Identification and characterization of a novel tight junction-associated family of proteins that interacts with a WW domain of MAGI-1

The membrane-associated guanylate kinase protein, MAGI-1, has been shown to be a component of epithelial tight junctions in both Madin-Darby canine kidney cells and in intestinal epithelium. Because we have previously observed MAGI-1 expression in glomerular visceral epithelial cells (podocytes) of the kidney, we screened a glomerular cDNA library to identify the potential binding partners of MAGI-1 and isolated a partial cDNA encoding a novel protein. The partial cDNA exhibited a high degree of identity to an uncharacterized human cDNA clone, KIAA0989, which encodes a protein of 780 amino acids and contains a predicted coiled-coil domain in the middle of the protein. In vitro binding assays using the partial cDNA as a GST fusion protein confirm the binding to full-length MAGI-1 expressed in HEK293 cells, as well as endogenous MAGI-1, and also identified the first WW domain of MAGI-1 as the domain responsible for binding to this novel protein. Although a conventional PPxY binding motif for WW domains was not present in the partial cDNA clone, a variant WW binding motif was identified, LPxY, and found to be necessary for interacting with MAGI-1. When expressed in Madin-Darby canine kidney cells, the full-length novel protein was found to colocalize with MAGI-1 at the tight junction of these cells and the coiled-coil domain was found to be necessary for this localization. Because of its interaction with MAGI-1 and its localization to cell-cell junctions, this novel protein has been given the name MAGI-1-associated coiled-coil tight junction protein (MASCOT).     

Interaction of two actin-binding proteins,synaptopodin and alpha-actinin-4, with the tight junction protein MAGI-1

In an attempt to find podocyte-expressed proteins that may interact with the tight junction protein MAGI-1, we screened a glomerulus-enriched cDNA library with a probe consisting of both WW domains of MAGI-1. One of the isolated clones contained two WW domain-binding motifs and was identified as a portion of the actin-bundling protein synaptopodin. In vitro binding assays confirmed this interaction between MAGI-1 and synaptopodin and identified the second WW domain of MAGI-1 to be responsible for the interaction. MAGI-1 and synaptopodin can also interact in vivo, as they can be immunoprecipitated together from HEK293 cell lysates. Another actin-bundling protein that is found in glomerular podocytes and shown to be mutated in an inheritable form of glomerulosclerosis is alpha-actinin-4. We show that alpha-actinin-4 is also capable of binding to MAGI-1 in in vitro binding assays and that this interaction is mediated by the fifth PDZ domain of MAGI-1 binding to the C terminus of alpha-actinin-4. Exogenously expressed synaptopodin and alpha-actinin-4 were found to colocalize along with endogenous MAGI-1 at the tight junction of Madin-Darby canine kidney cells. The interaction and colocalization of MAGI-1 with two actin-bundling proteins suggest that MAGI-1 may play a role in actin cytoskeleton dynamics within polarized epithelial cells.     

JAM4, a junctional cell adhesion molecule interacting with a tight junction protein,MAGI-1

MAGI-1 is a membrane-associated guanylate kinase protein at tight junctions in epithelial cells. It interacts with various molecules and functions as a scaffold protein at cell junctions. We report here a novel MAGI-1-binding protein that we named junctional adhesion molecule 4 (JAM4). JAM4 belongs to an immunoglobulin protein family. JAM4 was colocalized with ZO-1 in kidney glomeruli and in intestinal epithelial cells. Biochemical in vitro studies revealed that JAM4 bound to MAGI-1 but not to ZO-1, whereas JAM1 did not bind to MAGI-1. JAM4 and MAGI-1 interacted with each other and formed clusters in COS-7 cells when coexpressed. JAM4 mediated calcium-independent homophilic adhesion and was accumulated at cell-cell contacts when expressed in L cells. MAGI-1, ZO-1, and occludin were recruited to JAM4-based cell contacts. JAM4 also reduced the permeability of CHO cell monolayers. MAGI-1 strengthened JAM4-mediated cell adhesion in L cells and sealing effects in CHO cells. These findings suggest that JAM4 together with MAGI-1 provides an adhesion machinery at tight junctions, which may regulate the permeability of kidney glomerulus and small intestinal epithelial cells.     

