G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
calcium/calmodulin-dependent serine protein kinase (MAGUK family)
G00000865 (Mus musculus)

Databases (8)

ENSG00000147044 (Ensembl human gene)
8573 (Entrez Gene)
458 (G2Cdb plasticity & disease)
CASK (GeneCards)
300172 (OMIM)
Marker Symbol
HGNC:1497 (HGNC)
Protein Expression
1949 (human protein atlas)
Protein Sequence
O14936 (UniProt)

Synonyms (3)

  • CAGH39
  • FGS4
  • LIN2

Literature (60)

Pubmed - other

  • Characterisation of the interaction between syndecan-2, neurofibromin and CASK: dependence of interaction on syndecan dimerization.

    Volta M, Calza S, Roberts AM and Roberts RG

    Division of Medical & Molecular Genetics, Department of Neuroscience, Centre for the Cellular Basis of Behaviour, Institute of Psychiatry, King's College London, UK. manuela.volta@kcl.ac.uk

    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.

    Biochemical and biophysical research communications 2010;391;2;1216-21

  • Post-translational modification of CASK leads to its proteasome-dependent degradation.

    Sun Q and Kelly GM

    Department of Biology, University of Western Ontario, London, ON, Canada N6A 5B7.

    CASK is a member of the membrane-associated guanylate kinase family. In mammals it is an essential protein, as CASK knockout mice die after birth and its deletion in humans has developmental consequences. CASK plays a role in the transcription of genes required for forebrain development, and in the nervous systems of Drosophila and C. elegans, it participates in receptor localization at the plasma membrane. This role in organizing supramolecular protein complexes to appropriate subcellular regions is shared in mammals and is regulated by phosphorylation. CASK is a kinase and regulator of cell proliferation and adhesion, which adds to an expanding list of roles. In this study we report for the first time that CASK is degraded in a characteristic fashion in mammalian cells. We found that CASK is a long-lived protein despite the fact that it contains three putative PEST sequences. Finally, we provide detailed evidence that CASK degradation is mediated through a ubiquitin-proteasome pathway and this is phosphorylation-dependent. Together, these results provide evidence that post-translational modifications to CASK are major regulatory steps leading to its proteasomal degradation. This regulation not only has important implications on how CASK participates in its many disparate roles, but highlights how altering this regulation may contribute to the pathogenesis of human disease.

    The international journal of biochemistry & cell biology 2010;42;1;90-7

  • Calcium/calmodulin-dependent serine protein kinase and mental retardation.

    Hsueh YP

    Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan. yph@gate.sinica.edu.tw

    Calcium/calmodulin-dependent serine protein kinase (CASK) belongs to the membrane-associated guanylate kinase protein family. The members of this protein family function as multiple domain adaptor proteins originally identified at cell junctions and synapses. Insertional mutations or targeted disruption of the CASK gene in mice results in neonatal lethality, indicating an important role for CASK in development. Recently, several reports have also indicated that mutations in the human CASK gene result in X-linked malformations of the brain and mental retardation. At the molecular level, many studies indicate that CASK is critical for synapse formation at both presynaptic and postsynaptic junctions, and in the regulation of gene expression. The known molecular functions of CASK explain, at least partially, mental retardation and brain developmental defects in patients. In this review, recent findings about CASK are summarized and discussed.

    Annals of neurology 2009;66;4;438-43

  • Recurrent rearrangements in synaptic and neurodevelopmental genes and shared biologic pathways in schizophrenia, autism, and mental retardation.

    Guilmatre A, Dubourg C, Mosca AL, Legallic S, Goldenberg A, Drouin-Garraud V, Layet V, Rosier A, Briault S, Bonnet-Brilhault F, Laumonnier F, Odent S, Le Vacon G, Joly-Helas G, David V, Bendavid C, Pinoit JM, Henry C, Impallomeni C, Germano E, Tortorella G, Di Rosa G, Barthelemy C, Andres C, Faivre L, Frébourg T, Saugier Veber P and Campion D

    Institut National de la Santé et de la Recherche Médicale, Unité 614, Institut Hospitalo-Universitaire de Recherche Biomédicale, 76000 Rouen, France.

    Context: Results of comparative genomic hybridization studies have suggested that rare copy number variations (CNVs) at numerous loci are involved in the cause of mental retardation, autism spectrum disorders, and schizophrenia.

    Objectives: To provide an estimate of the collective frequency of a set of recurrent or overlapping CNVs in 3 different groups of cases compared with healthy control subjects and to assess whether each CNV is present in more than 1 clinical category.

    Design: Case-control study.

    Setting: Academic research.

    Participants: We investigated 28 candidate loci previously identified by comparative genomic hybridization studies for gene dosage alteration in 247 cases with mental retardation, in 260 cases with autism spectrum disorders, in 236 cases with schizophrenia or schizoaffective disorder, and in 236 controls.

    Collective and individual frequencies of the analyzed CNVs in cases compared with controls.

    Results: Recurrent or overlapping CNVs were found in cases at 39.3% of the selected loci. The collective frequency of CNVs at these loci is significantly increased in cases with autism, in cases with schizophrenia, and in cases with mental retardation compared with controls (P < .001, P = .01, and P = .001, respectively, Fisher exact test). Individual significance (P = .02 without correction for multiple testing) was reached for the association between autism and a 350-kilobase deletion located at 22q11 and spanning the PRODH and DGCR6 genes.

    Conclusions: Weakly to moderately recurrent CNVs (transmitted or occurring de novo) seem to be causative or contributory factors for these diseases. Most of these CNVs (which contain genes involved in neurotransmission or in synapse formation and maintenance) are present in the 3 pathologic conditions (schizophrenia, autism, and mental retardation), supporting the existence of shared biologic pathways in these neurodevelopmental disorders.

    Archives of general psychiatry 2009;66;9;947-56

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIDDK NIH HHS: K01 DK098285; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • Human calcium/calmodulin-dependent serine protein kinase regulates the expression of p21 via the E2A transcription factor.

    Sun R, Su Y, Zhao X, Qi J, Luo X, Yang Z, Yao Y, Luo X and Xia Z

    Burn Research Institute, Southwest Hospital, Third Military Medical University, Chongqing 400038, P.R. China.

    CASK (calcium/calmodulin-dependent serine protein kinase) is a kind of scaffolding protein that recruits or organizes other proteins at the plasma membrane to co-ordinate signal transduction pathways within the cytoplasm and nucleus. We have previously found that hCASK (human CASK) binds Id1 (inhibitor of DNA binding 1) through hCASK's GUK (guanylate kinase) domain and inhibits cell growth, probably via interactions with Id1. Overexpression of hCASK resulted in a reduced rate of cell growth, although inhibition of CASK via RNAi (RNA interference) promoted cell proliferation in ECV304 cells. This study revealed that hCASK regulates the protein and mRNA level of p21(wafi/cip1) (referred to throughout as p21), and activated the expression of p21 in a time-dependent manner. Two E-boxes in the proximal region at the TSS (transcription start site) play key roles in regulating hCASK-mediated p21 expression. We suggest that E2A (E12 and E47), a representative of the E proteins that binds the E-box elements, is a participant in the mediation of p21 expression by hCASK. The results of the present study suggest that hCASK regulation of cell growth might involve p21 expression, and that the bHLH (basic helix-loop-helix) transcription factor E2A probably participates in hCASK regulation of p21 expression. From these findings, we propose a novel proliferation signalling pathway mediated by hCASK.

    The Biochemical journal 2009;419;2;457-66

  • A missense mutation in CASK causes FG syndrome in an Italian family.

    Piluso G, D'Amico F, Saccone V, Bismuto E, Rotundo IL, Di Domenico M, Aurino S, Schwartz CE, Neri G and Nigro V

    Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli, Napoli 80138, Italy. giulio.piluso@unina2.it

    First described in 1974, FG syndrome (FGS) is an X-linked multiple congenital anomaly/mental retardation (MCA/MR) disorder, characterized by high clinical variability and genetic heterogeneity. Five loci (FGS1-5) have so far been linked to this phenotype on the X chromosome, but only one gene, MED12, has been identified to date. Mutations in this gene account for a restricted number of FGS patients with a more distinctive phenotype, referred to as the Opitz-Kaveggia phenotype. We report here that a p.R28L (c.83G-->T) missense mutation in CASK causes FGS phenotype in an Italian family previously mapped to Xp11.4-p11.3 (FGS4). The identified missense mutation cosegregates with the phenotype in this family and is absent in 1000 control X chromosomes of the same ethnic origin. An extensive analysis of CASK protein functions as well as structural and dynamic studies performed by molecular dynamics (MD) simulation did not reveal significant alterations induced by the p.R28L substitution. However, we observed a partial skipping of the exon 2 of CASK, presumably a consequence of improper recognition of exonic splicing enhancers (ESEs) induced by the c.83G-->T transversion. CASK is a multidomain scaffold protein highly expressed in the central nervous system (CNS) with specific localization to the synapses, where it forms large signaling complexes regulating neurotransmission. We suggest that the observed phenotype is most likely a consequence of an altered CASK expression profile during embryogenesis, brain development, and differentiation.

    Funded by: NICHD NIH HHS: HD 26202, R01 HD026202; Telethon: GGP06224

    American journal of human genetics 2009;84;2;162-77

  • Proteomic analysis reveals Hrs ubiquitin-interacting motif-mediated ubiquitin signaling in multiple cellular processes.

    Pridgeon JW, Webber EA, Sha D, Li L and Chin LS

    Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USA.

