Li Qingxia, Yang Wanfa, Wang Yue, Liu Wei
Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute , Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center , Shenzhen 518036 , China.
Biochemistry. 2018 Apr 10;57(14):2132-2139. doi: 10.1021/acs.biochem.8b00057. Epub 2018 Mar 28.
Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1 (ARAP1), Cbl-interacting protein of 85 kDa (CIN85), and casitas B-lineage lymphoma (Cbl) play important roles in epidermal growth factor receptor (EGFR) internalization and recycling. In previous studies, ARAP1 was found to interact with CIN85, and their interaction attenuated the ubiquitination of EGFR. However, the molecular mechanism was still unclear. In this study, we first biochemically and structurally characterized the interaction between ARAP1 and CIN85, and found that the CIN85 SH3B domain bound to the ARAP1 PXPXXRX (except P) XXR/H/K motif with high affinity and specificity. Based on this binding model, we further predicted other potential CIN85 binding partners and tested their interactions biochemically. Moreover, our swapping data and structure alignment analysis suggested that the β2-β3 loops of the CIN85 SH3 domains and the H87/E132 interaction were critical for ARAP1 binding specificity. Finally, our competitive analytical gel-filtration chromatography and isothermal titration calorimetry (ITC) results showed that ARAP1 could compete with Cbl for CIN85 binding, which provides a biochemical basis for the regulatory roles of ARAP1 in the CIN85-mediated EGFR internalizing process.
含Rho-GAP结构域、ANK重复序列和PH结构域的Arf-GAP蛋白1（ARAP1）、85 kDa的Cbl相互作用蛋白（CIN85）以及卡斯他滨B系淋巴瘤（Cbl）在表皮生长因子受体（EGFR）的内化和循环中发挥重要作用。在先前的研究中，发现ARAP1与CIN85相互作用，且它们的相互作用减弱了EGFR的泛素化。然而，其分子机制仍不清楚。在本研究中，我们首先对ARAP1与CIN85之间的相互作用进行了生化和结构表征，发现CIN85的SH3B结构域与ARAP1的PXPXXRX（除P外）XXR/H/K基序以高亲和力和特异性结合。基于此结合模型，我们进一步预测了其他潜在的CIN85结合伙伴，并对它们之间的相互作用进行了生化测试。此外，我们的交换数据和结构比对分析表明，CIN85 SH3结构域的β2-β3环以及H87/E132相互作用对ARAP1结合特异性至关重要。最后，我们的竞争性分析凝胶过滤色谱和等温滴定量热法（ITC）结果表明，ARAP1可与Cbl竞争CIN85的结合，这为ARAP1在CIN85介导的EGFR内化过程中的调节作用提供了生化基础。
