Farshori P, Kachar B
Section on Structural Cell Biology, National Institute on Deafness and Other Communication Disorders, National Institute of Health, Bethesda, MD 20892, USA.
J Membr Biol. 1999 Jul 15;170(2):147-56. doi: 10.1007/s002329900544.
We studied the expression, distribution, and phosphorylation of the tight junction (TJ) protein occludin in confluent MDCK cell monolayers following three procedures for opening and resealing of TJs. When Ca(2+) is transiently removed from the culture medium, the TJs open and the cells separate from each other, but the occludin band around each cell is retained. When Ca(2+) is reintroduced, the TJs reseal. When the monolayers are exposed to prolonged Ca(2+) starvation the cells maintain contact, but occludin disappears from the cell borders and can be detected only in a cytoplasmic compartment. When Ca(2+) is reintroduced, new TJs are assembled and the transepithelial electrical resistance (TER) is reestablished in about 20 hr. Monolayers treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) show a different pattern of TJ opening: the cell-cell contact is maintained but the TJ strand network, as seen in freeze-fracture replicas, becomes discontinuous. Occludin is still localized at the cell periphery, but in a pattern of distribution that matches the discontinuous TJ. These TJs do not reseal even 24 hr after removal of the TPA. Western blot analysis showed that the 62-65 kD double band of occludin did not change with these treatments. However, in vivo phosphorylation analysis showed that the TPA treatment reduced the phosphorylation levels of occludin, while the prolonged Ca(2+) starvation completely dephosphorylated the two occludin bands. In addition, a highly phosphorylated 71 kD band that immunoprecipitates with occludin is not present when TJ is opened by the Ca(2+) removal. Phosphoaminoacid analysis showed that the 62-65 kD occludin bands are phosphorylated on serine and threonine, while the 71 kD band was phosphorylated exclusively on serine. Our results provide further evidence that phosphorylation of occludin is an important step in regulating TJ formation and permeability.
我们研究了紧密连接（TJ）蛋白闭合蛋白在汇合的MDCK细胞单层中经三种TJ打开和重新封闭程序后的表达、分布及磷酸化情况。当从培养基中短暂去除Ca(2+)时，TJ打开，细胞彼此分离，但每个细胞周围的闭合蛋白条带得以保留。当重新引入Ca(2+)时，TJ重新封闭。当单层细胞暴露于长时间的Ca(2+)饥饿状态时，细胞保持接触，但闭合蛋白从细胞边界消失，仅能在细胞质区室中检测到。当重新引入Ca(2+)时，新的TJ组装形成，跨上皮电阻（TER）在约20小时内重新建立。用佛波酯12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）处理的单层细胞呈现出不同的TJ打开模式：细胞间接触得以维持，但如在冷冻蚀刻复制品中所见的TJ链网络变得不连续。闭合蛋白仍定位于细胞周边，但分布模式与不连续的TJ相匹配。即使在去除TPA后24小时，这些TJ也不会重新封闭。蛋白质免疫印迹分析表明，闭合蛋白的62 - 65 kD双条带在这些处理后没有变化。然而，体内磷酸化分析表明，TPA处理降低了闭合蛋白的磷酸化水平，而长时间的Ca(2+)饥饿使两条闭合蛋白条带完全去磷酸化。此外，当通过去除Ca(2+)打开TJ时，与闭合蛋白免疫沉淀的高度磷酸化的71 kD条带不存在。磷酸氨基酸分析表明，62 - 65 kD的闭合蛋白条带在丝氨酸和苏氨酸上磷酸化，而71 kD条带仅在丝氨酸上磷酸化。我们的结果进一步证明，闭合蛋白的磷酸化是调节TJ形成和通透性的重要步骤。
