Formation of complexes between Ca2+.calmodulin and the synapse-associated protein SAP97 requires the SH3 domain-guanylate kinase domain-connecting HOOK region

Mammalian synapse-associated protein SAP97, a structural and functional homolog of Drosophila Dlg, is a membrane-associated guanylate kinase (MAGUK) that is present at pre- and postsynaptic sites as well as in epithelial cell-cell contact sites. It is a multidomain scaffolding protein that shares with other members of the MAGUK protein family a characteristic modular organization composed of three sequential protein interaction motifs known as PDZ domains, followed by an Src homology 3 (SH3) domain, and an enzymatically inactive guanylate kinase (GK)-like domain. Specific binding partners are known for each domain, and different modes of intramolecular interactions have been proposed that particularly involve the SH3 and GK domains and the so-called HOOK region located between these two domains. We identified the HOOK region as a specific site for calmodulin binding and studied the dynamics of complex formation of recombinant calmodulin and SAP97 by surface plasmon resonance spectroscopy. Binding of various SAP97 deletion constructs to immobilized calmodulin was strictly calcium-dependent. From the rate constants of association and dissociation we determined an equilibrium dissociation constant K(d) of 122 nm for the association of calcium-saturated calmodulin and a SAP97 fragment, which encompassed the entire SH3-HOOK-GK module. Comparative structure-based sequence analysis of calmodulin binding regions from various target proteins predicts variable affinities for the interaction of calmodulin with members of the MAGUK protein family. Our findings suggest that calmodulin could regulate the intramolecular interaction between the SH3, HOOK, and GK domains of SAP97.     

Functional analysis of the nucleotide binding domain of membrane-associated guanylate kinases

Membrane-associated guanylate kinases (MAGUKs) regulate cellular adhesion and signal transduction at sites of cell-cell contact. MAGUKs are composed of modular protein-protein interaction motifs including L27, PDZ, Src homology (SH) 3, and guanylate kinase domains that aggregate adhesion molecules and receptors. Genetic analyses reveal that lethal mutations of MAGUKs often occur in the guanylate kinase domain, indicating a critical role for this domain. Here, we explored whether GMP binding to the guanylate kinase domain regulates MAGUK function. Surprisingly, and in contrast to previously published studies, we failed to detect GMP binding to the MAGUKs postsynaptic density-95 (PSD-95) and CASK. Two amino acid residues in the GMP binding pocket that differ between MAGUKs and authentic guanylate kinase explain this lack of binding, as swapping these residues largely prevent GMP binding to yeast guanylate kinase. Conversely, these mutations restore GMP binding but not catalytic activity to PSD-95. Protein ligands for the PSD-95 guanylate kinase domain, guanylate kinase-associated protein (GKAP) and MAP1A, appear not to interact with the canonical GMP binding pocket, and GMP binding does not influence the intramolecular SH3/guanylate kinase (GK) interaction within PSD-95. These studies indicate that MAGUK proteins have lost affinity for GMP but may have retained the guanylate kinase structure to accommodate a related regulatory ligand.     

Identification of an intramolecular interaction between the SH3 and guanylate kinase domains of PSD-95.

Postsynaptic density-95 (PSD-95/SAP-90) is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins that assemble protein complexes at synapses and other cell junctions. MAGUKs comprise multiple protein-protein interaction motifs including PDZ, SH3 and guanylate kinase (GK) domains, and these binding sites mediate the scaffolding function of MAGUK proteins. Synaptic binding partners for the PDZ and GK domains of PSD-95 have been identified, but the role of the SH3 domain remains elusive. We now report that the SH3 domain of PSD-95 mediates a specific interaction with the GK domain. The GK domain lacks a poly-proline motif that typically binds to SH3 domains; instead, SH3/GK binding is a bi-domain interaction that requires both intact motifs. Although isolated SH3 and GK domains can bind in trans, experiments with intact PSD-95 molecules indicate that intramolecular SH3/GK binding dominates and prevents intermolecular associations. SH3/GK binding is conserved in the related Drosophila MAGUK protein DLG but is not detectable for Caenorhabditis elegans LIN-2. Many previously identified genetic mutations of MAGUKs in invertebrates occur in the SH3 or GK domains, and all of these mutations disrupt intramolecular SH3/GK binding.     

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.     

