Cortactin Associates with the Cell-Cell Junction Protein ZO-1 in both Drosophila and Mouse

Abstract

Cortactin is an actin filament-binding protein localizing at cortical regions of cells and a prominent substrate for Src family protein-tyrosine kinases in response to multiple extracellular stimuli. Human cortactin has been identified as a protein product of a putative oncogene, EMS1. In this report, we describe the identification of a Drosophila homolog of cortactin as a molecule that interacts with Drosophila ZO-1 using yeast twohybrid screening. Drosophila cortactin is a 559-amino acid protein highly expressed in embryos, larvae, and pupae but relatively underexpressed in adult flies. Deletion and substitution mutant analyses revealed that the SH3 domain of Drosophila cortactin binds to a PXXP motif in the proline-rich domain of Drosophila ZO-1. Colocalization of these proteins at cell-cell junction sites was evident under a confocal laser-scanning microscope. In vivo association was confirmed by coimmunoprecipitation of cortactin and ZO-1 from Drosophila embryo lysates. We also demonstrate an association for each of the murine homologs by immunoprecipitation analyses of mouse tissue lysates. Our previous work has demonstrated the involvement of ZO-1 in a signaling pathway that regulates expression of the emc gene in Drosophila. The potential roles of the cortactin⅐ZO-1 complex in cell adhesion and cell signaling are discussed.Cell-cell adhesions are essential for the development of the multicellular organisms. Among the proteins composing the cell-cell adhesion complexes, members of the membrane-associated guanylate kinase homologs (MAGUKs) 1 are widely found in Hydra, Caenorhabditis elegans, Drosophila, and mammals (1-3). MAGUKs have distinctive domains including one or three copies of the PDZ domain, an SH3 domain, and a domain homologous to guanylate kinase (GUK) and implicated in both formation of cell-cell junctions and signal transduction. One of the most intensively characterized members of the MAGUKs is the mammalian ZO-1, which is known to associate with several cellular proteins including the components of cell-cell junctions (occludin, ␤-catenin, and ZO-2) and the components of cytoskeletal networks (␣-spectrin and actin filaments (F-actin)) (4 -9). While ZO-1 has been considered as a homolog of a Drosophila tumor suppresser Dlg, its biological functions in the cell-cell junction and signal transduction remain obscure (10, 11).We recently identified a new Drosophila MAGUK protein, Tamou, and reported its significant homology with ZO-1 (12). We will refer to Tamou as Drosophila ZO-1 (DZO-1) because we also found that the transgenes of mouse ZO-1 could replace the tam gene function in Drosophila.2 The DZO-1 tam1 mutant flies exhibit the supernumerary mechanosensory organs. This is a similar phenotype to that of an extramacrochaetae (emc) mutation. The emc gene encodes a helix-loop-helix type transcriptional regulator and negatively regulates specification of sensory organ precursor cells (13-16). We have previously shown that DZO-1 locates at cell-cell junctions and is involved in the signaling p...

UCS15A, a novel small molecule, SH3 domain-mediated protein–protein interaction blocking drug

“…In the experiment shown in Figure 4A, cortactin protein was immunoprecipitated from UCS15A treated HCT116 cells and the associated ZO1 protein was examined by immunoblot analyses with ZO1 antibody. As reported previously (Katsube et al, 1998), ZO1 associated with cortactin ( Figure 4a, upper panel, lane 2) and could be detected by immunoblotting with ZO1 antibody, which also recognized the 160 kilodalton related protein, ZO2 (Figure 4a, top panel, lanes 1 and 8). Upon UCS15A treatment, the level of cortactin-associated ZO1 diminished in a dose-dependent manner, especially between 2 and 5 mM and was virtually abolished at 10 mM of UCS15A (Figure 4a, upper panel).…”

“…To this end, the cortactin ± ZO1 complex, which involves the interaction of an SH3 domain with a typical proline-rich sequence, was chosen as an example of an SH3-mediated interaction that is important in cytoskeletal organization (reviewed in Tsukita et al, 1999). Previous studies have shown that the SH3 domain of the actin-binding protein, cortactin, interacts with a P-x-x-P motif in the prolinerich domain of ZO1 (Katsube et al, 1998). In the experiment shown in Figure 4A, cortactin protein was immunoprecipitated from UCS15A treated HCT116 cells and the associated ZO1 protein was examined by immunoblot analyses with ZO1 antibody.…”

Cortactin: coupling membrane dynamics to cortical actin assembly

“…The large degree of conservation in the SH3 domain suggests that this domain binds ligands conserved across a broad evolutionary spectrum, performing a similar function(s) in diverse species. The proline-rich segments in invertebrate cortactins are considerably longer than the similar regions in the vertebrate proteins, accounting in part for the low degree of homology (Katsube et al, 1998).…”

“…Tight junctions are associated with an apical cytoskeletal structure known as the terminal web (Fath et al, 1993). Cortactin is localized at the terminal web in human small bowel epithelium (Wu and Montone, 1998) and the SH3 domain associates with a proline-rich motif in ZO-1 (Katsube et al, 1998), a protein selectively localized to the epithelial tight junction (Stevenson et al, 1986). The ZO-1 proline-rich domain contains a homologous sequence to the consensus cortactin SH3 domainbinding motif (Figure 2d).…”

“…In addition to chicken, complete cDNA sequences have been reported for human (Schuuring et al, 1993), rat (Ohoka and Takai, 1998), mouse (Zhan et al, 1993;Miglarese et al, 1994), Drosophila (Katsube et al, 1998) and the sea urchin Strongylocentrouts purpuratus (Genebank accession #AF064260). All cortactins are structurally similar to each other, with the SH3 domain showing the greatest amount of evolutionary conservation while the prolinerich domain the least (Figure 1).…”