CARD11 and CARD14 are novel caspase recruitment domain (CARD)/membrane-associated guanylate kinase (MAGUK) family members that interact with BCL10 and activate NF-kappa B

The caspase recruitment domain (CARD) is a protein-binding module that mediates the assembly of CARD-containing proteins into apoptosis and NF-kappaB 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-kappaB through the IkappaB kinase complex in response to upstream stimuli. When expressed in cells, CARD11 and CARD14 activate NF-kappaB 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-kappaB signaling.     

The protein kinase C-responsive inhibitory domain of CARD11 functions in NF-kappaB activation to regulate the association of multiple signaling cofactors that differentially depend on Bcl10 and MALT1 for association

The activation of NF-κB by T-cell receptor (TCR) signaling is critical for T-cell activation during the adaptive immune response. CARD11 is a multidomain adapter that is required for TCR signaling to the IκB kinase (IKK) complex. During TCR signaling, the region in CARD11 between the coiled-coil and PDZ domains is phosphorylated by protein kinase Cθ (PKCθ) in a required step in NF-κB activation. In this report, we demonstrate that this region functions as an inhibitory domain (ID) that controls the association of CARD11 with multiple signaling cofactors, including Bcl10, TRAF6, TAK1, IKKγ, and caspase-8, through an interaction that requires both the caspase recruitment domain (CARD) and the coiled-coil domain. Consistent with the ID-mediated control of their association, we demonstrate that TRAF6 and caspase-8 associate with CARD11 in T cells in a signal-inducible manner. Using an RNA interference rescue assay, we demonstrate that the CARD, linker 1, coiled-coil, linker 3, SH3, linker 4, and GUK domains are each required for TCR signaling to NF-κB downstream of ID neutralization. Requirements for the CARD, linker 1, and coiled-coil domains in signaling are consistent with their roles in the association of CARD11 with Bcl10, TRAF6, TAK1, caspase-8, and IKKγ. Using Bcl10- and MALT1-deficient cells, we show that CARD11 can recruit signaling cofactors independently of one another in a signal-inducible manner.     

The dynamic distribution of CARD11 at the immunological synapse is regulated by the inhibitory kinesin GAKIN

T cell receptor (TCR) signaling to NF-κB is required for antigen-induced T cell activation. We conducted an expression-cloning screen for modifiers of CARD11, a critical adaptor in antigen receptor signaling, and identified the kinesin-3 family member GAKIN as a CARD11 inhibitor. GAKIN negatively regulates TCR signaling to NF-κB, associates with CARD11 in a signal-dependent manner and can compete with the required signaling protein, Bcl10, for association. In addition, GAKIN dynamically localizes to the immunological synapse and regulates the redistribution of CARD11 from the central region of the synapse to a distal region. We propose that CARD11 scaffold function and occupancy at the center of the synapse are negatively regulated by GAKIN to tune the output of antigen-receptor signaling.     

hCASK and hDlg associate in epithelia, and their src homology 3 and guanylate kinase domains participate in both intramolecular and intermolecular interactions

Membrane-associated guanylate kinase (MAGUK) proteins act as molecular scaffolds organizing multiprotein complexes at specialized regions of the plasma membrane. All MAGUKs contain a Src homology 3 (SH3) domain and a region homologous to yeast guanylate kinase (GUK). We showed previously that one MAGUK protein, human CASK (hCASK), is widely expressed and associated with epithelial basolateral plasma membranes. We now report that hCASK binds another MAGUK, human discs large (hDlg). Immunofluorescence microscopy demonstrates that hCASK and hDlg colocalize at basolateral membranes of epithelial cells in small and large intestine. These proteins co-precipitate from lysates of an intestinal cell line, Caco-2. The GUK domain of hCASK binds the SH3 domain of hDlg in both yeast two-hybrid and fusion protein binding assays, and it is required for interaction with hDlg in transfected HEK293 cells. In addition, the SH3 and GUK domains of each protein participate in intramolecular binding that in vitro predominates over intermolecular binding. The SH3 and GUK domains of human p55 display the same interactions in yeast two-hybrid assays as those of hCASK. Not all SH3-GUK interactions among these MAGUKs are permissible, however, implying specificity to SH3-GUK interactions in vivo. These results suggest MAGUK scaffold assembly may be regulated through effects on intramolecular SH3-GUK binding.     

