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Vol. 18, Issue 5, 1744-1755, May 2007
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*Medical Research Council Laboratory for Molecular Cell Biology, Cancer Research UK Oncogene and Signal Transduction Group, University College London, London WC1E 6BT, England; and Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology and Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium
Submitted November 1, 2006;
Revised January 16, 2007;
Accepted February 15, 2007
Monitoring Editor: Ben Margolis
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In epithelial sheets, cells are polarized with apical and basolateral membrane domains separated by tight junctions, specialized sites of intimate cell–cell contact that act as a barrier to the free diffusion of lipid and proteins (Yeaman et al., 1999). Numerous proteins and signal transduction pathways have been implicated in the establishment of cell polarity, but genetic analyses in Drosophila and Caenorhabditis elegans have identified three groups of highly conserved proteins that play a critical role in this process (Macara, 2004). These are: 1) Par3, Par6, and aPKC (Joberty et al., 2000; Lin et al., 2000; Wodarz et al., 2000; Petronczki and Knoblich, 2001; Tepass et al., 2001); 2) Crb3, PALS1 (MPP5), and PATJ, which are both located predominantly apically (Bachmann et al., 2001; Hong et al., 2001; Tepass et al., 2001; Medina et al., 2002); and 3) Dlg1, Scribble, and Lgl, located mostly on the basolateral surfaces (Bilder et al., 2000; Bilder and Perrimon, 2000; Bossinger et al., 2001; Tepass et al., 2001). With the exception of aPKC, members of these three groups of proteins lack catalytic activity. Instead they contain multiple protein–protein interaction domains, in particular PDZ, SH3, and guanylate kinase domains often referred to as membrane-associated guanylate kinase or MAGUK proteins (Funke et al., 2005), indicating a complex network of protein interactions controlling cell polarity.
Par3, Par6, and the activity of aPKC regulate the assembly of functional tight junctions in mammalian cells (Yamanaka et al., 2001; Suzuki et al., 2002). These proteins physically interact with each other. Par3, which contains three PDZ domains, for example, binds to the PDZ domain of Par6 (Joberty et al., 2000; Lin et al., 2000) and is able to associate with cell adhesion molecules such as Jam1 (Ebnet et al., 2001) or nectin (Takekuni et al., 2003). Both Par3 and Par6 can bind to aPKC, whereas the GTP-bound form of Cdc42 interacts with Par6 via a Cdc42/Rac-interactive binding (CRIB)-like motif to regulate aPKC activity (Izumi et al., 1998; Tabuse et al., 1998; Joberty et al., 2000; Lin et al., 2000; Qiu et al., 2000; Suzuki et al., 2001). The transmembrane protein Crb3 and its binding partners PALS1 (MPP5) and PATJ are also required for tight junction integrity (Roh et al., 2003; Straight et al., 2004; Shin et al., 2005). Crb3 binds through a carboxy-terminal motif to the PDZ domain of PALS1 (MPP5; Makarova et al., 2003; Roh et al., 2003) and in turn, PALS1 binds to PATJ through L27 domain interactions (Lemmers et al., 2002; Roh et al., 2002b). The multi-PDZ domain protein PATJ is able to associate with tight junction–associated proteins such as ZO-3 and claudin-1 (Roh et al., 2002a), providing a physical link to tight junctions. The third group of polarity proteins, the tumor suppressor proteins Dlg1, Scribble and Lgl have not been shown to physically associate with each other. However, mutations in any one of these genes disrupt apical-basal polarity in epithelial cells in Drosophila melanogaster (Woods et al., 1996; Bilder et al., 2000; Tanentzapf and Tepass, 2003; Bilder, 2004). shRNA-mediated depletion of hScrib leads to a delay in tight junction formation in MDCKII epithelial cells (Qin et al., 2005).
A key question is how the proteins in these three groups communicate with each other to promote epithelial polarity and tight junction formation. Based on genetic studies in D. melanogaster, there appears to be a complex interplay between these groups, which includes positive and negative feedback loops (Bilder et al., 2003; Tanentzapf and Tepass, 2003). The specification of the apical domain by Par proteins is thought to be an early event, which then leads to recruitment of the Crb group to this region. The Dlg group of proteins is recruited basolaterally, and their localization is restricted by antagonistic activities of the Crb group. However, it remains a major challenge to define the biochemical steps that generate a polarized epithelial cell monolayer.
Using a biochemical approach, we have identified a previously uncharacterized member of the MAGUK family, MPP7, as a novel protein that interacts with hDlg1. Using RNAi-mediated depletion in Caco2 cells, we show that both proteins are involved in the functional regulation of tight junction formation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture, Transfection, and RNA Interference
MCF7, HEK 293T, HeLa, and Caco-2 cells were cultured at 37°C in a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (Invitrogen-BRL), supplemented with 10% fetal calf serum and penicillin-streptomycin (100 IU/ml and 100 mg/ml, respectively). MCF10A cells were cultured at 37°C in a 5% CO2 and maintained in DMEM/F12 (Invitrogen-BRL) supplemented with 5% donor horse serum, 20 ng ml–1 EGF, 10 µg ml–1 insulin, 1 ng ml–1 cholera toxin, 100 µg ml–1 hydrocortisone, 50 U ml–1 penicillin, and 50 µg ml–1 streptomycin. MCF7 and MCF10A were transiently transfected using Lipofectamine 2000 (Invitrogen) and HEK 293T were transfected using GeneJuice (Novagen, Madison, WI). For small interfering RNA (siRNA) experiments, the following target sequences were used: MPP7-A (5'-GGAUACCAGUGGAGGAUAA-3', nt 569-587); MPP7-B (5'-UGAAUGAACUGAAACGAAA-3', nt 1142-1160); hDlg1-A (5'-CCACAAGUAUGUAUAUGAA-3', nt 1214-1232); hDlg1-B (5'-AGAAGUUACUCAUGAAGAA-3', nt 1140-1158); CASK-A (5'-GTAGCCAGCCATTATATGA-3', nt 349-367); and CASK-B (5'-GGCAACAAUUUGCUGUAAA-3', nt 104-122).
