CARD11 and CARD14 Are Novel Caspase Recruitment Domain
(CARD)/Membrane-associated Guanylate Kinase (MAGUK) Family Members that
Interact with BCL10 and Activate NF-
B*
John
Bertin
§,
Lin
Wang
,
Yin
Guo¶,
Michael D.
Jacobson
,
Jean-Luc
Poyet¶,
Srinivasa M.
Srinivasula¶,
Sarah
Merriam
,
Peter S.
DiStefano
, and
Emad
S.
Alnemri¶
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, November 20, 2000
 |
ABSTRACT |
The caspase recruitment domain (CARD)
is a protein-binding module that mediates the assembly of
CARD-containing proteins into apoptosis and NF-
B signaling
complexes. We report here that CARD protein 11 (CARD11) and CARD
protein 14 (CARD14) are novel CARD-containing proteins that belong to
the membrane-associated guanylate kinase (MAGUK) family, a class of
proteins that functions as molecular scaffolds for the assembly of
multiprotein complexes at specialized regions of the plasma membrane.
CARD11 and CARD14 have homologous structures consisting of an
N-terminal CARD domain, a central coiled-coil domain, and a C-terminal
tripartite domain comprised of a PDZ domain, an Src homology
3 domain, and a GUK domain with homology to guanylate kinase.
The CARD domains of both CARD11 and CARD14 associate specifically with
the CARD domain of BCL10, a signaling protein that activates
NF-
B through the I
B kinase complex in response to upstream
stimuli. When expressed in cells, CARD11 and CARD14 activate NF-
B
and induce the phosphorylation of BCL10. These findings suggest that
CARD11 and CARD14 are novel MAGUK family members that function as
upstream activators of BCL10 and NF-
B signaling.
 |
INTRODUCTION |
Modular protein interaction domains play an important role in
signal transduction by mediating the assembly of components into
specific signaling complexes (1). The interchange of protein modules
between signaling molecules has allowed nature to rapidly evolve new
signal transduction pathways that respond to specific stress and
developmental stimuli. The caspase recruitment domain (CARD)1 is a protein module
that participates in apoptosis signaling through protein-protein
interactions (2). CARD domains consist of six or seven antiparallel
-helices that form highly specific homophilic interactions between
signaling partners. CARD family members include the majority of class I
caspases, CED-4 family members Apaf-1 and CARD4 (Nod1), IAP family
members cIAP-1 and cIAP-2, RICK kinase, ARC, BCL10, RAIDD, ASC, CARD9,
and Iceberg (2-9). Confirming the selectivity of CARD-CARD
interactions, several CARD protein family members have been found to
assemble into discrete signaling complexes. For example, Apaf-1 and
caspase-9 assemble together in the presence of cytochrome c
and dATP resulting in caspase oligomerization and activation (10).
Other CARD proteins that segregate with discrete binding partners
include CARD4 with RICK, RAIDD with caspase-2, and CARD9 with BCL10 (3,
8, 11). The mechanisms by which upstream stimuli activate and/or assemble these CARD-CARD signaling complexes are not presently understood.
Recent studies have found that CARD proteins can also function as
components of signaling pathways that lead to activation of the
transcription factor NF-
B. CARD4, RICK, BCL10, and CARD9 induce
NF-
B activity through the IKK complex when overexpressed in cells
(3, 4, 6, 8, 12). NF-
B plays a central role in the activation of
genes involved in immunity, inflammation, and apoptosis (13, 14). In
unstimulated cells, NF-
B is sequestered in the cytoplasm through
interactions with inhibitory I
B proteins. In response to a variety
of signals including the cytokines interleukin-1 and tumor
necrosis factor
, bacterial lipopolysaccharide, and virus infection,
I
B
is phosphorylated and targeted to degradation by the
proteosome through covalent modification by ubiquitin. The degradation
of I
B
results in the translocation of NF-
B to the nucleus
where it binds to specific promoters and activates transcription.
Phosphorylation of I
B
is mediated by the IKK complex, which
consists of two catalytic subunits called IKK
and IKK
and one
regulatory subunit called IKK
. Although the mechanism by which
IKK
regulates IKK activity is presently unknown, it has been
proposed to link the IKKs to upstream regulatory molecules (15-17).
