Direct Interaction of CASK/LIN-2 and Syndecan Heparan Sulfate Proteoglycan and Their Overlapping Distribution in Neuronal Synapses

CASK, the rat homolog of a gene (LIN-2) required for vulval differentiation in Caenorhabditis elegans, is expressed in mammalian brain, but its function in neurons is unknown. CASK is distributed in a punctate somatodendritic pattern in neurons. By immunogold EM, CASK protein is concentrated in synapses, but is also present at nonsynaptic membranes and in intracellular compartments. This immunolocalization is consistent with biochemical studies showing the presence of CASK in soluble and synaptosomal membrane fractions and its enrichment in postsynaptic density fractions of rat brain. By yeast two-hybrid screening, a specific interaction was identified between the PDZ domain of CASK and the COOH terminal tail of syndecan-2, a cell surface heparan sulfate proteoglycan (HSPG). The interaction was confirmed by coimmunoprecipitation from heterologous cells. In brain, syndecan-2 localizes specifically at synaptic junctions where it shows overlapping distribution with CASK, consistent with an interaction between these proteins in synapses. Cell surface HSPGs can bind to extracellular matrix proteins, and are required for the action of various heparin-binding polypeptide growth/differentiation factors. The synaptic localization of CASK and syndecan suggests a potential role for these proteins in adhesion and signaling at neuronal synapses.     

MAGUIN, a novel neuronal membrane-associated guanylate kinase-interacting protein

Postsynaptic density (PSD)-95/Synapse-associated protein (SAP) 90 and synaptic scaffolding molecule (S-SCAM) are neuronal membrane-associated guanylate kinases. Because PSD-95/SAP90 and S-SCAM function as synaptic scaffolding proteins, identification of ligands for these proteins is important to elucidate the structure of synaptic junctions. Here, we report a novel protein interacting with the PDZ domains of PSD-95/SAP90 and S-SCAM and named it MAGUIN-1 (membrane-associated guanylate kinase-interacting protein-1). MAGUIN-1 has one sterile alpha motif, one PDZ, and one plekstrin homology domain. MAGUIN-1 is localized at the plasma membrane via the plekstrin homology domain and the C-terminal region and interacts with PSD-95/SAP90 and S-SCAM via a C-terminal PDZ domain-binding motif. MAGUIN-1 has a short isoform, MAGUIN-2, which lacks a PDZ domain-binding motif. MAGUINs are expressed in neurons and localized in the cell body and neurites and are coimmunoprecipitated with PSD-95/SAP90 and S-SCAM from rat crude synaptosome. MAGUIN-1 may play an important role with PSD-95/SAP90 and S-SCAM to assemble the components of synaptic junctions.     

Calcium channel structural determinants of synaptic transmission between identified invertebrate neurons

We report here that unlike what was suggested for many vertebrate neurons, synaptic transmission in Lymnaea stagnalis occurs independent of a physical interaction between presynaptic calcium channels and a functional complement of SNARE proteins. Instead, synaptic transmission in Lymnaea requires the expression of a C-terminal splice variant of the Lymnaea homolog to mammalian N- and P/Q-type calcium channels. We show that the alternately spliced region physically interacts with the scaffolding proteins Mint1 and CASK, and that synaptic transmission is abolished following RNA interference knockdown of CASK or after the injection of peptide sequences designed to disrupt the calcium channel-Mint1 interactions. Our data suggest that Mint1 and CASK may serve to localize the non-L-type channels at the active zone and that synaptic transmission in invertebrate neurons utilizes a mechanism for optimizing calcium entry, which occurs independently of a physical association between calcium channels and SNARE proteins.     

CASK Functions as a Mg2+-independent neurexin kinase

CASK is a unique MAGUK protein that contains an N-terminal CaM-kinase domain besides the typical MAGUK domains. The CASK CaM-kinase domain is presumed to be a catalytically inactive pseudokinase because it lacks the canonical DFG motif required for Mg2+ binding that is thought to be indispensable for kinase activity. Here we show, however, that CASK functions as an active protein kinase even without Mg2+ binding. High-resolution crystal structures reveal that the CASK CaM-kinase domain adopts a constitutively active conformation that binds ATP and catalyzes phosphotransfer without Mg2+. The CASK CaM-kinase domain phosphorylates itself and at least one physiological interactor, the synaptic protein neurexin-1, to which CASK is recruited via its PDZ domain. Thus, our data indicate that CASK combines the scaffolding activity of MAGUKs with an unusual kinase activity that phosphorylates substrates recuited by the scaffolding activity. Moreover, our study suggests that other pseudokinases (10% of the kinome) could also be catalytically active.     

