Systematic Identification of SHP2 Phosphatase Substrates

ABSTRACT: Phosphatases play essential roles in normal cell physiology and diseases like cancer. The challenges of studying phosphatases have limited our understanding of their substrates, molecular mechanisms, and unique functions within highly complicate networks. Here we introduce a novel strategy using substrate trapping mutant coupled with quantitative mass spectrometry to identify physiological substrates of protein tyrosine phosphatase Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) in a high-throughput manner. SHP2 plays a critical role in numerous cellular processes through the regulation of various signaling pathways. The method integrates three separate MS-based experiments; in vitro dephosphorylation assay, in vivo global phosphoproteomics, and pull down of substrate trapping mutant complex using HEK293SHP2KO cells. PTPN11-C459S/D425A is an optimized substrate trap mutant of SHP2. We identified eleven direct substrates, including both known and novel SHP2 substrates in EGFR signaling pathways, among which docking protein 1 (DOK1) was further validated as a new SHP2 substrate. This advanced workflow significantly improves the systemic identification of direct substrates of phosphatase, facilitating the comprehension of equally important roles of phosphatase signaling.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Acute Leukemia

SUBMITTER: Peipei Zhu  

LAB HEAD: Weiguo Andy Tao

PROVIDER: PXD028404 | Pride | 2022-09-20

REPOSITORIES: Pride