Regulated assembly of tight junctions by protein kinase C.
Regulated assembly of tight junctions by protein kinase C.
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DOI：
10.1073/pnas.92.13.6072
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发表时间：
1995-06
影响因子：
11.1
通讯作者：
R. O. Stuart;S. Nigam
中科院分区：
文献类型：
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作者：
R. O. Stuart;S. Nigam
我们之前已经证明蛋白质磷酸化在紧密连接的分类和组装中起重要作用。我们现在通过使用高度特异性和广谱抑制剂的组合，通过独立的机制，详细检查了蛋白激酶在细胞间连接生物发生中的作用。我们的数据表明，蛋白激酶C （PKC）是紧密连接的适当组装所必需的。低浓度的PKC特异性抑制剂calphostin C可显著抑制经上皮电阻的发展，这是紧密连接生物发生的功能指标。PKC抑制剂对紧密连接发展的影响，通过电阻来测量，与紧密连接蛋白，封闭带1 （ZO-1）对紧密连接的分选延迟相平行。免疫细胞化学分析表明，桥粒的组装和粘附体的连接没有明显的影响。此外，ZO-1在细胞-细胞接触开始后被磷酸化，用calphostin C处理可以阻止大约85%的磷酸化增加。此外，体外测量表明ZO-1可能是PKC的直接靶点。此外，在连接组装过程中，膜相关PKC活性增加了一倍以上，免疫细胞化学分析显示PKC zeta库似乎与ZO-1在紧密连接处共定位。在细胞-细胞接触和细胞-细胞接触缺失的情况下，通过共免疫沉淀检测到含有ZO-1、ZO-2、p130以及330和65 kda磷酸化蛋白的预形成复合物。330- kda和65-kDa磷酸化蛋白的身份仍有待确定，但65-kDa磷酸化蛋白可能是闭塞蛋白。该复合物的质量和ZO-1与Triton x -100不溶性细胞骨架的结合不依赖于PKC。
We have previously shown that protein phosphorylation plays an important role in the sorting and assembly of tight junctions. We have now examined in detail the role of protein kinases in intercellular junction biogenesis by using a combination of highly specific and broad-spectrum inhibitors that act by independent mechanisms. Our data indicate that protein kinase C (PKC) is required for the proper assembly of tight junctions. Low concentrations of the specific inhibitor of PKC, calphostin C, markedly inhibited development of transepithelial electrical resistance, a functional measure of tight-junction biogenesis. The effect of PKC inhibitors on the development of tight junctions, as measured by resistance, was paralleled by a delay in the sorting of the tight-junction protein, zona occludens 1 (ZO-1), to the tight junction. The assembly of desmosomes and the adherens junction were not detectably affected, as determined by immunocytochemical analysis. In addition, ZO-1 was phosphorylated subsequent to the initiation of cell-cell contact, and treatment with calphostin C prevented approximately 85% of the phosphorylation increase. Furthermore, in vitro measurements indicate that ZO-1 may be a direct target of PKC. Moreover, membrane-associated PKC activity more than doubled during junction assembly, and immunocytochemical analysis revealed a pool of PKC zeta that appeared to colocalize with ZO-1 at the tight junction. A preformed complex containing ZO-1, ZO-2, p130, as well as 330- and 65-kDa phosphoproteins was detected by coimmunoprecipitation in both the presence and absence of cell-cell contact. Identity of the 330- and 65-kDa phosphoproteins remains to be determined, but the 65-kDa protein may be occludin. The mass of this complex and the incorporation of ZO-1 into the Triton X-100-insoluble cytoskeleton were not PKC dependent.