Parkin and CASK/LIN-2 associate via a PDZ-mediated interaction and are co-localized in lipid rafts and postsynaptic densities in brain.

Mutations in the gene encoding parkin cause an autosomal recessive juvenile-onset form of Parkinson's disease. Parkin functions as a RING-type E3 ubiquitin-ligase, coordinating the transfer of ubiquitin to substrate proteins and thereby targeting them for degradation by the proteasome. We now report that the extreme C terminus of parkin, which is selectively truncated by a Parkinson's disease-causing mutation, functions as a class II PDZ-binding motif that binds CASK, the mammalian homolog of Caenorhabditis elegans Lin-2, but not other PDZ proteins in brain extracts. Importantly, parkin co-localizes with CASK at synapses in cultured cortical neurons as well as in postsynaptic densities and lipid rafts in brain. Further, parkin associates not only with CASK but also with other postsynaptic proteins in the N-methyl d-aspartate (NMDA) receptor-signaling complex, in rat brain in vivo. Finally, despite exhibiting E2-dependent ubiquitin ligase activity, rat brain parkin does not ubiquitinate CASK, suggesting that CASK may function in targeting or scaffolding parkin within the postsynaptic complex rather than as a direct substrate for parkin-mediated ubiquitination. These data implicate for the first time a PDZ-mediated interaction between parkin and CASK in neurodegeneration and possibly in ubiquitination of proteins involved in synaptic transmission and plasticity.     

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.     

Characterisation of the interaction between syndecan-2, neurofibromin and CASK: Dependence of interaction on syndecan dimerization

Neurofibromin and calcium/calmodulin-dependent serine protein kinase (CASK) are membrane-associated signalling and scaffolding proteins which are mutated in human genetic neurological disorders. Syndecan-2 is a highly glycosylated transmembrane protein whose intracellular C-terminus has previously been shown to interact with the post-synaptic density 95/discs large/zonula occludens-1 (PDZ) domain of CASK and with two separate regions of neurofibromin. These three proteins collaborate to orchestrate the induction of filopodia and dendritic spines. We have used systematic mutagenesis of the intracellular region of syndecan-2 and a quantitative yeast two-hybrid (Y2H) assay to study the determinants of their interactions. We show that syndecan’s interactions with both CASK and neurofibromin are dependent on syndecan homodimerization and that neurofibromin largely interacts with the membrane-proximal part of the dimeric syndecan intracellular domain, leaving the membrane-distal C-terminus free to interact with CASK. We conducted a phylogenetic study of syndecan sequences, finding correspondence between conserved residues and mutations affecting both dimerization and interactions; we also find that fish have a very different syndecan repertoire from tetrapods. Further Y2H screens reveal that syndecan-2 interacts with a third distinct region of neurofibromin, and that the multiple neurofibromin regions bind competitively, rather than co-operatively, to syndecan. We combine these results to propose a model for the ternary syndecan-neurofibromin-CASK complex.     

Interaction of S-SCAM with neural plakophilin-related Armadillo-repeat protein/delta-catenin

Synaptic scaffolding molecule (S-SCAM) is a multiple PDZ domain-containing protein, which interacts with neuroligin, a cell adhesion molecule, and the NMDA receptor. In this study, we searched for S-SCAM-interacting proteins and obtained a neuralplakophilin-related armadillo-repeat protein (NPRAP)/delta-catenin. NPRAP/delta-catenin bound to the last PDZ domain of S-SCAM via its carboxyl-terminus in three different cell-free assay systems, was coimmunoprecipitated with S-SCAM from rat crude synaptosomes, and was localized at the excitatory synapses in rat hippocampal neurons. NPRAP/delta-catenin may be implicated in the molecular organization of synaptic junctions through the interaction with S-SCAM.     