The CARD protein RICK binds directly to IKK
suggesting that it
functions as an adaptor molecule between the IKK complex and its
upstream binding partner CARD4 (17). Activation of the IKK complex may
occur through an oligomerization signal initiated by the
self-association of CARD4. Thus, CARD-CARD signaling complexes such as
CARD4/RICK can function as important mediators of NF-
B signaling.
BCL10 (also known as CLAP/CIPER/cE10/CARMEN) is an activator of
apoptosis and NF-
B signaling pathways that has been implicated in B
cell lymphomas of mucosa-associated lymphoid tissue (6, 12, 18-21).
BCL10 has a bipartite structure consisting of an N-terminal CARD and a
C-terminal domain that is rich in serine-threonine residues. Because
enforced oligomerization of the C terminus of BCL10 induces NF-
B
activation, the CARD domain has been proposed to function as an
oligomerization domain that transduces the activation signal to the IKK
complex through the C-terminal domain of BCL10 (12). The C terminus of
BCL10 may function in a manner analogous to the NF-
B activators RIP
and RICK and activate the IKK complex through binding and
oligomerization of IKK
(16, 17). We recently identified a novel CARD
NF-
B activator called CARD9 that assembles into a CARD-CARD
signaling complex with BCL10 (8). We report here the identity and
characterization of two additional CARD proteins, CARD11 and CARD14,
that assemble BCL10 into NF-
B signaling complexes. Unlike CARD9,
both CARD11 and CARD14 are members of the membrane-associated guanylate
kinase (MAGUK) family, a class of proteins that functions as molecular
scaffolds for the assembly of multiprotein complexes at the plasma
membrane. We propose that CARD11 and CARD14 form discrete CARD-CARD
signaling complexes with BCL10 and signal the activation of
NF-
B.
 |
EXPERIMENTAL PROCEDURES |
Expression Plasmids--
Plasmids expressing either CARD11 or
CARD14 with C-terminal Myc epitopes were constructed using pCMV-Tag 5A
(Stratagene). Constructs encoding epitope-tagged BCL10 were described
previously (12). For mammalian two-hybrid assays, pCMV-CARD11-CARD/AD
and pCMV-CARD14-CARD/AD plasmids were constructed by inserting the CARD
domain of CARD11 (residues 1-126) and CARD14 (residues 1-118) into
pCMV-AD (Stratagene). The panel of CARD domains used for the mammalian
two-hybrid screen was described previously (8).
Reporter Gene Assays--
For mammalian two-hybrid assays,
293T cells in six-well plates (35-mm wells) were transfected with the
following plasmids: 750 ng of pCMV-CARD11/AD or pCMV-CARD14/AD, 750 ng
of pCMV-BD fused to individual CARD domains, 250 ng of pFR-Luc firefly
reporter (Stratagene), and 250 ng of pRL-TK renilla reporter (Promega). For NF-
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.
Coimmunoprecipitation Assays--
293T cells transfected with
plasmids were lysed in 50 mM Tris, pH 8.0, 120 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40
buffer and incubated with a BCL10 monoclonal antibody (22). 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-Myc antibodies (Santa Cruz Biotechnology, Inc.).
In Vitro Binding Assays--
In vitro binding assays
between BCL10 and either CARD11 or CARD14 proteins were performed as
described previously (23). In brief, BCL10 wild type and L41R
mutant were expressed in DH5 Alpha bacteria as GST fusion proteins, and
equal amounts of protein were immobilized on glutathione-Sepharose
(Amersham Pharmacia Biotech). An equal amount of CARD11 or CARD14
protein labeled with [35S]methionine was incubated with
the protein-bound Sepharose beads in 100 µl of binding buffer (50 mM Tris-HCl, 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.
Immunostaining and Image Analysis--
Rat-1 cells were
transfected in poly-D-lysine-coated glass chamber
slides (BioCoat, Becton-Dickinson Labware) with plasmids expressing
HA-tagged BCL10 and either Myc-tagged CARD11 or CARD14 using FuGENE-6
(Roche Molecular Biochemicals) for 20 h. Cells were fixed in 4%
paraformaldehyde, permeabilized and blocked in a buffer containing
0.4% Triton X-100, and sequentially incubated with the following
primary and secondary antibodies: rabbit anti-HA polyclonal Y-11 (Santa
Cruz Biotechnology), mouse anti-Myc monoclonal 9E10 (Oncogene Research
Products), Alexa-488 goat anti-mouse IgG (Molecular Probes), and
Alexa-594 goat anti-rabbit IgG (Molecular Probes). No cross-reactivity
was observed between any of the antibodies (data not shown). Images
were acquired using a Nikon TE200 microscope with a 60× oil objective
and an Orca-I digital CCD camera (Hammamatsu, Inc.) driven by MetaMorph
software (Universal Imaging Corp.). Final images were prepared using
Adobe PhotoShop.
