Title: CARD11 and CARD14 Are Novel Caspase Recruitment Domain (CARD)/Membrane-associated Guanylate Kinase (MAGUK) Family Members that Interact with BCL10 and Activate NF-κB
Abstract: The caspase recruitment domain (CARD) is a protein-binding module that mediates the assembly of CARD-containing proteins into apoptosis and NF-κB 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-κB through the IκB kinase complex in response to upstream stimuli. When expressed in cells, CARD11 and CARD14 activate NF-κB 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-κB signaling. The caspase recruitment domain (CARD) is a protein-binding module that mediates the assembly of CARD-containing proteins into apoptosis and NF-κB 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-κB through the IκB kinase complex in response to upstream stimuli. When expressed in cells, CARD11 and CARD14 activate NF-κB 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-κB signaling. Modular protein interaction domains play an important role in signal transduction by mediating the assembly of components into specific signaling complexes (1Pawson T. Nash P. Genes Dev. 2000; 14: 1027-1047PubMed Google Scholar). 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 (2Hofmann K. Cell Mol. Life Sci. 1999; 55: 1113-1128Crossref PubMed Scopus (125) Google Scholar). CARD domains consist of six or seven antiparallel α-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 (2Hofmann K. Cell Mol. Life Sci. 1999; 55: 1113-1128Crossref PubMed Scopus (125) Google Scholar, 3Bertin J. Nir W.-J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H.W. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 4Inohara N. Koseki T. del Peso L. Hu Y. Yee C. Chen S. Carrio R. Merino J. Liu D. Ni J. Nunez G. J. Biol. Chem. 1999; 274: 14560-14567Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar, 5Masumoto J. Taniguchi S. Ayukawa K. Sarvotham H. Kishino T. Niikawa N. Hidaka E. Katsuyama T. Higuchi T. Sagara J. J. Biol. Chem. 1999; 274: 33835-33838Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 6Willis T.G. Jadayel D.M. Du M.Q. Peng H. Perry A.R. Abdul-Rauf M. Price H. Karran L. Majekodunmi O. Wlodarska I. Pan L. Crook T. Hamoudi R. Isaacson P.G. Dyer M.J. Cell. 1999; 96: 33-45Abstract Full Text Full Text PDF Scopus (580) Google Scholar, 7Inohara N. del Peso L. Koseki T. Chen S. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1998; 273: 12296-12300Google Scholar, 8Bertin J. Guo Y. Wang L. Srinivasula S.M. Jacobson M.D. Poyet J.-L. Merriam S. Du M.-Q. Dyer M.J.S. Robison K.E. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 275: 41082-41086Abstract Full Text Full Text PDF Scopus (194) Google Scholar, 9Humke E.W. Shriver S.K. Starovasnik M.A. Fairbrother W.J. Dixit V.M. Cell. 2000; 103: 99-111Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). 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 cand dATP resulting in caspase oligomerization and activation (10Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6317) Google Scholar). Other CARD proteins that segregate with discrete binding partners include CARD4 with RICK, RAIDD with caspase-2, and CARD9 with BCL10 (3Bertin J. Nir W.-J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H.W. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar,8Bertin J. Guo Y. Wang L. Srinivasula S.M. Jacobson M.D. Poyet J.-L. Merriam S. Du M.-Q. Dyer M.J.S. Robison K.E. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 275: 41082-41086Abstract Full Text Full Text PDF Scopus (194) Google Scholar, 11Duan H. Dixit V.M. Nature. 1997; 385: 86-89Crossref PubMed Scopus (471) Google Scholar). 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-κB. CARD4, RICK, BCL10, and CARD9 induce NF-κB activity through the IKK complex when overexpressed in cells (3Bertin J. Nir W.-J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H.W. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 4Inohara N. Koseki T. del Peso L. Hu Y. Yee C. Chen S. Carrio R. Merino J. Liu D. Ni J. Nunez G. J. Biol. Chem. 1999; 274: 14560-14567Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar, 6Willis T.G. Jadayel D.M. Du M.Q. Peng H. Perry A.R. Abdul-Rauf M. Price H. Karran L. Majekodunmi O. Wlodarska I. Pan L. Crook T. Hamoudi R. Isaacson P.G. Dyer M.J. Cell. 1999; 96: 33-45Abstract Full Text Full Text PDF Scopus (580) Google Scholar, 8Bertin J. Guo Y. Wang L. Srinivasula S.M. Jacobson M.D. Poyet J.-L. Merriam S. Du M.-Q. Dyer M.J.S. Robison K.E. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 275: 41082-41086Abstract Full Text Full Text PDF Scopus (194) Google Scholar, 12Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). NF-κB plays a central role in the activation of genes involved in immunity, inflammation, and apoptosis (13Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4657) Google Scholar, 14Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4155) Google Scholar). In unstimulated cells, NF-κB is sequestered in the cytoplasm through interactions with inhibitory IκB proteins. In response to a variety of signals including the cytokines interleukin-1 and tumor necrosis factor α, bacterial lipopolysaccharide, and virus infection, IκBα is phosphorylated and targeted to degradation by the proteosome through covalent modification by ubiquitin. The degradation of IκBα results in the translocation of NF-κB to the nucleus where it binds to specific promoters and activates transcription. Phosphorylation of IκBα is mediated by the IKK complex, which consists of two catalytic subunits called IKKα and IKKβ and one regulatory subunit called IKKγ. Although the mechanism by which IKKγ regulates IKK activity is presently unknown, it has been proposed to link the IKKs to upstream regulatory molecules (15Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 16Poyet J.