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

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 two-hybrid 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 Drosophilacortactin 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 Drosophilaembryo 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 emcgene in Drosophila. The potential roles of the cortactin·ZO-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)(2)(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)(14)(15)(16). We have previously shown that DZO-1 locates at cell-cell junctions and is involved in the signaling pathway, which activates the transcription of emc (12). Toward the elucidation of the DZO-1 functions in the signaling pathway, we performed a yeast two-hybrid screen to identify the Drosophila proteins that interact with DZO-1. One of the obtained cDNA clones is encoding a protein highly homologous to vertebrate cortactin.
Cortactin is an F-actin binding protein initially discovered as a prominent substrate for Src protein-tyrosine kinase (17,18). A human homolog was identified as a protein product of a putative oncogene, EMS1, and has been implicated in both cell adhesion and cell signaling (19). We now report the primary structure of Drosophila cortactin (DCortactin) and show multiple lines of evidence suggesting that DCortactin interacts with DZO-1. We also present results demonstrating the association between cortactin and ZO-1 in mouse tissues. In association with ZO-1, cortactin may play important roles in the formation and/or regulation of cell-cell adhesion and communication during growth, differentiation, and tumorigenesis.

EXPERIMENTAL PROCEDURES
Plasmids-pBD-DZO885-1367 was constructed as follows. The XhoI end of the 1.5-kb NcoI-XhoI fragment of the DZO-1 cDNA (12) was blunted and ligated to pAS2-1 (CLONTECH) digested with NcoI and SmaI. Deletion variants were constructed by double digesting pBD-DZO885-1367 with some pairs of restriction enzymes, one cut it within the DZO-1 coding region and another within the multi-cloning site, followed by blunt-ending before self-ligating. The SmaI, PinAI, Van91I, and AspI sites within the DZO-1 coding region and the BamHI, NcoI, and NdeI sites within the multi-cloning site were used for these construction. Point mutations were introduced into pBD-DZO1115-1253 by polymerase chain reaction-mediated site-directed mutagenesis (20). The synthetic oligonucleotide primers used for mutagenesis were as follows: 5Ј-gggtgctggcaccgccttgaagggt-3Ј and 5Ј-tcaaggcggtgccagcaccca-* This study was supported in part by grants from the Human Frontier Science Program Organization. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AB009998.
Yeast Two-hybrid Screen and ␤-Galactosidase Assay-Two-hybrid screening was performed using a Matchmaker two-hybrid system (CLONTECH). Each of the bait constructs and a Drosophila melanogaster (Canton-S wild type) 0-to 18-h embryo cDNA library constructed in pGAD10 (CLONTECH) were co-introduced simultaneously into yeast strain CG1945. Transformants were selected for growth on plates lacking histidine and supplemented with 5 mM 3-aminotriazole. Plasmids were recovered and reintroduced into yeast strain Y187 to confirm the interaction by quantitative ␤-galactosidase liquid assay utilizing a luminescent ␤-galactosidase detection kit (CLONTECH) and a Lumat LB9501 luminometer (Berthold, Bad Wildbad, Germany). The interaction between the DZO-1 C-terminal domain variants and the DCortactin SH3 domain was also tested by the same assay.
Molecular Analyses-Northern blot analysis was done as described previously (12). A Canton-S 4-to 8-h embryo cDNA library in a pNB40 plasmid vector was screened with a 32 P-labeled 1.8-kb EcoRI fragment from pcT407A. The nucleotide sequences of the plasmid clones were determined using an ABI Prism 377 DNA Sequencer (Perkin Elmer).
Western Blot Analysis-Samples separated by SDS-PAGE were transferred electrophoretically to an Immobilon-P membrane (Millipore Corp.). Immunodetection was performed with horseradish peroxidaseconjugated secondary antibody and enhanced chemiluminescent substrate (SuperSignal substrate, Pierce).
