POSSIBLE INVOLVEMENT OF PHOSPHORYLATION OF OCCLUDIN IN TIGHT JUNCTIONFORMATION

Occludin is an integral membrane protein localizing at tight junctions in epithelial and endothelial cells. Occludin from confluent culture MDCK I cells resolved as several (>10) bands between 62 and S2 kD in S DS-PAGE, of which two or three bands of the lowest M-r were predominan t. Among these bands, the lower predominant bands were essentially ext racted with 1% NP-40, whereas the other higher M-r bands were selectiv ely recovered in the NP-40-insoluble fraction. Alkaline phosphatase tr eatment converged these bands of occludin both in NP-40-soluble and -i nsoluble fractions into the lowest M-r band, and phosphoamino acid ana lyses identified phosphoserine (and phosphothreonine weakly) in the hi gher M-r bands of occludin. These findings indicated that phosphorylat ion causes an upward shift of occludin bands and that highly phosphory lated occludin resists NP-40 extraction. When cells were grown in low Ca medium, almost all occludin was NP-30 soluble. Switching from low t o normal Ca medium increased the amount of NP-40-insoluble occludin wi thin 10 min, followed by gradual upward shift of bands. This insolubil ization and the band shift correlated temporally with tight junction f ormation detected by immunofluorescence microscopy. Furthermore, we fo und that the anti-chicken occludin mAb, Oc-3, did not recognize the pr edominant lower M-r bands of occludin (non- or less phosphorylated for m) but was specific to the higher M-r bands (phosphorylated form) on i mmunoblotting. Immunofluorescence microscopy revealed that this mAb ma inly stained the tight junction proper of intestinal epithelial cells, whereas other anti-occludin mAbs, which can recognize the predominant lower M-r bands, labeled their basolateral membranes (and the cytopla sm) as well as tight junctions. Therefore, we conclude that non- or le ss phosphorylated occludin is distributed on the basolateral membranes and that highly phosphorylated occludin is selectively concentrated a t tight junctions as the NP-40-insoluble form. These findings suggest that the phosphorylation of occludin is a key step in tight junction a ssembly.