Activation of the Ca²⁺sensing receptor and the PKC/WNK4 downstream signaling cascade induces incorporation of ZO-2 to tight junctions and its separation from 14-3-3

Zonula occludens-2 (ZO-2) is a tight junction (TJ) cytoplasmic protein, whose localization varies according to cell density and Ca2+in the media. In cells cultured in low calcium (LC), ZO-2 displays a diffuse cytoplasmic distribution, but activation of the Ca2+sensing receptor (CaSR) with Gd3+triggers the appearance of ZO-2 at the cell borders. CaSR downstream signaling involves activation of protein kinase C, which phosphorylates and activates with no lysine kinase-4 that phosphorylates ZO-2 inducing its concentration at TJs. In LC, ZO-2 is protected from degradation by association to 14-3-3 proteins. When monolayers are transferred to normal calcium, the complexes ZO-2/14-3-3ζ and ZO-2/14-3-3σ move to the cell borders and dissociate. The 14-3-3 proteins are then degraded in proteosomes, whereas ZO-2 integrates to TJs. From the plasma membrane residual ZO-2 is endocyted and degradaded in lysosomes. The unique region 2 of ZO-2, and S261 located within a nuclear localization signal, are critical for the interaction with 14-3-3 ζ and σ and for the efficient nuclear importation of ZO-2. These results explain the molecular mechanism through which extracellular Ca2+triggers the appearance of ZO-2 at TJs in epithelial cells and reveal the novel interaction between ZO-2 and 14-3-3 proteins, which is critical for ZO-2 protection and intracellular traffic.