Possible Involvement of Phosphorylation of Occludin in Tight Junction Formation

Abstract

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 82 kD in SDS-PAGE, of which two or three bands of the lowest M r were predominant. Among these bands, the lower predominant bands were essentially extracted with 1% NP-40, whereas the other higher M r bands were selectively recovered in the NP-40–insoluble fraction. Alkaline phosphatase treatment converged these bands of occludin both in NP-40–soluble and -insoluble fractions into the lowest M r band, and phosphoamino acid analyses identified phosphoserine (and phosphothreonine weakly) in the higher M r bands of occludin. These findings indicated that phosphorylation causes an upward shift of occludin bands and that highly phosphorylated occludin resists NP-40 extraction. When cells were grown in low Ca medium, almost all occludin was NP-40 soluble. Switching from low to normal Ca medium increased the amount of NP-40–insoluble occludin within 10 min, followed by gradual upward shift of bands. This insolubilization and the band shift correlated temporally with tight junction formation detected by immunofluorescence microscopy. Furthermore, we found that the anti–chicken occludin mAb, Oc-3, did not recognize the predominant lower M r bands of occludin (non- or less phosphorylated form) but was specific to the higher M r bands (phosphorylated form) on immunoblotting. Immunofluorescence microscopy revealed that this mAb mainly 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 cytoplasm) as well as tight junctions. Therefore, we conclude that non- or less phosphorylated occludin is distributed on the basolateral membranes and that highly phosphorylated occludin is selectively concentrated at tight juctions as the NP-40–insoluble form. These findings suggest that the phosphorylation of occludin is a key step in tight junction assembly.

Tight junction properties of the immortalized human bronchial epithelial cell lines Calu‐3 and 16HBE14o‐

“…The most abundant of the forms had an apparent molecular mass of 62±66 kDa, close to the predicted molecular mass of the unmodified protein. Recent studies have suggested that occludin may undergo function-dependent modifications and that it may be the "heavy", possibly phosphorylated, forms of occludin that seal TJs [33,34]. The human airway cell lines expressed heavy forms of occludin (bands of~72±74 kDa were detectable in both cell lines with a protein of 94 kDa additionally present in Calu-3 cells), although the authors are unable to comment on their phosphorylation status.…”

Phosphorylation of claudin-4 by PKCε regulates tight junction barrier function in ovarian cancer cells

“…Interestingly, several studies have demonstrated the involvement of various kinases in the phosphorylation and regulation of claudin proteins [29][30][31][32][33][34][35][36][37], and we have recently shown that phosphorylation of claudin-3 by PKA can affect TJ properties in ovarian cancer cells [38]. Protein kinase C (PKC) isoforms are present in ovarian cancer and are known to modulate TJ function by phosphorylation of the proteins in the complex [24,34,[39][40][41][42][43], but it is unclear whether PKC can directly phosphorylate and regulate claudin proteins. We have previously shown that claudin-4 can be phosphorylated in ovarian cancer cells upon 12-OTetradecanoylophorbol-13-acetate (TPA) stimulation [38], but the exact PKC isoforms involved, the phosphorylation sites on claudin-4, and the consequences of this phosphorylation have remained unknown.…”

Cholesterol depletion alters detergent-specific solubility profiles of selected tight junction proteins and the phosphorylation of occludin

“…4. Test for the possibility that the early increase in TER, observed after addition of MBCD, might be associated with an increase of phosphorylated occludin, which has previously been shown to be present in epithelia with high, but not low TER [16].…”