    Despite the critical importance of protein ubiquitination in the regulation of diverse cellular processes, the molecular mechanisms by which cells recognize and transmit ubiquitin signals remain poorly understood. The endosomal sorting machinery component hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) contains a ubiquitin-interacting motif (UIM), which is believed to bind ubiquitinated membrane cargo proteins and mediate their sorting to the lysosomal degradation pathway. To gain insight into the role of Hrs UIM-mediated ubiquitin signaling in cells, we performed a proteomic screen for Hrs UIM-interacting ubiquitinated proteins in human brain by using an in vitro expression cloning screening approach. We have identified 48 ubiquitinated proteins that are specifically recognized by the UIM domain of Hrs. Among them, 12 are membrane proteins that are likely to be Hrs cargo proteins, and four are membrane protein-associated adaptor proteins whose ubiquitination may act as a signal to target their associated membrane cargo for Hrs-mediated endosomal sorting. Other classes of the identified proteins include components of the vesicular trafficking machinery, cell signaling molecules, proteins associated with the cytoskeleton and cytoskeleton-dependent transport, and enzymes involved in ubiquitination and metabolism, suggesting the involvement of Hrs UIM-mediated ubiquitin signaling in the regulation of multiple cellular processes. We have characterized the ubiquitination of two identified proteins, Munc18-1 and Hsc70, and their interaction with Hrs UIM, and provided functional evidence supporting a role for Hsc70 in the regulation of Hrs-mediated endosome-to-lysosome trafficking.

    Funded by: NIGMS NIH HHS: GM082828, R01 GM082828, R01 GM082828-01A1, R01 GM082828-02, R01 GM082828-02S1; NINDS NIH HHS: NS047575, NS050650, R01 NS047575, R01 NS047575-01, R01 NS047575-02, R01 NS047575-03, R01 NS047575-04, R01 NS050650, R01 NS050650-01A1, R01 NS050650-02, R01 NS050650-03, R01 NS050650-04, T32 NS007480, T32 NS007480-05, T32 NS007480-06, T32 NS007480-07, T32 NS007480-08, T32 NS007480-09, T32NS007480

    The FEBS journal 2009;276;1;118-31

  • Mutations of CASK cause an X-linked brain malformation phenotype with microcephaly and hypoplasia of the brainstem and cerebellum.

    Najm J, Horn D, Wimplinger I, Golden JA, Chizhikov VV, Sudi J, Christian SL, Ullmann R, Kuechler A, Haas CA, Flubacher A, Charnas LR, Uyanik G, Frank U, Klopocki E, Dobyns WB and Kutsche K

    Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, 20246 Hamburg, Germany.

    CASK is a multi-domain scaffolding protein that interacts with the transcription factor TBR1 and regulates expression of genes involved in cortical development such as RELN. Here we describe a previously unreported X-linked brain malformation syndrome caused by mutations of CASK. All five affected individuals with CASK mutations had congenital or postnatal microcephaly, disproportionate brainstem and cerebellar hypoplasia, and severe mental retardation.

    Funded by: NINDS NIH HHS: R01 NS046616, R01-NS050375

    Nature genetics 2008;40;9;1065-7

  • The CASK gene harbored in a deletion detected by array-CGH as a potential candidate for a gene causative of X-linked dominant mental retardation.

    Hayashi S, Mizuno S, Migita O, Okuyama T, Makita Y, Hata A, Imoto I and Inazawa J

    Department of Molecular Cytogenetics, Medical Research Institute and School of Biomedical Science, Tokyo Medical and Dental University, Tokyo, Japan.

    Here we report on a 5-year-old Japanese girl with developmental delay and microcephaly. Although she had a normal karyotype, a bacterial artificial chromosome-based array-comparative genome hybridization analysis detected a de novo 4.0-Mb heterozygous deletion at Xp11.3-p11.4 harboring nine genes. By comparison with a healthy carrier mother of a boy with atypical Norrie disease having a smaller deletion in the same region, we excluded four genes as candidates whose haploinsufficiency would be causative for developmental delay. Among the other five genes, CASK seems to be the most likely candidate for a causative gene, because it is strongly expressed in fetal brain and plays important roles in neural development and synaptic function. We confirmed that the expression of CASK mRNA was decreased in the patient compared with healthy controls and the patient's X-chromosomal inactivation was not skewed. These results suggested that the genetic deletion of CASK results in haploinsufficiency, which might be causative for the patient's developmental delay or mental retardation. (c) 2008 Wiley-Liss, Inc.

    American journal of medical genetics. Part A 2008;146A;16;2145-51

  • The MAGUK-family protein CASK is targeted to nuclei of the basal epidermis and controls keratinocyte proliferation.

    Ojeh N, Pekovic V, Jahoda C and Määttä A

    School of Biological and Biomedical Sciences, Durham University, Durham DH13LE, UK.

    The Ca2+/calmodulin-associated Ser/Thr kinase (CASK) binds syndecans and other cell-surface proteins through its PDZ domain and has been implicated in synaptic assembly, epithelial polarity and neuronal gene transcription. We show here that CASK regulates proliferation and adhesion of epidermal keratinocytes. CASK is localised in nuclei of basal keratinocytes in newborn rodent skin and developing hair follicles. Induction of differentiation shifts CASK to the cell membrane, whereas in keratinocytes that have been re-stimulated after serum starvation CASK localisation shifts away from membranes upon entry to S phase. Biochemical fractionation demonstrates that CASK has several subnuclear targets and is found in both nucleoplasmic and nucleoskeletal pools. Knockdown of CASK by RNA interference leads to increased proliferation in cultured keratinocytes and in organotypic skin raft cultures. Accelerated cell cycling in CASK knockdown cells is associated with upregulation of Myc and hyperphosphorylation of Rb. Moreover, CASK-knockdown cells show increased hyperproliferative response to KGF and TGFalpha, and accelerated attachment and spreading to the collagenous matrix. These functions are reflected in wound healing, where CASK is downregulated in migrating and proliferating wound-edge keratinocytes.

    Funded by: Medical Research Council: G0300353

    Journal of cell science 2008;121;Pt 16;2705-17

  • CASK Functions as a Mg2+-independent neurexin kinase.

    Mukherjee K, Sharma M, Urlaub H, Bourenkov GP, Jahn R, Südhof TC and Wahl MC

    Department of Neuroscience, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9111, USA. konark.mukherjee@utsouthwestern.edu

    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.

    Funded by: NIMH NIH HHS: R37 MH052804, R37 MH052804-08, R37 MH52804-08

    Cell 2008;133;2;328-39

  • Deletion of CASK in mice is lethal and impairs synaptic function.

    Atasoy D, Schoch S, Ho A, Nadasy KA, Liu X, Zhang W, Mukherjee K, Nosyreva ED, Fernandez-Chacon R, Missler M, Kavalali ET and Südhof TC

    Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390-9111, USA.

    CASK is an evolutionarily conserved multidomain protein composed of an N-terminal Ca2+/calmodulin-kinase domain, central PDZ and SH3 domains, and a C-terminal guanylate kinase domain. Many potential activities for CASK have been suggested, including functions in scaffolding the synapse, in organizing ion channels, and in regulating neuronal gene transcription. To better define the physiological importance of CASK, we have now analyzed CASK "knockdown" mice in which CASK expression was suppressed by approximately 70%, and CASK knockout (KO) mice, in which CASK expression was abolished. CASK knockdown mice are viable but smaller than WT mice, whereas CASK KO mice die at first day after birth. CASK KO mice exhibit no major developmental abnormalities apart from a partially penetrant cleft palate syndrome. In CASK-deficient neurons, the levels of the CASK-interacting proteins Mints, Veli/Mals, and neurexins are decreased, whereas the level of neuroligin 1 (which binds to neurexins that in turn bind to CASK) is increased. Neurons lacking CASK display overall normal electrical properties and form ultrastructurally normal synapses. However, glutamatergic spontaneous synaptic release events are increased, and GABAergic synaptic release events are decreased in CASK-deficient neurons. In contrast to spontaneous neurotransmitter release, evoked release exhibited no major changes. Our data suggest that CASK, the only member of the membrane-associated guanylate kinase protein family that contains a Ca2+/calmodulin-dependent kinase domain, is required for mouse survival and performs a selectively essential function without being in itself required for core activities of neurons, such as membrane excitability, Ca2+-triggered presynaptic release, or postsynaptic receptor functions.

    Funded by: NIMH NIH HHS: R37 MH052804, R37 MH52804-08

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;7;2525-30

  • Cell fate-specific regulation of EGF receptor trafficking during Caenorhabditis elegans vulval development.

    Stetak A, Hoier EF, Croce A, Cassata G, Di Fiore PP and Hajnal A

    Institute of Zoology, University of Zürich, Zürich, Switzerland.

    By controlling the subcellular localization of growth factor receptors, cells can modulate the activity of intracellular signal transduction pathways. During Caenorhabditis elegans vulval development, a ternary complex consisting of the LIN-7, LIN-2 and LIN-10 PDZ domain proteins localizes the epidermal growth factor receptor (EGFR) to the basolateral compartment of the vulval precursor cells (VPCs) to allow efficient receptor activation by the inductive EGF signal from the anchor cell. We have identified EGFR substrate protein-8 (EPS-8) as a novel component of the EGFR localization complex that links receptor trafficking to cell fate specification. EPS-8 expression is upregulated in the primary VPCs, where it creates a positive feedback loop in the EGFR/RAS/MAPK pathway. The membrane-associated guanylate kinase LIN-2 recruits EPS-8 into the receptor localization complex to retain the EGFR on the basolateral plasma membrane, and thus allow maximal receptor activation in the primary cell lineage. Low levels of EPS-8 in the neighboring secondary VPCs result in the rapid degradation of the EGFR, allowing these cells to adopt the secondary cell fate. Extracellular signals thus regulate EGFR trafficking in a cell type-specific manner to control pattern formation during organogenesis.

    The EMBO journal 2006;25;11;2347-57

  • Uncovering quantitative protein interaction networks for mouse PDZ domains using protein microarrays.

    Stiffler MA, Grantcharova VP, Sevecka M and MacBeath G

    Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.