Zonula occludens-1 function in the assembly of tight junctions in Madin-Darby canine kidney epithelial cells

Zonula occludens (ZO)-1 was the first tight junction protein to be cloned and has been implicated as an important scaffold protein. It contains multiple domains that bind a diverse set of junction proteins. However, the molecular functions of ZO-1 and related proteins such as ZO-2 and ZO-3 have remained unclear. We now show that gene silencing of ZO-1 causes a delay of approximately 3 h in tight junction formation in Madin-Darby canine kidney (MDCK) epithelial cells, but mature junctions seem functionally normal even in the continuing absence of ZO-1. Depletion of ZO-2, cingulin, or occludin, proteins that can interact with ZO-1, had no discernible effects on tight junctions. Rescue of junction assembly using murine ZO-1 mutants demonstrated that the ZO-1 C terminus is neither necessary nor sufficient for normal assembly. Moreover, mutation of the PDZ1 domain did not block rescue. However, point mutations in the Src homology 3 (SH3) domain almost completely prevented rescue. Surprisingly, the isolated SH3 domain of ZO-1 could also rescue junction assembly. These data reveal an unexpected function for the SH3 domain of ZO-1 in regulating tight junction assembly in epithelial cells and show that cingulin, occludin, or ZO-2 are not limiting for junction assembly in MDCK monolayers.     

MAGI-1 interacts with beta-catenin and is associated with cell-cell adhesion structures

The family of membrane-associated guanylate kinases (MAGUK) comprises peripheral membrane proteins involved in the formation of specialized cell-cell junctions. MAGUK proteins possess a conserved domain composition, containing PDZ, guanylate kinase, and SH3 or WW domains. MAGI-1 is a recently identified member of the MAGUK protein family. Three splice variantsof MAGI-1 have been characterized to date, including MAGI-1a, -1b, and -1c. MAGI-1b is predominantly associated with the crude membrane fraction. Here we show that the fifth PDZ domain of MAGI-1b is essential for membrane localization. We have also identified beta-catenin as a potential ligand for this PDZ domain. MAGI-1b forms complexes with beta-catenin and E-cadherin during the formation of cell-cell junctions in MDCK cells. In agreement with this observation, a significant portion of a GFP fusion of MAGI-1b localizes to the basolateral membrane of polarized MDCK cells.     

Localization of BAI-associated protein1/membrane-associated guanylate kinase-1 at adherens junctions in normal rat kidney cells: polarized targeting mediated by the carboxyl-terminal PDZ domains

Brain-specific angiogenesis inhibitor (BAI)-associated protein (BAP)1 (also called membrane-associated guanylate kinase [MAGI]-1) is composed of six PSD-95/Dlg-A/ZO-1 (PDZ) domains, two WW domains, and one guanylate kinase (GK) domain. We previously reported that BAP1 is localized at tight junctions in Madine Darby canine kidney (MDCK) cells and intestinal epithelial cells. Here, we have determined the localization of BAP1 in normal rat kidney (NRK) cells that do not form tight junctions. BAP1 was colocalized with E-cadherin along the lateral membrane, suggesting its localization at adherens junctions. Green fluorescent protein (GFP)-BAP1 was distributed in the cytosol in separate NRK cells, and accumulated to the cell-cell contacts when NRK cells have contact with each other. The GFP-BAP1 mutant containing either the first PDZ and GK domains or the WW and second PDZ domains was localized in the cytosol and the nucleus. The GFP-BAP1 mutant containing the second to fourth PDZ domains was distributed in the cytosol. The construct containing the fifth and sixth PDZ domains was localized at the cell-cell contacts along the lateral membrane and slightly in the nucleus, whereas the construct lacking the fifth and sixth PDZ domains was localized in the cytosol and in the nucleus. BAP1 was tyrosine-phosphorylated in vivo, but the tyrosine phosphorylation of BAP1 was not correlated with its localization. These results suggest that the signal in the carboxyl-terminal PDZ domains functions dominantly in vivo to target BAP1 to the lateral membrane, although potential nuclear localization signals exist in the N-terminal region of BAP1.     