Functional interactions between presynaptic calcium channels and the neurotransmitter release machinery

In vertebrates, the physical coupling between presynaptic calcium channels and synaptic vesicle release proteins enhances the efficiency of neurotransmission. Recent evidence indicates that these synaptic proteins may feedback directly on synaptic release by negatively regulating calcium entry, and indirectly through pathways involving second messenger molecules. Studies of individual neurons from both vertebrates and invertebrates have provided novel insights into the roles of scaffolding proteins in calcium channel targeting and neurotransmitter release. These studies require us to expand current models of synaptic transmission.     

Association of neuronal calcium channels with modular adaptor proteins.

Presynaptic voltage-gated calcium (Ca(2+)) channels mediate Ca(2+) influx into the presynaptic terminal that triggers synaptic vesicle fusion and neurotransmitter release. The immediate proximity of Ca(2+) channels to the synaptic vesicle release apparatus is critical for rapid and efficient synaptic transmission. In a series of biochemical experiments, we demonstrate a specific association of the cytosolic carboxyl terminus of the N-type Ca(2+) channel pore-forming alpha(1B) subunit with the modular adaptor proteins Mint1 and CASK. The carboxyl termini of alpha(1B) bind to the first PDZ domain of Mint1 (Mint1-1). The proline-rich region present in the carboxyl termini of alpha(1B) binds to the SH3 domain of CASK. Mint1-1 is specific for the E/D-X-W-C/S-COOH consensus, which defines a novel class of PDZ domains (class III). The Mint1-1 PDZ domain-binding motif is present only in the "long" carboxyl-terminal splice variants of N-type (alpha(1B)) and P/Q-type (alpha(1A)) Ca(2+) channels, but not in R-type (alpha(1E)) or L-type (alpha(1C)) Ca(2+) channels. Our results directly link presynaptic Ca(2+) channels to a macromolecular complex formed by modular adaptor proteins at synaptic junction and advance our understanding of coupling between cell adhesion and synaptic vesicle exocytosis.     

CASK and protein 4.1 support F-actin nucleation on neurexins

Rearrangements of the actin cytoskeleton are involved in a variety of cellular processes from locomotion of cells to morphological alterations of the cell surface. One important question is how local interactions of cells with the extracellular space are translated into alterations of their membrane organization. To address this problem, we studied CASK, a member of the membrane-associated guanylate kinase homologues family of adaptor proteins. CASK has been shown to bind the erythrocyte isoform of protein 4.1, a class of proteins that promote formation of actin/spectrin microfilaments. In neurons, CASK also interacts via its PDZ domain with the cytosolic C termini of neurexins, neuron-specific cell-surface proteins. We now show that CASK binds a brain-enriched isoform of protein 4.1, and nucleates local assembly of actin/spectrin filaments. These interactions can be reconstituted on the cytosolic tail of neurexins. Furthermore, CASK can be recovered with actin filaments prepared from rat brain extracts, and neurexins are recruited together with CASK and protein 4.1 into these actin filaments. Thus, analogous to the PDZ-domain protein p55 and glycophorin C at the erythrocyte membrane, a similar complex comprising CASK and neurexins exists in neurons. Our data suggest that intercellular junctions formed by neurexins, such as junctions initiated by beta-neurexins with neuroligins, are at least partially coupled to the actin cytoskeleton via an interaction with CASK and protein 4.1.     

PICK1 is required for the control of synaptic transmission by the metabotropic glutamate receptor 7

Both postsynaptic density and presynaptic active zone are structural matrix containing scaffolding proteins that are involved in the organization of the synapse. Little is known about the functional role of these proteins in the signaling of presynaptic receptors. Here we show that the interaction of the presynaptic metabotropic glutamate (mGlu) receptor subtype, mGlu7a, with the postsynaptic density-95 disc-large zona occludens 1 (PDZ) domain-containing protein, PICK1, is required for specific inhibition of P/Q-type Ca(2+) channels, in cultured cerebellar granule neurons. Furthermore, we show that activation of the presynaptic mGlu7a receptor inhibits synaptic transmission and this effect also requires the presence of PICK1. These results indicate that the scaffolding protein, PICK1, plays an essential role in the control of synaptic transmission by the mGlu7a receptor complex.     