Oligonucleotides were annealed and transfected using oligofectamine (Invitrogen) as described by the manufacturer. As a control, a scrambled sequence was chosen.
Retrovirus Construction and Infection
The retroviral vector pSUPER.retro was purchased from OligoEngine (Seattle, WA). The following oligonucleotides were designed: MPP7-A (5'-GATCCCCggataccagtggaggataaTTCAAGAGAttatcctccactggtatccTTTTTA-3', nt 569-587), MPP7-B (5'-GATCCCCggatgttcagcctcatacaTTCAAGAGA tgtatgaggctgaacttgaacatccTTTTTA-3', nt 1392-1410), hDlg1-A (5'-GATCCCCcagaagctgttcttccctcTTCAAGAGAgagggaagaacagcttctgTTTTTA-3', nt 488-506), hDlg1-B (5'-GATCCCCgcgttgaaagaagcagggtTTCAAGAGA accctgcttctttcaacgcTTTTTA-3', nt 883-901). Complementary oligonucleotides were annealed and cloned into the unique BglII-HindIII sites downstream of the H1 RNA promoter, and inserts were confirmed by sequencing. Amphotropic retroviruses were produced by transfection of DNA constructs into the retroviral packaging cell line 293-GPG using Lipofectamine (Invitrogen). The medium was changed 24 h after transfection, and 72 h after transfection viral supernatant was collected, filtered through a 0.45-µm membrane, and stored at –80°C. Caco-2 cells (5 x 105 cells/10-cm plate) were infected with viral supernatants supplemented with polybrene (8 µg/ml). Forty-eight hours after infection, Caco-2 cells were grown in selection media containing 6 µg/ml puromycin to select for cells stably expressing the retroviral vector. Puromycin-resistant Caco-2 cells were maintained in media containing 6 µg/ml puromycin.
Transepithelial Electrical Resistance Measurements and Calcium Switch
Caco-2 cells were grown to confluence on permeable, collagen-coated Transwell filters (Becton Dickinson, Lincoln Park, NJ; Cat. no. 354804) for 5 d, washed extensively with PBS, and then incubated in low-calcium medium overnight (16 h) to dissociate cell–cell contacts. The low-calcium medium was replaced the next day with normal growth medium (1.8 mM Ca2+), and transepithelial electrical resistance (TER) measurements were determined with a Millicell-ERS volt-ohm meter (Millipore, Billerica, MA), according to manufacturer's instructions, at various times afterward. Background resistance was determined using cell-free filters. Samples for each time point were measured in triplicate in a total of three independent experiments.
Antibody Reagents
Primary Antibodies were obtained as follows: mouse monoclonals to myc (Clone 9E10; Cancer Research UK, London, United Kingdom), VSV-G (clone PSD5; Cancer Research UK), FLAG (clone M2; Sigma-Aldrich), hDlg1 (clone 2D11; Santa Cruz Biotechnology, Santa Cruz, CA), E-cadherin (clone 4A2C7; Zymed, South San Francisco, CA), occludin (clone OC-3F10; Zymed), 
-catenin (clone 14; BD Transduction Laboratories, Lexington, CA), CASK (clone K56A; Chemicon, Temecula, CA), 
-tubulin (clone DM1a; Sigma-Aldrich), rabbit polyclonal antibodies to occludin (71-1500; Zymed), ZO-1 (61-7300; Zymed), CASK (71-5000; Zymed), FLAG (ab21536-100; Abcam, Cambridge, United Kingdom) and myc (MYC13-A; Alpha Diagnostics International, San Antonio, TX). A rabbit antibody against human MPP7 was raised against the peptide EVTPYRRQTNEKYR (amino acids 356-369) coupled to keyhole limpet hemocyanin. Antibodies were affinity-purified on the antigenic peptide covalently bound to SulfoLink Coupling Gel according to the manufacturer's instructions (Pierce Biotechnology, PERBIO Science GmbH, Bonn, Germany). The specificity of the MPP7 antibody is shown in the Supplementary Figure S1A. The antibody does not show cross-reactivity to other MAGUK proteins in total cell extracts and does not recognize ectopically expressed myc-MPP3 (the closest homolog of MPP7 of the p55-MAGUK subfamily).
