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.