    One of the principal challenges in systems biology is to uncover the networks of protein-protein interactions that underlie most biological processes. To date, experimental efforts directed at this problem have largely produced only qualitative networks that are replete with false positives and false negatives. Here, we describe a domain-centered approach--compatible with genome-wide investigations--that enables us to measure the equilibrium dissociation constant (K(D)) of recombinant PDZ domains for fluorescently labeled peptides that represent physiologically relevant binding partners. Using a pilot set of 22 PDZ domains, 4 PDZ domain clusters, and 20 peptides, we define a gold standard dataset by determining the K(D) for all 520 PDZ-peptide combinations using fluorescence polarization. We then show that microarrays of PDZ domains identify interactions of moderate to high affinity (K(D) < or = 10 microM) in a high-throughput format with a false positive rate of 14% and a false negative rate of 14%. By combining the throughput of protein microarrays with the fidelity of fluorescence polarization, our domain/peptide-based strategy yields a quantitative network that faithfully recapitulates 85% of previously reported interactions and uncovers new biophysical interactions, many of which occur between proteins that are co-expressed. From a broader perspective, the selectivity data produced by this effort reveal a strong concordance between protein sequence and protein function, supporting a model in which interaction networks evolve through small steps that do not involve dramatic rewiring of the network.

    Funded by: NIGMS NIH HHS: 1 R01 GM072872-01, 5 T32 GM07598-25, R01 GM072872, R01 GM072872-04, T32 GM007598

    Journal of the American Chemical Society 2006;128;17;5913-22

  • The role of the MAGUK protein CASK in neural development and synaptic function.

    Hsueh YP

    Institute of Molecular Biology, Academia Sinica, Taipei, 115, Taiwan, Republic of China. hsueh@imb.sinica.edu.tw

    CASK, which belongs to the family of membrane-associated guanylate kinase (MAGUK) proteins, is recognized as a multidomain scaffolding protein highly expressed in the mammalian nervous system. MAGUK proteins generally target to neuronal synapses and regulate trafficking, targeting, and signaling of ion channels. However, CASK is a unique MAGUK protein in several respects. It not only plays a role in synaptic protein targeting but also contributes to neural development and regulation of gene expression. Several CASK-interacting proteins have been identified from yeast two-hybrid screening and biochemical isolation. These proteins, whose interactions with CASK are reviewed here, include the Parkinson's disease molecule parkin, the adhesion molecule neurexin, syndecans, calcium channel proteins, the cytoplasmic adaptor protein Mint1, Veli/mLIN-7/MALS, SAP97, caskin and CIP98, transcription factor Tbr-1, and nucleosome assembly protein CINAP. More important, CASK may form different complexes with different binding partners and perform different functions. Among these interactions, CASK, Tbr-1, and CINAP can form a transcriptional complex regulating gene expression. Reelin and NMDAR subunit 2b (NR2b) genes have been identified as Tbr-1 target genes. Reelin is critical for neural development. NR2b is an important subunit of NMDAR, which plays important roles in neural function and neurological diseases. Regulation of reelin and NR2b expression suggests the potential roles of the Tbr-1-CASK-CINAP complex in neural activity, development, and disease. The functions of these CASK protein complexes are also discussed in detail in this review.

    Current medicinal chemistry 2006;13;16;1915-27

  • p38gamma regulates the localisation of SAP97 in the cytoskeleton by modulating its interaction with GKAP.

    Sabio G, Arthur JS, Kuma Y, Peggie M, Carr J, Murray-Tait V, Centeno F, Goedert M, Morrice NA and Cuenda A

    MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK.

    Activation of the p38 MAP kinase pathways is crucial for the adaptation of mammalian cells to changes in the osmolarity of the environment. Here we identify SAP97/hDlg, the mammalian homologue of the Drosophila tumour suppressor Dlg, as a physiological substrate for the p38gamma MAP kinase (SAPK3/p38gamma) isoform. SAP97/hDlg is a scaffold protein that forms multiprotein complexes with a variety of proteins and is targeted to the cytoskeleton by its association with the protein guanylate kinase-associated protein (GKAP). The SAPK3/p38gamma-catalysed phosphorylation of SAP97/hDlg triggers its dissociation from GKAP and therefore releases it from the cytoskeleton. This is likely to regulate the integrity of intercellular-junctional complexes, and cell shape and volume in response to osmotic stress.

    The EMBO journal 2005;24;6;1134-45

  • CASK inhibits ECV304 cell growth and interacts with Id1.

    Qi J, Su Y, Sun R, Zhang F, Luo X, Yang Z and Luo X

    Burn Research Institute, Southwest Hospital, Third Military Medical University, State Key Laboratory of Trauma, Burn and Combined Injury, Chongqing 400038, PR China.

    Calcium/calmodulin-dependent serine protein kinase (CASK) is generally known as a scaffold protein. Here we show that overexpression of CASK resulted in a reduced rate of cell growth, while inhibition of expression of endogenous CASK via RNA-mediated interference resulted in an increased rate of cell growth in ECV304 cells. To explore the molecular mechanism, we identified a novel CASK-interacting protein, inhibitor of differentiation 1 (Id1) with a yeast two-hybrid screening. Furthermore, endogenous CASK and Id1 proteins were co-precipitated from the lysates of ECV304 cells by immunoprecipitation. Mammalian two-hybrid protein-protein interaction assays indicated that CASK possessed a different binding activity for Id1 and its alternative splicing variant. It is known that Id proteins play important roles in regulation of cell proliferation and differentiation. Thus, we speculate that the regulation of cell growth mediated by CASK may be involved in Id1. Our findings indicate a novel function of CASK, the mechanism that remains to be further investigated.

    Biochemical and biophysical research communications 2005;328;2;517-21

  • A novel scaffold protein, TANC, possibly a rat homolog of Drosophila rolling pebbles (rols), forms a multiprotein complex with various postsynaptic density proteins.

    Suzuki T, Li W, Zhang JP, Tian QB, Sakagami H, Usuda N, Usada N, Kondo H, Fujii T and Endo S

    Department of Neuroplasticity, Institute on Ageing and Adaptation, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan. suzukit@sch.md.shinshu-u.ac.jp

    We cloned from the rat brain a novel gene, tanc (GenBank Accession No. AB098072), which encoded a protein containing three tetratricopeptide repeats (TPRs), ten ankyrin repeats and a coiled-coil region, and is possibly a rat homolog of Drosophila rolling pebbles (rols). The tanc gene was expressed widely in the adult rat brain. Subcellular distribution, immunohistochemical study of the brain and immunocytochemical studies of cultured neuronal cells indicated the postsynaptic localization of TANC protein of 200 kDa. Pull-down experiments showed that TANC protein bound PSD-95, SAP97, and Homer via its C-terminal PDZ-binding motif, -ESNV, and fodrin via both its ankyrin repeats and the TPRs together with the coiled-coil domain. TANC also bound the alpha subunit of Ca2+/calmodulin-dependent protein kinase II. An immunoprecipitation study showed TANC association with various postsynaptic proteins, including guanylate kinase-associated protein (GKAP), alpha-internexin, and N-methyl-D-aspartate (NMDA)-type glutamate receptor 2B and AMPA-type glutamate receptor (GluR1) subunits. These results suggest that TANC protein may work as a postsynaptic scaffold component by forming a multiprotein complex with various postsynaptic density proteins.

    The European journal of neuroscience 2005;21;2;339-50

  • Nephrin forms a complex with adherens junction proteins and CASK in podocytes and in Madin-Darby canine kidney cells expressing nephrin.

    Lehtonen S, Lehtonen E, Kudlicka K, Holthöfer H and Farquhar MG

    Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA.

    Mutations in the NPHS1 gene encoding nephrin lead to congenital nephrotic syndrome of the Finnish type. Nephrin is a key component of the glomerular slit diaphragms between epithelial foot processes, but its role in the pathogenesis of this disease is poorly understood. To further clarify the molecular mechanisms involved we investigated the interactions between nephrin and other components of the foot processes and filtration slits, especially adherens junction proteins, and searched for novel nephrin interacting proteins. Using co-immunoprecipitation and pull-down assays we show here that nephrin forms a multiprotein complex with cadherins and p120 catenin and with three scaffolding proteins, ZO-1, CD2AP, and CASK, in kidney glomeruli and when expressed in Madin-Darby canine kidney cells. CASK was identified as a novel binding partner of nephrin by mass spectrometry and was localized to podocytes in the glomerulus. CASK is a scaffolding protein that participates in maintenance of polarized epithelial cell architecture by linking membrane proteins and signaling molecules to the actin cytoskeleton. Our results support a model whereby the glomerular slit diaphragms are composed of cell adhesion molecules of the immunoglobulin and cadherin superfamilies that are connected to each other and to the actin cytoskeleton and signaling networks via the cytoplasmic scaffolding proteins CASK, CD2AP, and ZO-1.

    Funded by: NIDDK NIH HHS: DK 17724, R01 DK017724, R37 DK017724

    The American journal of pathology 2004;165;3;923-36

  • Protein trafficking and anchoring complexes revealed by proteomic analysis of inward rectifier potassium channel (Kir2.x)-associated proteins.

    Leonoudakis D, Conti LR, Anderson S, Radeke CM, McGuire LM, Adams ME, Froehner SC, Yates JR and Vandenberg CA

    Department of Molecular, Cellular, University of California, Santa Barbara, California 93106, USA.

