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Pleckstrin homology domain–containing A7 (PLEKHA7) is a cytoplasmic protein at adherens junctions that has been implicated in hypertension, glaucoma, and responses to Staphylococcus aureus α-toxin. Complex formation between PLEKHA7, PDZ domain–containing 11 (PDZD11), tetraspanin 33, and the α-toxin receptor ADAM metallopeptidase domain 10 (ADAM10) promotes junctional clustering of ADAM10 and α-toxin–mediated pore formation. However, how the N-terminal region of PDZD11 interacts with the N-terminal tandem WW domains of PLEKHA7 and how this interaction promotes tetraspanin 33 binding to the WW1 domain is unclear. Here, we used site-directed mutagenesis, glutathione S-transferase pulldown experiments, immunofluorescence, molecular modeling, and docking experiments to characterize the mechanisms driving these interactions. We found that Asp-30 of WW1 and His-75 of WW2 interact through a hydrogen bond and, together with Thr-35 of WW1, form a binding pocket that accommodates a polyproline stretch within the N-terminal PDZD11 region. By strengthening the interactions of the ternary complex, the WW2 domain stabilized the WW1 domain and cooperatively promoted the interaction with PDZD11. Modeling results indicated that, in turn, PDZD11 binding induces a conformational rearrangement, which strengthens the ternary complex, and contributes to enlarging a “hydrophobic hot spot” region on the WW1 domain. The last two lipophilic residues of tetraspanin 33, Trp-283 and Tyr-282, were required for its interaction with PLEKHA7. Docking of the tetraspanin 33 C terminus revealed that it fits into the hydrophobic hot spot region of the accessible surface of WW1. We conclude that communication between the two tandem WW domains of PLEKHA7 and the PLEKHA7–PDZD11 interaction modulate the ligand-binding properties of PLEKHA7.

Introduction PLEKHA7, ABCC5, and KALRN have been identified as susceptible genetic markers related to glaucoma. We aimed to investigate the association between the identified susceptible genetic markers PLEKHA7 rs11024102, ABCC5 rs17217796, and KALRN rs1392912 in the progression of primary angle-closure glaucoma (PACG) in Malay patients. Methods For this study, 163 Malay patients with PACG were recruited from April 2015 to April 2017 at Hospital Universiti Sains Malaysia and Hospital Raja Perempuan Zainab II, Kota Bharu. Venesection was performed. DNA was extracted using a commercial DNA extraction kit. The primer was optimized for rs11024102, rs17217796, and rs1392912 of the PLEKHA7, ABCC5, and KALRN genes, respectively. Polymerase chain reaction (PCR) was performed, and PCR products were purified. A DNA sequencer was used to identify polymorphisms. Progression was based on the agreement between the Advanced Glaucoma Intervention Study scoring system and the Hodapp-Parrish and Anderson staging system. The scoring was conducted on two reliable consecutive Humphrey visual fields (HVFs) during the recruitment period and two baseline HVFs obtained when the diagnosis was made. Based on the scoring, patients were grouped into progressed and non-progressed. A chi-square test was used to analyze the association between the genetic markers and the progression of PACG. Results One hundred and sixty-three Malay patients with PACG (58 men and 105 women) were recruited. Twenty-nine patients (18%) had visual field progression of PACG after a mean (SD) follow-up of 6.0 (1.0) years. The minor allele frequencies for PLEKHA7 rs11024102 (G/A), ABCC5 rs17217796 (C/G), and KALRN rs1392912 (A/G) were 0.44, 0.08, and 0.48, respectively. We found that rs11024102 (p=0.828), rs17217796 (p=0.865), and rs1392912 (p=0.684) were not associated with PACG progression in the Malay patients. Conclusion Although PLEKHA7 and ABCC5 were found to be genetic markers associated with the risk of PACG, they played no roles in PACG progression in the Malay population. Moreover, KALRN was not significantly associated with PACG progression. Other susceptible genetic markers may be responsible for PACG progression.

Copper homeostasis is crucial for cellular physiology and development, and its dysregulation leads to disease. The Menkes ATPase ATP7A plays a key role in copper efflux, by trafficking from the Golgi to the plasma membrane upon cell exposure to elevated copper, but the mechanisms that target ATP7A to the cell periphery are poorly understood. PDZD11 interacts with the C-terminus of ATP7A, which contains sequences involved in ATP7A trafficking, but the role of PDZD11 in ATP7A localization is unknown. Here we identify PLEKHA5 and PLEKHA6 as new interactors of PDZD11 that bind to the PDZD11 N-terminus through their WW domains similarly to the junctional protein PLEKHA7. Using CRISPR-KO kidney epithelial cells, we show by immunofluorescence microscopy that WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) recruit PDZD11 to distinct plasma membrane localizations and that they are required for the efficient anterograde targeting of ATP7A to the cell periphery in elevated copper conditions. Pull-down experiments show that WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A. However, WW-PLEKHAs and PDZD11 are not necessary for ATP7A Golgi localization in basal copper, ATP7A copper-induced exit from the Golgi, and ATP7A retrograde trafficking to the Golgi. Finally, measuring bioavailable and total cellular copper, metallothionein-1 expression, and cell viability shows that WW-PLEKHAs and PDZD11 are required for maintaining low intracellular copper levels when cells are exposed to elevated copper. These data indicate that WW-PLEKHAs-PDZD11 complexes regulate the localization and function of ATP7A to promote copper extrusion in elevated copper.

