Abstract
BackgroundCasein 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.ResultsHere, 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.ConclusionsThese 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.
Highlights
Casein kinase 2 (CK2) is a ubiquitously expressed Ser/Thr kinase with multiple functions in the regulation of cell proliferation and transformation
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 tight junctions (TJs)
Summary
Casein kinase 2 (CK2) is a ubiquitously expressed Ser/Thr kinase with multiple functions in the regulation of cell proliferation and transformation. Tight junctions (TJs) represent the most apical cell-cell contacts in epithelial and endothelial tissues and play a central role in the maintenance of tissue integrity. On the other hand the intracellular N- and C-terminal domains of these transmembrane proteins assemble TJ-associated proteins such as zonula occludens (ZO) proteins ZO-1, -2 and -3, 7H6, cingulin and symplekin which are essential for the association of TJs with the actin cytoskeleton and for assembly and maintenance of TJs [9] Some of these proteins reveal the typical dual function of Nacos (nuclear and adhesion complexes) proteins affecting adhesive activity and nuclear gene transcription [10]. Many TJ proteins are targets of protein kinases, which modulate assembly, stability and functional properties of TJs [4]
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