ANALYSIS OF THE DISTRIBUTION AND PHOSPHORYLATION STATE OF ZO-1 IN MDCK AND NONEPITHELIAL CELLS

We have examined the distribution and extent of phosphorylation of the tight junction-associated protein ZO-1 in the epithelial MDCK cell li ne, and in three cell types that do not form tight junctions: S180 (sa rcoma) cells, S180 cells transfected with E-cadherin (S180L), and prim ary cultures of astrocytes. In shortterm calcium chelation experiments on MDCK cells, removal of extracellular calcium caused cells to pull apart. However, ZO-1 remained concentrated at the plasma membrane and no change in ZO-1 phosphorylation was observed. Maintenance of MDCK ce lls in low calcium medium, conditions where no tight junctions are fou nd, resulted in altered ZO-1 distribution and lower total phosphorylat ion of the protein. In S180 cells, ZO-1 was diffusely distributed alon g the entire cell surface, with concentration of the antigen in motile regions of the cell. Cell-cell contact was not a prerequisite for ZO- 1 localization at the plasma membrane in this cell type, and the phosp hate content of ZO-1 was found to be lower in S180 cells relative to M DCK cells. Expression of E-cadherin in S180L cells did not alter eithe r the distribution or phosphorylation of ZO-1. In contrast to S180 cel ls, ZO-1 in primary cultures of astrocytes was concentrated at sites o f cell-cell contact, and the phosphorylation state was the same as tha t in control MDCK cells. Comparison of one-dimensional proteolytic dig ests of P-32-labeled ZO-1 revealed the phosphorylation of two peptides in control MDCK cells that was absent in both MDCK cells grown in low calcium and in S180 cells.