Exposure of mucosal epithelial cells to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is known to disrupt epithelial cell junctions by impairing stathmin-mediated microtubule depolymerization. However, the pathological significance of this process and its underlying molecular mechanism remain unclear. Here we show that treatment of epithelial cells with pseudotyped HIV-1 viral particles or recombinant gp120 protein results in the activation of protein kinase G 1 (PKG1). Examination of epithelial cells by immunofluorescence microscopy reveals that PKG1 activation mediates the epithelial barrier damage upon HIV-1 exposure. Immunoprecipitation experiments show that PKG1 interacts with stathmin and phosphorylates stathmin at serine 63 in the presence of gp120. Immunoprecipitation and immunofluorescence microscopy further demonstrate that PKG1-mediated phosphorylation of stathmin promotes its autophagic degradation by enhancing the interaction between stathmin and the autophagy adaptor protein p62. Collectively, these results suggest that HIV-1 exposure exploits the PKG1/stathmin axis to affect the microtubule cytoskeleton and thereby perturbs epithelial cell junctions. Our findings reveal a novel molecular mechanism by which exposure to HIV-1 increases epithelial permeability, which has implications for the development of effective strategies to prevent mucosal HIV-1 transmission.

Cadherins are homophilic adhesion molecules with important functions in cell-cell adhesion, tissue morphogenesis, and cancer. In epithelial cells, E-cadherin accumulates at areas of cell-cell contact, coalesces into macromolecular complexes to form the adherens junctions (AJs), and associates via accessory partners with a subcortical ring of actin to form the apical zonula adherens (ZA). As a master regulator of the epithelial phenotype, E-cadherin is essential for the overall maintenance and homeostasis of polarized epithelial monolayers. Its expression is regulated by a host of genetic and epigenetic mechanisms related to cancer, and its function is modulated by mechanical forces at the junctions, by direct binding and phosphorylation of accessory proteins collectively termed catenins, by endocytosis, recycling and degradation, as well as, by multiple signaling pathways and developmental processes, like the epithelial to mesenchymal transition (EMT). Nuclear signaling mediated by the cadherin associated proteins β-catenin and p120 promotes growth, migration and pluripotency. Receptor tyrosine kinase, PI3K/AKT, Rho GTPase, and HIPPO signaling, are all regulated by E-cadherin mediated cell-cell adhesion. Finally, the recruitment of the microprocessor complex to the ZA by PLEKHA7, and the subsequent regulation of a small subset of miRNAs provide an additional mechanism by which the state of epithelial cell-cell adhesion affects translation of target genes to maintain the homeostasis of polarized epithelial monolayers. Collectively, the data indicate that loss of E-cadherin function, especially at the ZA, is a common and crucial step in cancer progression.

Tight and adherens junctions play critical roles in the barrier, adhesion, and signaling functions of epithelial and endothelial cells. How the molecular organization of these junctions is tuned to the widely diverse physiological requirements of each tissue type is not well understood. Here, we address this question by examining the expression, localization, and interactions of major cytoplasmic plaque proteins of tight and adherens junctions in different cultured epithelial and endothelial cell lines. Immunoblotting and immunofluorescence analyses show that the expression profiles of cingulin, paracingulin, ZO-1, ZO-2, ZO-3, PLEKHA7, afadin, PDZD11, p120-catenin, and α-catenin, as well as the transmembrane junctional proteins occludin, E-cadherin, and VE-cadherin, are significantly diverse when comparing kidney cells (MDCK, mCCD), keratinocytes (HaCaT), lung carcinoma (A427, A549), and endothelium-derived cells (bEnd.3, meEC, H5V). Proximity ligation and co-immunoprecipitation assays show that PLEKHA7 and PDZD11 are significantly more associated with the tight junction proteins cingulin and ZO-1 in aortic endothelium-derived (meEC) cells but not kidney collecting duct epithelial (mCCD) cells. These results provide evidence that the cytoplasmic plaques of tight and adherens junctions are diverse in their composition and molecular architecture and establish a conceptual framework by which we can rationally address the mechanisms of tissue-dependent junction physiology and signaling by cytoplasmic junctional proteins.

