competitive regulation of e-cadherin juxtamembrane domain degradation by p120-catenin binding and hakai-mediated ubiquitination竞争监管的钙粘蛋白近膜域中p120-catenin绑定和降解hakai-mediated泛素化.pdfVIP

Competitive Regulation of E-Cadherin JuxtaMembrane Domain Degradation by p120-Catenin Binding and Hakai-Mediated Ubiquitination 1 1,2 Andrea Hartsock , W. James Nelson * 1 Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, United States of America, 2 Department of Biology, Stanford University, Stanford, California, United States of America Abstract 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-J