    Inward rectifier potassium (Kir) channels play important roles in the maintenance and control of cell excitability. Both intracellular trafficking and modulation of Kir channel activity are regulated by protein-protein interactions. We adopted a proteomics approach to identify proteins associated with Kir2 channels via the channel C-terminal PDZ binding motif. Detergent-solubilized rat brain and heart extracts were subjected to affinity chromatography using a Kir2.2 C-terminal matrix to purify channel-interacting proteins. Proteins were identified with multidimensional high pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry, N-terminal microsequencing, and immunoblotting with specific antibodies. We identified eight members of the MAGUK family of proteins (SAP97, PSD-95, Chapsyn-110, SAP102, CASK, Dlg2, Dlg3, and Pals2), two isoforms of Veli (Veli-1 and Veli-3), Mint1, and actin-binding LIM protein (abLIM) as Kir2.2-associated brain proteins. From heart extract purifications, SAP97, CASK, Veli-3, and Mint1 also were found to associate with Kir2 channels. Furthermore, we demonstrate for the first time that components of the dystrophin-associated protein complex, including alpha1-, beta1-, and beta2-syntrophin, dystrophin, and dystrobrevin, interact with Kir2 channels, as demonstrated by immunoaffinity purification and affinity chromatography from skeletal and cardiac muscle and brain. Affinity pull-down experiments revealed that Kir2.1, Kir2.2, Kir2.3, and Kir4.1 all bind to scaffolding proteins but with different affinities for the dystrophin-associated protein complex and SAP97, CASK, and Veli. Immunofluorescent localization studies demonstrated that Kir2.2 co-localizes with syntrophin, dystrophin, and dystrobrevin at skeletal muscle neuromuscular junctions. These results suggest that Kir2 channels associate with protein complexes that may be important to target and traffic channels to specific subcellular locations, as well as anchor and stabilize channels in the plasma membrane.

    Funded by: NINDS NIH HHS: NS33145, NS43377

    The Journal of biological chemistry 2004;279;21;22331-46

  • A multiprotein trafficking complex composed of SAP97, CASK, Veli, and Mint1 is associated with inward rectifier Kir2 potassium channels.

    Leonoudakis D, Conti LR, Radeke CM, McGuire LM and Vandenberg CA

    Department of Molecular, Cellular, and Developmental Biology, and Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA.

    Strong inward rectifier potassium (Kir2) channels are important in the control of cell excitability, and their functions are modulated by interactions with intracellular proteins. Here we identified a complex of scaffolding/trafficking proteins in brain that associate with Kir2.1, Kir2.2, and Kir2.3 channels. By using a combination of affinity interaction pulldown assays and co-immunoprecipitations from brain and transfected cells, we demonstrated that a complex composed of SAP97, CASK, Veli, and Mint1 associates with Kir2 channels via the C-terminal PDZ-binding motif. We further demonstrated by using in vitro protein interaction assays that SAP97, Veli-1, or Veli-3 binds directly to the Kir2.2 C terminus and recruits CASK. Co-immunoprecipitations indicated that specific Veli isoforms participate in forming distinct protein complexes in brain, where Veli-1 stably associates with CASK and SAP97, Veli-2 associates with CASK and Mint1, and Veli-3 associates with CASK, SAP97, and Mint1. Additionally, immunocytochemistry of rat cerebellum revealed overlapping expression of Kir2.2, SAP97, CASK, Mint1, with Veli-1 in the granule cell layer and Veli-3 in the molecular layer. We propose a model whereby Kir2.2 associates with distinct SAP97-CASK-Veli-Mint1 complexes. In one complex, SAP97 interacts directly with the Kir2 channels and recruits CASK, Veli, and Mint1. Alternatively, Veli-1 or Veli-3 interacts directly with the Kir2 channels and recruits CASK and SAP97; association of Mint1 with the complex requires Veli-3. Expression of Kir2.2 in polarized epithelial cells resulted in targeting of the channels to the basolateral membrane and co-localization with SAP97 and CASK, whereas a dominant interfering form of CASK caused the channels to mislocalize. Therefore, CASK appears to be a central protein of a macromolecular complex that participates in trafficking and plasma membrane localization of Kir2 channels.

    Funded by: NINDS NIH HHS: NS43377

    The Journal of biological chemistry 2004;279;18;19051-63

  • Transcriptional modification by a CASK-interacting nucleosome assembly protein.

    Wang GS, Hong CJ, Yen TY, Huang HY, Ou Y, Huang TN, Jung WG, Kuo TY, Sheng M, Wang TF and Hsueh YP

    Institute of Molecular Biology, Academia Sinica, National Yang-Ming University, Taipei, Taiwan, ROC.

    CASK acts as a coactivator for Tbr-1, an essential transcription factor in cerebral cortex development. Presently, the molecular mechanism of the CASK coactivation effect is unclear. Here, we report that CASK binds to another nuclear protein, CINAP, which binds histones and facilitates nucleosome assembly. CINAP, via its interaction with CASK, forms a complex with Tbr-1, regulating expression of the genes controlled by Tbr-1 and CASK, such as NR2b and reelin. A knockdown of endogenous CINAP in hippocampal neurons reduces the promoter activity of NR2b. Moreover, NMDA stimulation results in a reduction in the level of CINAP protein, via a proteasomal degradation pathway, correlating with a decrease in NR2b expression in neurons. This study suggests that reduction of the CINAP protein level by synaptic stimulation contributes to regulation of the transcriptional activity of the Tbr-1/CASK/CINAP protein complex and thus modifies expression of the NR2b gene.

    Neuron 2004;42;1;113-28

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Carom: a novel membrane-associated guanylate kinase-interacting protein with two SH3 domains.

    Ohno H, Hirabayashi S, Kansaku A, Yao I, Tajima M, Nishimura W, Ohnishi H, Mashima H, Fujita T, Omata M and Hata Y

    Department of Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.

    MAGI-1 and CASK are membrane-associated guanylate kinases of epithelial junctions. MAGI-1 is localized at tight junctions in polarized epithelial cells, whereas CASK is localized along the lateral membranes. We obtained the KIAA0769 gene product through the yeast two-hybrid screening using MAGI-1 as a bait and named it Carom. Carom has a coiled-coil domain in the middle region, and two src homology 3 domains and a PSD-95/Dlg-A/ZO-1 (PDZ)-binding motif in the C-terminal region. Carom binds to the fifth PDZ domain of MAGI-1 and the calmodulin kinase domain of CASK in vitro. MAGI-1 and CASK bind to the distinct sequences in the C-terminal region of Carom, but still compete with each other for Carom binding. The study using a stable transformant of Madine Darby canine kidney (MDCK) cells expressing GFP-Carom revealed that Carom was partially overlapped by MAGI-1 in MDCK cells, which have not yet established mature cell junctions, but became separated from MAGI-1 and colocalized with CASK in polarized cells. Carom was highly resistant to Triton X-100 extractions and recruited CASK to the Triton X-100-insoluble structures. Carom is a binding partner of CASK, which interacts with CASK in polarized epithelial cells and may link it to the cytoskeleton.

    Oncogene 2003;22;52;8422-31

  • CIP98, a novel PDZ domain protein, is expressed in the central nervous system and interacts with calmodulin-dependent serine kinase.

    Yap CC, Liang F, Yamazaki Y, Muto Y, Kishida H, Hayashida T, Hashikawa T and Yano R

    Laboratories for Cellular Information Processing, Brain Science Institute (BSI), RIKEN, Wako, Saitama, Japan.

    Receptors and various molecules in neurons are localized at precise locations to perform their respective functions, especially in synaptic sites. Among synaptic molecules, PDZ domain proteins play major roles in scaffolding and anchoring membrane proteins for efficient synaptic transmission. In the present study, we isolated CIP98, a novel protein (98 kDa) consisting of three PDZ domains and a proline-rich region, which is widely expressed in the central nervous system. In situ hybridization and immunohistochemical staining patterns demonstrate that CIP98 is expressed strongly in certain types of neurons, i.e. pyramidal cells in layers III-V of the cerebral cortex, projecting neurons in the thalamus and interneurons in the cerebellum. The results of immunocytochemical staining and electron microscopy revealed that CIP98 is localized both in dendrites and axons. Interestingly, CIP98 interacts with CASK (calmodulin-dependent serine kinase), a member of the membrane-associated guanylate kinase (MAGUK) family that plays important roles in the molecular organization of proteins at synapses. CIP98 was shown to co-localize with CASK along the dendritic processes of neurons. In view of its direct association with CASK, CIP98 may be involved in the formation of CASK scaffolding proteins complex to facilitate synaptic transmission in the CNS.

    Journal of neurochemistry 2003;85;1;123-34

  • Interaction of the plasma membrane Ca2+ pump 4b/CI with the Ca2+/calmodulin-dependent membrane-associated kinase CASK.

    Schuh K, Uldrijan S, Gambaryan S, Roethlein N and Neyses L

    Department of Medicine, University of Wuerzburg, D-97080 Wuerzburg, Germany.

    Spatial and temporal regulation of intracellular Ca(2+) is a key event in many signaling pathways. Plasma membrane Ca(2+)-ATPases (PMCAs) are major regulators of Ca(2+) homeostasis and bind to PDZ (PSD-95/Dlg/ZO-1) domains via their C termini. Various membrane-associated guanylate kinase family members have been identified as interaction partners of PMCAs. In particular, SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bind to the C-terminal tails of PMCA "b" splice variants. Additionally, it has been demonstrated that PMCA4b interacts with neuronal nitric-oxide synthase and that isoform 2b interacts with Na(+)/H(+) exchanger regulatory factor 2, both via a PDZ domain. CASK (calcium/calmodulin-dependent serine protein kinase) contains a calmodulin-dependent protein kinase-like domain followed by PDZ, SH3, and guanylate kinase-like domains. In adult brain CASK is located at neuronal synapses and interacts with various proteins, e.g. neurexin and Veli/LIN-7. In kidney it is localized to renal epithelia. Surprisingly, interaction with the Tbr-1 transcription factor, nuclear transport, binding to DNA T-elements (in a complex with Tbr-1), and transcriptional competence has been shown. Here we show that the C terminus of PMCA4b binds to CASK and that both proteins co-precipitate from brain and kidney tissue lysates. Immunofluorescence staining revealed co-expression of PMCA, CASK, and calbindin-d-28K in distal tubuli of rat kidney sections. To test if physical interaction of both proteins results in functional consequences we constructed a T-element-dependent reporter vector and investigated luciferase activity in HEK293 lysates, previously co-transfected with PMCA4b expression and control vectors. Expression of wild-type PMCA resulted in an 80% decrease in T-element-dependent transcriptional activity, whereas co-expression of a point-mutated PMCA, with nearly eliminated Ca(2+) pumping activity, had only a small influence on regulation of transcriptional activity. These results provide evidence of a new direct Ca(2+)-dependent link from the plasma membrane to the nucleus.