Immunofluorescence Microscopy
Immunofluorescence microscopy was performed on MCF7 cells or Caco-2 cells grown on coverslips. Cells were fixed with 3% [wt/vol] paraformaldehyde for 15 min at RT, quenched for 10 min with 50 mM ammonium chloride, and permeabilized with 0.1% [vol/vol] Triton X-100 for 5 min. Alternatively, cells were fixed in –20°C methanol for 6 min. Blocking for 30 min at RT and sequential incubation of primary and secondary antibodies for 1 h at RT were done in PBS containing 3% BSA. Primary antibodies used in this study were as follows: rabbit polyclonal, affinity-purified p-MPP7 (10 µg/ml), p--catenin (1:200), p-occludin (1:200), and p-FLAG (1:500) and mouse monoclonal m-hDlg1 (1:100), m-CASK (1:200), m-E-cadherin (1:200), m-occludin (1:200), and m-myc (1:100). Secondary antibodies conjugated to either Alexa 488, Alexa 568, or Texas Red were used to visualize antibody staining (1:1000, Molecular Probes, Eugene, OR). DNA was stained with Hoechst 33342 (1:20.000, Molecular Probes). Images were collected with a Zeiss Axioplan microscope using a 63x Plan Apochromat oil immersion objective (NA 1.4; Zeiss, Jena, Germany), standard filter sets and an ORCA-ER (Hamamatsu, Bridgewater, NJ) camera driven by Openlab software (Improvision, Lexington, MA). Confocal images were obtained with a MRC1024 (Bio-Rad, Richmond, CA) confocal OptiphotII (Nikon, Melville, NY) microscope using a 60x planapochromatic objective (NA 1.4). Images were cropped in Adobe Photoshop 7.0, sized, and placed in figures using Adobe Illustrator 10.0 (Adobe Systems, San Jose, CA).
Cell Lysates, Immune Precipitations, and Immunoblotting
MCF7, MCF10A, and HEK 293T cells were washed twice in ice-cold PBS, 1 mM phenylmethylsulfonyl fluoride (PMSF) and resuspended in lysis buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 0.5% [vol/vol] Triton X-100, 5 mM NaF, 20 mM -glycerophosphate, 100 µM Na3VO4, 1 mM PMSF, and protease inhibitor cocktail tablet [Roche]). Cell debris was pelleted by centrifugation at 14,000 rpm for 15 min at 4°C. Protein concentrations were determined using the Dc protein assay (Bio-Rad). For immune precipitations, equal amounts of cell extracts were precleared using protein G-Sepharose for 1 h at 4°C. They were then incubated with 1 µg of affinity-purified antibody for 2 h before 20 µl protein G- or A-Sepharose beads were added for 60 min. All incubations were performed on a rotating wheel. Immune complexes were spun down, washed three times with lysis-buffer containing 250 mM NaCl, and then boiled in SDS-PAGE sample buffer. For immunoblotting, proteins were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Membranes were stained with Ponceau S, blocked in 5% nonfat dry milk in PBS for 1 h at RT, and incubated overnight at 4°C with either rabbit polyclonal, affinity-purified primary MPP7 antibody (2 µg/ml), p-ZO-1 (1:1000), p-occludin (1:4000) or mouse monoclonal -hDlg1 antibody (1:500), m-CASK (1:1000), m-tubulin (1:4000), m-myc (1:500), m-FLAG (1:1000), m-E-cadherin, m--catenin, and m-VSV-G (all 1:1000). The membranes were washed with 0.05% Triton X-100/PBS, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce) in 5% nonfat dry milk in PBS for 1 h at room temperature, and then washed with 0.05% Triton X-100/PBS. Bound HRP-conjugated antibodies were visualized with the enhanced chemiluminescence (ECL) detection system (GE Healthcare, Buckingham, United Kingdom).
Purification of the hDlg1 Complex, Mass Spectrometry, and Gel Filtration Chromatography
For the purification of the hDlg1 complex, a total of 50 plates (150 mm) of MCF7 cells were grown to confluency. After washing the cells twice in ice-cold PBS containing 1 mM PMSF, they were resuspended in lysis buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 0.5% [vol/vol] Triton X-100, 5 mM NaF, 20 mM -glycerophosphate, 100 µM Na3VO4, 1 mM PMSF and protease inhibitor cocktail tablet [Roche]). Cell debris was pelleted by centrifugation at 14,000 rpm for 15 min at 4°C. The supernatant was then centrifuged at 40,000 rpm for 1 h at 4°C, and the remaining supernatant was precleared using protein G-Sepharose for 1 h at 4°C. For immune precipitations, equal amounts of MCF7 cell extracts were incubated with 8 µg m-hDlg1 antibody and control IgG1 for 14 h at 4°C, followed by the addition of protein G-Sepharose, rotating continuously. Immune complexes were washed four times with lysis buffer, 50 µl of reducing sample buffer was added, and then beads were heated at 95°C for 5 min, followed by the analysis on 7.5% minigels.
