title: hCASK and hDlg associate in epithelia, and their src homology 3 and guanylate kinase domains participate in both intramolecular and intermolecular interactions.
abstract: 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.
authors.affiliation: Departments of Internal Medicine, Cell Biology, and Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Nix
authors.forename: A H
authors.initials: AH
authors.lastname: Chishti
authors.forename: J M
authors.initials: JM
authors.lastname: Anderson
authors.forename: Z
authors.initials: Z
authors.lastname: Walther
mesh_terms: D001665:Binding Sites; D018938:Caco-2 Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D029721:Drosophila Proteins; D051528:Guanylate Kinases; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry*; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D025521:Tumor Suppressor Proteins; D018909:src Homology Domains
chemical_list: D029721:Drosophila Proteins; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; C070350:dlg1 protein, Drosophila; C106868:CASK kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases