Howarth A G, Singer K L, Stevenson B R
Department of Anatomy and Cell Biology, University of Alberta, Edmonton, Canada.
J Membr Biol. 1994 Feb;137(3):261-70. doi: 10.1007/BF00232594.
We have examined the distribution and extent of phosphorylation of the tight junction-associated protein ZO-1 in the epithelial MDCK cell line, and in three cell types that do not form tight junctions: S180 (sarcoma) cells, S180 cells transfected with E-cadherin (S180L), and primary cultures of astrocytes. In short-term 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 cells in low calcium medium, conditions where no tight junctions are found, resulted in altered ZO-1 distribution and lower total phosphorylation of the protein. In S180 cells, ZO-1 was diffusely distributed along 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 phosphate content of ZO-1 was found to be lower in S180 cells relative to MDCK cells. Expression of E-cadherin in S180L cells did not alter either the distribution or phosphorylation of ZO-1. In contrast to S180 cells, ZO-1 in primary cultures of astrocytes was concentrated at sites of cell-cell contact, and the phosphorylation state was the same as that in control MDCK cells. Comparison of one-dimensional proteolytic digests of 32P-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.
我们研究了紧密连接相关蛋白ZO-1在上皮性MDCK细胞系以及三种不形成紧密连接的细胞类型中的磷酸化分布及程度，这三种细胞类型分别是：S180（肉瘤）细胞、转染了E-钙黏蛋白的S180细胞（S180L）以及星形胶质细胞原代培养物。在对MDCK细胞进行的短期钙螯合实验中，去除细胞外钙会导致细胞分离。然而，ZO-1仍集中在质膜上，且未观察到ZO-1磷酸化的变化。将MDCK细胞维持在低钙培养基中（即不存在紧密连接的条件下），会导致ZO-1分布改变以及该蛋白的总磷酸化水平降低。在S180细胞中，ZO-1沿整个细胞表面呈弥散分布，抗原在细胞的运动区域聚集。在这种细胞类型中，细胞间接触并非ZO-1定位于质膜的先决条件，并且发现S180细胞中ZO-1的磷酸盐含量相对于MDCK细胞更低。E-钙黏蛋白在S180L细胞中的表达并未改变ZO-1的分布或磷酸化情况。与S180细胞不同，星形胶质细胞原代培养物中的ZO-1集中在细胞间接触部位，其磷酸化状态与对照MDCK细胞相同。对32P标记的ZO-1进行一维蛋白酶消化后的比较显示，对照MDCK细胞中有两个肽段发生了磷酸化，而在低钙条件下培养的MDCK细胞和S180细胞中均未出现这种情况。