PAPIN. A novel multiple PSD-95/Dlg-A/ZO-1 protein interacting with neural plakophilin-related armadillo repeat protein/delta-catenin and p0071

A neural plakophilin-related armadillo repeat protein (NPRAP)/delta-catenin interacts with one of Alzheimer disease-related gene products, presenilin 1. We have previously reported the interaction of NPRAP/delta-catenin with synaptic scaffolding molecule, which is involved in the assembly of synaptic components. NPRAP/delta-catenin also interacts with E-cadherin and beta-catenin and is implicated in the organization of cell-cell junctions. p0071, a ubiquitous isoform of NPRAP/delta-catenin, is localized at desmosomes in HeLa and A431 cells and at adherens junctions in Madin-Darby bovine kidney cells. We have identified here a novel protein interacting with NPRAP/delta-catenin and p0071 and named this protein plakophilin-related armadillo repeat protein-interacting PSD-95/Dlg-A/ZO-1 (PDZ) protein (PAPIN). PAPIN has six PDZ domains and binds to NPRAP/delta-catenin and p0071 via the second PDZ domain. PAPIN and p0071 are ubiquitously expressed in various tissues and are localized at cell-cell junctions in normal rat kidney cells and bronchial epithelial cells. PAPIN may be a scaffolding protein connecting components of epithelial junctions with p0071.     

PDZ Ligand Binding-Induced Conformational Coupling of the PDZ–SH3–GK Tandems in PSD-95 Family MAGUKs

Discs large (DLG) MAGUKs are abundantly expressed in glutamatergic synapses, crucial for synaptic transmission, and plasticity by anchoring various postsynaptic components including glutamate receptors, downstream scaffold proteins and signaling enzymes. Different DLG members have shared structures and functions, but also contain unique features. How DLG family proteins function individually and cooperatively is largely unknown. Here, we report that PSD-95 PDZ3 directly couples with SH3–GK tandem in a PDZ ligand binding-dependent manner, and the coupling can promote PSD-95 dimerization and multimerization. Aided by sortase-mediated protein ligation and selectively labeling, we elucidated the PDZ3/SH3–GK conformational coupling mechanism using NMR spectroscopy. We further demonstrated that PSD-93, but not SAP102, can also undergo PDZ3 ligand binding-induced conformational coupling with SH3–GK and form homo-oligomers. Interestingly, PSD-95 and PSD-93 can also form ligand binding-induced hetero-oligomers, suggesting a cooperative assembly mechanism for the mega-N-methyl-d-aspartate receptor synaptic signaling complex. Finally, we provide evidence showing that ligand binding-induced conformational coupling between PDZ and SH3–GK is a common feature for other MAGUKs including CASK and PALS1.     

TIP-1 has PDZ scaffold antagonist activity

PDZ proteins usually contain multiple protein-protein interaction domains and act as molecular scaffolds that are important for the generation and maintenance of cell polarity and cell signaling. Here, we identify and characterize TIP-1 as an atypical PDZ protein that is composed almost entirely of a single PDZ domain and functions as a negative regulator of PDZ-based scaffolding. We found that TIP-1 competes with the basolateral membrane mLin-7/CASK complex for interaction with the potassium channel Kir 2.3 in model renal epithelia. Consequently, polarized plasma membrane expression of Kir 2.3 is disrupted resulting in pronounced endosomal targeting of the channel, similar to the phenotype observed for mutant Kir 2.3 channels lacking the PDZ-binding motif. TIP-1 is ubiquitously expressed, raising the possibility that TIP-1 may play a similar role in regulating the expression of other membrane proteins containing a type I PDZ ligand.     

Synaptic scaffolding molecule (S-SCAM) membrane-associated guanylate kinase with inverted organization (MAGI)-2 is associated with cell adhesion molecules at inhibitory synapses in rat hippocampal neurons