A high-affinity Arg-X-X-Lys SH3 binding motif confers specificity for the interaction between Gads and SLP-76 in T cell signaling

A critical event in T cell receptor (TCR)-mediated signaling is the recruitment of hematopoietic-specific adaptor proteins that collect and transmit signals downstream of the TCR. Gads, a member of the Grb2 family of SH2 and SH3 domain-containing adaptors, mediates the formation of a complex between LAT and SLP-76 that is essential for signal propagation from the TCR. Here we examine the binding specificity of the Gads and Grb2 SH3 domains using peptide arrays and find that a nonproline-based R-X-X-K motif found in SLP-76 binds to the Gads carboxy-terminal SH3 domain with high affinity (K(D) = 240 +/- 45 nM). The Grb2 C-terminal SH3 domain also binds this motif, but with a 40-fold lower affinity than Gads. Single point mutations in either the relevant R (237) or K (240) completely abrogated SLP-76 association with Gads in vivo and impaired SLP-76 function. A chimeric Grb2 protein, possessing the C-terminal SH3 domain of Gads, was able to partially substitute for Gads in signaling downstream of the T cell receptor. These results provide a molecular explanation for the specific role of Gads in T cell receptor signaling, and identify a discrete subclass of SH3 domains whose binding is dependent on a core R-X-X-K motif.     

Card10 is a novel caspase recruitment domain/membrane-associated guanylate kinase family member that interacts with BCL10 and activates NF-kappa B

BCL10 belongs to the caspase recruitment domain (CARD) family of proteins that regulate apoptosis and NF-kappaB signaling pathways. Analysis of BCL10-deficient mice has revealed that BCL10 mediates NF-kappaB 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-kappaB 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-kappaB.     

Recruitment of the actin-binding protein HIP-55 to the immunological synapse regulates T cell receptor signaling and endocytosis

Actin cytoskeleton dynamics critically regulate T cell activation. We found that the cytoplasmic adaptor HIP-55, a Src/Syk-kinases substrate and member of the drebrin/Abp1 family of actin-binding proteins, localized to the T cell-antigen-presenting cell (APC) contact site in an antigen-dependent manner. Using green fluorescent protein fusion proteins, both Src homology 3 (SH3) and actin binding domains were found necessary for recruitment at the T cell-APC interface. HIP-55 was not implicated in conjugate formation and actin polymerization but regulated distal signaling events through binding and activation of hematopoietic progenitor kinase 1 (HPK1), a germinal center kinase (GCK) family kinase involved in negative signaling in T cells. Using RNA interference and overexpression experiments, the HIP-55-HPK1 complex was found to negatively regulate nuclear factor of activated T cell (NFAT) activation by the T cell antigen receptor. Moreover, we show that HIP-55, which partly co-localized with early endocytic compartments, promoted both basal and ligand-dependent T cell receptor (TCR) down-modulation, resulting in a decreased TCR expression. SH3 and actin-depolymerizing factor homology domains were required for this function. As controls, the expression of CD28 and the glycosylphosphatidylinositol-linked protein CD59 was not affected by HIP-55 overexpression. These results suggest that, in addition to binding to HPK1, HIP-55 might negatively regulate TCR signaling through down-regulation of TCR expression. Our findings show that HIP-55 is a key novel component of the immunological synapse that modulates T cell activation by connecting actin cytoskeleton and TCRs to gene activation and endocytic processes.     

Intramolecular interactions regulate SAP97 binding to GKAP

Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP-GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners.     

Differential effects of filipin and methyl-beta-cyclodextrin on B cell receptor signaling

The CED4/Apaf-1 family of proteins functions as critical regulators of apoptosis and NF-kappaB signaling pathways. A novel human member of this family, called CARD12, was identified that induces apoptosis when expressed in cells. CARD12 is most similar in structure to the CED4/Apaf-1 family member CARD4, and is comprised of an N-terminal caspase recruitment domain (CARD), a central nucleotide-binding site (NBS), and a C-terminal domain of leucine-rich repeats (LRR). The CARD domain of CARD12 interacts selectively with the CARD domain of ASC, a recently identified proapoptotic protein. In addition, CARD12 coprecipitates caspase-1, a caspase that participates in both apoptotic signaling and cytokine processing. CARD12 may assemble with proapoptotic CARD proteins to coordinate the activation of downstream apoptotic and inflammatory signaling pathways.     