-L. Srinivasula S.M. Lin J.H. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 17Inohara N. Koseki T. Lin J. del Peso L. Lucas P.C. Chen F.F. Ogura Y. Nunez G. J. Biol. Chem. 2000; 275: 27823-27831Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). The CARD protein RICK binds directly to IKKγ suggesting that it functions as an adaptor molecule between the IKK complex and its upstream binding partner CARD4 (17Inohara N. Koseki T. Lin J. del Peso L. Lucas P.C. Chen F.F. Ogura Y. Nunez G. J. Biol. Chem. 2000; 275: 27823-27831Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). 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-κB signaling. BCL10 (also known as CLAP/CIPER/cE10/CARMEN) is an activator of apoptosis and NF-κB signaling pathways that has been implicated in B cell lymphomas of mucosa-associated lymphoid tissue (6Willis T.G. Jadayel D.M. Du M.Q. Peng H. Perry A.R. Abdul-Rauf M. Price H. Karran L. Majekodunmi O. Wlodarska I. Pan L. Crook T. Hamoudi R. Isaacson P.G. Dyer M.J. Cell. 1999; 96: 33-45Abstract Full Text Full Text PDF Scopus (580) Google Scholar, 12Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 18Koseki T. Inohara N. Chen S. Carrio R. Merino J. Hottiger M.O. Nabel G.J. Nunez G. J. Biol. Chem. 1999; 274: 9955-9961Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 19Costanzo A. Guiet C. Vito P. J. Biol. Chem. 1999; 274: 20127-20132Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 20Thome M. Martinon F. Hofmann K. Rubio V. Steiner V. Schneider P. Mattmann C. Tschopp J. J. Biol. Chem. 1999; 274: 9962-9968Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 21Zhang Q. Siebert R. Yan M. Hinzmann B. Cui X. Xue L. Rakestraw K.M. Naeve C.W. Beckmann G. Weisenburger D.D. Sanger W.G. Nowotny H. Vesely M. Callet-Bauchu E. Salles G. Dixit V.M. Rosenthal A. Schlegelberger B. Morris S.W. Nat. Genet. 1999; 22: 63-68Crossref PubMed Scopus (337) Google Scholar). 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-κB 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 (12Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The C terminus of BCL10 may function in a manner analogous to the NF-κB activators RIP and RICK and activate the IKK complex through binding and oligomerization of IKKγ (16Poyet J.-L. Srinivasula S.M. Lin J.H. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 17Inohara N. Koseki T. Lin J. del Peso L. Lucas P.C. Chen F.F. Ogura Y. Nunez G. J. Biol. Chem. 2000; 275: 27823-27831Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). We recently identified a novel CARD NF-κB activator called CARD9 that assembles into a CARD-CARD signaling complex with BCL10 (8Bertin J. Guo Y. Wang L. Srinivasula S.M. Jacobson M.D. Poyet J.-L. Merriam S. Du M.-Q. Dyer M.J.S. Robison K.E. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 275: 41082-41086Abstract Full Text Full Text PDF Scopus (194) Google Scholar). We report here the identity and characterization of two additional CARD proteins, CARD11 and CARD14, that assemble BCL10 into NF-κB 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-κB. 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 (12Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). 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 (8Bertin J. Guo Y. Wang L. Srinivasula S.M. Jacobson M.D. Poyet J.-L. Merriam S. Du M.-Q. Dyer M.J.S. Robison K.E. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 275: 41082-41086Abstract Full Text Full Text PDF Scopus (194) Google Scholar). 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-κB assays, 293T cells were transfected with the following plasmids: 900 ng of pNF-κB 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. 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 (22Ye H. Dogan A. Karran L. Willis T.G. Chen L. Wlodarska I. Dyer M.J. Isaacson P.G. Du M.Q. Am. J. Pathol. 2000; 157: 1147-1154Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). 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 between BCL10 and either CARD11 or CARD14 proteins were performed as described previously (23Ahmad M. Srinivasula S.M. Wang L. Talanian R.V. Litwack G. Fernandes-Alnemri T. Alnemri E.S. Cancer Res. 1997; 57: 615-619PubMed Google Scholar). 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. 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. We searched public and proprietary data bases for novel members of the CARD family of apoptosis and NF-κB signaling proteins. Human CARD11 is a novel CARD family member of 1147 amino acids with a predicted molecular mass of 132.6 kDa (Fig.1 A). A second protein (1004 amino acids, 113.3 kDa) displaying significant similarity to CARD11 was also identified and designated CARD14 (Fig. 1 A). 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. 1 B). 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. 1 C). 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 (24Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Abstract Full Text PDF PubMed Scopus (1008) Google Scholar). The COILS2 program (25Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3536) Google Scholar) 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 (26Fanning A.S. Anderson J.M. Curr. Opin. Cell Biol. 1999; 11: 432-439Crossref PubMed Scopus (274) Google Scholar). The structure of CARD11 and CARD14 is most similar to CARD9 which contains an N-terminal CARD domain followed by multiple coiled-coil domains (8Bertin J. Guo Y. Wang L. Srinivasula S.M. Jacobson M.D. Poyet J.