PHOSPHORYLATION OF THE TIGHT JUNCTION PROTEIN CINGULIN AND THE EFFECTS OF PROTEIN-KINASE INHIBITORS AND ACTIVATORS IN MDCK EPITHELIAL-CELLS

In previous studies we have shown that protein kinase inhibitors and e xtracellular calcium can affect dramatically the assembly of tight jun ctions (TJ) and the localization of the TJ protein cingulin at sites o f cell-cell contact in renal epithelial (MDCK) cells. To characterize in more detail the relationships between kinase activity and junction organization, we have studied the effects of the protein kinase C agon ist phorbol myristate acetate (PMA) on the intracellular localization of cingulin, E-cadherin, desmoplakin and actin microfilaments in confl uent MDCK monolayers. To study cingulin phosphorylation, MDCK cells we re metabolically labelled with [P-32]orthophosphate and immunoprecipit ates were prepared with anti-cingulin antiserum. We show here that cin gulin is phosphorylated in vivo on serine, and its specific phosphoryl ation is not significantly changed by treatment of confluent MDCK mono layers with PMA, with the protein kinase inhibitor H-7, or with the ca lcium chelator EGTA. Metabolic labeling with a pulse of [S-35]methioni ne/cysteine showed that at normal extracellular calcium net cingulin b iosynthesis was not affected by PMA or H-7. During junction assembly b y calcium switch, H-7 did not change the specific phosphorylation of t he immunoprecipitated cingulin, however, it prevented the increase in the amount of cingulin in the immunoprecipitates, suggesting that H-7 may block tight junction assembly by interfering with cellular process es that lead to the accumulation and stabilization of TJ proteins at s ites of cell-cell contact.