The tight junction protein ZO-2 localizes to the nucleus and interacts with the heterogeneous nuclear ribonucleoprotein scaffold attachment factor-B

Zonula occludens proteins (ZOPs), currently comprising ZO-1, ZO-2, and ZO-3, belong to the family of membrane-associated guanylate kinase homologue (MAGUK) proteins that are involved in the organization of epithelial and endothelial intercellular junctions. ZOPs bind to the cytoplasmic C termini of junctional transmembrane proteins linking them to the actin cytoskeleton. They are characterized by several conserved modules, including three PDZ domains, one SH3 domain, and a guanylate kinase-like domain, elements indicating that ZOPs may serve multiple purposes. Interestingly, ZOPs contain some unique motifs not shared by other MAGUK family members, including nuclear localization and nuclear export signals and a leucine zipper-like sequence. Their potential involvement in cell growth and proliferation has been suggested earlier based on the observation that the N-terminal half of ZOPs displays significant similarity to the product of the Drosophila tumor suppressor gene lethal(1)discs-large (dlg). The nuclear targeting of ZOPs in subconfluent epithelial cell cultures is well documented, although the action of the junctional MAGUKs in the nucleus has remained elusive. Here we show for the first time that nuclear ZO-2 directly interacts with the DNA-binding protein scaffold attachment factor-B (SAF-B). Our results from two-hybrid assays and in vivo co-immunoprecipitation studies provide evidence to suggest that ZO-2 associates with the C-terminal portion of SAF-B via its PDZ-1 domain. We further demonstrate that enhanced green fluorescent protein (EGFP)- and DsRed-tagged ZO-2 and SAF-B fusion proteins partially co-localize in nuclei of transfected epithelial cells. As shown by laser confocal microscopy and epifluorescent analysis, nuclear ZO-2 is present in epithelial and endothelial cells, particularly in response to environmental stress conditions. Interestingly, no association of SAF-B with ZO-1 was found, which supports the notion that junctional MAGUKs serve nonredundant functions.     

Occludin and claudin-1 concentrate in the midbody of immortalized mouse hepatocytes during cell division.

It has been believed that epithelial cells maintain tight junctions at all times, including during cell division, to provide a continuous epithelial seal. However, changes in localization of integral tight junction proteins during cell division have not been examined. In this study, using SV40-immortalized mouse hepatocytes transfected with human Cx32 cDNA, in which tight junction strands and the endogenous tight junction proteins occludin, claudin-1, ZO-1, and ZO-2 were induced, we examined changes in localization of the tight junction proteins at all stages of cell division. All tight junction proteins were present between mitotic cells and neighboring cells throughout cell division. In late telophase, the integral tight junction proteins occludin and claudin-1, but not the cytoplasmic proteins ZO-1 and ZO-2, were concentrated in the midbody between the daughter cells and were observed at cell borders between the daugher and neighboring cells. These results indicate that the integral tight junction proteins are regulated in a different manner from the cytoplasmic proteins ZO-1 and ZO-2 during cytokinesis.     

A role for ZO-1 and PLEKHA7 in recruiting paracingulin to tight and adherens junctions of epithelial cells

Paracingulin is a 160-kDa protein localized in the cytoplasmic region of epithelial tight and adherens junctions, where it regulates RhoA and Rac1 activities by interacting with guanine nucleotide exchange factors. Here, we investigate the molecular mechanisms that control the recruitment of paracingulin to cell-cell junctions. We show that paracingulin forms a complex with the tight junction protein ZO-1, and the globular head domain of paracingulin interacts directly with ZO-1 through an N-terminal region containing a conserved ZIM (ZO-1-Interaction-Motif) sequence. Recruitment of paracingulin to cadherin-based cell-cell junctions in Rat1 fibroblasts requires the ZIM-containing region, whereas in epithelial cells removal of this region decreases the junctional localization of paracingulin at tight junctions but not at adherens junctions. Depletion of ZO-1, but not ZO-2, reduces paracingulin accumulation at tight junctions. A yeast two-hybrid screen identifies both ZO-1 and the adherens junction protein PLEKHA7 as paracingulin-binding proteins. Paracingulin forms a complex with PLEKHA7 and its interacting partner p120ctn, and the globular head domain of paracingulin interacts directly with a central region of PLEKHA7. Depletion of PLEKHA7 from Madin-Darby canine kidney cells results in the loss of junctional localization of paracingulin and a decrease in its expression. In summary, we characterize ZO-1 and PLEKHA7 as paracingulin-interacting proteins that are involved in its recruitment to epithelial tight and adherens junctions, respectively.     

