Large-Scale Phosphoproteomics Reveals Shp-2 Phosphatase-Dependent Regulators of Pdgf Receptor Signaling

Cell Rep. 2018 Mar 6;22(10):2784-2796. doi: 10.1016/j.celrep.2018.02.038.

Abstract

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.

Keywords: PDGF; Q exactive; SHP099; Shp-2; TiO2; label-free quantitation; mass spectrometry; orbitrap; phosphoproteomics; tyrosine phosphorylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / metabolism
  • Becaplermin / pharmacology
  • Enzyme Activation
  • Focal Adhesion Protein-Tyrosine Kinases / metabolism
  • Ligands
  • Mice
  • NIH 3T3 Cells
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Phosphotyrosine / metabolism
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / antagonists & inhibitors
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / metabolism*
  • Proteome / metabolism
  • Proteomics*
  • Receptors, Platelet-Derived Growth Factor / metabolism*
  • Signal Transduction*

Substances

  • Antibodies
  • Ligands
  • Phosphoproteins
  • Proteome
  • Becaplermin
  • Phosphotyrosine
  • Receptors, Platelet-Derived Growth Factor
  • Focal Adhesion Protein-Tyrosine Kinases
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11