Src homology 2 domains enhance tyrosine phosphorylation in vivo by protecting binding sites in their target proteins from dephosphorylation

J Biol Chem. 2018 Jan 12;293(2):623-637. doi: 10.1074/jbc.M117.794412. Epub 2017 Nov 21.


Phosphotyrosine (pTyr)-dependent signaling is critical for many cellular processes. It is highly dynamic, as signal output depends not only on phosphorylation and dephosphorylation rates but also on the rates of binding and dissociation of effectors containing phosphotyrosine-dependent binding modules such as Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains. Previous in vitro studies suggested that binding of SH2 and PTB domains can enhance protein phosphorylation by protecting the sites bound by these domains from phosphatase-mediated dephosphorylation. To test whether this occurs in vivo, we used the binding of growth factor receptor bound 2 (GRB2) to phosphorylated epidermal growth factor receptor (EGFR) as a model system. We analyzed the effects of SH2 domain overexpression on protein tyrosine phosphorylation by quantitative Western and far-Western blotting, mass spectrometry, and computational modeling. We found that SH2 overexpression results in a significant, dose-dependent increase in EGFR tyrosine phosphorylation, particularly of sites corresponding to the binding specificity of the overexpressed SH2 domain. Computational models using experimentally determined EGFR phosphorylation and dephosphorylation rates, and pTyr-EGFR and GRB2 concentrations, recapitulated the experimental findings. Surprisingly, both modeling and biochemical analyses suggested that SH2 domain overexpression does not result in a major decrease in the number of unbound phosphorylated SH2 domain-binding sites. Our results suggest that signaling via SH2 domain binding is buffered over a relatively wide range of effector concentrations and that SH2 domain proteins with overlapping binding specificities are unlikely to compete with one another for phosphosites in vivo.

Keywords: Src homology 2 domain (SH2 domain); crk; epidermal growth factor receptor (EGFR); growth factor receptor-bound protein 2 (GRB2); phosphorylation; phosphotyrosine.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Binding Sites
  • ErbB Receptors / metabolism*
  • GRB2 Adaptor Protein / metabolism*
  • Phosphorylation
  • Phosphotyrosine / metabolism
  • Protein Binding
  • Signal Transduction


  • GRB2 Adaptor Protein
  • Phosphotyrosine
  • ErbB Receptors