beta 1-adrenergic receptor association with the synaptic scaffolding protein membrane-associated guanylate kinase inverted-2 (MAGI-2). Differential regulation of receptor internalization by MAGI-2 and PSD-95

The beta1-adrenergic receptor (beta1AR) is known to be localized to synapses and to modulate synaptic plasticity in many brain regions, but the molecular mechanisms determining beta1AR subcellular localization are not fully understood. Using overlay and pull-down techniques, we found that the beta1AR carboxyl terminus associates with MAGI-2 (membrane-associated guanylate kinase inverted-2), a protein also known as S-SCAM (synaptic scaffolding molecule). MAGI-2 is a multidomain scaffolding protein that contains nine potential protein-protein interaction modules, including 6 PDZ domains, 2 WW domains, and a guanylate kinase-like domain. The beta1AR carboxyl terminus binds with high affinity to the first PDZ domain of MAGI-2, with the last few amino acids of the beta1AR carboxyl terminus being the key determinants of the interaction. In cells, the association of full-length beta1AR with MAGI-2 occurs constitutively and is enhanced by agonist stimulation of the receptor, as assessed by both co-immunoprecipitation experiments and immunofluorescence co-localization studies. Agonist-induced internalization of the beta1AR is markedly increased by co-expression with MAGI-2. Strikingly, this result is the opposite of the effect of co-expression with PSD-95, a previously reported binding partner of the beta1AR. Further cellular experiments revealed that MAGI-2 has no effect on beta1AR oligomerization but does promote association of beta1AR with the cytoplasmic signaling protein beta-catenin, a known MAGI-2 binding partner. These data reveal that MAGI-2 is a specific beta1AR binding partner that modulates beta1AR function and facilitates the physical association of the beta1AR with intracellular proteins involved in signal transduction and synaptic regulation.     

Connexin-occludin chimeras containing the ZO-binding domain of occludin localize at MDCK tight junctions and NRK cell contacts.

Occludin is a transmembrane protein of the tight junction that functions in creating both an intercellular permeability barrier and an intramembrane diffusion barrier. Creation of the barrier requires the precise localization of occludin, and a distinct family of transmembrane proteins called claudins, into continuous linear fibrils visible by freeze-fracture microscopy. Conflicting evidence exists regarding the relative importance of the transmembrane and extracellular versus the cytoplasmic domains in localizing occludin in fibrils. To specifically address whether occludin's COOH-terminal cytoplasmic domain is sufficient to target it into tight junction fibrils, we created chimeras with the transmembrane portions of connexin 32. Despite the gap junction targeting information present in their transmembrane and extracellular domains, these connexin-occludin chimeras localized within fibrils when expressed in MDCK cells, as assessed by immunofluorescence and immunogold freeze-fracture imaging. Localization of chimeras at tight junctions depends on the COOH-terminal ZO-binding domain and not on the membrane proximal domain of occludin. Furthermore, neither endogenous occludin nor claudin is required for targeting to ZO-1-containing cell-cell contacts, since in normal rat kidney fibroblasts targeting of chimeras again required only the ZO-binding domain. These results suggest an important role for cytoplasmic proteins, presumably ZO-1, ZO-2, and ZO-3, in localizing occludin in tight junction fibrils. Such a scaffolding and cytoskeletal coupling function for ZO MAGUKs is analogous to that of other members of the MAGUK family.     

Tight junction protein MAGI-1 is up-regulated by transfection with connexin 32 in an immortalized mouse hepatic cell line: cDNA microarray analysis

