immobilized on glutathione-Sepharose (Amersham Pharmacia Biotech). An equal amount of CARD10 protein, labeled with [35S]methionine, was incubated with the
protein-bound Sepharose beads in 100 µl of binding buffer (50 mM Tris-Cl, pH 7.6, 120 mM NaCl, 0.5% Brij,
and protease inhibitors) for 3 h. The beads were washed four times
with the same buffer and boiled in SDS sample buffer. The proteins were
then resolved on a 10% SDS gel and visualized by autoradiography.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (29K):
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Fig. 1.
The CARD domain of CARD10 interacts
selectively with the CARD of BCL10 by mammalian two-hybrid
analysis. A, 293T cells were transfected with
the mammalian two-hybrid reporter construct pFR-Luc (Stratagene), and
the CARD of CARD10 fused to the DNA-binding domain of GAL4 was screened
against a panel of individual CARDs fused to the activation domain of
murine NF- B. After 24 h, cells were collected and assayed for
relative luciferase activity as a measure of protein-protein
interaction. B, amino acid sequence of CARD10 (1032 residues). CARD, coiled-coil, PDZ, SH3, and GUK domains are
highlighted.
View larger version (81K):
[in a new window]
Fig. 2.
Domain structure and tissue distribution of
human CARD10. A, domain structure of CARD10 in
comparison to CARD11, CARD14, and CARD9 (10, 11) showing CARD,
coiled-coil, PDZ, SH3, and GUK domains. B, alignment of the
CARD of CARD10 (residues 31-117) with CARDs found in CARD11 (residues
19-105), CARD14 (residues 23-109), CARD9 (residues 14-100), BCL10
(residues 21-103), RAIDD (residues 9-95), and caspase-9 (residues
9-93). C, alignment of the PDZ domain of CARD10 (residues
607-680) with PDZ domains found in CARD11 (residues 660-742), CARD14
(residues 570-653), PSD-95 (repeat 3; residues 355-430), ZO-1
(repeat 3; residues 411-485), and ZO-2 (repeat 3; residues 511-587).
D, alignment of the SH3 domain of CARD10 (residues 704-772)
with SH3 domains found in CARD11 (residues 766-834), CARD14 (residues
676-744), PSD-95 (residues 471-540), ZO-1 (residues 504-571), and
ZO-2 (residues 604-668). E, alignment of the GUK domain of
CARD10 (residues 833-1018) with GUK domains found in CARD11 (residues
954-1142), CARD14 (residues 814-999), P55 (residues 269-460), and
PSD-95 (residues 564-761). F, tissue distribution of human
CARD10 transcripts. Expression was determined by Northern blot analysis
using CLONTECH human multiple tissue Northern
blots. PBL, peripheral blood leukocytes.
Our finding that the CARD domain of CARD10 interacts selectively with
the CARD domain of BCL10 by mammalian two-hybrid analysis suggests that
CARD10 is a specific signaling partner of BCL10. We therefore examined
the interactions between these two proteins when overexpressed in
cells. Immunoprecipitation of T7-tagged BCL10 quantitatively
co-precipitated FLAG-tagged CARD10 (Fig. 3A). This interaction was
dependent on the CARD domains of both proteins, because CARD10 failed
to associate with a variant BCL10 with a point mutation (L41R) that
disrupts CARD-mediated homodimerization (19). In addition, a CARD10
truncation mutant lacking its CARD domain failed to co-precipitate with
BCL10. We next tested whether CARD10 interacts with endogenous BCL10
when overexpressed in cells (Fig. 3B). Immunoprecipitation
of endogenous BCL10 co-precipitated FLAG-tagged CARD10 but not a
FLAG-tagged CARD10 truncation mutant lacking its CARD domain. We also
examined the interaction of radiolabeled CARD10 with GST-BCL10 in
vitro and found that CARD10 associates directly with BCL10 through
a CARD-CARD interaction (Fig. 3C).
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Our finding that CARD10 specifically associates with BCL10 prompted us
to examine whether CARD10 induces NF-B activity, using a luciferase
reporter gene system. When CARD10 was expressed in 293T cells, NF-
B
activity was induced 90-fold compared with empty vector (Fig.
4A). NF-
B activity was
dependent on the IKK complex, because dominant-negative versions of
IKK
and IKK
blocked the ability of CARD10 to induce the
activation of NF-
B (data not shown). To determine the role of
individual domains in NF-
B signaling, we constructed a series of N-
and C-terminal truncation mutants of CARD10 (Fig. 4B). The
N-terminal CARD domain of CARD10 was essential for NF-
B signaling,
because deletion of this domain eliminated the induction of NF-kB
activity (Fig. 4C). Immunoblot analysis revealed that the
truncation mutant was expressed at a level comparable with wt protein
that induces NF-
B activity 30- to 40-fold (compare Fig. 4,
A and C). Deletion of the C-terminal GUK domain
did not interfere with the ability of CARD10 to induce NF-
B
activity. However, further deletion of the SH3 and PDZ domains reduced
the levels of NF-
B activity to levels 2- to 3-fold less than that
obtained with wt protein. Immunoblot analysis revealed that the
C-terminal truncated proteins were expressed at levels similar to each
other and wt protein indicating that reductions in activity were not
because of reduced levels of expression (Fig. 4C). Thus, the
PDZ and SH3 domains are required for maximal activation of NF-kB
activity by CARD10.
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We have identified CARD10 as a specific regulator of BCL10. Our finding
that CARD10 both binds to BCL10 and signals NF-B activation through
its N-terminal CARD domain suggests that this molecule functions as an
upstream activator of BCL10. CARD10 is one of four CARD proteins
identified thus far that assemble with BCL10 and signal the activation
of NF-
B (10, 11). These molecules (CARD10, CARD9, CARD11, and
CARD14) likely function to transduce distinct upstream stimuli to the
activation of BCL10 and NF-
B. Interestingly, this subclass of CARD
proteins is related in both sequence and structure (Fig. 2). Besides
containing closely related N-terminal CARD domains that interact
specifically with BCL10, each molecule contains coiled-coiled domains
that could mediate self-association resulting in aggregation and
activation of BCL10 in response to upstream signals. BCL10 might then
engage and oligomerize IKK
resulting in the activation of the IKK
complex and NF-
B (17, 30; see Fig. 5).
Thus, CARD10 and the other BCL10 activators (CARD9, CARD11, and CARD14)
likely function in a manner analogous to Apaf-1 and CARD4 that induce
oligomerization and activation of their respective downstream CARD
binding partners. A unique feature of CARD10, CARD11, and CARD14 is the
presence of C-terminal PDZ/SH3/GUK domains that suggest a role for
these proteins in signal transduction by receptors at the plasma
membrane. A recent study implicating BCL10 as a mediator of antigen
receptor signaling in B and T cells (24) suggests that CARD10 and the
other CARD/MAGUK family members might function to recruit BCL10 to
receptor complexes.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA85421 (to E. S. A.).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) AY028896.
¶ To whom correspondence should be addressed: Millennium Pharmaceuticals, Inc., 640 Memorial Dr., Cambridge, MA 02139. Tel.: 617-679-7215; Fax: 617-679-7071; E-mail: bertin@mpi.com.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M102488200
2 J. Bertin, unpublished information.
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ABBREVIATIONS |
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The abbreviations used are: CARD, caspase recruitment domain; MAGUK, membrane-associated guanylate kinase; CMV, cytomegalovirus; wt, wild-type; GST, glutathione S-transferase.
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