Immunoprecipitation-Dechorionated 0-to 16-h Canton-S wild-type embryos or tissues of 1-3-day postnatal mice (ICR) were homogenized in 25 volumes of cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1.0% Nonidet P-40, 1 mM EDTA, 10 M benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 10 g/ml each of pepstatin A, leupeptin, and aprotinin). The homogenate was centrifuged at 15,000 ϫ g for 15 min to pellet debris. The supernatant was then spun at 140,000 ϫ g for 1 h at 4°C. The resulting supernatant was precleared by incubating with a 250-l settled volume of protein A-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden) per ml of supernatant for 1 h at 4°C. The beads were pelleted, and the supernatant was saved as precleared extract. 400 l of precleared extracts were incubated with 1-4 l of rabbit anti-DZO-1 antiserum, anti-DCortactin antiserum, or preimmune rabbit sera at 4°C for 3-12 h. Immune complexes were recovered by adding 20 l of protein A-Sepharose, followed by incubation at 4°C for 2-8 h with rocking. The beads were washed three times with cold lysis buffer, boiled in SDS sample buffer, and processed for Western analysis.

RESULTS
Isolation of DCortactin cDNA-By a yeast two-hybrid screen, a cDNA clone pcT407A was isolated as a clone encoding a polypeptide that interacts specifically with the C-terminal 483 amino acids region of DZO-1 (Fig. 1). Developmental Northern analysis of the Canton-S wild type, using a 0.85-kb insert of pcT407A as a probe, represented a 2.7-kb transcript in all developmental stages (Fig. 2). To isolate a full protein-coding cDNA, we screened a Canton-S wild-type embryo cDNA library by the same probe. Several positive clones were isolated, and the longest insert was sequenced. This insert has a single long open reading frame encoding a deduced protein of 559 amino acids with a calculated molecular mass of 61 kDa (Fig. 3). A data base search found that the deduced protein is a new member of a protein family consisting of cortactin and HS1 (17,19,(22)(23)(24)(25)(26) and is more closely related to cortactin than to HS1 (Fig. 4). Thus, we concluded that the isolated cDNA encodes a Drosophila homolog of cortactin (DCortactin).
The DCortactin SH3 Domain Binds to a PXXP Motif in the DZO-1 Proline-rich Domain-The cDNA clone pcT407A encodes the C-terminal 96 amino acids of DCortactin (Fig. 3,  boxed region). An SH3 domain of approximately 60 amino acids dominates this region, and no other significant structural motif is found. The SH3 domain is known to bind to a PXXP motif often found in proline-rich regions (27). To determine a region within DZO-1 sufficient to interact with the C-terminal SH3 domain of DCortactin, we constructed a series of deletion mutants of DZO885-1367 (Fig. 5). Their ability to interact with the DCortactin SH3 domain in yeast was analyzed by monitoring ␤-galactosidase activity. The 139-amino acid region extending from amino acid 1115 to 1253 (DZO1115-1253) was sufficient for the interaction. This region contains four isolated PXXP motifs and three overlapping motifs (PFKPVPPPKP). To identify the PXXP motif necessary for the interaction, we introduced a series of point mutations into DZO1115-1253. The  (Fig. 6). The preimmune serum did not react with them. 3 The expression of these proteins in adults was substantially less than those in other developmental stages. Western blotting with the rat antiserum gave almost the same profile. 3 The molecular weights of these proteins determined by SDS-PAGE are significantly larger than the predicted value (61 kDa). Similar observations of two forms of protein products with anomalous electrophoretic mobility have also been reported for vertebrate cortactin (17,19,24). The proline-rich domains may be responsible for their anomalous electrophoretic mobility.
To examine the cellular localization, DCortactin was immunostained in epithelial cells of imaginal discs. The typical honeycomb-like images indicated that the protein distributes in a cell-cell contact-associated manner. To clarify the subcellular localization, the double stainings of DCortactin with DZO-1, F-actin, and DE-cadherin were conducted using a laser-scanning confocal microscope. DE-cadherin is a component of the adherens junction and localizes at the apicolateral region of epithelial cell junctions (21,28). The distribution of DZO-1 partially overlaps with that of DE-cadherin and extends to the slightly basal region corresponding to the site of the septate junction (12). Colocalization of DCortactin, DZO-1, and DEcadherin was evident, while the staining area of DCortactin in the periplasm seemed slightly broader than those of DZO-1 and DE-cadherin (Fig. 7, c and i). Colocalization of DCortactin and F-actin in a periplasmic region was also observed (Fig. 7f). Regarding the apical-basal axis, the distribution of DCortactin extended from the basal half side of the adherens junction to the more baso-lateral region (Fig. 7, j, k, and l).