 |
RESULTS AND DISCUSSION |
We searched public and proprietary data bases for novel members of
the CARD family of apoptosis and NF-
B signaling proteins. Human
CARD11 is a novel CARD family member of 1147 amino acids with a
predicted molecular mass of 132.6 kDa (Fig.
1A). A second protein (1004 amino acids, 113.3 kDa) displaying significant similarity to CARD11 was
also identified and designated CARD14 (Fig. 1A). Analysis of
their amino acid sequences revealed that CARD11 and CARD14 were
homologous in structure and were comprised of at least five putative
functional domains (Fig. 1B). Both proteins contain an
N-terminal CARD domain and a central coiled-coil domain and possess a
C-terminal tripartite structure comprised of a PDZ domain, an SH3
domain, and a GUK domain with homology to guanylate kinase. Although
their CARD domains (residues 1-87) show significant similarity to
those found in other CARD family members, they are most similar to each
other (52% identity) and to the CARD of CARD9 (CARD11, 56% identity;
CARD14, 47% identity) (Fig. 1C). Adjacent to the N-terminal
CARD domains are extensive regions of heptad repeats found in
coiled-coil structures that function in protein oligomerization and
activation (24). The COILS2 program (25) predicts with a probability of
>80% at least two coiled-coil structures in CARD11 (residues 130-158
and 165-433) and five coiled-coil structures in CARD14 (residues
128-198, 205-238, 245-272, 281-330, and 356-409) that are
interrupted by regions with a lower coiled-coil potential. The
PDZ/SH3/GUK tripartite structure located at the C terminus of CARD11
and CARD14 are domains that have not been previously found in CARD
proteins (Fig. 1, D-F). These domains function as sites for
specific protein-protein interactions and classify CARD11 and CARD14 as
novel members of the MAGUK family of proteins that function to organize
signaling complexes at plasma membranes (26). The structure of CARD11
and CARD14 is most similar to CARD9 which contains an N-terminal CARD
domain followed by multiple coiled-coil domains (8). CARD11 and CARD14
maytherefore function in a manner similar to CARD9 and activate
downstream CARD proteins through their N-terminal CARD domains.
Although CARD11 and CARD14 have homologous structures, Northern blot
analysis revealed differences in expression profiles (Fig.
2). CARD11 is expressed as a
4.4-kilobase transcript in a variety of adult tissues including
thymus, spleen, liver, and peripheral blood leukocytes (Fig.
2A). CARD11 also showed abundant expression in specific
cancer cell lines, including promyelocytic leukemia HL-60 cells,
chronic myelogenous leukemia K562 cells, Burkitt's lymphoma Raji
cells, and colorectal adenocarcinoma SW480 cells (Fig. 2B).
In contrast, the 4.4-kilobase CARD14 transcript showed
expression only in placenta (Fig. 2C) and HeLa S3 cancer cells (Fig. 2D).

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Fig. 1.
Sequence and domain structure of human CARD11
and CARD14. A, amino acid alignment of CARD11 and
CARD14. Black shading indicates identical residues.
B, domain structure of CARD11 and CARD14 showing CARD,
coiled-coil, PDZ, SH3, and GUK domains. C, alignment of the
CARD of CARD11 (residues 19-105) and CARD14 (residues 23-109) with
CARDs found in CARD9 (residues 14-100), BCL10 (residues 21-103),
RAIDD (residues 9-95), and caspase-9 (residues 9-93). D,
alignment of the PDZ domain of CARD11 (residues 660-742) and CARD14
(residues 570-653) with PDZ domains found in PSD-95 (repeat 3, residues 355-430), ZO-1 (repeat 3, residues 411-485) and ZO-2 (repeat
3, residues 511-587). E, alignment of the SH3 domain of
CARD11 (residues 766-834) and CARD14 (residues 676-744) with SH3
domains found in PSD-95 (residues 471-540), ZO-1 (residues 504-571)
and ZO-2 (residues 604-668). F, alignment of the GUK domain
of CARD11 (residues 954-1142) and CARD14 (residues 814-999) with GUK
domains found in P55 (residues 269-460) and PSD-95 (residues
564-761).
