High-throughput phosphotyrosine profiling using SH2 domains

Mol Cell. 2007 Jun 22;26(6):899-915. doi: 10.1016/j.molcel.2007.05.031.


Protein tyrosine phosphorylation controls many aspects of signaling in multicellular organisms. One of the major consequences of tyrosine phosphorylation is the creation of binding sites for proteins containing Src homology 2 (SH2) domains. To profile the global tyrosine phosphorylation state of the cell, we have developed proteomic binding assays encompassing nearly the full complement of human SH2 domains. Here we provide a global view of SH2 domain binding to cellular proteins based on large-scale far-western analyses. We also use reverse-phase protein arrays to generate comprehensive, quantitative SH2 binding profiles for phosphopeptides, recombinant proteins, and entire proteomes. As an example, we profiled the adhesion-dependent SH2 binding interactions in fibroblasts and identified specific focal adhesion complex proteins whose tyrosine phosphorylation and binding to SH2 domains are modulated by adhesion. These results demonstrate that high-throughput comprehensive SH2 profiling provides valuable mechanistic insights into tyrosine kinase signaling pathways.

MeSH terms

  • Animals
  • Cell Adhesion / physiology
  • Fibroblasts / cytology
  • Fibroblasts / metabolism*
  • Focal Adhesions / physiology*
  • Humans
  • Mice
  • Multiprotein Complexes / metabolism
  • NIH 3T3 Cells
  • Peptides / metabolism
  • Phosphorylation
  • Phosphotyrosine / metabolism
  • Protein Array Analysis
  • Protein Processing, Post-Translational / physiology*
  • Protein-Tyrosine Kinases / metabolism
  • Proteome / metabolism*
  • Recombinant Proteins / metabolism
  • Signal Transduction / physiology*
  • src Homology Domains / physiology*


  • Multiprotein Complexes
  • Peptides
  • Proteome
  • Recombinant Proteins
  • Phosphotyrosine
  • Protein-Tyrosine Kinases