Phosphorylation of occludin correlates with occludin localization and function at the tight junction

Multiple forms of occluding were found in Madin-Darby canine kidney (MDCK) cells. In the absence of cell-to-cell contacts, achieved by incubating cells in low-calcium growth medium, a cluster of lower-molecular-weight (LMW) occluding bands (similar to 65,000-68,000) was present in both MDCK I and II cells. On formation of tight junctions, achieved by changing the low-calcium growth medium to normal-calcium growth medium, a cluster of higher-molecular-weight (HMW) bands (similar to 72,000-75,000 for MDCK I cells and similar to 70,000-73,000 for MDCK II cells) was also expressed. The HMW occluding bands could be eliminated by phosphatase treatment. Therefore, the HMW forms of occluding appeared to be the hyperphosphorylated product of the LMW forms. These HMW forms were Triton X-100 insoluble, which correlated with their localization at the tight junctions. Furthermore, depletion of tight junction-localized occluding by an occluding extracellular domian peptide (20) correlated with a decrease in the HMW forms of occluding. In conclusion, phosphorylation of occluding may be a mechanism by which occluding localization and function are regulated.