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Fig. 2.
Tissue distribution of human CARD11 and
CARD14 transcripts. The expression of CARD11 and CARD14 mRNA
in adult tissues (A) and (C) and various human
cancer cell lines (B) and (D) was determined by
Northern blot analysis using CLONTECH human
multiple tissue Northern blots. PBL, peripheral blood
leukocytes.
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Because CARD proteins have been implicated in NF-
B signaling
pathways, we determined whether CARD11 and CARD14 can induce NF-
B
activity using a luciferase reporter gene. When either CARD11 or CARD14
were expressed in 293T cells, NF-
B activity was induced 20-40-fold
compared with empty vector (Fig. 3,
A and B). NF-
B signaling occurred through the
IKK complex because dominant-negative versions of IKK-
and IKK-
blocked the abilities of CARD11 and CARD14 to induce NF-
B activity
(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 CARD11 and CARD14 (Fig. 3C). The
N-terminal CARD of both CARD11 and CARD14 was essential for NF-
B
signaling because deletion of this domain eliminated the induction of
NF-
B activity. (Fig. 3, D and E). Immunoblot
analysis revealed that the mutant proteins were expressed at levels
similar to wild type protein indicating that loss of function was not
due to reduced levels of expression. In contrast, the C-terminal PDZ,
SH3, and GUK domains were not required for NF-
B signaling because
deletion of these domains did not reduce the ability of CARD11 and
CARD14 to induce NF-
B activity. However, a CARD11 mutant lacking its
C-terminal PDZ, SH3, and GUK domains induced NF-
B activity to levels
4-5-fold greater than that obtained with wild type protein (Fig.
3D). Thus, the C-terminal domains may function to negatively
regulate induction of NF-
B signaling by CARD11.

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Fig. 3.
CARD11 and CARD14 activate
NF- B. Plasmids expressing CARD11 and
CARD14 were transfected into 293T cells, and relative luciferase
activities were measured to determine the induction of NF- B
activity. A and B, concentration-dependent
activation of NF- B activity by CARD11 and CARD14. C,
deletion mutants of CARD11 and CARD14 used to map domains involved in
the induction of NF- B activity. Bars indicate domains
expressed: construct 1 (CARD11, residues 1-1147; CARD14, residues
1-1004), construct 2 (CARD11, residues 127-1147; CARD14, residues
119-1004), construct 3 (CARD11, residues 1-468, CARD14, residues
1-436), construct 4 (CARD11, residues 1-759; CARD14, residues
1-669), construct 5 (CARD11, residues 1-869; CARD14, residues
1-807), construct 6 (CARD11, residues 469-1147, CARD14, residues
437-1004). D and E, induction of NF- B
activity by CARD11 and CARD14 deletion mutants (upper
panels). Lower panels in D and E
show protein levels of truncation mutants.
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The N-terminal CARDs of CARD11 and CARD14 likely interact with other
CARD-containing proteins to signal activation of NF-
B. To identify
the binding partners of CARD11 and CARD14, we performed a mammalian
two-hybrid analysis and screened their N-terminal CARDs for binding to
the CARD domains of 15 known proteins. The CARD of CARD11 interacted
with the CARD of BCL10 resulting in a 17-fold increase in relative
luciferase activity (Fig. 4A). Co-expression of CARD11-CARD with other CARD domains failed to activate
luciferase expression indicating that the CARD of CARD11 interacts
selectively with the CARD of BCL10. Likewise, the CARD of CARD14
interacted selectively with the CARD of BCL10 resulting in a 999-fold
increase in relative luciferase activity. These data suggest that both
CARD11 and CARD14 are signaling partners of the NF-
B activator
BCL10. We next tested whether CARD11 and CARD14 interact with
endogenous BCL10 when overexpressed in cells. Expression of either
Myc-tagged CARD11 or Myc-tagged CARD14 co-precipitated endogenous
BCL10, confirming that both CARD proteins interact with BCL10 (Fig.