The RNA interference (RNAi) machinery is an essential component of the cell, regulating miRNA biogenesis and function. RNAi complexes were thought to localize either in the nucleus, such as the microprocessor, or in the cytoplasm, such as the RNA-induced silencing complex (RISC). We recently revealed that the core microprocessor components DROSHA and DGCR8, as well as the main components of RISC, including Ago2, also associate with the apical adherens junctions of well-differentiated cultured epithelial cells. Here, we demonstrate that the localization of the core RNAi components is specific and predominant at apical areas of cell-cell contact of human normal colon epithelial tissues and normal primary colon epithelial cells. Importantly, the apical junctional localization of RNAi proteins is disrupted or lost in human colon tumors and in poorly differentiated colon cancer cell lines, correlating with the dysregulation of the adherens junction component PLEKHA7. We show that the restoration of PLEKHA7 expression at adherens junctions of aggressively tumorigenic colon cancer cells restores the junctional localization of RNAi components and suppresses cancer cell growth in vitro and in vivo. In summary, this work identifies the apical junctional localization of the RNAi machinery as a key feature of the differentiated colonic epithelium, with a putative tumor suppressing function.

Background The disruption of E-cadherin-mediated adhesion is considered an important driver of tumor progression. Nevertheless, numerous studies have demonstrated that E-cadherin promotes growth- or invasion-related signaling, contrary to the prevailing notion. During tumor progression, epithelial ovarian cancer (EOC) maintains E-cadherin expression and can positively affect EOC cell growth by contributing to PI3K/AKT activation. In polarized epithelia PLEKHA7, a regulator of the zonula adherens integrity, impinges E-cadherin functionality, but its role in EOCs has been never studied. Methods Ex-vivo EOC cells and cell lines were used to study E-cadherin contribution to growth and EGFR activation. The expression of the proteins involved was assessed by real time RT-PCR, immunohistochemistry and western blotting. Cells growth and drug susceptibility was monitored in different 3-dimensional (3D) systems. Recombinant lentivirus-mediated gene expression, western blotting, immunoprecipitation and confocal microscopy were applied to investigate the biological impact of PLEKHA7 on E-cadherin behaviour. The clinical impact of PLEKHA7 was determined in publicly available datasets. Results We show that E-cadherin expression contributes to growth of EOC cells and forms a complex with EGFR thus positively affecting ligand-dependent EGFR/CDK5 signaling. Accordingly, 3D cultures of E-cadherin-expressing EOC cells are sensitive to the CDK5 inhibitor roscovitine combined with cisplatin. We determined that PLEKHA7 overexpression reduces the formation of E-cadherin-EGFR complex, EGFR activation and cell tumorigenicity. Clinically, PLEKHA7 mRNA is statistically decreased in high grade EOCs respect to low malignant potential and low grade EOCs and correlates with better EOC patient outcome. Conclusions These data represent a significant step towards untangling the role of E-cadherin in EOCs by assessing its positive effects on EGFR/CDK5 signaling and its contribution to cell growth. Hence, the inhibition of this signaling using a CDK5 inhibitor exerts a synergistic effect with cisplatin prompting on the design of new therapeutic strategies to inhibit growth of EOC cells. We assessed for the first time in EOC cells that PLEKHA7 induces changes in the asset of E-cadherin-containing cell-cell contacts thus inhibiting E-cadherin/EGFR crosstalk and leading to a less aggressive tumor phenotype. Accordingly, PLEKHA7 levels are lower in high grade EOC patient tumors and EOC patients with better outcomes display higher PLEKHA7 levels. Electronic supplementary material The online version of this article (10.1186/s13046-018-0796-1) contains supplementary material, which is available to authorized users.

E-cadherin and p120 catenin (p120) are essential for epithelial homeostasis, but can also exert pro-tumorigenic activities. Here, we resolve this apparent paradox by identifying two spatially and functionally distinct junctional complexes in non-transformed polarized epithelial cells: one growth suppressing at the apical zonula adherens (ZA), defined by the p120 partner PLEKHA7 and a non-nuclear subset of the core microprocessor components DROSHA and DGCR8, and one growth promoting at basolateral areas of cell-cell contact containing tyrosine-phosphorylated p120 and active Src. Recruitment of DROSHA and DGCR8 to the ZA is PLEKHA7 dependent. The PLEKHA7-microprocessor complex co-precipitates with primary microRNAs (pri-miRNAs) and possesses pri-miRNA processing activity. PLEKHA7 regulates the levels of select miRNAs, in particular processing of miR-30b, to suppress expression of cell transforming markers promoted by the basolateral complex, including SNAI1, MYC and CCND1. Our work identifies a mechanism through which adhesion complexes regulate cellular behaviour and reveals their surprising association with the microprocessor.