Coomassie-stained proteins of interest were excised and in-gel digested with trypsin as described (Gevaert and Vandekerckhove, 2005). The generated peptide mixture was dried, redissolved in 20 µl of 0.1% formic acid in 2/98 (vol/vol) acetonitrile/water, and 10 µl was applied for nano-LC-MS/MS analysis using an Ultimate (Dionex, Amsterdam, The Netherlands) HPLC system in-line connected to an Esquire HCT ion trap (Bruker Daltonics, Bremen, Germany). Peptides were first trapped on a trapping column (PepMap C18 column, 0.3 mm ID x 5 mm, Dionex) and after back-flushing, they were loaded on a 75 µm ID x 150-mm reverse-phase column (PepMap C18, Dionex). The peptides were eluted with a linear solvent gradient over 50 min ending in 0.1% of formic acid in acetonitrile/water (7/3, vol/vol). Using data-dependent acquisition, only multiple charged ions with intensities above a threshold of 100,000 were selected for further fragmentation. For MS/MS analysis, a MS/MS fragmentation amplitude of 0.7 V and a scan time of 40 ms were used. The fragmentation spectra were converted to Mascot generic files (mgf) using the Automation Engine software (version 3.2, Bruker) and searched using MASCOT (http://www.matrixscience.com) against the human IPI database (http://www.ebi.ac.uk/IPI/IPIhelp.html). Only spectra that exceeded Mascot's threshold score for identify (set at the 95% confidence level) were reported for further manual validation.
Gel filtration chromatography was carried out on a Superose-6 column by FPLC (Pharmacia Biotech, Newcastle upon Tyne, UK). The column was calibrated with standards of known Stokes radii as indicated in Figure 1. Two milligrams of MCF7 cell extracts were chromatographed over the column in lysis buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 5 mM NaF, 20 mM -glycerophosphate, 100 µM Na3VO4, 1 mM PMSF, 1 mM benzamidine, 1 µg/ml aprotinin, and 1 µg/ml leupeptin). Equal volumes of each fraction were analyzed by SDS-PAGE and immunoblotting.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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140-kDa bands as hDlg1 splice variants and revealed that the other two proteins were CASK and MPP7, respectively (Figure 1B, lane 1, and Supplementary Figure S2). CASK and MPP7 were not present when immunoprecipitation was performed with a control antibody (Figure 1B, lane 2). The identity of these proteins was confirmed by coimmunoprecipitation and Western blot analysis with specific antibodies (Figure 1C, lane 1, and Supplementary Figure S1A).
hDlg1, CASK, and MPP7 are all members of the MAGUK family of proteins, the defining features of which are at least one PDZ domain, followed by an SH3 domain and a catalytically inactive guanylate kinase domain (Funke et al., 2005). CASK contains an additional amino-terminal region homologous to calcium/calmodulin-dependent protein kinase II and has previously been shown to interact with hDlg1 (Nix et al., 2000; Lee et al., 2002). MPP7 is a so far uncharacterized member of the p55 subfamily of MAGUKs (Funke et al., 2005). Importantly, we could recover both proteins associated with hDlg1 in immunoprecipitates from a wide range of epithelial cell lines derived from different tissues (Figure 1C, lanes 2–6) independently of whether the cells were polarized or not (data not shown).
To characterize further the hDlg1 complexes, we first examined whether the interaction between hDlg1, MPP7, and CASK represented one or more distinct complexes. Size exclusion chromatographic fractionation of MCF7 lysates followed by Western blotting revealed that cellular hDlg1 elutes at a size of 500–600 kDa, suggesting one or more multimolecular complexes (Figure 1D, top). A fraction of MPP7 and CASK also eluted at a similar position to hDlg1 (Figure 1D, middle and bottom), though the majority of the cellular pool of these two proteins eluted elsewhere. Several MPP7 splice variants eluted mainly at around 200 kDa, whereas the bulk of CASK eluted as a 300-kDa complex (Figure 1D, middle and bottom). The same comigration pattern of hDlg1, MPP7, and CASK was obtained using cell extracts of unpolarized (i.e., in low calcium) MCF7 cells (data not shown). Interestingly, the elution profile of CASK and MPP7 in the hDlg1 region was slightly different, suggesting the existence of two distinct hDlg1 complexes.
To examine this further, myc-tagged MPP7 or VSVG-tagged CASK were ectopically expressed in HEK 293T cells. Immunoprecipitation with an anti-myc antibody followed by Western blot analysis revealed an interaction between myc-MPP7 and endogenous hDlg1, but not between myc-MPP7 and endogenous CASK (Figure 1E, lane 3). In contrast, anti-VSVG antibodies coprecipitated VSVG-CASK and endogenous hDlg1, but not endogenous MPP7 (Figure 1E, lane 8). Moreover, in the presence of myc-MPP7, the amount of endogenous CASK associated with endogenous hDlg1 was reduced, whereas in the presence of VSVG-CASK significantly less endogenous MPP7 could be detected in an hDlg1 immunoprecipitate (Figure 1E, lanes 2, 7, and 11). We conclude that CASK and MPP7 compete for the same or overlapping binding sites on hDlg1.
