Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, United States of America.
PLoS One. 2012;7(5):e37476. doi: 10.1371/journal.pone.0037476. Epub 2012 May 31.
p120-Catenin binding to, and Hakai-mediated ubiquitination of the E-cadherin juxtamembrane domain (JMD) are thought to be involved in regulating E-cadherin internalization and degradation. However, the relationship between these two pathways is not understood. We targeted the E-cadherin JMD to mitochondria (WT-JMD) to isolate this domain from the plasma membrane and internalization, and to examine protein modifications and degradation. WT-JMD localized to mitochondria, but did not accumulate there except when proteasome activity was inhibited. We found WT-JMD was ubiquitinated, and arginine substitution of lysines at position 5 (K5R) and 83 (K83R) resulted in the stable accumulation of mutant JMD at mitochondria. p120-Catenin did not localize, or bind to WT-JMD even upon proteasome inhibition, whereas the K5,83R-JMD mutant bound and localized p120-catenin to mitochondria. Mutation of the p120-catenin binding site in combination with these lysine mutations inhibited p120-catenin binding, but did not decrease JMD stability or its accumulation at mitochondria. Thus, increased stability of JMD lysine mutants was due to inhibition of ubiquitination and not to p120-catenin binding. Finally, mutation of these critical lysines in full length E-cadherin had similar effects on protein stability as WT-JMD. Our results indicate that ubiquitination of the JMD inhibits p120-catenin binding, and targets E-cadherin for degradation.
p120 连环蛋白与 E-钙黏蛋白的近膜结构域（JMD）的 Hakai 介导泛素化被认为参与调节 E-钙黏蛋白的内化和降解。然而，这两条途径之间的关系尚不清楚。我们将 E-钙黏蛋白 JMD 靶向线粒体（WT-JMD），将该结构域从质膜和内化中分离出来，并检查蛋白质修饰和降解。WT-JMD 定位于线粒体，但除了蛋白酶体活性受到抑制时，不会在那里积累。我们发现 WT-JMD 被泛素化，并且赖氨酸位置 5（K5R）和 83（K83R）的精氨酸取代导致突变 JMD 在线粒体中稳定积累。p120 连环蛋白即使在蛋白酶体抑制下也不会定位或与 WT-JMD 结合，而 K5、83R-JMD 突变体则结合并将 p120 连环蛋白定位于线粒体。p120 连环蛋白结合位点的突变与这些赖氨酸突变的结合抑制了 p120 连环蛋白的结合，但并没有降低 JMD 的稳定性或其在线粒体中的积累。因此，JMD 赖氨酸突变体的稳定性增加是由于泛素化的抑制而不是 p120 连环蛋白的结合。最后，在全长 E-钙黏蛋白中突变这些关键赖氨酸对蛋白质稳定性的影响与 WT-JMD 相似。我们的结果表明，JMD 的泛素化抑制了 p120 连环蛋白的结合，并将 E-钙黏蛋白靶向降解。