Adherens junction (AJ) is a specialized cell-cell junction structure that plays a role in mechanically connecting adjacent cells to resist strong contractile forces and to maintain tissue structure, particularly in the epithelium. AJ is mainly comprised of cell adhesion molecules cadherin and nectin and their associating cytoplasmic proteins including β-catenin, α-catenin, p120ctn, and afadin. Our series of studies have revealed that nectin first forms cell-cell adhesion and then recruits cadherin to form AJ. The recruitment of cadherin by nectin is mediated by the binding of α-catenin and p120ctn to afadin. Recent studies showed that PLEKHA7 binds to p120ctn, which is associated with E-cadherin, and maintains the integrity of AJ in epithelial cells. In this study, we showed that PLEKHA7 bound to afadin in addition to p120ctn and was recruited to the nectin-3α-based cell-cell adhesion site in a manner dependent on afadin, but not on p120ctn. The binding of PLEKHA7 to afadin was required for the proper formation of AJ, but not for the formation of tight junction, in EpH4 mouse mammary gland epithelial cells. These results indicate that PLEKHA7 plays a cooperative role with nectin and afadin in the proper formation of AJ in epithelial cells. Background: Afadin is an important regulator of cell-cell adhesion. Results: PLEKHA7 binds to afadin and this binding is required for the proper formation of adherens junction (AJ). Conclusion: PLEKHA7 plays a cooperative role with nectin and afadin in the proper formation of AJ in epithelial cells. Significance: The result indicates a novel regulatory mechanism for the formation of cell-cell adhesion.

Kourtidis et al. demonstrate that cadherin complexes at the zonula adherens recruit a diverse set of mRNAs to a junction-associated RISC, via PLEKHA7. In this way, PLEKHA7 regulates expression of critical regulators of epithelial homeostasis, such as JUN, MYC, and SOX2, through Ago2 and miRNA-mediated silencing., Cumulative evidence demonstrates that most RNAs exhibit specific subcellular distribution. However, the mechanisms regulating this phenomenon and its functional consequences are still under investigation. Here, we reveal that cadherin complexes at the apical zonula adherens (ZA) of epithelial adherens junctions recruit the core components of the RNA-induced silencing complex (RISC) Ago2, GW182, and PABPC1, as well as a set of 522 messenger RNAs (mRNAs) and 28 mature microRNAs (miRNAs or miRs), via PLEKHA7. Top canonical pathways represented by these mRNAs include Wnt/β-catenin, TGF-β, and stem cell signaling. We specifically demonstrate the presence and silencing of MYC, JUN, and SOX2 mRNAs by miR-24 and miR-200c at the ZA. PLEKHA7 knockdown dissociates RISC from the ZA, decreases loading of the ZA-associated mRNAs and miRNAs to Ago2, and results in a corresponding increase of MYC, JUN, and SOX2 protein expression. The present work reveals a mechanism that directly links junction integrity to the silencing of a set of mRNAs that critically affect epithelial homeostasis.

PLEKHA7 is a recently characterized component of the cytoplasmic region of epithelial adherens junctions (AJ). It comprises two WW domains, a pleckstrin-homology domain, and proline-rich and coiled-coil domains. PLEKHA7 interacts with cytoplasmic components of the AJ (p120-catenin, paracingulin, afadin), stabilizes the E-cadherin complex by linking it to the minus ends of noncentrosomal microtubules, and stabilizes junctional nectins through the newly identified interactor PDZD11. Similarly to afadin, and unlike E-cadherin and p120-catenin, the localization of PLEKHA7 at AJ is strictly zonular (in the zonula adhaerens subdomain of AJ), and does not extend along the basolateral contacts. Genome-wide association studies and experiments on animal and cellular models show that although PLEKHA7 is not required for organism viability, it is implicated in cardiovascular physiology, hypertension, primary angle closure glaucoma, susceptibility to staphylococcal α-toxin, and epithelial morphogenesis and growth. Thus, PLEKHA7 is a cytoskeletal adaptor protein important for AJ organization, and at the center of junction-associated signaling pathways which fine-tune important pathophysiological processes.

Cingulin, paracingulin, and PLEKHA7 are proteins localized in the cytoplasmic region of the apical junctional complex of vertebrate epithelial cells. Cingulin has been detected at tight junctions (TJs), whereas paracingulin has been detected at both TJs and adherens junctions (AJs) and PLEKHA7 has been detected at AJs. One function of cingulin and paracingulin is to regulate the activity of Rho family GTPases at junctions through their direct interaction with guanidine exchange factors of RhoA and Rac1. Cingulin also contributes to the regulation of transcription of several genes in different types of cultured cells, in part through its ability to modulate RhoA activity. PLEKHA7, together with paracingulin, is part of a protein complex that links E-cadherin to the microtubule cytoskeleton at AJs. In this paper, we review the current knowledge about these proteins, including their discovery, the characterization of their expression, localization, structure, molecular interactions, and their roles in different developmental and disease model systems.