The unique-5 and -6 motifs of ZO-1 regulate tight junction strand localization and scaffolding properties

The proper cellular location and sealing of tight junctions is assumed to depend on scaffolding properties of ZO-1, a member of the MAGUK protein family. ZO-1 contains a conserved SH3-GUK module that is separated by a variable region (unique-5), which in other MAGUKs has proven regulatory functions. To identify motifs in ZO-1 critical for its putative scaffolding functions, we focused on the SH3-GUK module including unique-5 (U5) and unique-6 (U6), a motif immediately C-terminal of the GUK domain. In vitro binding studies reveal U5 is sufficient for occludin binding; U6 reduces the affinity of this binding. In cultured cells, U5 is required for targeting ZO-1 to tight junctions and removal of U6 results in ectopically displaced junction strands containing the modified ZO-1, occludin, and claudin on the lateral cell membrane. These results provide evidence that ZO-1 can control the location of tight junction transmembrane proteins and reveals complex protein binding and targeting signals within its SH3-U5-GUK-U6 region. We review these findings in the context of regulated scaffolding functions of other MAGUK proteins.     

The Src homology 3 domain is required for junctional adhesion molecule binding to the third PDZ domain of the scaffolding protein ZO-1

Tight junctions are cell-cell contacts that regulate the paracellular flux of solutes and prevent pathogen entry across cell layers. The assembly and permeability of this barrier are dependent on the zonula occludens (ZO) membrane-associated guanylate kinase (MAGUK) proteins ZO-1, -2, and -3. MAGUK proteins are characterized by a core motif of protein-binding domains that include a PDZ domain, a Src homology 3 (SH3) domain, and a region of homology to guanylate kinase (GUK); the structure of this core motif has never been determined for any MAGUK. To better understand how ZO proteins organize the assembly of protein complexes we have crystallized the entire PDZ3-SH3-GUK core motif of ZO-1. We have also crystallized this core motif in complex with the cytoplasmic tail of the ZO-1 PDZ3 ligand, junctional adhesion molecule A (JAM-A) to determine how the activity of different domains is coordinated. Our study shows a new feature for PDZ class II ligand binding that implicates the two highly conserved Phe(-2) and Ser(-3) residues of JAM. Our x-ray structures and NMR experiments also show for the first time a role for adjacent domains in the binding of ligands to PDZ domains in the MAGUK proteins family.     

Mice lacking the CARD of CARMA1 exhibit defective B lymphocyte development and impaired proliferation of their B and T lymphocytes

CARMA1 (originally called CARD11) is a membrane-associated guanylate kinase family member that is required for T cell receptor (TCR)-induced NF-kappa B activation in T cell leukemia lines. It uses its N-terminal caspase activation and recruitment domain (CARD) to interact with the CARD in the downstream adaptor Bcl-10. We show that primary B and T lymphocytes from knock-in mice expressing only a CARDless form of CARMA1 (Delta CARD) are defective at mitogen-induced NF-kappa B activation and fail to proliferate. CARMA1 mutant mice exhibited normal T but impaired B cell development; CD5(+) peritoneal B cells were absent, and serum immunoglobulin levels were markedly reduced. A lacZ reporter gene knocked into the CARMA1 locus confirmed lymphocyte-specific expression of CARMA1. Thus, CARMA1 has an essential role in mediating B and T lymphocyte proliferation and requires its CARD to engage downstream signaling components.     

CARMA1 coiled-coil domain is involved in the oligomerization and subcellular localization of CARMA1 and is required for T cell receptor-induced NF-kappaB activation

T lymphocyte (T cell) activation and proliferation is induced by the activation of multiple signal transduction pathways. Earlier studies indicate that CARMA1, a Caspase Recruitment Domain (CARD) and Membrane-associated GUanylate Kinase domain (MAGUK)-containing scaffold protein, plays an essential role in NF-kappaB activation induced by the costimulation of T cell receptor (TCR) and CD28 molecules. However, the molecular mechanism by which CARMA1 mediates TCR-CD28 costimulation-induced NF-kappaB activation is not fully understood. Here we show that CARMA1 is constitutively oligomerized. This oligomerization of CARMA1 is through its Coiled-coil domain. Disruption of the predicted structure of the Coiled-coil domain of CARMA1 impaired its oligomerization and, importantly, abrogated CARMA1-mediated NF-kappaB activation. Interestingly, disruption of the CC1 domain abrogates CARMA1 localization, whereas disruption of the CC2 domain seems to inhibit CARMA1 self-association. Together, our results demonstrate that the oligomerization of CARMA1 is required for TCR-induced NF-kappaB activation.     

