The tight junction protein ZO-2 associates with Jun, Fos and C/EBP transcription factors in epithelial cells

Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (CINVESTAV), México DF, 07000, Mexico.
Experimental Cell Research (Impact Factor: 3.25). 02/2004; 292(1):51-66. DOI: 10.1016/j.yexcr.2003.08.007
Source: PubMed


ZO-2 is a membrane-associated guanylate kinase (MAGUK) protein present at the tight junction (TJ) of epithelial cells. While confluent monolayers have ZO-2 at their cellular borders, sparse cultures conspicuously show ZO-2 at the nuclei. To study the role of nuclear ZO-2, we tested by pull-down assays and gel shift analysis the interaction between ZO-2 GST fusion proteins and different transcription factors. We identified the existence of a specific interaction of ZO-2 with Fos, Jun and C/EBP (CCAAT/enhancer binding protein). To analyze if this association is present "in vivo", we performed immunoprecipitation and immunolocalization experiments, which revealed an interaction of ZO-2 with Jun, Fos and C/EBP not only at the nucleus but also at the TJ region. To test if the association of ZO-2 with AP-1 (activator protein-1) modulates gene transcription, we performed reporter gene assays employing chloramphenicol acetyltransferase (CAT) constructs with promoters under the control of AP-1 sites. We observed that the co-transfected ZO-2 down-regulates CAT expression in a dose-dependent manner. Since ZO-2 is a multidomain protein, we proceeded to determine which region of the molecule is responsible for the modulation of gene expression, and observed that both the amino and the carboxyl domains are capable of inhibiting gene transcription.

