PHOSPHORYLATION OF GROWTH-FACTOR RECEPTOR-BINDING PROTEIN-2 BY PP60(C-SRC) TYROSINE KINASE

Growth factor receptor binding protein-2 (GRB2) couples growth factor receptor activation to the p21-ras nucleotide exchange factor son-of-s evenless. Both GRB2 and son-of-sevenless display phosphorylation in ce lls treated with growth factors and may be subject to feed back regula tion in mitogen-stimulated cells, Herein, we demonstrate that pp60(c-s rc) can utilize GRB2 as a substrate. NM 3T3 fibroblasts overexpressing pp60(v-src) contained high levels of phosphorylated GRB2. In comparis on, control fibroblasts contained phosphorylated GRB2 only after stimu lation with platelet-derived growth factor. Analysis of GRB2 immune co mplexes isolated from fibroblasts stimulated with PDGF or transformed by pp60(v-src) revealed a kinase activity capable of phosphorylating G RB2 in. vitro. Incubation of native or recombinant GRB2 with purified pp60(c-src) provided additional support for pp60(c-src) as the kinase for GRB2. Deletion mutants of GRB2 demonstrated that pp60(c-src) phosp horylated 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 pp60(c-src). We conclude that Src finds GRB2 a suitable substrate in vitro and may ph osphorylate GRB2 in cells responding to platelet-derived growth factor . (C) 1997 Academic Press.