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Three groups of evolutionarily conserved proteins have been implicated in the establishment of epithelial cell polarity: the apically-localized proteins of the Par (Par3-Par6-aPKC-Cdc42) and Crumbs groups (Crb3-PALS1-PATJ) and the basolaterally localized proteins of the Dlg group (Dlg1-Scribble-Lgl). During epithelial morphogenesis, these proteins participate in a complex network of interdependent interactions that define the position and functional organization of adherens junctions and tight junctions. However, the biochemical pathways through which they control polarity are poorly understood. In this study, we identify an interaction between endogenous hDlg1 and MPP7, a previously uncharacterized MAGUK-p55 subfamily member. We find that MPP7 targets to the lateral surface of epithelial cells via its L27N domain, through an interaction with hDlg1. Loss of either hDlg1 or MPP7 from epithelial Caco-2 cells results in a significant defect in the assembly and maintenance of functional tight junctions. We conclude that the formation of a complex between hDlg1 and MPP7 promotes epithelial cell polarity and tight junction formation.
The establishment of cell polarity and the formation of multicellular tissues and organs are fundamentally important in the development of higher eukaryotes (Schock and Perrimon, 2002 ). 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.
All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) or Merck (Rahway, NJ) unless otherwise specified. Cell culture media and sera were obtained from Invitrogen (Carlsbad, CA) and Mycoplex (A15–043) (PAA, Pasching, Austria), respectively. Cytochalasin D was from Sigma. DNA primers were synthesized by Sigma-Genosys, and synthetic RNA duplexes by Dharmacon Research (Boulder, CO). Restriction enzymes were purchased from New England Biolabs (Beverly, MA), and reagents for the purification of DNA from QIAGEN (Chatsworth, CA). Full-length human cDNAs for MPP7 (IRATp970B0732D6), MPP5 (DKFZp451E015Q2), and MPP3 (IRAKp961D21119Q2) were obtained from the Deutsches Ressourcenzentrum fuer Genomforschung (RZPD; GmbH, Berlin, Germany). Full-length human Crb3 was amplified from a Caco-2 cDNA library using Pfu polymerase (Stratagene, La Jolla, CA) and oligonucleotides 5′-GCC-ACCGGATCCATGGCGAACCCCGGGCTGGGGCTG-3′ and 5′-CCGAAT-TCCTCAGATGAGCCGCTCTTCCGGCGG-3′. VSVG-tagged CASK constructs were obtained by Z. Walther (Yale University, New Haven, CT). Point mutants were constructed using the QuikChange mutagenesis protocol (Stratagene). All constructs were confirmed by DNA sequencing (MWG-Biotech, Ebersberg, Germany). Mammalian expression constructs were made in pRK5-myc and pRK5-FLAG.
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.
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 × 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.
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.
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), β-catenin (clone 14; BD Transduction Laboratories, Lexington, CA), CASK (clone K56A; Chemicon, Temecula, CA), α-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 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 pα-MPP7 (10 μg/ml), pα-α-catenin (1:200), pα-occludin (1:200), and pα-FLAG (1:500) and mouse monoclonal mα-hDlg1 (1:100), mα-CASK (1:200), mα-E-cadherin (1:200), mα-occludin (1:200), and mα-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 63× 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 60× 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).
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 β-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), pα-ZO-1 (1:1000), pα-occludin (1:4000) or mouse monoclonal α-hDlg1 antibody (1:500), mα-CASK (1:1000), mα-tubulin (1:4000), mα-myc (1:500), mα-FLAG (1:1000), mα-E-cadherin, mα-β-catenin, and mα-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).
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 β-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 mα-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 × 5 mm, Dionex) and after back-flushing, they were loaded on a 75 μm ID × 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 β-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.
To elucidate the function of cell polarity proteins in mammalian epithelial cells, we searched biochemically for proteins interacting with hDlg1 (human Dlg1/SAP97 (synapse-associated protein 97, Figure 1A). hDlg1 protein-containing complexes were immuno-affinity-purified from MCF7 breast epithelial tumor cell extracts using a specific antibody (Supplementary Figure S1A in the Supplementary Data) and fractionated by gel electrophoresis. Two coprecipitating proteins of 110 and 65 kDa were visible on gels, in addition to a triplet of bands at the size expected for hDlg1 splice variants (Figure 1B, lane 1). Mass spectrometric analyses confirmed the identity of the ~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).