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Available from: Esther Lopez-Bayghen
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    • "The cZO-2 amino-terminal segment containing PDZ1, PDZ2, and PDZ3 domains (nucleotides [nt] 398–2165) and the cZO-2 carboxyl-terminal (AP) segment containing the acidic and proline-rich regions (nt 3029– 3923) were introduced into the HisMaxB vector as reported previously (Betanzos et al., 2004; Jaramillo et al., 2004). The following point mutants derived from these constructs were made with the QuikChange Multisite-Directed Mutagenesis Kit (200513; Stratagene, Santa Clara, CA), according to the manufacturer's instructions: 1) pGW1- HA–ZO-2: S257E/S259E/S261E, S257A, and S257E and 2) HisMax-ZO-2-amino S257A. "
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    ABSTRACT: Zona Occludens 2 (ZO-2) has a dual localization. In confluent epithelia, ZO-2 is present at tight junctions (TJs) while in sparse proliferating cells is also found at the nucleus. Previously we demonstrated that in sparse cultures, newly synthesized ZO-2 travels to the nucleus before reaching the plasma membrane and now found that in confluent cultures newly synthesized ZO-2 goes directly to the plasma membrane. We observe that EGF induces through AKT activation the phosphorylation of the kinase for SR repeats SRPK1, which in turn phosphorylates ZO-2 that contains 16 SR repeats. This phosphorylation induces ZO-2 entry into the nucleus and accumulation in speckles. ZO-2 departure from the nucleus requires an intact S257 and stabilizing the β-O-linked N-acetylglucocylation (O-GlcNAc) of S257 with PUGNAc, an inhibitor of O-GlcNAcase, triggers nuclear exportation and proteosomal degradation of ZO-2. At the plasma membrane ZO-2 is not O-GlcNAc and instead as TJ mature, becomes phosphorylated at S257 by PKCζ. This late phosphorylation of S257 is required for the correct cytoarchitecture to develop as cells transfected with ZO-2 mutants S257A or S257E form aberrant cysts with multiple lumens. These results reveal novel posttranslational modifications of ZO-2 that regulate the intracellular fate of this protein.
    Full-text · Article · Jun 2013 · Molecular biology of the cell
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    • "In some experiments, actin was stained with TRITC-phalloidin and nuclei were labeled with DAPI for 5 min. After washing, cells were mounted using Vectashield (Vector) [32] and analysed by laser confocal microscopy (Leica TCS_SP5_MO). "
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    ABSTRACT: Entamoeba histolytica, the protozoan responsible for human amoebiasis, causes between 30,000 and 100,000 deaths per year worldwide. Amoebiasis is characterized by intestinal epithelial damage provoking severe diarrhea. However, the molecular mechanisms by which this protozoan causes epithelial damage are poorly understood. Here, we studied the initial molecular interactions between the E. histolytica EhCPADH112 virulence complex and epithelial MDCK and Caco-2 cells. By confocal microscopy, we discovered that after contact with trophozoites or trophozoite extracts (TE), EhCPADH112 and proteins forming this complex (EhCP112 and EhADH112) co-localize with occludin and claudin-1 at tight junctions (TJ). Immunoprecipitation assays revealed interaction between EhCPADH112 and occludin, claudin-1, ZO-1 and ZO-2. Overlay assays confirmed an interaction of EhCP112 and EhADH112 with occludin and claudin-1, whereas only EhADH112 interacted also with ZO-2. We observed degradation of all mentioned TJ proteins after incubation with TE. Importantly, inhibiting proteolytic activity or blocking the complex with a specific antibody not only prevented TJ protein degradation but also epithelial barrier disruption. Furthermore, we discovered that TE treatment induces autophagy and apoptosis in MDCK cells that could contribute to the observed barrier disruption. Our results suggest a model in which epithelial damage caused by E. histolytica is initiated by the interaction of EhCP112 and EhADH112 with TJ proteins followed by their degradation. Disruption of TJs then induces increased paracellular permeability, thus facilitating the entry of more proteases and other parasite molecules leading eventually to tissue destruction.
    Full-text · Article · Jun 2013 · PLoS ONE
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    • "Several nuclear localisation and export signals (NLS and NES) have been described for ZO-2 [9]. The nuclear localization of ZO proteins may be functionally linked to the description that ZO-1 and ZO-2 associate with proteins involved in the regulation of cell proliferation such as the transcription factors Jun, Fos, C/EBP (CCAAT/enhancer-binding protein) [10], ZONAB/DbpA (ZO-1 associated nucleic-acid-binding protein/DNA binding protein A) [11] and KyoT2 [12]. ZO-2 has also been shown to modulate AP-1- regulated gene transcription [13]. "
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    ABSTRACT: We have previously characterized ubinuclein (Ubn-1) as a NACos (Nuclear and Adherent junction Complex components) protein which interacts with viral or cellular transcription factors and the tight junction (TJ) protein ZO-1. The purpose of the present study was to get more insights on the binding partners of Ubn-1, notably those present in the epithelial junctions. Using an in vivo assay of fluorescent protein-complementation assay (PCA), we demonstrated that the N-terminal domains of the Ubn-1 and ZO-1 proteins triggered a functional interaction inside the cell. Indeed, expression of both complementary fragments of venus fused to the N-terminal parts of Ubn-1 and ZO-1 was able to reconstitute a fluorescent venus protein. Furthermore, nuclear expression of the chimeric Ubn-1 triggered nuclear localization of the chimeric ZO-1. We could localize this interaction to the PDZ2 domain of ZO-1 using an in vitro pull-down assay. More precisely, a 184-amino acid region (from amino acids 39 to 223) at the N-terminal region of Ubn-1 was responsible for the interaction with the PDZ2 domain of ZO-1. Co-imunoprecipitation and confocal microscopy experiments also revealed the tight junction protein cingulin as a new interacting partner of Ubn-1. A proteomic approach based on mass spectrometry analysis (MS) was then undertaken to identify further binding partners of GST-Ubn-1 fusion protein in different subcellular fractions of human epithelial HT29 cells. LYRIC (Lysine-rich CEACAM1-associated protein) and RACK-1 (receptor for activated C-kinase) proteins were validated as bona fide interacting partners of Ubn-1. Altogether, these results suggest that Ubn-1 is a scaffold protein influencing protein subcellular localization and is involved in several processes such as cell-cell contact signalling or modulation of gene activity.
    Full-text · Article · Mar 2012 · Experimental Cell Research
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