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Platelet-derived growth factor stimulates phosphorylation of growth factor receptor-binding protein-2 in vascular
C W Benjamin1, D A Linseman, D A Jones
1Upjohn Laboratories, Kalamazoo, Michigan 49001.
Abstract:
Growth factor receptor-binding protein-2 (GRB2) couples growth factor receptor activation to the p21ras nucleotide exchange factor son-of-sevenless (SOS). Son-of-sevenless can serve as a substrate for mitogen-activated protein kinases and may be subject to feed back regulation in mitogen-stimulated cells. Herein, we demonstrate phosphorylation on GRB2 in rat A10 vascular smooth muscle cells exposed to platelet-derived growth factor (PDGF). Lysates from smooth cells stimulated with PDGF revealed a shift in the electrophoretic mobility of GRB2. Further investigation confirmed that phosphorylation on GRB2 accompanied this mobility shift. Phosphorylation on GRB2 was time-dependent and correlated with PDGF receptor activation. The time-course for phosphorylation of GRB2 and subsequent decay corresponded with other events characteristic of platelet-derived growth factor signaling. GRB2 was not phosphorylated in cells treated with phorbol 12-myristate 13-acetate, and down-regulation of protein kinase C failed to attenuate phosphorylation on GRB2 in response to growth factor. Analysis of GRB2 immune complexes revealed a kinase activity capable of phosphorylating GRB2 in vitro and demonstrated that the kinase activated in response to PDGF may physically associate with GRB2 signaling complexes.
Insights
Platelet-derived growth factor (PDGF) stimulates phosphorylation of Growth factor receptor-binding protein-2 (GRB2) in vascular smooth muscle cells. This phosphorylation is linked to PDGF receptor activation and involves a GRB2-associated kinase.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Growth factor receptor-binding protein-2 (GRB2) is a key adaptor protein linking growth factor receptor activation to downstream signaling pathways, including the p21ras/son-of-sevenless (SOS) pathway.
- Son-of-sevenless (SOS) can be phosphorylated by mitogen-activated protein kinases, suggesting potential feedback regulation in cellular responses to mitogens.
Purpose of the Study:
- To investigate the phosphorylation of GRB2 in response to platelet-derived growth factor (PDGF) in rat A10 vascular smooth muscle cells.
- To elucidate the relationship between PDGF receptor activation and GRB2 phosphorylation.
- To identify the kinase responsible for PDGF-induced GRB2 phosphorylation.
Main Methods:
- Stimulation of rat A10 vascular smooth muscle cells with PDGF.
- Analysis of GRB2 electrophoretic mobility shifts via Western blotting.
- Time-course studies of GRB2 phosphorylation and PDGF receptor activation.
- Investigation of GRB2 phosphorylation in response to phorbol 12-myristate 13-acetate and protein kinase C down-regulation.
- In vitro kinase assays using GRB2 immune complexes.
Main Results:
- PDGF stimulation induced a time-dependent shift in GRB2 electrophoretic mobility, indicative of phosphorylation.
- GRB2 phosphorylation correlated directly with PDGF receptor activation and followed a characteristic signaling time-course.
- GRB2 was not phosphorylated by phorbol 12-myristate 13-acetate, and protein kinase C inhibition did not affect PDGF-induced GRB2 phosphorylation.
- A GRB2-associated kinase activity capable of phosphorylating GRB2 in vitro was identified and found to be activated by PDGF.
Conclusions:
- PDGF signaling in vascular smooth muscle cells involves the phosphorylation of the adaptor protein GRB2.
- The phosphorylation is mediated by a PDGF-activated kinase that may associate with GRB2 signaling complexes.
- This finding provides new insights into the regulation of GRB2 function in growth factor signaling pathways.