Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants

Biochem J. 2013 Sep 15;454(3):501-13. doi: 10.1042/BJ20121750.

Abstract

Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Carcinoma, Squamous Cell / enzymology
  • Carcinoma, Squamous Cell / genetics*
  • Cluster Analysis
  • Collagen Type I / metabolism
  • Discoidin Domain Receptors
  • HEK293 Cells
  • Humans
  • Lung Neoplasms / enzymology
  • Lung Neoplasms / genetics*
  • Molecular Sequence Data
  • Mutation, Missense
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein Processing, Post-Translational*
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11 / metabolism*
  • Proteomics
  • Receptor Protein-Tyrosine Kinases / genetics
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Receptors, Collagen / metabolism
  • Receptors, Mitogen / genetics
  • Receptors, Mitogen / metabolism*
  • Signal Transduction
  • Tandem Mass Spectrometry
  • src-Family Kinases / metabolism

Substances

  • Collagen Type I
  • Phosphoproteins
  • Receptors, Collagen
  • Receptors, Mitogen
  • Discoidin Domain Receptors
  • Receptor Protein-Tyrosine Kinases
  • src-Family Kinases
  • PTPN11 protein, human
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11