The occludin and ZO-1 complex, defined by small angle X-ray scattering and NMR, has implications for modulating tight junction permeability

Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, H171, 500 University Drive, Hershey, PA 17033, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/2012; 109(27):10855-60. DOI: 10.1073/pnas.1121390109
Source: PubMed


Tight junctions (TJs) are dynamic cellular structures that are critical for compartmentalizing environments within tissues and regulating transport of small molecules, ions, and fluids. Phosphorylation-dependent binding of the transmembrane protein occludin to the structural organizing protein ZO-1 contributes to the regulation of barrier properties; however, the details of their interaction are controversial. Using small angle X-ray scattering (SAXS), NMR chemical shift perturbation, cross-saturation, in vitro binding, and site-directed mutagenesis experiments. we define the interface between the ZO-1 PDZ3-SH3-U5-GuK (PSG) and occludin coiled-coil (CC) domains. The interface is comprised of basic residues in PSG and an acidic region in CC. Complex formation is blocked by a peptide (REESEEYM) that corresponds to CC residues 468-475 and includes a previously uncharacterized phosphosite, with the phosphorylated version having a larger effect. Furthermore, mutation of E470 and E472 reduces cell border localization of occludin. Together, these results localize the interaction to an acidic region in CC and a predominantly basic helix V within the ZO-1 GuK domain. This model has important implications for the phosphorylation-dependent regulation of the occludin:ZO-1 complex.

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    • "This is also documented by an enhanced TX-100 insolubility of the phospho-mimetic Occ-T400E/T404E/S408E protein. According to a recent study by Tash et al. defining the primary ZO-1 binding site within residues 468–475 of occludin, CK2-dependent phosphorylation of occludin cannot exert its effects on complex formation directly on this primary interaction site but may act on a postulated secondary site [45]. Interestingly, when we analyzed the binding of ZO-2 protein in pull-down and co-immunoprecipitation experiments, association of ZO-2 was significantly attenuated in the phospho-mimetic Occ-T400E/T404E/S408E-transfected cells, whereas in Occ-T400A/T404A/S408A-transfected cells a minor but not significant increase in ZO-2 binding was detectable compared to wildtype occludin. "
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    ABSTRACT: Background Casein kinase 2 (CK2) is a ubiquitously expressed Ser/Thr kinase with multiple functions in the regulation of cell proliferation and transformation. In targeting adherens and tight junctions (TJs), CK2 modulates the strength and dynamics of epithelial cell-cell contacts. Occludin previously was identified as a substrate of CK2, however the functional consequences of CK2-dependent occludin phosphorylation on TJ function were unknown. Results Here, we present evidence that phosphorylation of a Thr400-XXX-Thr404-XXX-Ser408 motif in the C-terminal cytoplasmic tail of human occludin regulates assembly/disassembly and barrier properties of TJs. In contrast to wildtype and T400A/T404A/S408A-mutated occludin, a phospho-mimetic Occ-T400E/T404E/S408E construct was impaired in binding to ZO-2. Interestingly, pre-phosphorylation of a GST-Occ C-terminal domain fusion protein attenuated binding to ZO-2, whereas, binding to ZO-1 was not affected. Moreover, Occ-T400E/T404E/S408E showed delayed reassembly into TJs in Ca2+-switch experiments. Stable expression of Occ-T400E/T404E/S408E in MDCK C11 cells augments barrier properties in enhancing paracellular resistance in two-path impedance spectroscopy, whereas expression of wildtype and Occ-T400A/T404A/S408A did not affect transepithelial resistance. Conclusions These results suggest an important role of CK2 in epithelial tight junction regulation. The occludin sequence motif at amino acids 400–408 apparently represents a hotspot for Ser/Thr-kinase phosphorylation and depending on the residue(s) which are phosphorylated it differentially modulates the functional properties of the TJ.
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    ABSTRACT: A comprehensive investigation of the morphology of human airway epithelial tight junctions was carried out by freeze-fracture electron microscopy using quantitative methods designed to analyze a range of junctional characteristics. Extrapulmonary bronchi that appeared grossly normal were taken at sites distant from tumor in lungs resected for pulmonary carcinoma. The absence of cellular atypia in the samples was confirmed by histology. Airway levels I (main bronchus; n = 7 subjects) and II (lobar bronchus; n = 5 subjects) were compared with respect to junctional depth, strand number, and junctional complexity. Junctional complexity was assessed by frequency of strand interconnection and numbers of strands per interconnection. Comparisons between airway levels I and II for these parameters showed that there were no significant differences in strand number or junctional complexity between the two airway levels. However, junctional depth was slightly but significantly reduced at level II compared with level I (P less than 0.01). The arrangement of strands varied considerably from one junction to the next, irrespective of the cell types involved. "Parallel" and "network" patterns of junctions were observed; the existence of gradations between these two patterns indicated that they represent opposite extremes of a single junctional form rather than distinct categories of junction. These results have allowed us to establish a data pool for normal human bronchi from which the structure of epithelial cell junctions in bronchial diseases can be compared.
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    ABSTRACT: Tight junctions (TJs) are protein complexes comprised of claudins, which anchor them in the membrane and numerous cytosolic scaffolding proteins including MAGI, MUPP1, cingulin and members of the Zonula Occludens (ZO) family. Originally, their main function was thought to be as a paracellular barrier. More recently, however, additional roles in signal transduction, differentiation and proliferation have been reported. Dysregulation is associated with a wide range of disease states, including diabetic retinopathy, irritable bowel disease and some cancers. ZO proteins and occludin form a protein complex that appears to act as a master regulator of TJ assembly/disassembly. Recent studies have highlighted the structural character of the primary ZO-1:occludin interaction and identified regions on occludin that control association and disassociation of TJ in a phosphorylation-dependent manner. We hypothesize that regions within ZO-1 in the so-called U5 and U6 regions behave in a similar manner.
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