An Integrated Proteomic Strategy to Identify SHP2 Substrates

Protein phosphatases play an essential role in normal cell physiology and the development of diseases such as cancer. The innate challenges associated with studying protein phosphatases have limited our understanding of their substrates, molecular mechanisms, and unique functions within highly coordinated networks. Here, we introduce a novel strategy using substrate-trapping mutants coupled with quantitative proteomics methods to identify physiological substrates of Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) in a high-throughput manner. The technique integrates three parallel mass spectrometry-based proteomics experiments, including affinity isolation of substrate-trapping mutant complex using wild-type and SHP2 KO cells, in vivo global quantitative phosphoproteomics, and in vitro phosphatase reaction. We confidently identified 18 direct substrates of SHP2 in the epidermal growth factor receptor signaling pathways, including both known and novel SHP2 substrates. Docking protein 1 was further validated using biochemical assays as a novel SHP2 substrate, providing a mechanism for SHP2-mediated Ras activation. This advanced workflow improves the systemic identification of direct substrates of protein phosphatases, facilitating our understanding of the equally important roles of protein phosphatases in cellular signaling.蛋白磷酸酶（protein phosphatases）在正常细胞生理活动以及癌症等疾病的发生发展中发挥不可或缺的关键作用。长期以来，研究蛋白磷酸酶所面临的固有挑战限制了我们对其底物、分子机制以及其在高度协同的细胞信号网络中独特功能的认知。本研究提出一种全新策略：将底物捕获突变体（substrate-trapping mutant）与定量蛋白质组学（quantitative proteomics）技术相结合，以高通量方式鉴定含Src同源2结构域的蛋白酪氨酸磷酸酶2（Src homology 2 containing protein tyrosine phosphatase 2，SHP2）的生理性底物。该技术整合了三项基于质谱的平行蛋白质组学实验：分别利用野生型细胞与SHP2敲除（KO，knockout）细胞完成底物捕获突变体复合物的亲和分离、开展体内全局定量磷酸化蛋白质组学（quantitative phosphoproteomics）分析，以及进行体外磷酸酶反应实验。本研究可靠鉴定出表皮生长因子受体信号通路（epidermal growth factor receptor signaling pathway）中的18种SHP2直接底物，涵盖已知与全新两类SHP2底物。研究人员进一步通过生化实验验证了停靠蛋白1（docking protein 1）为全新SHP2底物，该发现为SHP2介导的Ras蛋白（Ras）激活提供了分子机制。这套先进的实验流程优化了蛋白磷酸酶直接底物的系统性鉴定方法，有助于我们更全面地理解蛋白磷酸酶在细胞信号转导中发挥的同等重要的作用。