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 extracellular calcium can affect dramatically the assembly of tight junctions (TJ) and the localization of the TJ protein cingulin at sites of 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 agonist phorbol myristate acetate (PMA) on the intracellular localization of cingulin, E-cadherin, desmoplakin and actin microfilaments in confluent MDCK monolayers. To study cingulin phosphorylation, MDCK cells were metabolically labelled with [32P]orthophosphate and immunoprecipitates were prepared with anti-cingulin antiserum. We show here that cingulin is phosphorylated in vivo on serine, and its specific phosphorylation is not significantly changed by treatment of confluent MDCK monolayers with PMA, with the protein kinase inhibitor H-7, or with the calcium chelator EGTA. Metabolic labeling with a pulse of [35S]methionine/cysteine showed that at normal extracellular calcium net cingulin biosynthesis was not affected by PMA or H-7. During junction assembly by calcium switch, H-7 did not change the specific phosphorylation of the 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 processes that lead to the accumulation and stabilization of TJ proteins at sites of cell-cell contact.