Tong Jiefei, Taylor Paul, Moran Michael F
From the ‡The Hospital For Sick Children, Program in Molecular Structure and Function, Princess Margaret Cancer Centre, and Department of Molecular Genetics, University of Toronto. Peter Gilgan Centre for Research and Learning, 686 Bay Street, Toronto M5G 0A4, Canada.
From the ‡The Hospital For Sick Children, Program in Molecular Structure and Function, Princess Margaret Cancer Centre, and Department of Molecular Genetics, University of Toronto. Peter Gilgan Centre for Research and Learning, 686 Bay Street, Toronto M5G 0A4, Canada
Mol Cell Proteomics. 2014 Jul;13(7):1644-58. doi: 10.1074/mcp.M114.038596. Epub 2014 May 5.
Aberrant expression, activation, and stabilization of epidermal growth factor receptor (EGFR) are causally associated with several human cancers. Post-translational modifications and protein-protein interactions directly modulate the signaling and trafficking of the EGFR. Activated EGFR is internalized by endocytosis and then either recycled back to the cell surface or degraded in the lysosome. EGFR internalization and recycling also occur in response to stresses that activate p38 MAP kinase. Mass spectrometry was applied to comprehensively analyze the phosphorylation, ubiquitination, and protein-protein interactions of wild type and endocytosis-defective EGFR variants before and after internalization in response to EGF ligand and stress. Prior to internalization, EGF-stimulated EGFR accumulated ubiquitin at 7 K residues and phosphorylation at 7 Y sites and at S(1104). Following internalization, these modifications diminished and there was an accumulation of S/T phosphorylations. EGFR internalization and many but not all of the EGF-induced S/T phosphorylations were also stimulated by anisomycin-induced cell stress, which was not associated with receptor ubiquitination or elevated Y phosphorylation. EGFR protein interactions were dramatically modulated by ligand, internalization, and stress. In response to EGF, different E3 ubiquitin ligases became maximally associated with EGFR before (CBL, HUWE1, and UBR4) or after (ITCH) internalization, whereas CBLB was distinctively most highly EGFR associated following anisomycin treatment. Adaptin subunits of AP-1 and AP-2 clathrin adaptor complexes also became EGFR associated in response to EGF and anisomycin stress. Mutations preventing EGFR phosphorylation at Y(998) or in the S(1039) region abolished or greatly reduced EGFR interactions with AP-2 and AP-1, and impaired receptor trafficking. These results provide new insight into spatial, temporal, and mechanistic aspects of EGFR regulation.
表皮生长因子受体（EGFR）的异常表达、激活和稳定与多种人类癌症存在因果关联。翻译后修饰和蛋白质-蛋白质相互作用直接调节EGFR的信号传导和运输。激活的EGFR通过内吞作用内化，然后要么循环回到细胞表面，要么在溶酶体中降解。EGFR的内化和循环也会在激活p38丝裂原活化蛋白激酶的应激反应中发生。应用质谱法全面分析野生型和内吞缺陷型EGFR变体在响应表皮生长因子（EGF）配体和应激而内化前后的磷酸化、泛素化以及蛋白质-蛋白质相互作用。在内化之前，EGF刺激的EGFR在7个赖氨酸（K）残基处积累泛素，在7个酪氨酸（Y）位点以及丝氨酸（S1104）处发生磷酸化。内化之后，这些修饰减少，并且出现苏氨酸/丝氨酸（S/T）磷酸化的积累。EGFR的内化以及许多但并非所有EGF诱导的S/T磷酸化也受到茴香霉素诱导的细胞应激的刺激，这种应激与受体泛素化或酪氨酸磷酸化升高无关。EGFR的蛋白质相互作用受到配体、内化和应激的显著调节。响应EGF时，不同的E3泛素连接酶在内化之前（CBL、HUWE1和UBR4）或之后（ITCH）与EGFR最大程度地结合，而在茴香霉素处理后，CBLB与EGFR的结合尤为显著。网格蛋白衔接复合物AP-1和AP-2的衔接蛋白亚基也会在响应EGF和茴香霉素应激时与EGFR结合。阻止EGFR在酪氨酸（Y998）或丝氨酸（S1039）区域磷酸化的突变消除或大大减少了EGFR与AP-2和AP-1的相互作用，并损害了受体运输。这些结果为EGFR调节的空间、时间和机制方面提供了新的见解。