    The Journal of biological chemistry 2003;278;11;9778-83

  • Association of the type 1 inositol (1,4,5)-trisphosphate receptor with 4.1N protein in neurons.

    Maximov A, Tang TS and Bezprozvanny I

    Department of Physiology, UT Southwestern Medical Center at Dallas, 75390, Dallas, TX, USA.

    The type 1 inositol (1,4,5)-trisphosphate receptor (InsP(3)R1) is an intracellular calcium (Ca(2+)) release channel that plays an important role in neuronal function. In yeast two-hybrid screen of rat brain cDNA library with the InsP(3)R1 carboxy-terminal bait we isolated multiple clones of neuronal cytoskeletal protein 4.1N. We mapped the 4.1N-interaction site to a short fragment (50 amino acids) within the carboxy-terminal tail of the InsP(3)R1 and the InsP(3)R1-interaction site to the carboxy-terminal domain (CTD) of 4.1N. We established that InsP(3)R1 carboxy-terminal binds selectively to the CTDDelta alternatively spliced form of the 4.1N protein. In biochemical experiments we demonstrated that 4.1N and InsP(3)R1 specifically associate in vitro. We showed that both 4.1N and InsP(3)R1 were enriched in synaptic locations and immunoprecipitated the 4.1N-InsP(3)R1 complex from rat brain synaptosomes. In biochemical experiments we demonstrated a possibility of InsP(3)R1-4.1N-CASK-syndecan-2 quaternary complex formation. From our findings we hypothesize that InsP(3)R1-4.1N association may play a role in InsP(3)R1 localization or Ca(2+) signaling in neurons.

    Funded by: NINDS NIH HHS: R01 NS38082

    Molecular and cellular neurosciences 2003;22;2;271-83

  • Genetic heterogeneity of FG syndrome: a fourth locus (FGS4) maps to Xp11.4-p11.3 in an Italian family.

    Piluso G, Carella M, D'Avanzo M, Santinelli R, Carrano EM, D'Avanzo A, D'Adamo AP, Gasparini P and Nigro V

    Dipartimento di Patologia Generale, Facoltà di Medicina e Chirurgia, Seconda Università degli Studi di Napoli, Via Luigi De Crecchio 7, 80138 Naples, Italy.

    FG syndrome (FGS, MIM 305450) is a rare X-linked recessive disorder comprising mental retardation and multiple malformations. Various families have been described to date, increasing our knowledge of the phenotype variability and making the clinical diagnosis complex, especially in sporadic patients. The first locus for FG syndrome (FGS1) was linked to chromosome region Xq12-q21.31, but other families have been excluded from this locus. The genetic heterogeneity of FG syndrome has been confirmed by analysis of an X chromosome inversion [inv(X)(q11q28)] in an affected boy and in his mentally retarded maternal uncle, suggesting that an additional locus for FG syndrome (FGS2, MIM 300321) is located at either Xq11 or Xq28. Recently, a third locus (FGS3) has been mapped to Xp22.3. We have identified and clinically characterized an Italian FG family, including 31 members with three affected males in two generations and two obligate carriers. We have excluded linkage to known FGS loci, whereas an extensive study of the whole X chromosome has yielded a maximum LOD score (Z(max)) of 2.66 (recombination fraction=0) for markers between DXS8113 and sWXD805. This new locus for FG syndrome corresponds to a region of approximately 4.6 Mb on the X chromosome.

    Funded by: Telethon: TGM03Z02, TGM06S01

    Human genetics 2003;112;2;124-30

  • Identification of multiple binding partners for the amino-terminal domain of synapse-associated protein 97.

    Karnak D, Lee S and Margolis B

    Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

    Multiprotein complexes mediate static and dynamic functions to establish and maintain cell polarity in both epithelial cells and neurons. Membrane-associated guanylate kinase (MAGUK) proteins are thought to be scaffolding molecules in these processes and bind multiple proteins via their obligate postsynaptic density (PSD)-95/Disc Large/Zona Occludens-1, Src homology 3, and guanylate kinase-like domains. Subsets of MAGUK proteins have additional protein-protein interaction domains. An additional domain we identified in SAP97 called the MAGUK recruitment (MRE) domain binds the LIN-2,7 amino-terminal (L27N) domain of mLIN-2/CASK, a MAGUK known to bind mLIN-7. Here we show that SAP97 binds two other mLIN-7 binding MAGUK proteins. One of these MAGUK proteins, DLG3, coimmunoprecipitates with SAP97 in lysates from rat brain and transfected Madin-Darby canine kidney cells. This interaction requires the MRE domain of SAP97 and surprisingly, both the L27N and L27 carboxyl-terminal (L27C) domains of DLG3. We also demonstrate that SAP97 can interact with the MAGUK protein, DLG2, but not the highly related protein, PALS2. The ability of SAP97 to interact with multiple MAGUK proteins is likely to be important for the targeting of specific protein complexes in polarized cells.

    Funded by: NIDDK NIH HHS: 2-P50-DK39255; NIGMS NIH HHS: 5-T32-GM07544, GM08353

    The Journal of biological chemistry 2002;277;48;46730-5

  • Coordinated folding and association of the LIN-2, -7 (L27) domain. An obligate heterodimerization involved in assembly of signaling and cell polarity complexes.

    Harris BZ, Venkatasubrahmanyam S and Lim WA

    Program in Biological Sciences, University of California, San Francisco, California 94143-0450, USA.

    LIN-2, -7 (L27) homology domains are putative protein-protein interaction modules found in several scaffold proteins involved in the assembly of polarized cell-signaling structures. These specific interaction pairs are well conserved across metazoan species, from worms to man. We have expressed and purified L27 domains from multiple species and find that certain domains from proteins such as Caenorhabditis elegans LIN-2 and LIN-7 can specifically heterodimerize. Biophysical analysis of interacting L27 domains demonstrates that the domains interact with a 1:1 stoichiometry. Circular dichroism studies reveal that the domains appear to function as an obligate heterodimer; individually the domains are largely unfolded, but when associated they show a significant increase in helicity, as well as a cooperative unfolding transition. These novel obligate interacting pairs are likely to play a key role in regulating the organization of signaling proteins at polarized cell structures.

    The Journal of biological chemistry 2002;277;38;34902-8

  • SynCAM, a synaptic adhesion molecule that drives synapse assembly.

    Biederer T, Sara Y, Mozhayeva M, Atasoy D, Liu X, Kavalali ET and Südhof TC

    Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Thomas.Biederer@UTSouthwestern.edu

    Synapses, the junctions between nerve cells through which they communicate, are formed by the coordinated assembly and tight attachment of pre- and postsynaptic specializations. We now show that SynCAM is a brain-specific, immunoglobulin domain-containing protein that binds to intracellular PDZ-domain proteins and functions as a homophilic cell adhesion molecule at the synapse. Expression of the isolated cytoplasmic tail of SynCAM in neurons inhibited synapse assembly. Conversely, expression of full-length SynCAM in nonneuronal cells induced synapse formation by cocultured hippocampal neurons with normal release properties. Glutamatergic synaptic transmission was reconstituted in these nonneuronal cells by coexpressing glutamate receptors with SynCAM, which suggests that a single type of adhesion molecule and glutamate receptor are sufficient for a functional postsynaptic response.

    Science (New York, N.Y.) 2002;297;5586;1525-31

  • Postsynaptic targeting of alternative postsynaptic density-95 isoforms by distinct mechanisms.

    Chetkovich DM, Bunn RC, Kuo SH, Kawasaki Y, Kohwi M and Bredt DS

    Department of Physiology, University of California, San Francisco, San Francisco, California 94143-0444, USA.

    Members of the postsynaptic density-95 (PSD95)/synapse-associated protein-90 (SAP90) family of scaffolding proteins contain a common set of modular protein interaction motifs including PDZ (postsynaptic density-95/Discs large/zona occludens-1), Src homology 3, and guanylate kinase domains, which regulate signaling and plasticity at excitatory synapses. We report that N-terminal alternative splicing of PSD95 generates an isoform, PSD95beta that contains an additional "L27" motif, which is also present in SAP97. Using yeast two hybrid and coimmunoprecipitation assays, we demonstrate that this N-terminal L27 domain of PSD-95beta, binds to an L27 domain in the membrane-associated guanylate kinase calcium/calmodulin-dependent serine kinase, and to Hrs, an endosomal ATPase that regulates vesicular trafficking. By transfecting heterologous cells and hippocampal neurons, we find that interactions with the L27 domain regulate synaptic clustering of PSD95beta. Disrupting Hrs-regulated early endosomal sorting in hippocampal neurons selectively blocks synaptic clustering of PSD95beta but does not interfere with trafficking of the palmitoylated isoform, PSD95alpha. These studies identify molecular and functional heterogeneity in synaptic PSD95 complexes and reveal critical roles for L27 domain interactions and Hrs regulated vesicular trafficking in postsynaptic protein clustering.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;15;6415-25

  • Recruitment of the kainate receptor subunit glutamate receptor 6 by cadherin/catenin complexes.

    Coussen F, Normand E, Marchal C, Costet P, Choquet D, Lambert M, Mège RM and Mulle C

    Centre National de la Recherche Scientifique Unité Mixte de Recherche 5091, Institut François Magendie, Paris 75005, France.

