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）在正常细胞生理及癌症等疾病的发生发展中发挥着至关重要的作用。长期以来，研究蛋白磷酸酶所面临的固有难题，限制了我们对其底物、分子机制以及其在高度协调的细胞网络中独特功能的认知。在此研究中，我们提出了一种全新策略：将底物捕获突变体与定量蛋白质组学方法相结合，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）的生理底物。该技术整合了三项平行的基于质谱的蛋白质组学实验，分别为利用野生型细胞与SHP2敲除（knockout, KO）细胞亲和分离底物捕获突变体复合物、体内全局定量磷酸蛋白质组学分析，以及体外磷酸酶反应实验。我们可靠鉴定出表皮生长因子受体信号通路中的18种SHP2直接底物，其中既包含已知的SHP2底物，也涵盖了全新的底物。我们进一步通过生化实验验证了对接蛋白1（Docking protein 1）为新型SHP2底物，这为SHP2介导的Ras激活机制提供了实验依据。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，有助于我们更深入地理解蛋白磷酸酶在细胞信号传导中的同等重要的调控作用。