Gap junctions are considered to play a crucial role in differentiation of epithelial cells, including hepatocytes. Recently, we found that Cx32 but not Cx26 was closely related to tight junctional proteins in primary cultured rat hepatocytes (Kojima et al., Exp Cell Res 263:193–201, 2001) and that Cx32 formation and/or Cx32-mediated intercellular communication could induce expression and function of tight junctions in a mouse hepatic cell line (Kojima et al., Exp Cell Res 276:40–51, 2002). In this study, to investigate the mechanisms of induction of tight junctions by transfection with Cx32, we performed cDNA microarray analysis of Cx32 transfectants, compared with parental cells derived from Cx32-deficient hepatocytes. In cDNA microarray analysis, a 2.5-fold increase in expression of membrane-associated guanylate kinase with inverted orientation-1 (MAGI-1), which is known to be localized at adherens and tight junction regions, was observed. High expression of MAGI-1 in Cx32 transfectants was confirmed by Western blotting and RT-PCR. MAGI-1 was colocalized with occludin, claudin-2, ZO-1, and F-actin, but not with E-cadherin in the apical-most regions at cell borders of Cx32 transfectants, similar to junctional adhesion molecule-1 (JAM-1), which may play a crucial role in formation and assembly of tight junctions. Treatment with the gap junction blocker 18-glycyrrhetinic acid did not affect expression of MAGI-1 and JAM-1 in Cx32 transfectants. These results suggest that Cx32 expression is in part related to induction of tight junctions through modulation of MAGI-1 expression in an immortalized mouse hepatic cell line.This work was supported in by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science Technology and the Ministry of Health, Labour and Welfare of Japan, and by the Kato Memorial Bioscience Foundation, the Uehara Memorial Foundation, the Suhara Memorial Foundation, the Smoking Research Foundation, and the Long-Range Research Initiative Project of the Japan Chemical Industry Association.     

hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells

dCrumbs is an apical organizer crucial for the maintenance of epithelial polarity in Drosophila (1). It is known that dCrumbs interacts with Discs lost (Dlt), a protein with four PDZ (PSD95/Discs Large/ZO-1) domains (2), and Stardust (Sdt), a protein of the MAGUK (membrane-associated guanylate kinase) family (3, 4). We have searched for potential homologs of Dlt in human epithelial cells and characterized one of them in intestinal epithelial cells. Human INAD-like (hINADl) contains 8 PDZ domains, is concentrated in tight junctions, and is also found at the apical plasma membrane. Overexpression of hINADl disrupted the tight junctions localization of ZO-1 and 3. We also identified a partial cDNA coding the transmembrane and cytoplasmic domains of a new human crumbs (CRB3) expressed in Caco-2 cells. This CRB3 was able to interact through its C-terminal end with the N-terminal domain of hINADl. Taken together, the data indicate that hINADl is likely to represent a Dlt homolog in mammalian epithelial cells and might be involved in regulating the integrity of tight junctions. We thus propose to rename hINADl PATJ for protein associated to tight junctions.     

Endothelial adhesion molecule ESAM binds directly to the multidomain adaptor MAGI-1 and recruits it to cell contacts

Endothelial cell-selective adhesion molecule (ESAM) is an immunoglobulin-like transmembrane protein associated with endothelial tight junctions (TJ). Based on a yeast two-hybrid screen, we have identified the membrane-associated guanylate kinase protein MAGI-1 as an intracellular binding partner of ESAM. MAGI-1 is a multidomain adaptor protein, which binds to transmembrane, cytoskeletal, and signaling molecules, and has been localized to tight junctions in epithelial cells. MAGI-1 associates with the very C-terminal sequence of ESAM most likely through a PDZ domain-mediated interaction. The direct interaction between ESAM and MAGI-1 was confirmed by pull-down experiments. The two proteins formed stable complexes in transfected Chinese hamster ovary (CHO) cells, which could be immunoisolated. We found MAGI-1 to be associated with cell-cell contacts in human umbilical vein endothelial cells (HUVECs) and in mouse endothelium, where it colocalizes with ESAM. In CHO cells, recruitment of MAGI-1 to cell contacts required the presence of ESAM. Hence, ESAM may be involved in anchoring MAGI-1 at endothelial tight junctions.     

The carboxyl terminus of zona occludens-3 binds and recruits a mammalian homologue of discs lost to tight junctions

Mammalian homologues of the Drosophila polarity proteins Stardust, Discs Lost, and Crumbs have been identified as Pals1, Pals1-associated tight junction protein (PATJ), and human Crumbs homologue 1 (CRB1), respectively. We have previously demonstrated that PATJ, Pals1, and CRB1 can form a tripartite tight junction complex in epithelial cells and that PATJ recruits Pals1 to tight junctions. Here, we observed that the Pals1/PATJ interaction was not crucial for the ultimate targeting of PATJ itself to tight junctions. This prompted us to examine if any of the 10 post-synaptic density-95/Discs Large/zona occludens-1 (PDZ) domains of PATJ could bind to the carboxyl termini of known tight junction constituents. We found that the 6th and 8th PDZ domains of PATJ can interact with the carboxyl termini of zona occludens-3 (ZO-3) and claudin 1, respectively. PATJ missing the 6th PDZ domain was found to mislocalize away from cell contacts. Surprisingly, deleting the 8th PDZ domain had little effect on PATJ localization. Finally, reciprocal co-immunoprecipitation experiments revealed that full-length ZO-3 can associate with PATJ. Hence, the PATJ/ZO-3 interaction is likely important for recruiting PATJ and its associated proteins to tight junctions.     