A previous report demonstrated that the interaction between hDlg1 and CASK is mediated through L27 domains present on both proteins (Lee et al., 2002). To map the interaction sites of MPP7 with hDlg1, myc-tagged MPP7 deletion constructs were expressed in HEK 293T cells, and anti-myc immunoprecipitations probed on Western blots for endogenous hDlg1. Full-length myc-MPP7 could readily be coprecipitated with endogenous hDlg1 and vice versa (Figure 1F, lane 1, and data not shown); however, removal of the L27N domain of MPP7 abolished the interaction (Figure 1F, lane 2), indicating that this protein motif is required for binding to hDlg1. In agreement with this, a fragment comprising the L27N and L27C domains only of MPP7 was sufficient to specifically associate with endogenous hDlg1 (Figure 1F, lane 8). These results point to an interaction between the L27 domain of hDlg1 and the L27 domains of MPP7. To test this, key hydrophobic residues were substituted with a polar hydrophilic residue within the L27N (L38S) or L27C (L95S) domains of MPP7. myc-MPP7 and myc-MPP7/L95S coprecipitated with endogenous hDlg1 when expressed in HEK 293T cells using either myc or hDlg1 antibodies (Figure 1G, lanes 1 and 3), whereas myc-MPP7/L38S did not (Figure 1G, lane 2). We conclude that hDlg1 exists in two discrete complexes with either MPP7 or CASK in epithelial cells and that both proteins use L27 domains to interact with hDlg1.
Because hDlg1 forms two separate protein complexes with MPP7 and CASK, we made use of Caco-2 cells to examine their subcellular localization. Unlike MCF7 cells, Caco-2 cells are capable of establishing a highly polarized morphology in tissue culture conditions. We raised an antibody to MPP7 for immunofluorescence studies and found that endogenous MPP7 associates primarily with lateral membranes and is largely excluded from the basal site in polarized Caco-2 cells (Figure 2, A and B). hDlg1 is both lateral and basal and therefore colocalizes with MPP7 along the lateral side (Figure 2A). In contrast, the basolateral protein CASK only partially overlaps with MPP7 at the lateral site (Figure 2B), but colocalizes with hDlg1 along the basal membrane (data not shown, Lee et al., 2002). Interestingly, MPP7 is found slightly more apical along the lateral membrane than hDlg1 (Figure 2A, right panel, inset, arrowhead). In addition, both MPP7 and hDlg1 overlap with markers for adherens junctions (Figure 2, C and D) and tight junctions (Figure 2, E and F). MPP7 staining is specific, because almost no signal is detectable in MPP7 siRNA treated cells (Supplementary Figure S1, B and C).
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). Treatment of MCF7 cells with cytochalasin D for 1 h to disassemble the actin cytoskeleton induced a cytoplasmic relocalization of both endogenous hDlg1 and MPP7 (Figure 2G). Next, a series of myc-tagged MPP7 deletion constructs were introduced into MCF7 cells and analyzed by immunofluorescence microscopy with anti-myc antibodies (Figures 3A and Supplementary Figure S3). Full-length myc-MPP7 localized to the plasma membrane similarly to endogenous protein. However, deletion of the amino-terminal, hDlg1-interacting L27N domain abolished membrane localization of MPP7 (Figures 3A and Supplementary Figure S3). Surprisingly, further analysis of deletion constructs revealed a second domain required for membrane localization of MPP7. A mutant lacking the SH3-HOOK domain also did not localize to the plasma membrane despite containing the L27N domain (Figures 3A and Supplementary Figure S3). We conclude that both the hDlg1-interacting L27N domain and the SH3-HOOK regions are required for targeting of MPP7 to the plasma membrane. To examine whether the interaction with hDlg1 is required for membrane localization, gene silencing by siRNA was used in MCF7 cells. Immunofluorescence microscopy and immunoblotting demonstrated efficient and uniform hDlg1 depletion (Figure 3, B and C). In the absence of hDlg1, endogenous MPP7 no longer localizes to the plasma membrane (Figure 3B, middle row), although strong membrane staining can be seen in control cells and in untransfected cells (Figure 3B, top row). Components of adherens junctions, such as E-cadherin, and tight junction markers, such as occludin, appeared to localize normally at the plasma membrane in the absence of hDlg1 (Figure 3D; data not shown). In contrast, depletion of MPP7 by siRNA did not interfere with the membrane localization of hDlg1 (Figure 3B, bottom). Thus, the recruitment of MPP7 to the plasma membrane is dependent on hDlg1.
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). To examine whether Crb3 might also affect MPP7 localization, FLAG-tagged Crb3 together with myc-MPP7 were cotransfected into MCF10A cells and analyzed by immunofluorescence microscopy. In Crb3-expressing cells, myc-MPP7 was recruited to the plasma membrane (Figure 4C, first column), whereas it remained cytosolic when expressed with a Crb3 construct lacking the C-terminal PDZ-binding motif (Figure 4, B and C, second column). Importantly, the localization of endogenous hDlg1 remained unaffected in MCF10A cells expressing FLAG-Crb3-
ERLI, illustrating a requirement of Crb3 in plasma membrane localization of MPP7 (Figure 4D). myc-MPP3, the closest homolog of MPP7 in the p55 MAGUK family, was not recruited to the plasma membrane in a Crb3-dependent manner in MCF10A cells, demonstrating a specific effect of Crb3 on MPP7 (Figure 4E, compare first and second columns). As shown previously, both the L27N and the SH3-HOOK domains are required for membrane targeting of MPP7 (Figure 3A, second column). In agreement, transfection of deletion constructs of MPP7 (Figure 3A, third column) with full-length Crb3 revealed that indeed the SH3-HOOK domain of MPP7 is required for Crb3-mediated membrane targeting (data not shown). In summary, these data suggest that plasma membrane localization of MPP7 is dependent on its L27N domain-mediated binding with hDlg1 and on a Crb3-dependent recruitment via the SH3-HOOK domain.
