Phosphorylation of growth factor receptor binding protein-2 by pp60c-src tyrosine kinase.
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.
Jones
D A
DA
Cardiovascular Pharmacology, Pharmacia and Upjohn Inc., Kalamazoo, Michigan 49001, USA.
Benjamin
C W
CW
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002461:Cell Line, Transformed; D066246:ErbB Receptors / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D051379:Mice; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src)