Identification of a novel protein, LYRIC, localized to tight junctions of polarized epithelial cells

Tight junctions (TJ) are multiprotein complexes that function to regulate paracellular transport of molecules through epithelial and endothelial cell layers. Many new tight junction-associated proteins have been identified in the past few years, and their functional roles and interactions have just begun to be elucidated. In this paper, we describe a novel protein LYsine-RIch CEACAM1 co-isolated (LYRIC) that is widely expressed and highly conserved between species. LYRIC has no conserved domains that would indicate function and does not appear to be a member of a larger protein family. Data from analysis of rat and human tissue sections and cell lines show that LYRIC colocalizes with tight junction proteins ZO-1 and occludin in polarized epithelial cells, suggesting that LYRIC is part of the tight junction complex. LYRIC dissociates from ZO-1 when junctional complexes are disrupted, and as tight junctions reform, ZO-1 relocalizes before LYRIC. These results suggest that LYRIC is most likely not a structural component required for TJ formation, but rather is recruited during the maturation of the tight junction complex.     

Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia

The Discs large (Dlg) protein of Drosophila is the prototypic member of a growing family of proteins termed membrane-associated guanylate kinase homologs (MAGUKs). The MAGUKs are composed of a series of peptide domains that include one or three DHR/PDZs, an SH3, and a region homologous to guanylate kinase (GUK). We have previously shown that the product of this gene, the Dlg protein, is localized at the septate junctions between epithelial cells, and that mutations in the gene cause neoplastic overgrowth of the imaginal discs. The dlg locus is therefore defined as a tumor suppressor gene. In this paper, we show that the Dlg protein is localized on the cytoplasmic face of the septate junction and is required for the maintenance of this structure. It is also required for proper organization of the cytoskeleton, for the differential localization of membrane proteins, and for apicobasal polarity of epithelial cells. However, these other functions can be uncoupled from Dlg's role as a tumor suppressor since mutations in two domains of the protein, the SH3 and GUK, cause loss of normal cell proliferation control without affecting the other functions of the protein. These results suggest that, besides regulating cellular proliferation, the Dlg protein is a critical component of the septate junctions and is required for maintaining apicobasal polarity in Drosophila epithelium.     

A systematic analysis of human papillomavirus (HPV) E6 PDZ substrates identifies MAGI-1 as a major target of HPV type 16 (HPV-16) and HPV-18 whose loss accompanies disruption of tight junctions

The E6 proteins from high-risk, cancer-causing types of human papillomavirus (HPV) are characterized by the presence of a PDZ (PSD95/Dlg/ZO-1) binding motif in their extreme carboxy termini, through which they interact with a number of cellular PDZ domain-containing substrates. In order to ascertain how many of these are degraded by E6 in vivo, we performed an extensive analysis of the effects of E6 ablation on the expression levels of a number of previously reported E6 PDZ substrates. Using HPV type 16 (HPV-16)-positive CaSKi cells and HPV-18-positive HeLa cells, we have found that MAGI-1 is a major degradation target of both HPV-16 and HPV-18 E6. In contrast, hDlg, hScrib, PTPN3, TIP2, FAP1, and PSD95 all exhibit various degrees of susceptibility to E6-induced degradation, and a high degree of HPV type specificity is observed for certain substrates. We also show that E6 preferentially targets MAGI-1 within the nucleus and at membrane sites. One of the direct consequences of MAGI-1 degradation is a loss of tight-junction integrity, as determined by mislocalization of the tight-junction protein ZO-1. Ablation of E6 expression restores tight junctions, and this restoration is dependent on the presence of MAGI-1. These results demonstrate that oncogenic HPV E6 proteins disrupt cellular tight junctions through the degradation of MAGI-1, and they provide further evidence of how the PDZ binding potential of E6 can contribute to HPV-induced malignancy.