DCortactin Associates with DZO-1 in Drosophila-To examine the in vivo association of DZO-1 and DCortactin, we immunoprecipitated Canton-S wild-type embryo lysates with the rabbit anti-DCortactin antiserum, the rabbit anti-DZO-1 antiserum, or the respective preimmune sera and analyzed the precipitates by Western blotting (Fig. 8). Western blotting with the affinity-purified anti-DZO-1 antibody detected a 160-kDa protein in the precipitate of the anti-DZO-1 antiserum (lane 1). This protein was coprecipitated by the anti-DCortactin anti-

FIG. 5. The C-terminal SH3 domain of DCortactin interacts with a PXXP motif in the proline-rich domain of DZO-1.
Schematic representation of the DZO-1 C-terminal region variants used for the two-hybrid assay to identify the domain necessary for the interaction with the DCortactin C-terminal region. Each plasmid expressing one of these variants fused to the GAL4 DNA binding domain was co-introduced into yeast strain Y187 with pcT407A, and ␤-galactosidase reporter gene induction was measured. Numbers refer to the amino acid residues that define the boundaries of each construct. DZO885-1367 corresponds to the original construct used for the yeast two-hybrid screen as a bait. PXXP motifs are represented by P or P*. Three overlapping PXXP motifs are clustered (PFKPVPPPKP) at the site represented by P*. In DZO1115-1253M1, PFKPVPPPKP motif starting at amino acid residue 1192 was substituted to PFKAVPAPKP. In DZO1115-1253M2-5, PXXP motifs starting at amino acid residues 1230, 1239, 1118, and 1130, respectively, were substituted to AXXA. The sites of modified PXXP motifs are represented by A or A*. ␤-Galactosidase activities relative to the activity for original DZO885-1367 construct are shown by the symbols ϩϩ (Ͼ50%), ϩ (10 -50%), and Ϫ (Ͻ10%).  5 and 6). These results clearly proved that DCortactin associates with DZO-1 in Dro-sophila embryo cells. The absence of the DCortactin 110-kDa form in the precipitate of the anti-DZO-1 antiserum is thought to be due to the instability of that form in lysate because it also disappeared in the diluted lysate (lane 4). However, we cannot rule out the possibility that the 110-kDa form cannot associate with DZO-1.
Cortactin Associates with ZO-1 in Mouse-We found that rabbit anti-DCortactin antibody could also react with a bacterially expressed protein containing the mouse cortactin 37amino acid repeat domain fused to maltose binding protein. 3 Using this antibody, Western blot analysis of tissue lysates from a 4-day postnatal mouse detected 80-and 85-kDa proteins (Fig. 9a). These proteins are fairly abundant in brain and testis but not so in liver and kidney. Western blot analysis using the rat anti-DCortactin antiserum also yielded the same pattern as that using an anti-chicken p80/85 (cortactin) monoclonal antibody, 3 which was reported to cross-react with mouse cortactin (17,24). These results indicate that both the rabbit and rat anti-DCortactin antibodies can cross-react with mouse cortactin.
To examine the interaction between mouse cortactin and mouse ZO-1, we conducted immunoprecipitation analysis of brain and testis lysates with the rabbit anti-DCortactin antiserum. Western blotting of a mouse tissue lysate with antimouse ZO-1 monoclonal antibody showed one faint and two prominent bands with molecular masses of 210, 200, and 190 kDa, respectively (Fig. 9b, lane 1). A 200-kDa protein was detected in the immunoprecipitates of the anti-DCortactin antiserum from brain and testis lysates of 1-3-day postnatal mice (lanes 3 and 5) but not in those of the pre-immune serum ( lanes  2 and 4). These results clearly show that mouse cortactin associates with mouse ZO-1 in vivo.