SUMMARYThe vertebrate heart is one of the first organs to form, and its early function and morphogenesis are crucial for continued embryonic development. Here we analyze the effects of loss of Heart adaptor protein 1 (Hadp1), which we show is required for normal function and morphogenesis of the embryonic zebrafish heart. Hadp1 is a pleckstrin homology (PH)-domain-containing protein whose expression is enriched in embryonic cardiomyocytes. Knockdown of hadp1 in zebrafish embryos reduced cardiac contractility and altered late myocyte differentiation. By using optical mapping and submaximal levels of hadp1 knockdown, we observed profound effects on Ca2+ handling and on action potential duration in the absence of morphological defects, suggesting that Hadp1 plays a major role in the regulation of intracellular Ca2+ handling in the heart. Hadp1 interacts with phosphatidylinositol 4-phosphate [PI4P; also known as PtdIns(4)P] derivatives via its PH domain, and its subcellular localization is dependent upon this motif. Pharmacological blockade of the synthesis of PI4P derivatives in vivo phenocopied the loss of hadp1 in zebrafish. Collectively, these results demonstrate that hadp1 is required for normal cardiac function and morphogenesis during embryogenesis, and suggest that hadp1 modulates Ca2+ handling in the heart through its interaction with phosphatidylinositols.

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.

OBJECTIVES/SPECIFIC AIMS: The goal of this study is to test the hypothesis that the adherens junctions of colon epithelial cells regulate lncRNAs levels and function via the microprocessor and RISC complexes to suppress expression of pro-tumorigenic markers and aberrant cell behavior. METHODS/STUDY POPULATION: To test this hypothesis, we used colon epithelial cancer cell lines. We performed RNA-seq following knockdown of PLEKHA7, a key component of the adherens junctions, to identify changes in lncRNA expression and downstream mRNA levels. We confirmed junctional localization of affected lncRNAs from the RNA-seq and those that we found in our preliminary study by using in situ hybridization (ISH). RESULTS/ANTICIPATED RESULTS: RNA-seq identified junction-associated lncRNAs whose expression levels are regulated by PLEKHA7. The top upregulated lncRNA upon PLEKHA7 depletion was MIR17HG, an oncogenic host transcript of a cluster of miRNAs. These mature miRNAs also co-precipitate with PLEKHA7. PLEKHA7 knockdown results in increased levels of MIR17HG, but only a subset of its hosted miRNAs (miR-19a,b). Notably, miR-19a and mir-19b are highly upregulated in colon cancer. Our data suggest that 2 PLEKHA7-associated miRNAs, miR-203a and miR-372, mediate suppression MIR17HG. Re-expression of PLEKHA7 in aggressive colon cancer cells that lack PLEKHA7 suppressed expression of MIR17HG, as well as anchorage independent growth of these cells. DISCUSSION/SIGNIFICANCE OF IMPACT: Our data point towards a novel mechanism of lncRNA regulation that tethers epithelial tissue integrity with pro-tumorigenic cell transformation. Reducing elevated MIR17HG levels, is a potential therapeutic approach to suppress the tumorigenic behavior of cells that have lost their junctional integrity and homeostasis. identify a network of miRNA-mRNA-lncRNA interactions that could be exploited for further mechanistic studies, as well as for diagnostic and therapeutic purposes in the future.

SummaryEpithelial cells contain noncentrosomal microtubules (MTs), whose minus ends are oriented apically. In contrast with the well-known interactions of the minus ends with the centrosome, little is known about the termination site of the noncentrosomal minus ends. Here we show that a population of MT minus ends is anchored at the zonula adherens (ZA), the apical-most part of the cadherin-based adherens junction, via a protein that we have termed Nezha. We initially identified PLEKHA7 as a ZA component and subsequently detected Nezha as a partner for PLEKHA7. Nezha bound MTs at their minus ends and tethered them to the ZA. Furthermore, we found that a minus end-directed motor, KIFC3, was concentrated at the ZA in a PLEKHA7/Nezha/MT-dependent manner; and depletion of any of these proteins resulted in disorganization of the ZA. We propose that the PLEKHA7/Nezha/MT complex regulates the ZA integrity by recruiting KIFC3 to this junctional site.

