Department of Cell & Applied Biology, Faculty of Science, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.
Cell Signal. 2011 Feb;23(2):458-67. doi: 10.1016/j.cellsig.2010.10.023. Epub 2010 Oct 30.
Overexpression and poor downregulation of ErbB receptor tyrosine kinases are associated with enhanced signaling and tumorigenesis. Attenuation of EGF-receptor (EGFR) signaling is mediated by endocytosis and ubiquitination by the E3-ligase Cbl. En route to lysosomes, but before incorporation of the EGFR into internal vesicles of MVBs, the EGFR undergoes Usp8-mediated deubiquitination. ErbB2 displays enhanced recycling back to the cell surface, and therefore we hypothesized that Usp8 is not part of the ErbB2 trafficking pathway. Here, we demonstrate, in the context of a chimeric EGFR-ErbB2 receptor, that (i) EGF induces pY1091 Cbl binding site-dependent K63-polyubiquitination of EGFR-ErbB2, (ii) Cbl is tyrosine phosphorylated upon stimulation of EGFR-ErbB2 wt and Y1091F mutant receptor, (iii) EGF-induced activation of EGFR-ErbB2 induces Usp8 tyrosine phosphorylation, and (iv) ubiquitination of the EGFR-ErbB2 wt and Y1091F mutant is enhanced upon coexpression of catalytically inactive Usp8-C748A in the presence and absence of EGF. We further show that Usp8 tyrosine phosphorylation upon stimulation of EGFR-ErbB2 is (a) independent of Y1091, (b) dependent on Src- and EGFR-ErbB2-kinase activity, (c) enhanced upon coexpression of Usp8-C748A, and (d) partly dependent on the Microtubule Interacting and Transport (MIT) domain of Usp8. Our findings demonstrate that Usp8 is part of the ErbB2 endosomal trafficking pathway.
表皮生长因子受体（EGFR）酪氨酸激酶的过度表达和下调不足与信号增强和肿瘤发生有关。通过 E3 连接酶 Cbl 的内吞作用和泛素化来调节 EGF 受体（EGFR）信号。在溶酶体中，但在 EGFR 被纳入 MVBs 的内部囊泡之前，EGFR 经历 Usp8 介导的去泛素化。ErbB2 显示出增强的再循环回到细胞表面，因此我们假设 Usp8 不是 ErbB2 运输途径的一部分。在这里，我们在嵌合 EGFR-ErbB2 受体的背景下证明：（i）EGF 诱导 pY1091 Cbl 结合位点依赖性 K63-多泛素化的 EGFR-ErbB2，（ii）Cbl 在刺激 EGFR-ErbB2 wt 和 Y1091F 突变受体时被酪氨酸磷酸化，（iii）EGF 诱导的 EGFR-ErbB2 激活诱导 Usp8 酪氨酸磷酸化，和（iv）在存在和不存在 EGF 的情况下共表达催化失活的 Usp8-C748A 时，EGFR-ErbB2 wt 和 Y1091F 突变体的泛素化增强。我们进一步表明，EGFR-ErbB2 刺激时 Usp8 的酪氨酸磷酸化（a）独立于 Y1091，（b）依赖于Src 和 EGFR-ErbB2-激酶活性，（c）在共表达 Usp8-C748A 时增强，和（d）部分依赖于 Usp8 的微管相互作用和运输（MIT）结构域。我们的研究结果表明，Usp8 是 ErbB2 内体运输途径的一部分。
