Phosphorylation of Numb regulates its interaction with the clathrin‐associated adaptor AP‐2
Phosphorylation of Numb regulates its interaction with the clathrin‐associated adaptor AP‐2
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DOI：
10.1016/j.febslet.2006.09.043
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发表时间：
2006-10
期刊：
影响因子：
3.5
通讯作者：
H. Tokumitsu;N. Hatano;Shigeyuki Yokokura;Yuka Sueyoshi;N. Nozaki;R. Kobayashi
中科院分区：
文献类型：
--
作者：
H. Tokumitsu;N. Hatano;Shigeyuki Yokokura;Yuka Sueyoshi;N. Nozaki;R. Kobayashi
Numb被认为通过直接与网格蛋白相关的接头复合物AP-2相互作用参与网格蛋白依赖的内吞作用，尽管潜在的机制尚不清楚。在体外和体内，Numb也已知在ser264位点磷酸化。在这里，我们发现Numb在体外被Ca2+/钙调素依赖性蛋白激酶I磷酸化Ser283。在转染的COS-7细胞中也观察到这种磷酸化，表明其生理相关性。下拉实验表明，Numb的磷酸化破坏了其与AP-2复合物的结合，同时在体外募集14-3-3蛋白。通过对Numb突变体的实验发现，最初的ser264磷酸化和随后的ser283磷酸化都足以消除Numb与AP-2的结合，并促进与14-3-3蛋白的相互作用。这些发现提示了一种新的机制来调节麻木介导的内吞作用，即通过直接磷酸化。
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 Ser264in vitro and in vivo. Here, we found that Numb is phosphorylated in vitro by Ca2+/calmodulin-dependent protein kinase I on Ser283. 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 Ser264and the subsequent phosphorylation of Ser283are 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.