Functional modularity of the beta-subunit of voltage-gated Ca2+ channels

The beta-subunit of voltage-gated Ca(2+) channels plays a dual role in chaperoning the channels to the plasma membrane and modulating their gating. It contains five distinct modular domains/regions, including the variable N- and C-terminus, a conserved Src homology 3 (SH3) domain, a conserved guanylate kinase (GK) domain, and a connecting variable and flexible HOOK region. Recent crystallographic studies revealed a highly conserved interaction between the GK domain and alpha interaction domain (AID), the high-affinity binding site in the pore-forming alpha(1) subunit. Here we show that the AID-GK domain interaction is necessary for beta-subunit-stimulated Ca(2+) channel surface expression and that the GK domain alone can carry out this function. We also examined the role of each region of all four beta-subunit subfamilies in modulating P/Q-type Ca(2+) channel gating and demonstrate that the beta-subunit functions modularly. Our results support a model that the conserved AID-GK domain interaction anchors the beta-subunit to the alpha(1) subunit, enabling alpha(1)-beta pair-specific low-affinity interactions involving the N-terminus and the HOOK region, which confer on each of the four beta-subunit subfamilies its distinctive modulatory properties.     

Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.     

GKAP, a Novel Synaptic Protein That Interacts with the Guanylate Kinase-like Domain of the PSD-95/SAP90 Family of Channel Clustering Molecules

The molecular mechanisms underlying the organization of ion channels and signaling molecules at the synaptic junction are largely unknown. Recently, members of the PSD-95/SAP90 family of synaptic MAGUK (membrane-associated guanylate kinase) proteins have been shown to interact, via their NH₂-terminal PDZ domains, with certain ion channels (NMDA receptors and K⁺ channels), thereby promoting the clustering of these proteins. Although the function of the NH₂-terminal PDZ domains is relatively well characterized, the function of the Src homology 3 (SH3) domain and the guanylate kinase-like (GK) domain in the COOH-terminal half of PSD-95 has remained obscure. We now report the isolation of a novel synaptic protein, termed GKAP for guanylate kinase-associated protein, that binds directly to the GK domain of the four known members of the mammalian PSD-95 family. GKAP shows a unique domain structure and appears to be a major constituent of the postsynaptic density. GKAP colocalizes and coimmunoprecipitates with PSD-95 in vivo, and coclusters with PSD-95 and K⁺ channels/ NMDA receptors in heterologous cells. Given their apparent lack of guanylate kinase enzymatic activity, the fact that the GK domain can act as a site for protein– protein interaction has implications for the function of diverse GK-containing proteins (such as p55, ZO-1, and LIN-2/CASK).     

Interaction of NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase protein, with calmodulin and PSD-95/SAP90. A possible regulatory role in molecular clustering at synaptic sites

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends of N-methyl-D-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic alpha-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a Kd value of 44 nM. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses.     

CASK point mutation regulates protein-protein interactions and NR2b promoter activity

Mutations in the CASK gene result in mental retardation and microcephaly in humans, suggesting an important role for CASK in brain. CASK gene knockout in mice causes neonatal lethality, making further elucidation in mouse models difficult. Because CASK was originally identified as a multidomain adaptor protein, identifying a point mutation interrupting a specific protein interaction would be useful in dissecting its molecular function. Here, a Thr-to-Ala mutation in the rat CASK guanylate kinase (GK) domain was shown to reduce interactions among CASK and Tbr-1 and CINAP, two critical brain proteins. The effect is specific: this mutation does not affect CASK dimerization that occurs via the GK domain. The Tbr-1-CASK-CINAP complex regulates expression of the NMDA receptor subunit 2b (NR2b), and we show that this point mutation also affects NR2b promoter activity. The identification of this mutation may make it possible to further dissect the function of CASK in brain.     

Isotope effects in the study of enzymatic phosphoryl transfer reactions

CASK, a member of the membrane-associated guanylate kinase (MAGUK) superfamily, binds to the carboxyl-terminus of beta-neurexins on the intracellular side of the presynaptic membrane. The guanylate kinase-like (GUK) domains of MAGUKs lack kinase activities, but might be important for mediating specific protein-protein interaction. By a yeast two-hybrid approach, we identified an interaction between the GUK domain of CASK and the C2B domain of rabphilin3a, a presynaptic protein involved in synaptic vesicle exocytosis. The interaction was confirmed by in vitro GST pull-down and co-immunoprecipitation assays. It was proposed that presynaptic vesicles might be guided to the vicinity of points of exocytosis defined by beta-neurexins via the interaction between rabphilin3a-CASK-beta-neurexins.     

Structural characterization of the intramolecular interaction between the SH3 and guanylate kinase domains of PSD-95.