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; Hong et al., 2001; Roh et al., 2002b, 2003). To examine whether the interaction between MPP7 and Crb3 is directly or indirectly mediated by PALS1 (MPP5), wild-type and deletion constructs of MPP7 were examined for their ability to interact with MPP5. HEK 293T cells were cotransfected with myc-tagged MPP7 and FLAG-tagged MPP5, and, as shown in Figure 5B (lane 3), myc-MPP7 can be coprecipitated with FLAG-MPP5 and vice versa. Importantly, MPP3, another member of the p55 subfamily of MAGUKs, did not interact with myc-MPP7, suggesting a specific interaction between MPP7 and PALS1 (MPP5; Figure 5B, lane 2). Moreover, PALS1 (MPP5) associated much more efficiently with Crb3 than MPP7 (Figure 5C, compare lanes 5 and 7), supporting the idea that MPP7 binding to Crb3 is indirectly mediated via its interaction with PALS1 (MPP5). Importantly, the interaction with PALS1 (MPP5) required the SH3-HOOK domain, but not the PDZ domain of MPP7 (Figures 3A, fourth column, and 5D, compare lanes 4, 6, and 7), excluding a potential unspecific interaction with the PDZ-binding motif of Crb3.
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-catenin (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Wodarz, 2005). The protein is located basolaterally in polarized epithelia (Bilder and Perrimon, 2000), but its biochemical role is not at all clear. Interestingly, in migrating primary astrocytes Dlg1 localizes to the leading edge in a Par6/aPKC-dependent manner, where it interacts with the microtubule-bound adenomatous polyposis coli (APC) tumor suppressor protein to regulate microtubule cytoskeleton polarity (Etienne-Manneville et al., 2005). Here we identify MPP7, a member of the p55 subfamily of MAGUK proteins, as a new binding partner of Dlg1 in human epithelial cells. We find that the N-terminal L27 domain in MPP7 interacts with hDlg1. Through its SH3-HOOK domain, MPP7 also interacts with PALS1 (MPP5), a member of the Crb3/PALS1 (MPP5)/PATJ group of apically localized polarity proteins, and in turn PALS1 (MPP5) interacts with Crb3 (Bachmann et al., 2001; Hong et al., 2001; Roh et al., 2002b, 2003). In Caco-2 cells, we find that both hDlg1 and MPP7 show partial colocalization with Occludin and ZO-1, suggesting that the interaction of these polarity complexes might occur at the level of cell junctions. However, it remains unclear whether all these proteins are simultaneously present in one complex.
We also provide evidence that MPP7 can interact with Crb3 likely through its interaction with MPP5 (PALS1). A recent report on the identification of new polarity complexes showed that MPP7 can be coprecipitated with PALS1 (MPP5) and PATJ (Wells et al., 2006), thus validating our data. This raises the possibility that MPP7 might act as a bridge between the Dlg and Crb groups of polarity proteins. However, we have been unable to detect any Crb3 or PALS1 (MPP5) in hDlg1 immunoprecipitates (data not shown) and so this remains speculative. It is possible that the interaction between MPP7 and the Crb3/PALS1 (MPP5) complex is transient and that MPP7 is recruited to the plasma membrane through an interaction between its SH3-HOOK domain and PALS1 (MPP5) and is then retained at the cell membrane through binding to hDlg1. This would account for our observation that hDlg1 is required, but not sufficient, for the localization of MPP7 to the plasma membrane. Such a two-step mechanism for plasma membrane recruitment is already known for several polarity proteins, e.g., Dlg1 (Thomas et al., 2000) and Scribble (Albertson et al., 2004; Zeitler et al., 2004).
Finally, we provide evidence that the interaction of hDlg1 and MPP7 is functionally important in tight junction formation. Retroviral-shRNA mediated depletion of either MPP7 or hDlg1 compromised functional tight junctions assembly in Caco-2 cells after a calcium switch, as revealed by quantitative TER. It is clear, however, that although the loss of hDlg1 or MPP7 in Caco-2 cells leads to a defect in tight junction functionality, it does not lead to significant mislocalization of tight junction markers (occludin, ZO-1), or adherens junction proteins (E-cadherin, -catenin). A similar conclusion has been reached in hScrib-depleted cells (Qin et al., 2005). This may suggest that MPP7 and the Dlg/Scrib group of polarity proteins are not required for cell junction assembly, but rather for some functional activity perhaps related to its dynamic stability. Alternatively, possible redundancy in the activities and/or protein–protein interactions associated with the numerous polarity proteins, coupled with the relatively unsophisticated nature of these 2D tissue culture assays may account for the lack of dramatic phenotypes.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Present address: Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 572, New York, NY 10021.
Address correspondence to: Alan Hall (halla{at}mskcc.org)
Abbreviations used: PDZ, PSD-95, ZO-1, and Discs-large; MPP, membrane-palmitoylated protein.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bachmann, A., Schneider, M., Theilenberg, E., Grawe, F., Knust, E. (2001). Drosophila Stardust is a partner of Crumbs in the control of epithelial cell polarity. Nature 414, 638–643.[CrossRef][Medline]
Bilder, D. (2004). Epithelial polarity and proliferation control: links from the Drosophila neoplastic tumor suppressors. Genes Dev 18, 1909–1925.
