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CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity.

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.

SUBMITTER: Dorfel MJ

PROVIDER: S-EPMC3695765 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity.

Dörfel Max J MJ Westphal Julie K JK Bellmann Christian C Krug Susanne M SM Cording Jimmi J Mittag Sonnhild S Tauber Rudolf R Fromm Michael M Blasig Ingolf E IE Huber Otmar O

Cell communication and signaling : CCS 20130610 1

<h4>Background</h4>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.<h4>Results</h4>Here, we present evidence tha ...[more]

PMID: 23758859

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Project description:BACKGROUND & AIMS:Inflammatory bowel diseases (IBD) increase risk for colorectal cancer. Mutations in the Mediterranean fever gene (MEFV or pyrin) are associated with hereditary autoinflammatory disease and severe IBD. Expression of MEFV, a sensor protein that the initiates assembly of the inflammasome complex, is increased in colon biopsies from patients with IBD. We investigated the role of pyrin in intestinal homeostasis in mice. METHODS:Mefv-/- mice and C57/BL6 mice (controls) were given azoxymethane followed by multiple rounds of dextran sodium sulfate (DSS) to induce colitis and tumorigenesis. In some experiments, Mefv-/- mice were given injections of recombinant interleukin 18 (rIL18) or saline (control) during DSS administration. Colon tissues were collected at different time points during colitis development and analyzed by histology, immunohistochemistry, immunoblots, or ELISAs (to measure cytokines). Spleen and mesenteric lymph node were collected, processed, and analyzed by flow cytometry. Colon epithelial permeability was measured in mice with colitis by gavage of fluorescent dextran and quantification of serum levels. RESULTS:MEFV was expressed in colons of control mice and expression increased during chronic and acute inflammation; high levels were detected in colon tumor and adjacent non-tumor tissues. Mefv-/- mice developed more severe colitis than control mice, with a greater extent of epithelial hyperplasia and a larger tumor burden. Levels of inflammatory cytokines (IL6) and chemokines were significantly higher in colons of Mefv-/- mice than control mice following colitis induction, whereas the level IL18, which depends on the inflammasome for maturation and release, was significantly lower in colons of Mefv-/- mice. Mefv-/- mice had increased epithelial permeability following administration of DSS than control mice, and loss of the tight junction proteins occludin and claudin-2 from intercellular junctions. STAT3 was activated (phosphorylated) in inflamed colon tissues from Mefv-/-, which also had increased expression of stem cell markers (OLFM4, BMI1, and MSI1) compared with colons from control mice. Administration of rIL18 to Mefv-/- mice reduced epithelial permeability, intestinal inflammation, the severity of colitis, and colon tumorigenesis. CONCLUSIONS:In studies with DSS-induced colitis, we found that pyrin (MEFV) is required for inflammasome activation and IL18 maturation, which promote intestinal barrier integrity and prevent colon inflammation and tumorigenesis. Strategies to increase activity of MEFV or IL18 might be developed for the treatment of IBD and prevention of colitis-associated tumorigenesis.

Dataset Information

CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity.

Publications

CK2-dependent phosphorylation of occludin regulates the interaction with ZO-proteins and tight junction integrity.

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