PSD-95/SAP90 is a member of the MAGUK superfamily. In excitatory synapses, PSD-95 clusters receptors and ion channels at specific sites in the postsynaptic membrane and organizes downstream signaling and cytoskeletal molecules. We have determined the crystal structures of the apo and GMP-bound forms to 2.3 and 2.0 A resolutions, respectively, of a fragment containing the SH3, HOOK, and guanylate kinase (GK) domains of PSD-95. We observe an intramolecular interaction between the SH3 and GK domains involving the formation of a beta sheet including residues N- and C-terminal to the GK domain. Based on amino acid conservation and mutational data available in the literature, we propose that this intramolecular interaction is a common feature among MAGUK proteins.     

Calcium channel function regulated by the SH3-GK module in beta subunits

beta subunits of voltage-gated calcium channels (VGCCs) regulate channel trafficking and function, thereby shaping the intensity and duration of intracellular changes in calcium. beta subunits share limited sequence homology with the Src homology 3-guanylate kinase (SH3-GK) module of membrane-associated guanylate kinases (MAGUKs). Here, we show biochemical similarities between beta subunits and MAGUKs, revealing important aspects of beta subunit structure and function. Similar to MAGUKs, an SH3-GK interaction within beta subunits can occur both intramolecularly and intermolecularly. Mutations that disrupt the SH3-GK interaction in beta subunits alter channel inactivation and can inhibit binding between the alpha(1) and beta subunits. Coexpression of beta subunits with complementary mutations in their SH3 and GK domains rescues these deficits through intermolecular beta subunit assembly. In MAGUKs, the SH3-GK module controls protein scaffolding. In beta subunits, this module regulates the inactivation of VGCCs and provides an additional mechanism for tuning calcium responsiveness.     

Interdomain interactions in the tumor suppressor discs large regulate binding to the synaptic protein GukHolder

Multidomain scaffolding proteins are central components of many signaling pathways and are commonly found at membrane specializations. Here we have shown that multiple interdomain interactions in the scaffold Discs Large (Dlg) regulate binding to the synaptic protein GukHolder (GukH). GukH binds the Src homology 3 (SH3) and guanylate kinase-like (GK) protein interaction domains of Dlg, whereas an intramolecular interaction between the two domains inhibits association with GukH. Regulation occurs through a PDZ domain adjacent to the SH3 that allows GukH to interact with the composite SH3-GK binding site, but PDZ ligands inhibit GukH binding such that Dlg forms mutually exclusive PDZ ligand and GukH cellular complexes. The PDZ-SH3-GK module is a common feature of membrane associate guanylate kinase scaffolds such as Dlg, and these results indicate that its supramodular architecture leads to regulation of Dlg complexes.     

SUMOylation of the MAGUK protein CASK regulates dendritic spinogenesis

Membrane-associated guanylate kinase (MAGUK) proteins interact with several synaptogenesis-triggering adhesion molecules. However, direct evidence for the involvement of MAGUK proteins in synapse formation is lacking. In this study, we investigate the function of calcium/calmodulin-dependent serine protein kinase (CASK), a MAGUK protein, in dendritic spine formation by RNA interference. Knockdown of CASK in cultured hippocampal neurons reduces spine density and shrinks dendritic spines. Our analysis of the time course of RNA interference and CASK overexpression experiments further suggests that CASK stabilizes or maintains spine morphology. Experiments using only the CASK PDZ domain or a mutant lacking the protein 4.1-binding site indicate an involvement of CASK in linking transmembrane adhesion molecules and the actin cytoskeleton. We also find that CASK is SUMOylated. Conjugation of small ubiquitin-like modifier 1 (SUMO1) to CASK reduces the interaction between CASK and protein 4.1. Overexpression of a CASK-SUMO1 fusion construct, which mimicks CASK SUMOylation, impairs spine formation. Our study suggests that CASK contributes to spinogenesis and that this is controlled by SUMOylation.     

Structure of the SH3-guanylate kinase module from PSD-95 suggests a mechanism for regulated assembly of MAGUK scaffolding proteins.

Membrane-associated guanylate kinases (MAGUKs), such as PSD-95, are modular scaffolds that organize signaling complexes at synapses and other cell junctions. MAGUKs contain PDZ domains, which recruit signaling proteins, as well as a Src homology 3 (SH3) and a guanylate kinase-like (GK) domain, implicated in scaffold oligomerization. The crystal structure of the SH3-GK module from PSD-95 reveals that these domains form an integrated unit: the SH3 fold comprises noncontiguous sequence elements divided by a hinge region and the GK domain. These elements compose two subdomains that can assemble in either an intra- or intermolecular fashion to complete the SH3 fold. We propose a model for MAGUK oligomerization in which complementary SH3 subdomains associate by 3D domain swapping. This model provides a possible mechanism for ligand regulation of oligomerization.     