CARD9 is a novel caspase recruitment domain-containing protein that interacts with BCL10/CLAP and activates NF-kappa B

BCL10/CLAP is an activator of apoptosis and NF-kappaB signaling pathways and has been implicated in B cell lymphomas of mucosa-associated lymphoid tissue. Although its role in apoptosis remains to be determined, BCL10 likely activates NF-kappaB through the IKK complex in response to upstream stimuli. The N-terminal caspase recruitment domain (CARD) of BCL10 has been proposed to function as an activation domain that mediates homophilic interactions with an upstream CARD-containing NF-kappaB activator. To identify upstream signaling partners of BCL10, we performed a mammalian two-hybrid analysis and identified CARD9 as a novel CARD-containing protein that interacts selectively with the CARD activation domain of BCL10. When expressed in cells, CARD9 binds to BCL10 and activates NF-kappaB. Furthermore, endogenous CARD9 is found associated with BCL10 suggesting that both proteins form a pre-existing signaling complex within cells. CARD9 also self-associates and contains extensive coiled-coil motifs that may function as oligomerization domains. We propose here that CARD9 is an upstream activator of BCL10 and NF-kappaB signaling.     

MAGUK-Controlled Ubiquitination of CARMA1 Modulates Lymphocyte NF-κB Activity

The adaptor protein CARMA1 is required for antigen receptor-triggered activation of IKK and JNK in lymphocytes. Once activated, the events that subsequently turn off the CARMA1 signalosome are unknown. In this study, we found that antigen receptor-activated CARMA1 underwent lysine 48 (K48) polyubiquitination and proteasome-dependent degradation. The MAGUK region of CARMA1 was an essential player in this event; the SH3 and GUK domains contained the main ubiquitin acceptor sites, and deletion of a Hook domain (an important structure for maintaining inactive MAGUK proteins) between SH3 and GUK was sufficient to induce constitutive ubiquitination of CARMA1. A similar deletion promoted the ubiquitination of PSD-95 and Dlgh1, suggesting that a conserved mechanism may control the turnover of other MAGUK family protein complexes. Functionally, we demonstrated that elimination of MAGUK ubiquitination sites in CARMA1 resulted in elevated basal and inducible NF-κB and JNK activation as a result of defective K48 ubiquitination and increased persistence of this ubiquitination-deficient CARMA1 protein in activated lymphocytes. The coordination of degradation with the full activation of the CARMA1 molecule likely provides an intrinsic feedback control mechanism to balance lymphocyte activation upon antigenic stimulation.The CARD-containing MAGUK protein 1 (CARMA1, or CARD11) is regarded as an orchestrator of both T-cell-dependent and T-cell-independent immune responses due to its requirement in the activation of IKK and JNK signaling pathways downstream of antigen receptor (AR) ligation in B and T cells (3, 6, 8, 17). CARMA1 overexpression and/or mutations have also been associated with lymphomagenesis, as it promotes sustained activation of NF-κB-dependent cell survival (10, 16, 18). Structurally, CARMA1 is a multidomain adaptor protein containing a caspase recruitment (CARD) and a coiled-coil (CC) domain linked upstream of a region that is related to the MAGUK family of proteins. This MAGUK region contains a postsynaptic density 95/disc large/zona occludens 1 (PDZ), a SRC homology 3 (SH3), and a guanylate kinase-like (GUK) domain (2, 20). In addition, CARMA1 contains a flexible serine/threonine-rich linker that bridges the CC and MAGUK domains. Phosphorylation of this linker by protein kinase Cβ (PKCβ) or PKCθ controls the activation status of CARMA1 (13, 29, 30); thus, this region has been designated as the PKC-regulated domain (PRD) (22). It is likely that PRD phosphorylation destabilizes an inhibitory conformation in CARMA1 that exposes the various interaction domains required to assemble its downstream signaling components. Consistent with this model, deletion of the PRD results in a constitutively active CARMA1, resulting in high basal NF-κB activation (14, 30).Proximal downstream adaptors of CARMA1 include BCL10 and MALT1 (22). Genetic deletion of any of these proteins in cellular and animal models has revealed the importance of this pathway to immune cell function. While AR-induced activation of early signaling pathways, such as protein tyrosine phosphorylation, intracellular Ca²⁺ flux, and activation of extracellular signal-regulated kinase (ERK) and Akt are intact in CARMA1-, BCL10-, or MALT1-deficient lymphocytes, activation of NF-κB and JNK signaling pathways is markedly impaired (6, 24-26). This is manifested in defective lymphocyte proliferation and survival and in reduced immune responses.While the events leading to the activation of the IKK signaling complex downstream of CARMA1 have been well characterized, the signals that down-modulate this pathway are less well understood. Studies of BCL10 turnover have yielded some possible mechanisms (34). After cell activation, BCL10 is posttranslationally modified by both phosphorylation (possibly via IKKβ or CaMKII) and polyubiquitination (polyUb) and undergoes degradation that results in the down-modulation of NF-κB activity. Several ubiquitin protein ligases (E3s), including Itch, NEDD4, cIAP2, and βTrCP, have been reported to drive BCL10 ubiquitination in lymphocytes and to promote its degradation through either lysosomal or proteasomal pathways (7, 12, 27, 37). Overexpression of such E3s downregulates BCL10-dependent pathways, including NF-κB and the production of interleukin-2. Interestingly, antigen receptor-induced phosphorylation and degradation of BCL10 is not observed in the absence of CARMA1 (12), indicating a role for CARMA1 in BCL10 turnover.In this report, we demonstrate that endogenous CARMA1 is directly ubiquitinated and degraded by the proteasome in AR-activated lymphocytes. Structure-function analyses showed that the primary targets for ubiquitination within CARMA1 were localized within the MAGUK region. Mutation of all lysine residues (potential ubiquitin modification targets) to arginines within the MAGUK of CARMA1 produced a hyperactive molecule that promoted high NF-κB and JNK activation levels. Unlike the wild-type (WT) CARMA1 molecule, a lysine-to-arginine mutant CARMA1 was not modified by polyUb chains upon cell activation and had a resulting increase in protein stability. We identified a region between the SH3 and GUK domains that is highly similar to a region (termed the Hook region) that regulates the conformation of the MAGUK protein PSD-95. Notably, deletion of this Hook region was sufficient to trigger polyUb of CARMA1 as well as PSD-95 and Dlgh1, other MAGUK family proteins. These data suggest that activation of CARMA1 initiates a feedback mechanism controlled by the MAGUK domain that triggers ubiquitination and degradation of the CARMA1 signalosome, thereby limiting NF-κB and JNK signaling.     