5A, lanes 1 and
3). We also examined the interaction of radiolabeled CARD11
and CARD14 with GST-BCL10 in vitro and found that both
proteins associate directly with BCL10 through their N-terminal CARD
domains (Fig. 5B, lane 3). Confirming the importance of the BCL10 CARD domain, radiolabeled CARD11 and CARD14 did
not associate with a variant of BCL10 (L41R) that is unable to
homodimerize (Fig. 5B, lane 4; Ref. 12).

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Fig. 4.
The CARD domains of CARD11 and CARD14
interact selectively with the CARD of BCL10 by mammalian two-hybrid
analysis. 293T cells were transfected with the mammalian
two-hybrid reporter construct pFR-Luc (Stratagene), and combinations of
plasmids expressing CARD domains fused to either the activation domain
of the mouse protein NF- B or the DNA-binding domain of the yeast
protein Gal4. After 24 h, cells were collected and assayed
for relative luciferase activity. CARD domains of CARD11 (A)
and CARD14 (B) fused to the activation domain and screened
against a panel of individual CARDs fused to the DNA-binding
domain.
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Fig. 5.
CARD11 and CARD14 interact with BCL10.
A, 293T cells were transfected with plasmids expressing
either Myc-tagged CARD11 or CARD14. Cell extracts were
immunoprecipitated (IP) with either BCL10 antibodies
(lanes 1, 3 and 5) or control T7
monoclonal antibodies (lanes 2, 4 and
6), and immunoblotted (WB) with anti-Myc
antibodies to detect epitope-tagged CARD11 and CARD14. B,
in vitro interaction of CARD11 and CARD14 with GST-BCL10.
35S-labeled protein (lane 1, 10% input) was
precipitated with glutathione-Sepharose beads bound to an equal amount
of GST (lane 2), GST-BCL10 (lane 3), and
GST-BCL10-L41R (lane 4) and then analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography. The point
mutation L41R within the CARD domain abrogates CARD-CARD
interactions.
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The cellular localization of CARD14 was also examined. When
epitope-tagged CARD14 and BCL10 were expressed alone, the two proteins
displayed distinctly different patterns of cellular localization. As
observed previously (8, 27), BCL10 exhibited either a discrete pattern
of cytoplasmic filaments and aggregates or a diffuse whole-cell
distribution (data not shown). In contrast, CARD14 exhibited a
predominantly cytoplasmic, slightly punctate distribution but did not
form large aggregates or filaments (Fig. 6A). When these two proteins
were co-expressed in the same cell, however, some of the CARD14 was
found to co-localize with the BCL10 filaments or aggregates (Fig. 6,
B-D). This finding is consistent with an intracellular
interaction between CARD14 and BCL10 and suggests that CARD14 is
recruited to a cytoplasmic signaling complex with BCL10. To test
whether the CARD domain of CARD14 was required for this interaction, we
examined the localization of a CARD14 truncation mutant lacking the
N-terminal CARD domain (CARD14/
CARD). When expressed alone,
CARD14/
CARD formed aggregates and showed a punctate distribution
(Fig. 6E). When co-expressed with BCL10, however,
CARD14/
CARD did not co-localize with BCL10 (Fig. 6, F-H). Deletion of the C-terminal PDZ/SH3/GUK domain also
resulted in co-localization with BCL10 (data not shown), indicating
that the CARD and coiled-coil domains are sufficient for the
interaction between CARD14 and BCL10 and that this interaction requires
an intact CARD domain. A similar CARD-dependent
co-localization was observed between CARD11 and BCL10 (data not
shown).

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Fig. 6.
CARD14 recruitment to BCL10 filaments
requires an intact CARD domain. HA-tagged BCL10 and Myc-tagged
CARD14 proteins were expressed in Rat-1 cells. A and
E show CARD14 and CARD14/ CARD when expressed
alone. B-D and F-H show
the immunostaining patterns observed when each protein was co-expressed
with BCL10. Note the strong co-localization of CARD14 to BCL10
filaments (B-D) and the lack of co-localization
of CARD14/ CARD with BCL10 even in a cell in which BCL10
formed filaments (F-H).
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BCL10 migrates in SDS gels as a triplet ranging in size from 29 to 32 kDa due to phosphorylation of its C-terminal domain (12, 18). Treatment
of cell lysates with calf intestinal alkaline phosphatase eliminates
the slower migrating forms demonstrating that the fastest migrating
band represents unphosphorylated BCL10 (12). Because phosphorylation
can play a critical role in signal transduction, we examined whether
co-expression of either CARD11 or CARD14 induces the phosphorylation of
BCL10 (Fig. 7, upper panel).
