hCASK and hDlg Associate in Epithelia, and Their Src Homology 3 and Guanylate Kinase Domains Participate in Both Intramolecular and Intermolecular Interactions

doi:10.1074/jbc.M002078200

hCASK and hDlg Associate in Epithelia, and Their Src Homology 3 and Guanylate Kinase Domains Participate in Both Intramolecular and Intermolecular Interactions*

From the Departments of ^‡Internal Medicine,^¶Cell Biology, and ^‖Pathology, Yale University School of Medicine, New Haven, Connecticut 06520 and the ^§Division of Hematology Research, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135

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, humandiscs 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 thatin 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.

Footnotes

↵* This work was supported by NCI, National Institutes of Health Grant CA66263 (to J. M. A. and A. H. C.), National Institutes of Health Grant DK34989 (Yale Digestive Diseases Research Core), and National Institutes of Health Postdoctoral Fellowships T32 DK07017 and F32 DK09955 (to Z. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵** To whom correspondence should be addressed: Dept. of Internal Medicine, Yale University School of Medicine, 333 Cedar St., 1080 LMP, P.O. Box 208019, New Haven, CT 06520-8019. E-mail: zenta.walther@yale.edu.
Published, JBC Papers in Press, September 18, 2000, DOI 10.1074/jbc.M002078200
Abbreviations:

MAGUK

membrane associate guanylate kinase homologs

ZO

zonula occludens

PSD

post synaptic density

PDZ

PSD-95/Dlg/ZO-1

SH3

Src homology 3

GUK

guanylate kinase-like

hCASK

human CASK

hDlg

human discs large

NE-dlg

neuroendocrine discs large

PCR

polymerase chain reaction

VSV-G

vesicular stomatitis virus glycoprotein

CB

calmodulin binding region

GST

glutathioneS-transferase

MBP

maltose-binding protein

PAGE

polyacrylamide gel electrophoresis

aa

amino acid(s)
- Received March 13, 2000.
- Revision received August 28, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.