    Kainate receptors modulate synaptic transmission by acting either at presynaptic or at postsynaptic sites. The precise localization of kainate receptors as well as the mechanisms of targeting and stabilization of these receptors in neurons are largely unknown. We have generated transgenic mice expressing the kainate receptor subunit glutamate receptor 6 (GluR6) bearing an extracellular myc epitope (myc-GluR6), in forebrain neurons, in which it assembles with endogenous kainate receptor subunits. In transgenic mice crossed with GluR6-deficient mice, myc-GluR6 efficiently rescues the missing subunit. Immunoprecipitation of transgenic brain extracts with anti-myc antibodies demonstrates an interaction with cadherins, beta-catenin, and p120 catenin, as well as with the associated proteins calcium calmodulin-dependent serine kinase and Velis, but not with alpha-catenin. In glutathione S-transferase-pulldown experiments, beta-catenin interacts, although indirectly, with the last 14 aa of GluR6. Transfected myc-GluR6 colocalizes with beta-catenin at cell-cell junctions in non-neuronal cells. Finally, activation of N-cadherins by ligand-covered latex beads recruits GluR6 to cadherin/catenin complexes. These results suggest an important role for cadherin/catenin complexes in the stabilization of kainate receptors at the synaptic membrane during synapse formation and remodeling.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;15;6426-36

  • CASK participates in alternative tripartite complexes in which Mint 1 competes for binding with caskin 1, a novel CASK-binding protein.

    Tabuchi K, Biederer T, Butz S and Sudhof TC

    The Center for Basic Neuroscience, Department of Molecular Genetics, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    CASK, an adaptor protein of the plasma membrane, is composed of an N-terminal calcium/calmodulin-dependent protein (CaM) kinase domain, central PSD-95, Dlg, and ZO-1/2 domain (PDZ) and Src homology 3 (SH3) domains, and a C-terminal guanylate kinase sequence. The CaM kinase domain of CASK binds to Mint 1, and the region between the CaM kinase and PDZ domains interacts with Velis, resulting in a tight tripartite complex. CASK, Velis, and Mint 1 are evolutionarily conserved in Caenorhabditis elegans, in which homologous genes (called lin-2, lin-7, and lin-10) are required for vulva development. We now demonstrate that the N-terminal CaM kinase domain of CASK binds to a novel brain-specific adaptor protein called Caskin 1. Caskin 1 and a closely related isoform, Caskin 2, are multidomain proteins containing six N-terminal ankyrin repeats, a single SH3 domain, and two sterile alpha motif domains followed by a long proline-rich sequence and a short conserved C-terminal domain. Unlike CASK and Mint 1, no Caskin homolog was detected in C. elegans. Immunoprecipitations showed that Caskin 1, like Mint 1, is stably bound to CASK in the brain. Affinity chromatography experiments demonstrated that Caskin 1 coassembles with CASK on the immobilized cytoplasmic tail of neurexin 1, suggesting that CASK and Caskin 1 coat the cytoplasmic tails of neurexins and other cell-surface proteins. Detailed mapping studies revealed that Caskin 1 and Mint 1 bind to the same site on the N-terminal CaM kinase domain of CASK and compete with each other for CASK binding. Our data suggest that in the vertebrate brain, CASK and Velis form alternative tripartite complexes with either Mint 1 or Caskin 1 that may couple CASK to distinct downstream effectors.

    Funded by: NIMH NIH HHS: R37-MH52804-06

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;11;4264-73

  • Caspr3 and caspr4, two novel members of the caspr family are expressed in the nervous system and interact with PDZ domains.

    Spiegel I, Salomon D, Erne B, Schaeren-Wiemers N and Peles E

    Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.

    The NCP family of cell-recognition molecules represents a distinct subgroup of the neurexins that includes Caspr and Caspr2, as well as Drosophila Neurexin-IV and axotactin. Here, we report the identification of Caspr3 and Caspr4, two new NCPs expressed in nervous system. Caspr3 was detected along axons in the corpus callosum, spinal cord, basket cells in the cerebellum and in peripheral nerves, as well as in oligodendrocytes. In contrast, expression of Caspr4 was more restricted to specific neuronal subpopulations in the olfactory bulb, hippocampus, deep cerebellar nuclei, and the substantia nigra. Similar to the neurexins, the cytoplasmic tails of Caspr3 and Caspr4 interacted differentially with PDZ domain-containing proteins of the CASK/Lin2-Veli/Lin7-Mint1/Lin10 complex. The structural organization and distinct cellular distribution of Caspr3 and Caspr4 suggest a potential role of these proteins in cell recognition within the nervous system.

    Molecular and cellular neurosciences 2002;20;2;283-97

  • Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach.

    Bécamel C, Alonso G, Galéotti N, Demey E, Jouin P, Ullmer C, Dumuis A, Bockaert J and Marin P

    CNRS UPR9023, CCIPE 141 rue de la Cardonille, F-34094 Montpellier Cedex 05, France.

    Membrane-bound receptors such as tyrosine kinases and ionotropic receptors are associated with large protein networks structured by protein-protein interactions involving multidomain proteins. Although these networks have emerged as a general mechanism of cellular signalling, much less is known about the protein complexes associated with G-protein-coupled receptors (GPCRs). Using a proteomic approach based on peptide affinity chromatography followed by mass spectrometry and immunoblotting, we have identified 15 proteins that interact with the C- terminal tail of the 5-hydroxytryptamine 2C (5-HT(2C)) receptor, a GPCR. These proteins include several synaptic multidomain proteins containing one or several PDZ domains (PSD95 and the proteins of the tripartite complex Veli3-CASK-Mint1), proteins of the actin/spectrin cytoskeleton and signalling proteins. Coimmunoprecipitation experiments showed that 5-HT(2C) receptors interact with PSD95 and the Veli3-CASK-Mint1 complex in vivo. Electron microscopy also indicated a synaptic enrichment of Veli3 and 5-HT(2C) receptors and their colocalization in microvilli of choroidal cells. These results indicate that the 5-HT(2C) receptor is associated with protein networks that are important for its synaptic localization and its coupling to the signalling machinery.

    The EMBO journal 2002;21;10;2332-42

  • CASK and its target gene Reelin were co-upregulated in human esophageal carcinoma.

    Wang Q, Lu J, Yang C, Wang X, Cheng L, Hu G, Sun Y, Zhang X, Wu M and Liu Z

    National Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, PR China.

    Calcium/calmodulin-dependent serine protein kinase (CASK) showed overexpression in human esophageal carcinoma by suppression subtractive hybridization. The upregulation of CASK gene and its target gene Reelin in human esophageal carcinoma tissues versus corresponding normal tissues was revealed by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry or Western blot. Moreover, RT-PCR results indicated that the expression patterns of CASK and Reelin in human gastric carcinoma and colon carcinoma were different with those in esophageal carcinoma. Therefore, it suggested that CASK and Reelin were associated with tumorigenesis of esophagus and they were co-upregulated in human esophageal carcinoma.

    Cancer letters 2002;179;1;71-7

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

    Fallon L, Moreau F, Croft BG, Labib N, Gu WJ and Fon EA

    Centre for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.

    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.

    The Journal of biological chemistry 2002;277;1;486-91

  • Basolateral membrane expression of the Kir 2.3 channel is coordinated by PDZ interaction with Lin-7/CASK complex.

    Olsen O, Liu H, Wade JB, Merot J and Welling PA

    Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

    The basolateral membrane sorting determinant of an inwardly rectifying potassium channel, Kir 2.3, is comprised of a unique arrangement of trafficking motifs containing tandem, conceivably overlapping, biosynthetic targeting and PDZ-based signals. In the present study, we elucidate a mechanism by which a PDZ interaction coordinates one step in a basolateral membrane sorting program. In contrast to apical missorting of channels lacking the entire sorting domain, deletion of the PDZ binding motif caused channels to accumulate into an endosomal compartment. Here, we identify a new human ortholog of a Caenorhabditis elegans PDZ protein, hLin-7b, that interacts with the COOH-terminal tail of Kir 2.3 in renal epithelia. hLin-7b associates with the channel as a part of a multimeric complex on the basolateral membrane similar to a basolateral membrane complex in C. elegans vulva progenitor cells. Coexpression of hLin-7b with Kir 2.3 dramatically increases channel activity by stabilizing plasma membrane expression. The discovery identifies one component of the sorting machinery and provides evidence for a retention mechanism in a hierarchical basolateral trafficking program.

    Funded by: NIDDK NIH HHS: DK-32839, DK-54231

    American journal of physiology. Cell physiology 2002;282;1;C183-95

  • Bipartite interaction between neurofibromatosis type I protein (neurofibromin) and syndecan transmembrane heparan sulfate proteoglycans.

    Hsueh YP, Roberts AM, Volta M, Sheng M and Roberts RG

    Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, 115, Republic of China. yph@gate.sinica.edu.tw

    The neurofibromatosis type 1 (NF1) gene encodes a large tumor suppressor protein (neurofibromin). Although it is known to possess Ras GTPase-activating protein (GAP) activity, the cellular role of neurofibromin remains unclear. Here we used yeast two-hybrid screening to identify neurofibromin-interacting proteins. Syndecan-2, a transmembrane heparan sulfate proteoglycan (HSPG), was isolated as a binding partner for two distinct regions of the neurofibromin protein. We subsequently found that neurofibromin can bind all four mammalian syndecans. NF1 interaction requires the transmembrane domain and a membrane-proximal region of the cytoplasmic tail of syndecan, but not the C terminus of syndecan known to bind to CASK, a membrane-associated guanylate kinase (MAGUK). Neurofibromin, syndecans, and CASK have overlapping subcellular distributions in axons and synapses of neurons, as shown by biochemical fractionation and immunostaining. Moreover, neurofibromin exists in a complex with syndecan and CASK in vivo, as evidenced by their coimmunoprecipitation from rat brain. Our findings suggest that interaction with different members of the syndecan family may be a mechanism for localizing neurofibromin to specialized domains of the plasma membrane.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;11;3764-70

  • The scaffolding protein CASK mediates the interaction between rabphilin3a and beta-neurexins.

    Zhang Y, Luan Z, Liu A and Hu G

    Max-Planck Guest Laboratory, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 320, Yue-Yang Road, 200031, Shanghai, PR China.

    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.

    FEBS letters 2001;497;2-3;99-102

  • Association of junctional adhesion molecule with calcium/calmodulin-dependent serine protein kinase (CASK/LIN-2) in human epithelial caco-2 cells.

    Martinez-Estrada OM, Villa A, Breviario F, Orsenigo F, Dejana E and Bazzoni G

    Istituto di Ricerche Farmacologiche Mario Negri, 20157 Milan, Italy.

    We report here that junctional adhesion molecule (JAM) interacts with calcium/calmodulin-dependent serine protein kinase (CASK), a protein related to membrane-associated guanylate kinases. In Caco-2 cells, JAM and CASK were coprecipitated and found to colocalize at intercellular contacts along the lateral surface of the plasma membrane. Association of JAM with CASK requires the PSD95/dlg/ZO-1 (PDZ) domain of CASK and the putative PDZ-binding motif Phe-Leu-Val(COOH) in the cytoplasmic tail of JAM. Temporal dissociation in the junctional localization of the two proteins suggests that the association with CASK is not required for recruiting JAM to intercellular junctions. Compared with mature intercellular contacts, junction assembly was characterized by both enhanced solubility of CASK in Triton X-100 and reduced amounts of Triton-insoluble JAM-CASK complexes. We propose that JAM association with CASK is modulated during junction assembly, when CASK is partially released from its cytoskeletal associations.

    The Journal of biological chemistry 2001;276;12;9291-6

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

    Nix SL, Chishti AH, Anderson JM and Walther Z

    Departments of Internal Medicine, Cell Biology, and Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

    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.

    Funded by: NCI NIH HHS: CA66263; NIDDK NIH HHS: DK34989, T32DK07017; ...

    The Journal of biological chemistry 2000;275;52;41192-200

  • Mints as adaptors. Direct binding to neurexins and recruitment of munc18.

    Biederer T and Südhof TC

    Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    Mint1 (X11/human Lin-10) and Mint2 are neuronal adaptor proteins that bind to Munc18-1 (n/rb-sec1), a protein essential for synaptic vesicle exocytosis. Mint1 has previously been characterized in a complex with CASK, another adaptor protein that in turn interacts with neurexins. Neurexins are neuron-specific cell surface proteins that act as receptors for the excitatory neurotoxin alpha-latrotoxin. Hence, one possible function for Mint1 is to mediate the recruitment of Munc18 to neurexins. In agreement with this hypothesis, we now show that the cytoplasmic tail of neurexins captures Munc18 via a multiprotein complex that involves Mint1. Furthermore, we demonstrate that both Mint1 and Mint2 can directly bind to neurexins in a PDZ domain-mediated interaction. Various Mint and/or CASK-containing complexes can be assembled on neurexins, and we demonstrate that Mint1 can bind to Munc18 and CASK simultaneously. Our data support a model whereby one of the functions of Mints is to localize the vesicle fusion protein Munc18 to those sites at the plasma membrane that are defined by neurexins, presumably in the vicinity of points of exocytosis.

    The Journal of biological chemistry 2000;275;51;39803-6

  • Mapping and expression analysis of the human CASK gene.

    Stevenson D, Laverty HG, Wenwieser S, Douglas M and Wilson JB

    Robertson Laboratory, Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, 54 Dumbarton Road, Glasgow G11 6NU, UK.

    Mammalian genome : official journal of the International Mammalian Genome Society 2000;11;10;934-7

  • Junctional adhesion molecule interacts with the PDZ domain-containing proteins AF-6 and ZO-1.

    Ebnet K, Schulz CU, Meyer Zu Brickwedde MK, Pendl GG and Vestweber D

    Institute of Cell Biology, ZMBE, University of Muenster, D-48149 Muenster, Germany.

    We have identified the PDZ domain protein AF-6 as an intracellular binding partner of the junctional adhesion molecule (JAM), an integral membrane protein located at cell contacts. Binding of AF-6 to JAM required the presence of the intact C terminus of JAM, which represents a classical type II PDZ domain-binding motif. Although JAM did not interact with the single PDZ domains of ZO-1 or of CASK, we found that a ZO-1 fragment containing PDZ domains 2 and 3 bound to JAM in vitro in a PDZ domain-dependent manner. AF-6 as well as ZO-1 could be coprecipitated with JAM from endothelial cell extracts, demonstrating the association of the endogenously expressed molecules in vivo. Targeting of JAM to sites of cell contacts could be affected by the loss of the PDZ domain-binding C terminus. Full-length mouse JAM co-distributed with endogenous AF-6 in human Caco-2 cells at sites of cell contact independent of whether adjacent cells expressed mouse JAM as an extracellular binding partner. In contrast, truncated JAM lacking the PDZ domain-binding C terminus did not co-distribute with endogenous AF-6, but was restricted to cell contacts between cells expressing mouse JAM. Our results suggest that JAM can be recruited to intercellular junctions by its interaction with the PDZ domain-containing proteins AF-6 and possibly ZO-1.

    The Journal of biological chemistry 2000;275;36;27979-88

  • Nuclear translocation and transcription regulation by the membrane-associated guanylate kinase CASK/LIN-2.

    Hsueh YP, Wang TF, Yang FC and Sheng M

    Howard Hughes Medical Institute and Department of Neurobiology, Massachusetts General Hospital and Harvard Medical School, Boston 02114, USA.

    Membrane-associated guanylate kinases (MAGUKs) contain multiple protein-binding domains that allow them to assemble specific multiprotein complexes in particular regions of the cell. CASK/LIN-2, a MAGUK required for EGF receptor localization and signalling in Caenorhabditis elegans, contains a calmodulin-dependent protein kinase-like domain followed by PDZ, SH3 and guanylate kinase-like domains. In adult rat brain, CASK is concentrated at neuronal synapses and binds to the cell-surface proteins neurexin and syndecan and the cytoplasmic proteins Mint/LIN-10 and Veli/LIN-7. Here we report that, through its guanylate kinase domain, CASK interacts with Tbr-1, a T-box transcription factor that is involved in forebrain development. CASK enters the nucleus and binds to a specific DNA sequence (the T-element) in a complex with Tbr-1. CASK acts as a coactivator of Tbr-1 to induce transcription of T-element containing genes, including reelin, a gene that is essential for cerebrocortical development. Our findings show that a MAGUK which is usually associated with cell junctions has a transcription regulation function.

    Nature 2000;404;6775;298-302

  • Regulated expression and subcellular localization of syndecan heparan sulfate proteoglycans and the syndecan-binding protein CASK/LIN-2 during rat brain development.

    Hsueh YP and Sheng M

    Howard Hughes Medical Institute and Department of Neurobiology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.

    The syndecan family of cell surface heparan sulfate proteoglycans interacts via their cytoplasmic C-terminal tail with the PDZ domain of CASK/LIN-2, a membrane-associated guanylate kinase homolog. The syndecan-CASK interaction may be involved in intercellular signaling and/or cell adhesion. Here we show that syndecan-1 to syndecan-4 have distinctive mRNA distributions in adult rat brain by in situ hybridization, with syndecan-2 and -3 being the major syndecans expressed in neurons of the forebrain. At the protein level, syndecan-2 and -3 are differentially localized within neurons; syndecan-3 is concentrated in axons, whereas syndecan-2 is localized in synapses. The synaptic accumulation of syndecan-2 occurs late in synapse development. CASK is a cytoplasmic-binding partner for syndecans, and its subcellular distribution changes strikingly during development, shifting from a primarily axonal distribution in the first 2 postnatal weeks to a somatodendritic distribution in adult brain. This change in CASK distribution correlates temporally and spatially with the expression patterns of syndecan-3 and -2, consistent with the association of both of these syndecans with CASK in vivo. In support of this, we were able to coimmunoprecipitate a complex of CASK and syndecan-3 from brain extracts. Our results indicate that specific syndecans are differentially expressed in various cell types of the brain and are targeted to distinct subcellular compartments in neurons, where they may serve specialized functions. Moreover, CASK is appropriately expressed and localized to interact with both syndecan-2 and -3 in different compartments of the neuron throughout postnatal development.

    Funded by: NINDS NIH HHS: NS35050

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;17;7415-25

  • Association of neuronal calcium channels with modular adaptor proteins.

    Maximov A, Südhof TC and Bezprozvanny I

    Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.

    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.

    The Journal of biological chemistry 1999;274;35;24453-6

  • Molecular analysis of the X11-mLin-2/CASK complex in brain.

    Borg JP, Lõpez-Figueroa MO, de Taddèo-Borg M, Kroon DE, Turner RS, Watson SJ and Margolis B

    Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.

    A heterotrimeric complex containing Lin-10/X11alpha, Lin-2/CASK, and Lin-7 is evolutionarily conserved from worms to mammals. In Caenorhabditis elegans, it localizes Let-23, a receptor tyrosine kinase, to the basolateral side of vulval epithelium, a step crucial for proper vulva development. In mammals, the complex may also participate in receptor targeting in neurons. Accordingly, phosphotyrosine binding (PTB) and postsynaptic density-95/Discs large/Zona Occludens-1 domains found in X11alpha and mLin-2/CASK bind to cell-surface proteins, including amyloid precursor protein, neurexins, and syndecans. In this paper, we have further analyzed the X11alpha-mLin-2/CASK association that is mediated by a novel protein-protein interaction. We show that the mLin-2/CASK calmodulin kinase II (CKII) domain directly binds to a 63 amino acids peptide located between the Munc-18-1 binding site and the PTB domain in X11alpha. Ca2+/calmodulin association with mLin-2/CASK does not modify the X11alpha-mLin-2 interaction. A region containing the mLin-2/CASK guanylate kinase domain also interacts with X11alpha but with a lower affinity than the CKII domain. Immunostaining of X11alpha in the brain shows that the protein is expressed in areas shown previously to be positive for mLin-2/CASK staining. Together, our data demonstrate that the X11alpha-mLin-2 complex contacts many partners, creating a macrocomplex suitable for receptor targeting at the neuronal plasma membrane.