Bilder, D., Li, M., Perrimon, N. (2000). Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science 289, 113–116.
Bilder, D. and Perrimon, N. (2000). Localization of apical epithelial determinants by the basolateral PDZ protein Scribble. Nature 403, 676–680.[CrossRef][Medline]
Bilder, D., Schober, M., Perrimon, N. (2003). Integrated activity of PDZ protein complexes regulates epithelial polarity. Nat. Cell Biol 5, 53–58.[CrossRef][Medline]
Bossinger, O., Klebes, A., Segbert, C., Theres, C., Knust, E. (2001). Zonula adherens formation in Caenorhabditis elegans requires dlg-1, the homologue of the Drosophila gene discs large. Dev. Biol 230, 29–42.[CrossRef][Medline]
Ebnet, K., Suzuki, A., Horikoshi, Y., Hirose, T., Meyer Zu Brickwedde, M. K., Ohno, S., Vestweber, D. (2001). The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM). EMBO J 20, 3738–3748.[CrossRef][Medline]
Etienne-Manneville, S., Manneville, J. B., Nicholls, S., Ferenczi, M. A., Hall, A. (2005). Cdc42 and Par6-PKCzeta regulate the spatially localized association of Dlg1 and APC to control cell polarization. J. Cell Biol 170, 895–901.
Fogg, V. C., Liu, C. J., Margolis, B. (2005). Multiple regions of Crumbs3 are required for tight junction formation in MCF10A cells. J. Cell Sci 118, 2859–2869.
Funke, L., Dakoji, S., Bredt, D. S. (2005). Membrane-associated guanylate kinases regulate adhesion and plasticity at cell junctions. Annu. Rev. Biochem 74, 219–245.[CrossRef][Medline]
Gevaert, K. and Vandekerckhove, J. (2005). In-gel digestion of protein spots for mass spectrometry. In: Cell Biology Handbook: A Laboratory Manual, ed. J. Celis. 3rd ed. San Diego: Academic Press, Vol 4, 379–382 Chapter 48.
Hong, Y., Stronach, B., Perrimon, N., Jan, L. Y., Jan, Y. N. (2001). Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts. Nature 414, 634–638.[CrossRef][Medline]
Izumi, Y., Hirose, T., Tamai, Y., Hirai, S., Nagashima, Y., Fujimoto, T., Tabuse, Y., Kemphues, K. J., Ohno, S. (1998). An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3. J. Cell Biol 143, 95–106.
Joberty, G., Petersen, C., Gao, L., Macara, I. G. (2000). The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nat. Cell Biol 2, 531–539.[CrossRef][Medline]
Lee, S., Fan, S., Makarova, O., Straight, S., Margolis, B. (2002). A novel and conserved protein-protein interaction domain of mammalian Lin-2/CASK binds and recruits SAP97 to the lateral surface of epithelia. Mol. Cell. Biol 22, 1778–1791.
Lemmers, C., Medina, E., Delgrossi, M. H., Michel, D., Arsanto, J. P., Le Bivic, A. (2002). hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells. J. Biol. Chem 277, 25408–25415.
Lin, D., Edwards, A. S., Fawcett, J. P., Mbamalu, G., Scott, J. D., Pawson, T. (2000). A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity. Nat. Cell Biol 2, 540–547.[CrossRef][Medline]
Macara, I. G. (2004). Parsing the polarity code. Nat. Rev. Mol. Cell Biol 5, 220–231.[CrossRef][Medline]
Makarova, O., Roh, M. H., Liu, C. J., Laurinec, S., Margolis, B. (2003). Mammalian Crumbs3 is a small transmembrane protein linked to protein associated with Lin-7 (Pals1). Gene 302, 21–29.[CrossRef][Medline]
Medina, E., Lemmers, C., Lane-Guermonprez, L., Le Bivic, A. (2002). Role of the Crumbs complex in the regulation of junction formation in Drosophila and mammalian epithelial cells. Biol. Cell 94, 305–313.[CrossRef][Medline]
Nix, S. L., Chishti, A. H., Anderson, J. M., Walther, Z. (2000). hCASK and hDlg associate in epithelia, and their src homology 3 and guanylate kinase domains participate in both intramolecular and intermolecular interactions. J. Biol. Chem 275, 41192–41200.
Petronczki, M. and Knoblich, J. A. (2001). DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila. Nat. Cell Biol 3, 43–49.[CrossRef][Medline]
Qin, Y., Capaldo, C., Gumbiner, B. M., Macara, I. G. (2005). The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin. J. Cell Biol 171, 1061–1071.
Qiu, R. G., Abo, A., Steven Martin, G. (2000). A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKCzeta signaling and cell transformation. Curr. Biol 10, 697–707.[CrossRef][Medline]
Reuver, S. M. and Garner, C. C. (1998). E-cadherin mediated cell adhesion recruits SAP97 into the cortical cytoskeleton. J. Cell Sci 111, Pt 81071–1080.[Abstract]
Roh, M. H., Fan, S., Liu, C. J., Margolis, B. (2003). The Crumbs3-Pals1 complex participates in the establishment of polarity in mammalian epithelial cells. J. Cell Sci 116, 2895–2906.
Roh, M. H., Liu, C. J., Laurinec, S., Margolis, B. (2002a). The carboxyl terminus of zona occludens-3 binds and recruits a mammalian homologue of discs lost to tight junctions. J. Biol. Chem 277, 27501–27509.