When expressed alone, the majority of HA-tagged BCL10 exists in the
unphoshorylated form (Fig. 7, lane 1, lower
band). However, co-expression of either CARD11 or CARD14 markedly
increased the amount of phosphorylated BCL10 represented by the slower
migrating bands (Fig. 7, lanes 3 and middle and
upper bands). The induction of BCL10 phosphorylation is
dependent on the N-terminal CARD of CARD11 and CARD14 because
co-expression of truncated mutants lacking these domains has no effect
on BCL10 phosphorylation levels (Fig. 7, lanes 5 and
9). Immunoblot analysis revealed that the Myc-tagged truncation mutants were expressed at levels similar to wild type protein suggesting that loss of function is not due to reduced levels
of expression (Fig. 7, lower panel). Taken together, these data suggest that CARD11 and CARD14 stimulate phosphorylation of BCL10
in a CARD-dependent manner.

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Fig. 7.
CARD11 and CARD14 stimulate phosphorylation
of BCL10. 293T cells were transfected with expression constructs
encoding HA-BCL10 and either Myc-tagged CARD11 or CARD14. Cell lysates
were collected and immunoblotted (WB) with HA and Myc
antibodies to detect BCL10 (upper panel), CARD11, and CARD14
proteins (lower panel).
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We have identified CARD11 and CARD14 as specific regulators of
BCL10 function. Our finding that CARD11 and CARD14 bind to BCL10
through a CARD-CARD interaction suggests that these molecules function
as upstream activators of BCL10. CARD9 also binds to the CARD
activation domain of BCL10 and signals NF-
B activation (8). Thus,
CARD11, CARD14, and CARD9 constitute a subclass of CARD proteins that
likely functions to transduce distinct upstream stimuli to the
activation of BCL10 and NF-
B. In response to upstream signals, the
coiled-coil domains could mediate self-association of CARD11 and CARD14
resulting in the aggregation and activation of BCL10. BCL10 might then
engage and oligomerize IKK
resulting in the activation of the IKK
complex and NF-
B (16, 17). Thus, CARD11 and CARD14 could act in a
manner analogous to Apaf-1 and CARD4 that functions to induce
oligomerization and activation of the respective downstream
CARD-binding partners. Our data also indicate that CARD11 and CARD14
induce the phosphorylation of BCL10 suggesting that signal transduction
may involve the participation of a serine/threonine kinase. A unique
feature of CARD11 and CARD14 is the presence of C-terminal
PDZ/SH3/GUK domains. These domains may function in an analogous manner
to the C-terminal leucine-rich repeat domain of CARD4 and the
WD-40 domain of Apaf-1 to regulate protein activation by upstream
signals (8, 10). PDZ/SH3/GUK domains identify MAGUK family members, a
class of proteins that associate with the plasma membrane through
interactions with transmembrane proteins (e.g. ion
channels), cytoskeletal components, and signal transduction proteins
(26). Interestingly, the PDZ domain found in many MAGUK proteins has
been shown to interact with the intracellular domains of specific
receptors. Thus, CARD11 and CARD14 may function as scaffolding proteins
to assemble a multiprotein complex at the intracellular domains of
receptors that signal the activation of NF-
B.
 |
FOOTNOTES |
*
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) AF322641 and AF322642.
§
To whom correspondence may be addressed: Millennium
Pharmaceuticals, Inc., 640 Memorial Dr., Cambridge, MA 02139. Tel:
617-679-7215; Fax: 617-679-7071; E-mail: bertin@mpi.com.
To whom correspondence may be addressed: Kimmel Cancer Inst.,
Thomas Jefferson Univ., Bluemle Life Sciences Bldg., Rm. 904, 233 S. 10th St., Philadelphia, PA 19107. Tel: 215-503-4632; Fax: 215-923-1098; E-mail: E Alnemri@lac.jci.tju.edu.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M010512200
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ABBREVIATIONS |
The abbreviations used are:
CARD, caspase
recruitment domain;
IKK, I
B kinase;
GUK, guanylate kinase;
MAGUK, membrane-associated GUK;
GST, glutathione S-transferase;
SH3, Src homology 3;
HA, hemagglutinin.
 |
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