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

Max J Dörfel1, Julie K Westphal, Christian Bellmann

  • 1Institute of Biochemistry II, Jena University Hospital, Friedrich-Schiller-University Jena, Nonnenplan 2, 07743 Jena, Germany. otmar.huber@mti.uni-jena.de.

Cell Communication and Signaling : CCS
|June 14, 2013
PubMed
Abstract

Insights

Casein kinase 2 (CK2) phosphorylation of occludin at amino acids 400-408 regulates epithelial tight junction (TJ) assembly and barrier function. This phosphorylation impacts occludin

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Casein kinase 2 (CK2) is a kinase regulating cell proliferation and transformation.
  • CK2 influences epithelial cell-cell contacts by targeting adherens and tight junctions (TJs).
  • Occludin, a TJ protein, is a known substrate of CK2, but the functional impact of its phosphorylation was unclear.

Purpose of the Study:

  • To investigate the functional consequences of CK2-dependent occludin phosphorylation on TJ assembly and barrier properties.
  • To identify the specific phosphorylation sites in occludin targeted by CK2.
  • To elucidate how occludin phosphorylation affects its interactions with TJ-associated proteins.

Main Methods:

  • Site-directed mutagenesis to create occludin phosphorylation mutants (T400A/T404A/S408A) and phospho-mimetic mutants (T400E/T404E/S408E).
  • Co-immunoprecipitation assays to assess binding of occludin to ZO-1 and ZO-2.
  • Ca2+-switch experiments to monitor TJ reassembly.
  • Stable expression of occludin variants in MDCK C11 cells and measurement of transepithelial electrical resistance (TER) using two-path impedance spectroscopy.

Main Results:

  • Phosphorylation of a Thr400-XXX-Thr404-XXX-Ser408 motif in occludin's C-terminal tail regulates TJ assembly/disassembly and barrier function.
  • A phospho-mimetic occludin mutant (Occ-T400E/T404E/S408E) showed impaired binding to ZO-2, while binding to ZO-1 was unaffected.
  • The phospho-mimetic mutant exhibited delayed TJ reassembly and augmented paracellular resistance, indicating enhanced barrier properties.
  • Mutating the phosphorylation sites (T400A/T404A/S408A) did not affect TJ reassembly or barrier function compared to wild-type occludin.

Conclusions:

  • CK2 plays a significant role in regulating epithelial tight junction function through occludin phosphorylation.
  • The occludin motif (amino acids 400-408) is a critical site for Ser/Thr-kinase phosphorylation.
  • Phosphorylation at this motif differentially modulates TJ functional properties, impacting barrier integrity.

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