Casein kinase 2 (CK2) phosphorylates occludin at Ser408 to increase intra‐tight junction diffusion and reduce paracellular barrier function

CK2 phosphorylates the carboxy‐terminal cytoplasmic tail of the tight junction protein occludin. We sought to determine the consequences of CK2‐mediated occludin phosphorylation and identify critical target residues(s). The CK2 inhibitors TBB and DMAT each elevated transepithelial resistance (TER) of Caco‐2 monolayers. This regulation was CK2‐ and occludin‐dependent, as it did not occur after siRNA knockdown (KD) of either protein. To assess the functional relevance of previously identified CK2 targets Thr404 and Ser408, wild type or T408D/S408D occludin was expressed in occludin KD cell lines. Both constructs restored TER, which was reduced by occludin KD, but only wild type occludin re‐established the TER response to CK2 inhibition. Fluorescence recovery after photobleaching (FRAP) showed a reduced occludin mobile fraction after either chemical‐ or siRNA‐mediated CK2 inhibition, which was associated with increased occludin density at the tight junction. DMAT similarly reduced occludin FRAP in vivo. In vitro, T404A, T404D, and S408D occludin mutants had FRAP behavior indistinguishable from wild type occludin, but S408D was unaffected by CK2 inhibition. Conversely, S408A occludin FRAP at the tight junction was reduced, relative to wild type, and was unaffected by CK2 inhibition. Thus, CK2‐mediated phosphorylation of occludin S408 accelerates tight junction remodeling and increases permeability.