To examine the potential interdependency for plasma membrane localization of MPP7 and hDlg1, we returned to a more easily transfectable epithelial cell line, MCF7. First, it has previously been reported that the association of hDlg1 with the plasma membrane is dependent on the cortical actin cytoskeleton (Reuver and Garner, 1998 ). 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.
Our observation that the SH3-HOOK domain in MPP7 functions as an additional membrane-targeting domain prompted us to investigate the underlying mechanism. We hypothesized that proteins other than hDlg1 might function through the SH3-HOOK domain. In fact, although myc-MPP7 similarly localized to the plasma membrane of several cell types, we noticed that it did not localize to the membrane when expressed in MCF10A mammary breast epithelial cells, despite high level expression and normal plasma membrane localization of endogenous hDlg1 in these cells (Figure 4A). A recent report showed that MCF10A fail to form tight junctions, because they express very low levels of the apical polarity transmembrane determinant, Crb3 (Fogg et al., 2005 ). 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-ΔERLI, 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.
To provide further evidence for an interaction between MPP7 and Crb3, HEK 293T cells were transfected with myc-tagged MPP7 and FLAG-tagged wild-type and mutant forms of Crb3. Myc-MPP7 was not detected in immune precipitates with the PDZ-binding-site mutant of Crb3, whereas it readily coprecipitated with the wild-type, the FERM-, and the RP-motif mutants (Figure 5A). Previous studies have shown that the PDZ-binding motif of Crb3 binds to PALS1/MPP5, another member of the p55 subfamily of MAGUK proteins (Bachmann et al., 2001 ; 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.
Because hDlg1 and MPP7 show overlapping localization to tight junctions, we directly investigated whether these proteins are required for the functional assembly of epithelial tight junctions. To examine this, pools of Caco-2 cells stably depleted of either hDlg1 or MPP7 by infection with retroviral-mediated shRNA were established. Western blot analysis confirmed that protein expression of the two proteins was attenuated (Figure 6A). To assess tight junction function, we measured transepithelial electrical resistance (TER) as a quantitative readout for tight junction integrity in combination with a calcium-switch assay. hDlg1 and MPP7-depleted Caco-2 cells were plated on collagen-coated filters and grown to confluence over 5 d to establish polarity. The medium was then replaced with a low-calcium medium, and the cells were left overnight to disrupt cell–cell contacts. After this time, normal growth medium was readded and TER was measured over a 3-d time course. Control cells rapidly reformed tight junctions within 10–15 h after returning to normal growth medium (Supplementary Figure S4, A and B), whereas there was a delay in the formation of tight junctions in the hDlg1-depleted cell lines (Figures 6B and Supplementary Figure S4A). shRNA-mediated elimination of MPP7 led to a significant retardation of the development of TER and the extent of TER inhibition correlated with the degree of MPP7 suppression (Figure 6, A and C, and Supplementary Figure S4B). Moreover, the start and end point TER-values remained significantly lower than of control cells, suggesting that MPP7 may also be involved in tight junction maintenance. To examine whether the defects observed in Caco-2 cells lacking hDlg1 or MPP7 correlated with improper localization of cell junction proteins, cells were fixed and stained after the calcium-switch assays. In low-calcium medium, cell–cell junctions were completely disrupted as shown in Figure 6D (t = 0 h). However, we found no obvious mislocalization of tight junction markers, such as occludin and ZO-1 (Figure 6D and data not shown), or adherens junction markers, such as E-cadherin and β-catenin (data not shown).
In Drosophila, Dlg1 plays an important role in the establishment of cell polarity during epithelial morphogenesis and asymmetric cell division in neuroblasts (Macara, 2004 ; 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, β-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.
We are grateful to Zenta Walther (Yale) for the VSVG-CASK construct, James Staddon (EISAI Research Laboratories, London, United Kingdom) for help with gel filtration chromatography and members of the laboratory for valuable discussions. V.M.S. is supported by an European Union Marie-Curie fellowship, and A.H. is generously supported by Cancer Research UK. K.G. is a Postdoctoral Fellow of the Fund for Scientific Research, Flanders, Belgium (F.W.O. Vlaanderen). The project was supported by research grants from the Fund for Scientific Research, Flanders, Belgium (project number G.0008.03) and the GBOU-research initiative (project number 20204) of the Flanders Institute of Science and Technology (IWT) to K.G. and J.V.
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-11-0980) on March 1, 2007.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).