The effects of Src tyrosine kinase activation in subconfluent temperature sensitive (ts)-Src-transformed Madin-Darby canine kidney (MDCK) cells were analyzed by shifting them from nonpermissive (40.5 degrees C) to permissive (35 degrees C) temperature. Already, in 15 minutes, adherens junction components were released from the lateral walls and accumulated to basal surfaces. Simultaneously, membranous actin staining vanished, actin bundles appeared at the basal surface, and the cells flattened. The only component phosphorylated and translocated after the shift to 35 degrees C was p120ctn. The epithelial-mesenchymal transition could be inhibited by a specific inhibitor of Src kinase, PP2, or by inhibiting endocytosis. Therefore, Src activation was responsible for the transition, but not because of phosphorylation of adherens junction components but by way of activation of endocytic machinery and RhoGTPase. Expression of an RacGEF, Tiam-1 (T-lymphoma invasion and metastasis gene 1), prevented flattening of Src-transformed MDCK cells at 35 degrees C and resulted in accumulation of cadherin to lateral membranes. In the case where the Src-MDCK cells were cultivated at 35 degrees C and shifted for short time periods to 40.5 degrees C, cadherin rapidly returned to lateral membranes, whereas actin and p120ctn followed hours afterward. This further supports the view that cadherin internalization is the primary target of Src kinase. We also looked at the cell morphology and distribution of cadherin and Tiam-1 in cells grown in three-dimensional gels composed of collagen and laminin or in Matrigel. At nonpermissive temperature, both Src-MDCK and Tiam-1-transfected Src-MDCK cells exhibited nonpolarized morphology in collagen I, a loose cluster in the mixture of collagen I and laminin, and a differentiated cyst in Matrigel. In growth factor-depleted Matrigel, the Src-MDCK cells grew in nondifferentiated clusters, whereas Tiam-1-transfected cells went to apoptosis. The differentiated phenotype of both cell lines could be rescued by Matrigel-conditioned medium, platelet-derived growth factor, or cholera toxin. Concomitantly, both cadherin and Tiam-1 were recruited to lateral membranes. Therefore, cadherin and Tiam-1 seem to be the key players in the differentiation process of MDCK cells.

中文翻译：

---

SRC 诱导的 madin-darby 犬肾细胞粘附连接的分解依赖于钙粘蛋白的内吞作用并被 Tiam-1 拮抗。

Src 酪氨酸激酶活化对亚融合温度敏感 (ts)-Src 转化的 Madin-Darby 犬肾 (MDCK) 细胞的影响通过将它们从非允许（40.5 摄氏度）转移到允许（35 摄氏度）温度进行分析。已经，在 15 分钟内，粘附连接组件从侧壁释放并积累到基底表面。同时，膜肌动蛋白染色消失，肌动蛋白束出现在基底表面，细胞扁平化。转移到 35 摄氏度后磷酸化和易位的唯一成分是 p120ctn。上皮-间充质转化可被 Src 激酶的特异性抑制剂 PP2 或通过抑制内吞作用抑制。因此，Src 激活负责转换，但不是因为粘附连接成分的磷酸化，而是通过激活内吞机制和 RhoGTPase。RacGEF、Tiam-1（T 淋巴瘤侵袭和转移基因 1）的表达阻止了 Src 转化的 MDCK 细胞在 35 摄氏度时变平，并导致钙粘蛋白积累到侧膜。在 Src-MDCK 细胞在 35 摄氏度下培养并短时间转移到 40.5 摄氏度的情况下，钙粘蛋白迅速返回到侧膜，而肌动蛋白和 p120ctn 则在数小时后返回。这进一步支持了钙粘蛋白内化是 Src 激酶的主要目标的观点。我们还研究了在由胶原蛋白和层粘连蛋白组成的三维凝胶或基质胶中生长的细胞中钙粘蛋白和 Tiam-1 的细胞形态和分布。在不允许的温度下，Src-MDCK 和 Tiam-1 转染的 Src-MDCK 细胞都在胶原蛋白 I 中表现出非极化形态，在胶原蛋白 I 和层粘连蛋白的混合物中表现出松散的簇状结构，在基质胶中表现出分化的囊肿。在生长因子耗尽的 Matrigel 中，Src-MDCK 细胞在未分化的簇中生长，而 Tiam-1 转染的细胞发生凋亡。两种细胞系的分化表型都可以通过 Matrigel 条件培养基、血小板衍生生长因子或霍乱毒素来挽救。同时，钙粘蛋白和 Tiam-1 都被募集到侧膜。因此，钙粘蛋白和 Tiam-1 似乎是 MDCK 细胞分化过程中的关键参与者。和 Matrigel 中的分化囊肿。在生长因子耗尽的 Matrigel 中，Src-MDCK 细胞在未分化的簇中生长，而 Tiam-1 转染的细胞发生凋亡。两种细胞系的分化表型都可以通过 Matrigel 条件培养基、血小板衍生生长因子或霍乱毒素来挽救。同时，钙粘蛋白和 Tiam-1 都被募集到侧膜。因此，钙粘蛋白和 Tiam-1 似乎是 MDCK 细胞分化过程中的关键参与者。和 Matrigel 中的分化囊肿。在生长因子耗尽的 Matrigel 中，Src-MDCK 细胞在未分化的簇中生长，而 Tiam-1 转染的细胞发生凋亡。两种细胞系的分化表型都可以通过 Matrigel 条件培养基、血小板衍生生长因子或霍乱毒素来挽救。同时，钙粘蛋白和 Tiam-1 都被募集到侧膜。因此，钙粘蛋白和 Tiam-1 似乎是 MDCK 细胞分化过程中的关键参与者。钙粘蛋白和 Tiam-1 均被募集到侧膜。因此，钙粘蛋白和 Tiam-1 似乎是 MDCK 细胞分化过程中的关键参与者。钙粘蛋白和 Tiam-1 均被募集到侧膜。因此，钙粘蛋白和 Tiam-1 似乎是 MDCK 细胞分化过程中的关键参与者。