    Funded by: NIA NIH HHS: P50 AG008671; NIMH NIH HHS: MH 42251

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;4;1307-16

  • Identification of an evolutionarily conserved heterotrimeric protein complex involved in protein targeting.

    Borg JP, Straight SW, Kaech SM, de Taddéo-Borg M, Kroon DE, Karnak D, Turner RS, Kim SK and Margolis B

    Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.

    In Caenorhabditis elegans, lin-2, lin-7, and lin-10 genetically interact to control the trafficking of the Let-23 growth factor receptor to the basolateral surface of body epithelia. The human homologue of the lin-10 gene has recently been identified as a member of the X11 gene family. The X11 proteins contain one phosphotyrosine binding (PTB) and two PSD-95.Dlg.ZO-1 (PDZ) domains as well as an extended amino terminus. We have previously shown that the PTB domain of X11alpha (also known as Mint1) can bind to the amyloid precursor protein (APP) in a phosphotyrosine-independent fashion and can markedly inhibit the processing of APP to the amyloid beta (Abeta) peptide. Here, we report that X11alpha directly binds to the mammalian homologue of Lin-2 (mLin-2), also known as CASK. This binding is mediated by direct interaction between the Calmodulin Kinase II (CKII)-like domain of mLin-2 and the amino terminus of X11alpha. Furthermore, we can detect direct interactions between mLin-2 and mammalian Lin-7 (mLin-7). In mouse brain, we have identified a heterotrimeric complex that contains mLin-2, mLin-7, and X11alpha and that is likely important for the localization of proteins in polarized cells. This complex may play an important role in the trafficking and processing of APP in neurons.

    Funded by: NIA NIH HHS: P50 AG008671

    The Journal of biological chemistry 1998;273;48;31633-6

  • A tripartite protein complex with the potential to couple synaptic vesicle exocytosis to cell adhesion in brain.

    Butz S, Okamoto M and Südhof TC

    Center for Basic Neuroscience, Department of Molecular Genetics, Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center at Dallas, 75235, USA.

    We identify a complex of three proteins in brain that has the potential to couple synaptic vesicle exocytosis to neuronal cell adhesion. The three proteins are: (1) CASK, a protein related to MAGUKs (membrane-associated guanylate kinases); (2) Mint1, a putative vesicular trafficking protein; and (3) Veli1, -2, and -3, vertebrate homologs of C. elegans LIN-7. CASK, Mint1, and Velis form a tight, salt-resistant complex that can be readily isolated. CASK, Mint1, and Velis contain PDZ domains in addition to other modules. However, no PDZ domains are involved in complex formation, leaving them free to recruit cell adhesion molecules, receptors, and channels to the complex. We propose that the tripartite complex acts as a nucleation site for the assembly of proteins involved in synaptic vesicle exocytosis and synaptic junctions.

    Funded by: NIMH NIH HHS: R01-MH52804

    Cell 1998;94;6;773-82

  • The location of human CASK at Xp11.4 identifies this gene as a candidate for X-linked optic atrophy.

    Dimitratos SD, Stathakis DG, Nelson CA, Woods DF and Bryant PJ

    Developmental Biology Center, University of California, Irvine 92697-2275, USA. sdimitra@uci.edu

    Funded by: NCI NIH HHS: P01 CA66263

    Genomics 1998;51;2;308-9

  • Direct interaction of CASK/LIN-2 and syndecan heparan sulfate proteoglycan and their overlapping distribution in neuronal synapses.

    Hsueh YP, Yang FC, Kharazia V, Naisbitt S, Cohen AR, Weinberg RJ and Sheng M

    Howard Hughes Medical Institute and Department of Neurobiology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.

    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.

    Funded by: NINDS NIH HHS: NS29879

    The Journal of cell biology 1998;142;1;139-51

  • Human CASK/LIN-2 binds syndecan-2 and protein 4.1 and localizes to the basolateral membrane of epithelial cells.

    Cohen AR, Woods DF, Marfatia SM, Walther Z, Chishti AH, Anderson JM and Wood DF

    Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06520, USA.

    In Caenorhabditis elegans, mutations in the lin-2 gene inactivate the LET-23 receptor tyrosine kinase/Ras/MAP kinase pathway required for vulval cell differentiation. One function of LIN-2 is to localize LET-23 to the basal membrane domain of vulval precursor cells. LIN-2 belongs to the membrane-associated guanylate kinase family of proteins. We have cloned and characterized the human homolog of LIN-2, termed hCASK, and Northern and Western blot analyses reveal that it is ubiquitously expressed. Indirect immunofluorescence localizes CASK to distinct lateral and/or basal plasma membrane domains in different epithelial cell types. We detect in a yeast two-hybrid screen that the PDZ domain of hCASK binds to the heparan sulfate proteoglycan syndecan-2. This interaction is confirmed using in vitro binding assays and immunofluorescent colocalization. Furthermore, we demonstrate that hCASK binds the actin-binding protein 4.1. Syndecans are known to bind extracellular matrix, and to form coreceptor complexes with receptor tyrosine kinases. We speculate that CASK mediates a link between the extracellular matrix and the actin cytoskeleton via its interaction with syndecan and with protein 4.1. Like other membrane-associated guanylate kinases, its multidomain structure enables it to act as a scaffold at the membrane, potentially recruiting multiple proteins and coordinating signal transduction.

    Funded by: NIDDK NIH HHS: DK38979

    The Journal of cell biology 1998;142;1;129-38

  • Crystal structure of the hCASK PDZ domain reveals the structural basis of class II PDZ domain target recognition.

    Daniels DL, Cohen AR, Anderson JM and Brünger AT

    The Howard Hughes Medical Institute and Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.

    PDZ domain containing proteins assist formation of cell-cell junctions and localization of membrane protein receptors and ion channels. PDZ domains interact with the C-terminal residues of a particular target membrane protein. Based on their binding specificities and sequence homologies, PDZ domains fall into two classes. The first crystal structure of a class II PDZ domain, that of hCASK, has been solved by multi-wavelength anomalous dispersion phasing. Complex formation with the C-terminus of a neighboring non-crystallographically related PDZ domain reveals interactions between hCASK and its ligand. Class II PDZ domains differ from class I domains by formation of a second hydrophobic binding pocket. The C-terminal carboxylate binding loop of the PDZ domain is structurally conserved in both classes suggesting a generalized carboxylate binding motif (h-Gly-h) where h is a hydrophobic residue.

    Nature structural biology 1998;5;4;317-25

  • cDNAs with long CAG trinucleotide repeats from human brain.

    Margolis RL, Abraham MR, Gatchell SB, Li SH, Kidwai AS, Breschel TS, Stine OC, Callahan C, McInnis MG and Ross CA

    Laboratory of Molecular Neurobiology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA. rmargoli@welchlink.welch.jhu.edu

    Twelve diseases, most with neuropsychiatric features, arise from trinucleotide repeat expansion mutations. Expansion mutations may also cause a number of other disorders, including several additional forms of spinocerebellar ataxia, bipolar affective disorder, schizophrenia, and autism. To obtain candiate genes for these disorders, cDNA libraries from adult and fetal human brain were screened at high stringency for clones containing CAG repeats. Nineteen cDNAs were isolated and mapped to chromosomes 1, 2, 4, 6, 7, 8, 9, 12, 16, 19, 20, and X. The clones contain between 4 and 17 consecutive CAG, CTG, TCG, or GCA triplets. Clone H44 encodes 40 consecutive glutamines, more than any other entry in the nonredundant GenBank protein database and well within the range that causes neuronal degeneration in several of the glutamine expansion diseases. Eight cDNAs encode 15 or more consecutive glutamine residues, suggesting that the gene products may function as transcription factors, with a potential role in the regulation of neurodevelopment or neuroplasticity. In particular, the conceptual translation of clone CTG3a contains 18 consecutive glutamines and is 45% identical to the C-terminal 306 residues of the mouse numb gene product. These genes are therefore candidates for diseases featuring anticipation, neurodegeneration, or abnormalities of neurodevelopment.

    Funded by: NIMH NIH HHS: MH02175-10A1, MH50763; NINDS NIH HHS: NS34172

    Human genetics 1997;100;1;114-22

  • CASK: a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein kinase domain identified by interaction with neurexins.

    Hata Y, Butz S and Südhof TC

    Department of Molecular Genetics, The University of Texa Southwestern Medical Center at Dallas, 75235, USA.

    Neurexins are neuronal cell surface proteins with hundreds of isoforms. In yeast two-hybrid screens for intracellular molecules interacting with different neurexins, we identified a single interacting protein called CASK. CASK is composed of an N-terminal Ca2+, calmodulin-dependent protein kinase sequence and a C-terminal region that is similar to the intercellular junction proteins dlg-A, PSD95/SAP90, SAP97, Z01, and Z02 and that contains DHR-, SH3-, and guanylate kinase domains. CASK is enriched in brain in synaptic plasma membranes but is also detectable at low levels in all tissues tested. The cytoplasmic domains of all three neurexins bind CASK in a salt-labile interaction. In neurexin I, this interaction is dependent on the C-terminal three residues. Thus, CASK is a membrane-associated protein that combines domains found in Ca2+ - activated protein kinases and in proteins specific for intercellular junctions, suggesting that it may be a signaling molecule operating at the plasma membrane, possibly in conjunction with neurexins.

    Funded by: NIMH NIH HHS: R01-MH52804

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1996;16;8;2488-94

Gene lists (6)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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