Phosphorylation of growth factor receptor binding protein-2 by pp60c-src tyrosine kinase

Arch Biochem Biophys. 1997 Jan 15;337(2):143-8. doi: 10.1006/abbi.1996.9789.

Abstract

Growth factor receptor binding protein-2 (GRB2) couples growth factor receptor activation to the p21-ras nucleotide exchange factor son-of-sevenless. Both GRB2 and son-of-sevenless display phosphorylation in cells treated with growth factors and may be subject to feed back regulation in mitogen-stimulated cells. Herein, we demonstrate that pp60c-src can utilize GRB2 as a substrate. NIH 3T3 fibroblasts overexpressing pp60v-src contained high levels of phosphorylated GRB2. In comparison, control fibroblasts contained phosphorylated GRB2 only after stimulation with platelet-derived growth factor. Analysis of GRB2 immune complexes isolated from fibroblasts stimulated with PDGF or transformed by pp60v-src revealed a kinase activity capable of phosphorylating GRB2 in vitro. Incubation of native or recombinant GRB2 with purified pp60c-src provided additional support for pp60c-src as the kinase for GRB2. Deletion mutants of GRB2 demonstrated that pp60c-src phosphorylated GRB2 on a tyrosine residue (residue 160) located between the SH2 domain and carboxyl terminal SH3 domain. Mutation of tyrosine 160 to phenylalanine abolished phosphorylation of GRB2 by pp60c-src. We conclude that Src finds GRB2 a suitable substrate in vitro and may phosphorylate GRB2 in cells responding to platelet-derived growth factor.

MeSH terms

  • 3T3 Cells
  • Adaptor Proteins, Signal Transducing*
  • Animals
  • Cell Line, Transformed
  • ErbB Receptors / metabolism
  • GRB2 Adaptor Protein
  • Mice
  • Phosphorylation
  • Phosphotyrosine / metabolism
  • Platelet-Derived Growth Factor / pharmacology
  • Proteins / metabolism*
  • Proto-Oncogene Proteins pp60(c-src) / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • src Homology Domains

Substances

  • Adaptor Proteins, Signal Transducing
  • GRB2 Adaptor Protein
  • Grb2 protein, mouse
  • Platelet-Derived Growth Factor
  • Proteins
  • Recombinant Fusion Proteins
  • Phosphotyrosine
  • ErbB Receptors
  • Proto-Oncogene Proteins pp60(c-src)