The transition of sessile epithelial cells to a migratory, mesenchymal phenotype is essential for metazoan development and tissue repair, but this program is exploited by tumor cells in order to escape the confines of the primary organ site, evade immunosurveillance, and resist chemo-radiation. In addition, epithelial-to-mesenchymal transition (EMT) confers stem-like properties that increase efficiency of colonization of distant organs. This review evaluates the role of cell–cell junctions in suppressing EMT and maintaining a quiescent epithelium. We discuss the conflicting data on junctional signaling in cancer and recent developments that resolve some of these conflicts. We focus on evidence from breast cancer, but include other organ sites where appropriate. Current and potential strategies for inhibition of EMT are discussed.

E-cadherin-p120 catenin complexes are essential for adherens junction (AJ) formation and for the maintenance of the normal epithelial phenotype. PLEKHA7 was originally identified as a member of this complex that tethers microtubules to the AJs and supports their overall integrity. Recently, we revealed that PLEKHA7 regulates cellular behavior via miRNAs by associating with the microprocessor complex at the apical zonula adherens (ZA). We have also identified a new set of PLEKHA7 interacting partners at the apical ZA, via proteomics. Our analysis shows that the main groups of proteins associating with PLEKHA7 are cytoskeletal-related and RNA-binding proteins. Here, we provide extended evidence for association of PLEKHA7 with several of these proteins. We also show that PLEKHA7 loss activates the actin regulator cofilin in a p120-dependent manner, providing an explanation for the effects of PLEKHA7 on the cortical actin ring. Interestingly, PLEKHA7 regulates the levels and associates with PP1α, a phosphatase responsible for cofilin activation. Finally, we clarify the mode of regulation of the oncogenic miR-19a by PLEKHA7. Overall, our findings support a multi-layered role of PLEKHA7 in converging cytoskeletal dynamics and miRNA-mediated growth regulation at the ZA, with potentially critical implications in cancer that warrant further investigation.

PLEKHA7 is a junctional protein implicated in stabilization of the cadherin protein complex, hypertension, cardiac contractility, glaucoma, microRNA processing, and susceptibility to bacterial toxins. To gain insight into the molecular basis for the functions of PLEKHA7, we looked for new PLEKHA7 interactors. Here, we report the identification of PDZ domain-containing protein 11 (PDZD11) as a new interactor of PLEKHA7 by yeast two-hybrid screening and by mass spectrometry analysis of PLEKHA7 immunoprecipitates. We show that PDZD11 (17 kDa) is expressed in epithelial and endothelial cells, where it forms a complex with PLEKHA7, as determined by co-immunoprecipitation analysis. The N-terminal Trp-Trp (WW) domain of PLEKHA7 interacts directly with the N-terminal 44 amino acids of PDZD11, as shown by GST-pulldown assays. Immunofluorescence analysis shows that PDZD11 is localized at adherens junctions in a PLEKHA7-dependent manner, because its junctional localization is abolished by knock-out of PLEKHA7, and is rescued by re-expression of exogenous PLEKHA7. The junctional recruitment of nectin-1 and nectin-3 and their protein levels are decreased via proteasome-mediated degradation in epithelial cells where either PDZD11 or PLEKHA7 have been knocked-out. PDZD11 forms a complex with nectin-1 and nectin-3, and its PDZ domain interacts directly with the PDZ-binding motif of nectin-1. PDZD11 is required for the efficient assembly of apical junctions of epithelial cells at early time points in the calcium-switch model. These results show that the PLEKHA7-PDZD11 complex stabilizes nectins to promote efficient early junction assembly and uncover a new molecular mechanism through which PLEKHA7 recruits PDZ-binding membrane proteins to epithelial adherens junctions.

Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7(-/-) mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.

PLEKHA7 is a junctional protein, which participates in a complex that stabilizes E-cadherin at the zonula adhaerens. Since E-cadherin is involved in epithelial morphogenesis, signaling, and tumor progression, we explored PLEKHA7 expression in cancer. PLEKHA7 expression was assessed in invasive ductal and lobular carcinomas of the breast by immunohistochemistry, immunofluorescence and quantitative RT-PCR. PLEKHA7 was detected at epithelial junctions of normal mammary ducts and lobules, and of tubular and micropapillary structures within G1 and G2 ductal carcinomas. At these junctions, the localization of PLEKHA7 was along the circumferential belt (zonula adhaerens), and only partially overlapping with that of E-cadherin, p120ctn and ZO-1, as shown previously in rodent tissues. PLEKHA7 immunolabeling was strongly decreased in G3 ductal carcinomas and undetectable in lobular carcinomas. PLEKHA7 mRNA was detected in both ductal and lobular carcinomas, with no observed correlation between mRNA levels and tumor type or grade. In summary, PLEKHA7 is a junctional marker of epithelial cells within tubular structures both in normal breast tissue and ductal carcinomas, and since PLEKHA7 protein but not mRNA expression is strongly decreased or lost in high grade ductal carcinomas and in lobular carcinomas, loss of PLEKHA7 is a newly characterized feature of these carcinomas.

