A Chimeric Pre-ubiquitinated EGF Receptor is Constitutively Endocytosed in a Clathrin-Dependent, but Kinase-Independent Manner
A Chimeric Pre-ubiquitinated EGF Receptor is Constitutively Endocytosed in a Clathrin-Dependent, but Kinase-Independent Manner
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DOI：
10.1111/j.1600-0854.2011.01162.x
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发表时间：
2011-04-01
期刊：
影响因子：
4.5
通讯作者：
Madshus, Inger Helene
中科院分区：
文献类型：
--
作者：
Bertelsen, Vibeke;Sak, Malgorzata Magdalena;Madshus, Inger Helene
表皮生长因子受体(EGFR)激酶活性和泛素化在EGFR内吞作用中的作用一直存在争议。据报道，接头蛋白和泛素连接酶Cb1是必需的。我们现在一致地报道，siRNA介导的c-Cbl和Cbl-b的敲除通过抑制EGFR的募集而显著减缓了激活的野生型(Wt)EGFR对cathrin依赖的内化。然而，一个由wt-EGFR、一个C端连接子和四个线性连接的泛素组成的嵌合蛋白被发现与Eps15和epsin 1相互作用，并以依赖于笼蛋白的方式组成内吞。有趣的是，这种融合蛋白的内吞作用不需要EGF的结合。也不需要激酶活性，融合蛋白在EGFR激酶抑制剂存在的情况下被内吞，这有效地抵消了酪氨酸磷酸化。这表明泛素化优先于在将EGFR重新募集到笼状蛋白包裹的凹坑中对激酶活性的要求。
The roles of EGF receptor (EGFR) kinase activity and ubiquitination in EGFR endocytosis have been controversial. The adaptor protein and ubiquitin ligase Cbl has reportedly been required. Consistently, we now report that siRNA-mediated knock-down of c-Cbl and Cbl-b significantly slowed clathrin-dependent internalization of activated wild-type (wt) EGFR by inhibiting recruitment of the EGFR to clathrin-coated pits. However, a chimeric protein consisting of wt-EGFR, a C-terminal linker and four linearly connected ubiquitins was found to interact with Eps15 and epsin 1 and to be constitutively endocytosed in a clathrin-dependent manner. Interestingly, endocytosis of this fusion protein did not require binding of EGF. Nor was kinase activity required, and the fusion protein was endocytosed in the presence of an EGFR kinase inhibitor, which efficiently counteracted tyrosine phosphorylation. This demonstrates that ubiquitination over-rides the requirement for kinase activity in recruitment of the EGFR to clathrin-coated pits.