-L. Merriam S. Du M.-Q. Dyer M.J.S. Robison K.E. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 275: 41082-41086Abstract Full Text Full Text PDF Scopus (194) Google Scholar). 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.2 A). 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. 2 B). In contrast, the 4.4-kilobase CARD14 transcript showed expression only in placenta (Fig. 2 C) and HeLa S3 cancer cells (Fig. 2 D).Figure 2Tissue 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because CARD proteins have been implicated in NF-κB signaling pathways, we determined whether CARD11 and CARD14 can induce NF-κB activity using a luciferase reporter gene. When either CARD11 or CARD14 were expressed in 293T cells, NF-κB activity was induced 20–40-fold compared with empty vector (Fig. 3,A and B). NF-κB signaling occurred through the IKK complex because dominant-negative versions of IKK-γ and IKK-β blocked the abilities of CARD11 and CARD14 to induce NF-κB activity (data not shown). To determine the role of individual domains in NF-κB signaling, we constructed a series of N- and C-terminal truncation mutants of CARD11 and CARD14 (Fig. 3 C). The N-terminal CARD of both CARD11 and CARD14 was essential for NF-κB signaling because deletion of this domain eliminated the induction of NF-κB 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-κB signaling because deletion of these domains did not reduce the ability of CARD11 and CARD14 to induce NF-κB activity. However, a CARD11 mutant lacking its C-terminal PDZ, SH3, and GUK domains induced NF-κB activity to levels 4–5-fold greater than that obtained with wild type protein (Fig.3 D). Thus, the C-terminal domains may function to negatively regulate induction of NF-κB signaling by CARD11. The N-terminal CARDs of CARD11 and CARD14 likely interact with other CARD-containing proteins to signal activation of NF-κB. 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. 4 A). 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-κB 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.5 A, lanes 1 and3). 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. 5 B, 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. 5 B, lane 4; Ref. 12Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar).Figure 5CARD11 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 and6), 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 (8Bertin J. Guo Y. Wang L. Srinivasula S.M. Jacobson M.D. Poyet J.-L. Merriam S. Du M.-Q. Dyer M.J.S. Robison K.E. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 275: 41082-41086Abstract Full Text Full Text PDF Scopus (194) Google Scholar, 27Guiet C. Vito P. J. Cell Biol. 2000; 148: 1131-1140Crossref PubMed Scopus (69) Google Scholar), 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.6 A). 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/ΔCARD). When expressed alone, CARD14/ΔCARD formed aggregates and showed a punctate distribution (Fig. 6 E). When co-expressed with BCL10, however, CARD14/ΔCARD 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). BCL10 migrates in SDS gels as a triplet ranging in size from 29 to 32 kDa due to phosphorylation of its C-terminal domain (12Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 18Koseki T. Inohara N. Chen S. Carrio R. Merino J. Hottiger M.O. Nabel G.J. Nunez G. J. Biol. Chem. 1999; 274: 9955-9961Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Treatment of cell lysates with calf intestinal alkaline phosphatase eliminates the slower migrating forms demonstrating that the fastest migrating band represents unphosphorylated BCL10 (12Srinivasula S.M. Ahmad M. Lin J.H. Poyet J.L. Fernandes-Alnemri T. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 1999; 274: 17946-17954Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). 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 andupper 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 and9). 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. 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-κB activation (8Bertin J. Guo Y. Wang L. Srinivasula S.M. Jacobson M.D. Poyet J.-L. Merriam S. Du M.-Q. Dyer M.J.S. Robison K.E. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 275: 41082-41086Abstract Full Text Full Text PDF Scopus (194) Google Scholar). 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-κB. 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 IKKγ resulting in the activation of the IKK complex and NF-κB (16Poyet J.-L. Srinivasula S.M. Lin J.H. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 17Inohara N. Koseki T. Lin J. del Peso L. Lucas P.C. Chen F.F. Ogura Y. Nunez G. J. Biol. Chem. 2000; 275: 27823-27831Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). 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 (8Bertin J. Guo Y. Wang L. Srinivasula S.M. Jacobson M.D. Poyet J.-L. Merriam S. Du M.-Q. Dyer M.J.S. Robison K.E. DiStefano P.S. Alnemri E.S. J. Biol. Chem. 2001; 275: 41082-41086Abstract Full Text Full Text PDF Scopus (194) Google Scholar, 10Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6317) Google Scholar). 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 (26Fanning A.S. Anderson J.M. Curr. Opin. Cell Biol. 1999; 11: 432-439Crossref PubMed Scopus (274) Google Scholar). 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-κB. caspase recruitment domain IκB kinase guanylate kinase membrane-associated GUK glutathione S-transferase Src homology 3 hemagglutinin