Phosphorylation of Numb regulates its interaction with the clathrin-associated adaptor AP-2 |
| |
Authors: | Tokumitsu Hiroshi Hatano Naoya Yokokura Shigeyuki Sueyoshi Yuka Nozaki Naohito Kobayashi Ryoji |
| |
Affiliation: | Department of Signal Transduction Sciences, Faculty of Medicine, Kagawa University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. tokumit@med.kagawa-u.ac.jp |
| |
Abstract: | Numb is thought to participate in clathrin-dependent endocytosis by directly interacting with the clathrin-associated adaptor complex AP-2, although the underlying mechanisms are unknown. Numb is also known to be phosphorylated at Ser(264)in vitro and in vivo. Here, we found that Numb is phosphorylated in vitro by Ca(2+)/calmodulin-dependent protein kinase I on Ser(283). This phosphorylation was also observed in transfected COS-7 cells, indicating its physiological relevance. Pull-down experiments showed that the phosphorylation of Numb impaired its binding to the AP-2 complex and simultaneously recruited 14-3-3 proteins in vitro. Based on experiments using Numb mutants, both the initial phosphorylation of Ser(264) and the subsequent phosphorylation of Ser(283) are sufficient to abolish the binding of Numb to AP-2 and to promote the interaction with 14-3-3 protein. These findings suggest a novel mechanism for the regulation of Numb-mediated endocytosis, namely through direct phosphorylation. |
| |
Keywords: | CaM-K, Ca2+/CaM-dependent protein kinase CaM, calmodulin LC, liquid chromatography MS/MS, tandem mass spectrometry GST, glutathione S-transferase |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|