The structure of the PDZ3-SH3-GuK tandem of ZO-1 protein suggests a supramodular organization of the membrane-associated guanylate kinase (MAGUK) family scaffold protein core

Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tethering membrane receptors, adhesion molecules, and macromolecular signaling complexes for tissue developments, cell-cell communications, and intracellular signal transductions. The defining feature of the MAGUK family scaffolds is that each member contains a conserved core consisting of a PSD-95/Dlg/ZO-1 (PDZ) domain, an Src homology 3 (SH3) domain, and a catalytically inactive guanylate kinase (GuK) domain arranged in tandem, although the structural features and functional implications of the PDZ-SH3-GuK tandem arrangement are unclear. The structure of the ZO-1 PDZ3-SH3-GuK tandem solved in this study reveals that the PDZ domain directly interacts with the SH3-GuK module, forming a structural supramodule with distinct target binding properties with respect to the isolated domains. Structure-based sequence analysis suggests that the PDZ-SH3-GuK tandems of other members of the MAGUK family also form supramodules.     

Scaffold proteins (MAGUK, Shank and Homer) in postsynaptic density in the central nervous system

The postsynaptic density (PSD) is a dynamic multi-protein complex attached to the postsynaptic membrane composed of several hundred proteins such as receptors and channels, scaffolding and adaptor proteins, cell-adhesion proteins, cytoskeletal proteins, G-proteins and their modulators and signaling molecules including kinases and phosphtases. This review focuses on the prominent PSD scaffolds proteins such as members of the MAGUK (membrane-associated guanylyl kinase), Shank (SH3 domain and ankyrin repeat-containing protein) and Homer families. These molecules interact simultaneously with different kinds of receptors and modulate their function by linking the receptors to downstream signaling events. For example PSD 95, a main member of MAGUK family, interacts directly with carboxyl termini of NMDA receptor subunits and clusters them to the postsynaptic membrane. In addition, PSD 95 is involved in binding and organizing proteins connected with NMDAR signaling. Based on the modular character and ability to form multiproteins interactions, MAGUK, Shank and Homer are perfectly suited to act as a major scaffold in postsynaptic density.