Despite its low cellular abundance, phosphotyrosine (pTyr) regulates numerous cell signaling pathways in health and disease. We applied comprehensive phosphoproteomics to unravel differential regulators of receptor tyrosine kinase (RTK)-initiated signaling networks upon activation by Pdgf-ββ, Fgf-2, or Igf-1 and identified more than 40,000 phosphorylation sites, including many phosphotyrosine sites without additional enrichment. The analysis revealed RTK-specific regulation of hundreds of pTyr sites on key signaling molecules. We found the tyrosine phosphatase Shp-2 to be the master regulator of Pdgfr pTyr signaling. Application of a recently introduced allosteric Shp-2 inhibitor revealed global regulation of the Pdgf-dependent tyrosine phosphoproteome, which significantly impaired cell migration. In addition, we present a list of hundreds of Shp-2-dependent targets and putative substrates, including Rasa1 and Cortactin with increased pTyr and Gab1 and Erk1/2 with decreased pTyr. Our study demonstrates that large-scale quantitative phosphoproteomics can precisely dissect tightly regulated kinase-phosphatase signaling networks.

SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines, yet is frequently mutated in human cancer. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by SHP099. The dynamics of phosphotyrosine abundance at more than 400 tyrosine residues reveal six distinct response signatures following SHP099 treatment and washout. Remarkably, in addition to newly identified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ2, another class of sites shows reduced phosphotyrosine abundance upon SHP2 inhibition. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight the distinct roles of the scaffolding and catalytic activities of SHP2 in effecting a transmembrane signaling response.

The protein tyrosine phosphatase SHP2 is crucial for oncogenic transformation of acute myeloid leukemia (AML) cells expressing mutated receptor tyrosine kinases (RTK). SHP2 is required for full RAS-ERK activation to promote cell proliferation and survival programs. Allosteric SHP2 inhibitors act by stabilizing SHP2 in its auto-inhibited conformation and are currently being tested in clinical trials for tumors with overactivation of the RAS/ERK pathway, alone and in various drug combinations. In this study, we established cells with acquired resistance to the allosteric SHP2 inhibitor SHP099 from two FLT3-ITD-positive AML cell lines. Label-free and isobaric labeling quantitative mass spectrometry-based phosphoproteomics of these resistant models demonstrated that AML cells can restore phosphorylated ERK (pERK) in the presence of SHP099, thus developing adaptive resistance. Mechanistically, SHP2 inhibition induced tyrosine phosphorylation and feedback-driven activation of the FLT3 receptor, which in turn phosphorylated SHP2 on tyrosine 62. This phosphorylation stabilized SHP2 in its open conformation, preventing SHP099 binding and conferring resistance. Combinatorial inhibition of SHP2 and MEK or FLT3 prevented pERK rebound and resistant cell growth. The same mechanism was observed in a FLT3-mutated B-ALL cell line and in the inv(16)/KitD816Y AML mouse model, but allosteric inhibition of Shp2 did not impair the clonogenic ability of normal bone marrow progenitors. Together, these results support the future use of SHP2 inhibitor combinations for clinical applications.