Roh, M. H., Makarova, O., Liu, C. J., Shin, K., Lee, S., Laurinec, S., Goyal, M., Wiggins, R., Margolis, B. (2002b). The Maguk protein, Pals1, functions as an adapter, linking mammalian homologues of Crumbs and Discs Lost. J. Cell Biol 157, 161–172.
Schock, F. and Perrimon, N. (2002). Molecular mechanisms of epithelial morphogenesis. Annu. Rev. Cell Dev. Biol 18, 463–493.[CrossRef][Medline]
Shin, K., Straight, S., Margolis, B. (2005). PATJ regulates tight junction formation and polarity in mammalian epithelial cells. J. Cell Biol 168, 705–711.
Straight, S. W., Shin, K., Fogg, V. C., Fan, S., Liu, C. J., Roh, M., Margolis, B. (2004). Loss of PALS1 expression leads to tight junction and polarity defects. Mol. Biol. Cell 15, 1981–1990.
Suzuki, A., Ishiyama, C., Hashiba, K., Shimizu, M., Ebnet, K., Ohno, S. (2002). aPKC kinase activity is required for the asymmetric differentiation of the premature junctional complex during epithelial cell polarization. J. Cell Sci 115, 3565–3573.
Suzuki, A., Yamanaka, T., Hirose, T., Manabe, N., Mizuno, K., Shimizu, M., Akimoto, K., Izumi, Y., Ohnishi, T., Ohno, S. (2001). Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures. J. Cell Biol 152, 1183–1196.
Tabuse, Y., Izumi, Y., Piano, F., Kemphues, K. J., Miwa, J., Ohno, S. (1998). Atypical protein kinase C cooperates with PAR-3 to establish embryonic polarity in Caenorhabditis elegans. Development 125, 3607–3614.[Abstract]
Takekuni, K., Ikeda, W., Fujito, T., Morimoto, K., Takeuchi, M., Monden, M., Takai, Y. (2003). Direct binding of cell polarity protein PAR-3 to cell-cell adhesion molecule nectin at neuroepithelial cells of developing mouse. J. Biol. Chem 278, 5497–5500.
Tanentzapf, G. and Tepass, U. (2003). Interactions between the crumbs, lethal giant larvae and bazooka pathways in epithelial polarization. Nat. Cell Biol 5, 46–52.[CrossRef][Medline]
Tepass, U., Tanentzapf, G., Ward, R., Fehon, R. (2001). Epithelial cell polarity and cell junctions in Drosophila. Annu. Rev. Genet 35, 747–784.[CrossRef][Medline]
Thomas, U., Ebitsch, S., Gorczyca, M., Koh, Y. H., Hough, C. D., Woods, D., Gundelfinger, E. D., Budnik, V. (2000). Synaptic targeting and localization of discs-large is a stepwise process controlled by different domains of the protein. Curr. Biol 10, 1108–1117.[CrossRef][Medline]
Wells, C. D., et al. (2006). A. Rich1/Amot complex regulates the Cdc42 GTPase and apical-polarity proteins in epithelial cells. Cell 125, 535–548.[CrossRef][Medline]
Wodarz, A. (2005). Molecular control of cell polarity and asymmetric cell division in Drosophila neuroblasts. Curr. Opin. Cell Biol 17, 475–481.[CrossRef][Medline]
Wodarz, A., Ramrath, A., Grimm, A., Knust, E. (2000). Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelia and neuroblasts. J. Cell Biol 150, 1361–1374.
Woods, D. F., Hough, C., Peel, D., Callaini, G., Bryant, P. J. (1996). Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia. J. Cell Biol 134, 1469–1482.
Yamanaka, T., et al. (2001). PAR-6 regulates aPKC activity in a novel way and mediates cell-cell contact-induced formation of the epithelial junctional complex. Genes Cells 6, 721–731.[Abstract]
Yeaman, C., Grindstaff, K. K., Nelson, W. J. (1999). New perspectives on mechanisms involved in generating epithelial cell polarity. Physiol. Rev 79, 73–98.
Zeitler, J., Hsu, C. P., Dionne, H., Bilder, D. (2004). Domains controlling cell polarity and proliferation in the Drosophila tumor suppressor Scribble. J. Cell Biol 167, 1137–1146.
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/cm2. Results were plotted as the relative values of TER (for hDlg1-A- and hDlg1-B-shRNA) compared with the TER of Ctrl-shRNA (set TER = 1 for every time point). Values have been corrected for background and represent the mean of data taken in three independent experiments (and per experiment in duplicate). Error bars, SD between individual measurements. (C) Transepithelial electrical resistance (TER) measurements of Caco-2 cells stably expressing Ctrl-, MPP7-A- and MPP7-B-shRNA as indicated. Measurements and cell culture was performed as described in B. (D) Caco-2 cell lines were seeded onto Transwell filters and grown at confluence for 5 d. After incubation in low-calcium medium for 16 h, the cells were incubated in normal growth medium and at different times (shown for t = 0, 2, 5, and 10 h) were fixed, permeabilized, and immunostained with anti-occludin antibodies. For better visualization of cell borders and cytoplasmic aggregates of tight junction components, immunostaining is shown in the inverted mode.