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Phosphorylation of growth factor receptor binding protein-2 by pp60c-src tyrosine kinase
1Cardiovascular Pharmacology, Pharmacia and Upjohn Inc., Kalamazoo, Michigan 49001, USA.
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.
Insights
pp60c-src phosphorylates Growth factor receptor binding protein-2 (GRB2), a key signaling molecule. This phosphorylation occurs on tyrosine 160 and may happen in cells responding to platelet-derived growth factor.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncogenesis
Background:
- Growth factor receptor binding protein-2 (GRB2) is crucial for coupling growth factor receptor activation to downstream signaling pathways like Ras.
- Both GRB2 and its binding partner, son-of-sevenless, are phosphorylated in response to growth factors and may be involved in feedback regulation.
- The kinase responsible for GRB2 phosphorylation in response to growth factors has not been definitively identified.
Purpose of the Study:
- To investigate whether pp60c-src can phosphorylate GRB2.
- To identify the specific site of GRB2 phosphorylation by pp60c-src.
- To determine if pp60c-src is the kinase responsible for GRB2 phosphorylation in growth factor-stimulated cells.
Main Methods:
- NIH 3T3 fibroblasts overexpressing pp60v-src were used to assess GRB2 phosphorylation levels.
- GRB2 immune complexes from platelet-derived growth factor (PDGF)-stimulated or pp60v-src-transformed fibroblasts were analyzed for kinase activity.
- Native and recombinant GRB2 were incubated with purified pp60c-src.
- Deletion mutants and site-directed mutagenesis of GRB2 were employed to map the phosphorylation site.
Main Results:
- NIH 3T3 fibroblasts overexpressing pp60v-src exhibited high levels of phosphorylated GRB2.
- Control fibroblasts showed phosphorylated GRB2 only after PDGF stimulation.
- Kinase activity capable of phosphorylating GRB2 was detected in GRB2 immune complexes from stimulated or transformed cells.
- pp60c-src directly phosphorylated GRB2 on tyrosine residue 160, located between the SH2 and carboxyl-terminal SH3 domains.
- Mutation of tyrosine 160 to phenylalanine abolished pp60c-src-mediated GRB2 phosphorylation.
Conclusions:
- pp60c-src phosphorylates GRB2 on tyrosine 160 in vitro.
- pp60c-src is a suitable substrate for GRB2 phosphorylation.
- pp60c-src may phosphorylate GRB2 in cells stimulated by PDGF.