Synaptic scaffolding molecule (S-SCAM) is a synaptic protein, which harbors five or six PSD-95/Discs large/ZO-1 (PDZ), a guanylate kinase and two WW domains. It interacts with NMDA receptor subunits, neuroligin and beta-catenin, and is involved in the accumulation of neuroligin at excitatory synapses. In this study, we have demonstrated S-SCAM is localized at inhibitory synapses in rat primary cultured hippocampal neurons. We have identified beta-dystroglycan (beta-DG) as a binding partner for S-SCAM at inhibitory synapses. WW domains of S-SCAM bind to three sequences of beta-DG. We have also revealed that S-SCAM can interact with neuroligin 2, which is known to be exclusively localized at inhibitory synapses. The WW domains and the second PDZ domain of S-SCAM are involved in the interaction with neuroligin 2. Beta-DG, neuroligin 2 and S-SCAM form a tripartite complex in vitro. Neuroligin 2 is detected in the immunoprecipitates by anti-beta-DG antibody from rat brain. S-SCAM, beta-DG and neuroligin 2 are partially co-localized in rat hippocampal neurons. These data suggest that S-SCAM is associated with beta-DG and neuroligin 2 at inhibitory synapses, and functions as a linker between the dystrophin glycoprotein complex and the neurexin-neuroligin complex.     

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).     

Autism and Intellectual Disability-Associated KIRREL3 Interacts with Neuronal Proteins MAP1B and MYO16 with Potential Roles in Neurodevelopment

Cell-adhesion molecules of the immunoglobulin superfamily play critical roles in brain development, as well as in maintaining synaptic plasticity, the dysfunction of which is known to cause cognitive impairment. Recently dysfunction of KIRREL3, a synaptic molecule of the immunoglobulin superfamily, has been implicated in several neurodevelopmental conditions including intellectual disability, autism spectrum disorder, and in the neurocognitive delay associated with Jacobsen syndrome. However, the molecular mechanisms of its physiological actions remain largely unknown. Using a yeast two-hybrid screen, we found that the KIRREL3 extracellular domain interacts with brain expressed proteins MAP1B and MYO16 and its intracellular domain can potentially interact with ATP1B1, UFC1, and SHMT2. The interactions were confirmed by co-immunoprecipitation and colocalization analyses of proteins expressed in human embryonic kidney cells, mouse neuronal cells, and rat primary neuronal cells. Furthermore, we show KIRREL3 colocalization with the marker for the Golgi apparatus and synaptic vesicles. Previously, we have shown that KIRREL3 interacts with the X-linked intellectual disability associated synaptic scaffolding protein CASK through its cytoplasmic domain. In addition, we found a genomic deletion encompassing MAP1B in one patient with intellectual disability, microcephaly and seizures and deletions encompassing MYO16 in two unrelated patients with intellectual disability, autism and microcephaly. MAP1B has been previously implicated in synaptogenesis and is involved in the development of the actin-based membrane skeleton. MYO16 is expressed in hippocampal neurons and also indirectly affects actin cytoskeleton through its interaction with WAVE1 complex. We speculate KIRREL3 interacting proteins are potential candidates for intellectual disability and autism spectrum disorder. Moreover, our findings provide further insight into understanding the molecular mechanisms underlying the physiological action of KIRREL3 and its role in neurodevelopment.     

gamma-Syntrophin scaffolding is spatially and functionally distinct from that of the alpha/beta syntrophins

The syntrophins are a family of scaffolding proteins with multiple protein interaction domains that link signaling proteins to dystrophin family members. Each of the three most characterized syntrophins (alpha, beta1, beta2) contains a PDZ domain that binds a unique set of signaling proteins including kinases, ion and water channels, and neuronal nitric oxide synthase (nNOS). The PDZ domains of the gamma-syntrophins do not bind nNOS. In vitro pull-down assays show that the gamma-syntrophins can bind dystrophin but have unique preferences for the syntrophin binding sites of dystrophin family members. Despite their ability to bind dystrophin in vitro, neither gamma-syntrophin isoform co-localizes with dystrophin in skeletal muscle. Furthermore, gamma-syntrophins do not co-purify with dystrophin isolated from mouse tissue. These data suggest that the interaction of gamma-syntrophin with dystrophin is transient and potentially subject to regulatory mechanisms. gamma1-Syntrophin is highly expressed in brain and is specifically localized in hippocampal pyramidal neurons, Purkinje neurons in cerebellum, and cortical neurons. gamma2-Syntrophin is expressed in many tissues including skeletal muscle where it is found only in the subsynaptic space beneath the neuromuscular junction. In both neurons and muscle, gamma-syntrophin isoforms localize to the endoplasmic reticulum where they may form a scaffold for signaling and trafficking.