Conserved Interaction of Cortactin and ZO-1-We have identified
DCortactin as a protein that interacts with the C-terminal proline-rich domain of DZO-1 by a yeast two-hybrid system. Interaction of these proteins was supported by colocalization at cell-cell junction sites in wing disc epithelial cells and confirmed by coimmunoprecipitation from embryo lysates (Figs. 7 and 8). We showed that mouse cortactin associates with mouse ZO-1 in brain and testis of 1-3-day postnatal mice as DCortactin does with DZO-1 in Drosophila embryo (Fig. 9b). This conservation suggests that the interaction of these proteins has a functional significance.  2 and 4). The precipitates were analyzed with anti-mouse ZO-1 monoclonal antibody. To localize mouse ZO-1, diluted kidney lysate of a 4-day postnatal mouse was loaded on lane 1.
Recently, the SH3 domain of vertebrate cortactin was found to bind preferentially to peptides sharing the consensus motif ϩPP⌿PXKPXWL (ϩ and ⌿ represent basic and aliphatic residues, respectively) (29). A search of the proline-rich domain of DZO-1 revealed that the KPVPPPKPKNY sequence (amino acid residues 1194 -1204) is the most likely ligand sequence for the SH3 domain of cortactin. This is consistent with our result showing that proline residues at amino acids 1195 and 1198 (PFKPVPPPKP) are necessary for the interaction with DCortactin in yeast (Fig. 5).
Possible Function of Cortactin in the Signaling Pathway-We have previously shown that DZO-1 is involved in a signaling pathway that activates transcription of emc and proposed that mammalian ZO-1 is involved in a similar signaling pathway that activates transcription of Id genes (12). Id genes encode transcriptional regulators homologous to emc and are known to play an important role in the regulation of fate determination, proliferation, and transformation in several cell lineages (30 -33).
Cortactin, a substrate for Src protein-tyrosine kinase, is phosphorylated in response to multiple extracellular stimuli and implicated in cell signaling (17, 18, 23, 34 -38). It is known that cortactin is up-regulated during osteoclast differentiation and megakaryocyte maturation (39,40). DCortactin is not as abundant in adult flies as in embryos, larvae, and pupae, in which developmental stages cells are actively proliferating (Fig. 6). Further, the result of our preliminary experiment indicates that cortactin is abundant in highly proliferating cultured human cells but not in adult human tissues. 3 These observations suggest that cortactin is involved in both cell differentiation and proliferation.
Human cortactin encoding gene EMS1 is amplified and overexpressed in subsets of breast carcinomas (19,(41)(42)(43). Oncogenic effects of Id-1 overexpression in mammary epithelial cells were demonstrated by the analysis using a cell line (44). We hypothesize that cortactin is included in the ZO-1 signaling pathway regulating the expression of emc/Id gene(s).
Possible Function of Cortactin in Cell-Cell Junctions-Cortactin is an F-actin binding protein primarily localizing at cortical structures and thought to be important for microfilament-membrane interactions (18,34). Cortactin also has an F-actin cross-linking activity and is implicated in cytoskeleton reorganization (45). On the other hand, ZO-1 is a component of cell-cell junctions (4,5,7) and is thought to work in a structural linkage between cell-cell junctions and cytoskeletal networks (6,8,9). As we have shown, subcellular localization of DCortactin overlaps well with F-actin as well as with DZO-1 at cell-cell junction sites in wing disc cells (Fig. 7). Thus, cortactin may be included in the structural linkage in association with ZO-1. The tyrosine phosphorylation of cortactin by Src, which affects the F-actin binding and cross-linking activities, may regulate the cell-cell junction formation (45).
Perspectives-We found that the DCortactin gene is situated on the chromosome segment between 93B3 and 93B7. 3 The isolation of mutant alleles of DCortactin gene is currently under progress using deficiency lines encompassing this region. Molecular genetic studies of DCortactin will provide further information on the conserved roles of cortactin in cell adhesion and signaling required for cell fate determination and cell proliferation.