For more than a century, Staphylococcus aureus α-toxin, also known as α-hemolysin or Hla, has been recognized as an important virulence factor (1). α-Toxin exhibits many roles in the virulence of this formidable pathogen. In the past few years, our understanding of these roles has increased immensely. This wave of discovery began with the identification of α-toxin as a critical determinant of community-associated methicillin-resistant S. aureus pneumonia (2, 3). These studies were then followed by the identification of the metalloprotease a disintegrin and metalloprotease 10 (ADAM10) as the receptor for α-toxin (4), and the discovery that α-toxin targeting of ADAM10 has a critical role in the pathogenesis of S. aureus pneumonia and skin infection (5, 6), through the disruption of epithelial barriers. These studies demonstrated that α-toxin disrupts barriers not only by lysing cells but also by the more subtle mechanism of activating ADAM10 to cleave the ectodomains of E-cadherin molecules, breaking adherens junctions and causing dramatic changes of the actin cytoskeleton (4–6). The pathogenic functions of α-toxin have been further extended to include several roles in sepsis, including disruption of endothelial barriers and interactions with platelets and leukocytes (7, 8). Further, α-toxin interacts with the development of adaptive immunity and has a role in recurrent skin and soft tissue infections (9). In PNAS, Popov et al. (10) deepen our understanding of the interaction between α-toxin and cellular junctions, finding that several components of the adherens junctions, including pleckstrin-homology domain containing protein 7 (PLEKHA7), are necessary for cell death upon intoxication with this toxin.

The pleckstrin-homology-domain-containing protein PLEKHA7 was recently identified as a protein linking the E-cadherin-p120 ctn complex to the microtubule cytoskeleton. Here we characterize the expression, tissue distribution and subcellular localization of PLEKHA7 by immunoblotting, immunofluorescence microscopy, immunoelectron microscopy, and northern blotting in mammalian tissues. Anti-PLEKHA7 antibodies label the junctional regions of cultured kidney epithelial cells by immunofluorescence microscopy, and major polypeptides of M(r) approximately 135 kDa and approximately 145 kDa by immunoblotting of lysates of cells and tissues. Two PLEKHA7 transcripts ( approximately 5.5 kb and approximately 6.5 kb) are detected in epithelial tissues. PLEKHA7 is detected at epithelial junctions in sections of kidney, liver, pancreas, intestine, retina, and cornea, and its tissue distribution and subcellular localization are distinct from ZO-1. For example, PLEKHA7 is not detected within kidney glomeruli. Similarly to E-cadherin, p120 ctn, beta-catenin and alpha-catenin, PLEKHA7 is concentrated in the apical junctional belt, but unlike these adherens junction markers, and similarly to afadin, PLEKHA7 is not localized along the lateral region of polarized epithelial cells. Immunoelectron microscopy definitively establishes that PLEKHA7 is localized at the adherens junctions in colonic epithelial cells, at a mean distance of 28 nm from the plasma membrane. In summary, we show that PLEKHA7 is a cytoplasmic component of the epithelial adherens junction belt, with a subcellular localization and tissue distribution that is distinct from that of ZO-1 and most AJ proteins, and we provide the first description of its distribution and localization in several tissues.

PLEKHA7 is a recently identified protein of the epithelial zonula adhaerens (ZA), and is part of a protein complex that stabilizes the ZA, by linking it to microtubules. Since the ZA is important in the assembly and disassembly of tight junctions (TJ), we asked whether PLEKHA7 is involved in modulating epithelial TJ barrier function. We generated clonal MDCK cell lines in which one of four different constructs of PLEKHA7 was inducibly expressed. All constructs were localized at junctions, but constructs lacking the C-terminal region were also distributed diffusely in the cytoplasm. Inducible expression of PLEKHA7 constructs did not affect the expression and localization of TJ proteins, the steady-state value of transepithelial resistance (TER), the development of TER during the calcium switch, and the flux of large molecules across confluent monolayers. In contrast, expression of three out of four constructs resulted both in enhanced recruitment of E-cadherin and associated proteins at the apical ZA and at lateral puncta adherentia (PA), a decreased TER at 18 h after assembly at normal calcium, and an attenuation in the fall in TER after extracellular calcium removal. This latter effect was inhibited when cells were treated with nocodazole. Immunoprecipitation analysis showed that PLEKHA7 forms a complex with the cytoplasmic TJ proteins ZO-1 and cingulin, and this association does not depend on the integrity of microtubules. These results suggest that PLEKHA7 modulates the dynamics of assembly and disassembly of the TJ barrier, through E-cadherin protein complex- and microtubule-dependent mechanisms.