The HOOK-domain between the SH3 and the GK domains of Cavbeta subunits contains key determinants controlling calcium channel inactivation

Ca(v)beta subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3)-domain and a guanylate kinase-like (GK)-domain. The SH3-domain is split, with its final (fifth) beta-strand separated from the rest of the domain by an intervening sequence termed the HOOK-domain, whose sequence varies between Ca(v)beta subunits. Here we have been guided by the recent structural studies of Ca(v)beta subunits in the design of specific truncated constructs, with the goal of investigating the role of the HOOK-domain of Ca(v)beta subunits in the modulation of inactivation of N-type calcium channels. We have coexpressed the beta subunit constructs with Ca(v)2.2 and alpha(2)delta-2, using the N-terminally palmitoylated beta(2a) subunit, because it supports very little voltage-dependent inactivation, and made comparisons with beta(1b) domains. Deletion of the variable region of the beta(2a) HOOK-domain resulted in currents with a rapidly inactivating component, and additional mutation of the beta(2a) palmitoylation motif further enhanced inactivation. The isolated GK-domain of beta(2a) alone enhanced current amplitude, but the currents were rapidly and completely inactivating. When the beta(2a)-GK-domain construct was extended proximally, by including the HOOK-domain and the epsilon-strand of the SH3-domain, inactivation was about four-fold slower than in the absence of the HOOK domain. When the SH3-domain of beta(2a) truncated prior to the HOOK-domain was coexpressed with the (HOOK+epsilonSH3+GK)-domain of beta(2a), all the properties of beta(2a) were restored, in terms of loss of inactivation. Furthermore, removal of the HOOK sequence from the (HOOK+epsilonSH3+GK)-beta(2a) construct increased inactivation. Together, these results provide evidence that the HOOK domain is an important determinant of inactivation.     

The interaction between PSD-95 and Ca2+/calmodulin is enhanced by PDZ-binding proteins

In this study, we evaluate the interaction between the postsynaptic scaffolding protein, PSD-95, and calmodulin. Surface plasmon resonance spectroscopy was used to characterize the binding of PSD-95 to calmodulin that had been immobilized on a sensor chip. Additionally, soluble calmodulin was found to inhibit the binding of PSD-95 to immobilized calmodulin. The HOOK region of PSD-95, which is located between the src homology 3 domain and the guanylate kinase-like domain, was determined to be involved in the binding of PSD-95 to calmodulin. We also found that C-terminal peptides from proteins such as CRIPT and the N-methyl-d-aspartate receptor NR2B subunit, which associate with the PDZ domain of PSD-95, enhanced the affinity of PSD-95 for calmodulin. The binding of ligands to the PDZ domain may change the conformation of PSD-95 and affect the interaction between PSD-95 and calmodulin.     

GAKIN, a novel kinesin-like protein associates with the human homologue of the Drosophila discs large tumor suppressor in T lymphocytes

Reorganization of the cortical cytoskeleton is a hallmark of T lymphocyte activation. Upon binding to antigen presenting cells, the T cells rapidly undergo cytoskeletal re-organization thus forming a cap at the cell-cell contact site leading to receptor clustering, protein segregation, and cellular polarization. Previously, we reported cloning of the human lymphocyte homologue of the Drosophila Discs Large tumor suppressor protein (hDlg). Here we show that a novel protein termed GAKIN binds to the guanylate kinase-like domain of hDlg. Affinity protein purification, peptide sequencing, and cloning of GAKIN cDNA from Jurkat J77 lymphocytes identified GAKIN as a novel member of the kinesin superfamily of motor proteins. GAKIN mRNA is ubiquitously expressed, and the predicted amino acid sequence shares significant sequence similarity with the Drosophila kinesin-73 motor protein. GAKIN sequence contains a motor domain at the NH(2) terminus, a central stalk domain, and a putative microtubule-interacting sequence called the CAP-Gly domain at the COOH terminus. Among the MAGUK superfamily of proteins examined, GAKIN binds to the guanylate kinase-like domain of PSD-95 but not of p55. The hDlg and GAKIN are localized mainly in the cytoplasm of resting T lymphocytes, however, upon CD2 receptor cross-linking the hDlg can translocate to the lymphocyte cap. We propose that the GAKIN-hDlg interaction lays the foundation for a general paradigm of coupling MAGUKs to the microtubule-based cytoskeleton, and that this interaction may be functionally important for the intracellular trafficking of MAGUKs and associated protein complexes in vivo.