title: 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.
authors.affiliation: Institute of Biochemistry II, Jena University Hospital, Friedrich-Schiller-University Jena, Nonnenplan 2, 07743 Jena, Germany. otmar.huber@mti.uni-jena.de.
authors.forename: Max J
authors.initials: MJ
authors.lastname: Dörfel
authors.forename: Julie K
authors.initials: JK
authors.lastname: Westphal
authors.forename: Christian
authors.initials: C
authors.lastname: Bellmann
authors.forename: Susanne M
authors.initials: SM
authors.lastname: Krug
authors.forename: Jimmi
authors.initials: J
authors.lastname: Cording
authors.forename: Sonnhild
authors.initials: S
authors.lastname: Mittag
authors.forename: Rudolf
authors.initials: R
authors.lastname: Tauber
authors.forename: Michael
authors.initials: M
authors.lastname: Fromm
authors.forename: Ingolf E
authors.initials: IE
authors.lastname: Blasig
authors.forename: Otmar
authors.initials: O
authors.lastname: Huber