KRAS harbors oncogenic mutations that yield a constitutively active protein in approximately 40% of colorectal cancer. Randomized clinical trials have indicated that the wild-type KRAS (wt-KRAS) is predictive for resistance to anti-EGFR therapy and there is currently no effective therapy for mutant KRAS (mut-KRAS) tumors that is associated with a poor prognosis. Therefore, mut-KRAS tumors are an unmet medical need in colorectal and other cancers. In a search for target genes that when down-regulated selectively inhibit the growth of cells with mut-KRAS but not the cells without mut-KRAS, we used a global synthetic lethal small interfering RNA screen of MiaPaCa-2 and HCT-116 cells (mut-KRAS). The screen identified the gene encoding PLEKHA7 (pleckstrin homology (PH) domain containing, family A member 7). Other PLEKHA family members did not show this effect. PLEKHA7 is a component of the epithelial adherens junction belt that links E-cadherin to the cytoskeleton, and through which KRAS may signal its effects on cell proliferation, invasion and metastasis. PLEKHA7 and KRAS levels were measured by RT-PCR in a panel of 23 colon cancer cell lines (10 wt-KRAS and 13 mut-KRAS) and showed a positive correlation with KRAS levels (R2 = 0.943), and a Wilcoxon Rank test showed that the median expression of PLEKHA7 was higher in cell lines with mut-KRAS than with wt-KRAS (P=0.02). Targeted knockdown of PLEKHA7 using siRNA resulted in growth inhibition for mut-KRAS cells (HCT116, DLD-1, SK-CO-1, SW480, HCT15, and LOVO), but not for wt-KRAS cells (SW48, CACO-2, SW1417, and HT29). Paradoxically, targeted knock-down of KRAS caused an increase in PLEKHA7 mRNA levels in mut-KRAS cells. PLEKHA7 contains a PH-domain in a three-dimensional protein fold that binds to plasma membrane phosphoinositide-3-phosphates (PIP3) formed by phosphatidyl-3-phosphate kinase. We have previously shown that PH-domains can be selectively targeted by small molecules, blocking the binding to PIP3, and inhibiting the function of PH-domain proteins such as Akt and PDPK1. The findings of the present study imply that PLEKHA7 offers a potentially druggable target for inhibiting mut-KRAS-related signaling and cancer-related functions, providing an approach for inhibiting cancer progression of colorectal cancer with KRAS mutation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A199.

We previously identified PLEKHA7 and other junctional proteins as host factors mediating death by S. aureus α-toxin, but the mechanism through which junctions promote toxicity was unclear. Using cell biological and biochemical methods, we now show that ADAM10 is docked to junctions by its transmembrane partner Tspan33, whose cytoplasmic C terminus binds to the WW domain of PLEKHA7 in the presence of PDZD11. ADAM10 is locked at junctions through binding of its cytoplasmic C terminus to afadin. Junctionally clustered ADAM10 supports the efficient formation of stable toxin pores. Instead, disruption of the PLEKHA7-PDZD11 complex inhibits ADAM10 and toxin junctional clustering. This promotes toxin pore removal from the cell surface through an actin- and macropinocytosis-dependent process, resulting in cell recovery from initial injury and survival. These results uncover a dock-and-lock molecular mechanism to target ADAM10 to junctions and provide a paradigm for how junctions regulate transmembrane receptors through their clustering.

PLEKHA5, PLEKHA6, and PLEKHA7 (WW-PLEKHAs) are members of the PLEKHA family of proteins that interact with PDZD11 through their tandem WW domains. WW-PLEKHAs contribute to the trafficking and retention of transmembrane proteins, including nectins, Tspan33, and the copper pump ATP7A, at cell-cell junctions and lateral membranes. However, the structural basis for the distinct subcellular localizations of PLEKHA5, PLEKHA6, and PLEKHA7 is not clear. Here we expressed mutant and chimeric proteins of WW-PLEKHAs in cultured cells to clarify the role of their structural domains in their localization. We found that the WW-mediated interaction between PLEKHA5 and PDZD11 is required for their respective association with cytoplasmic microtubules. The PH domain of PLEKHA5 is required for its localization along the lateral plasma membrane and promotes the lateral localization of PLEKHA7 in a chimeric molecule. Although the PH domain of PLEKHA7 is not required for its localization at the adherens junctions (AJ), it promotes a AJ localization of chimeric proteins. The C-terminal region of PLEKHA6 and PLEKHA7 and the coiled-coil region of PLEKHA7 promote their localization at AJ of epithelial cells. These observations indicate that the localizations of WW-PLEKHAs at specific subcellular sites, where they recruit PDZD11, are the result of multiple cooperative protein-lipid and protein-protein interactions and provide a rational basis for the identification of additional proteins involved in trafficking and sorting of WW-PLEKHAs.