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Association of Fyn with the activated platelet-derived growth factor receptor: requirements for binding and
G M Twamley1, R M Kypta, B Hall
1Differentiation Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Abstract:
Three members of the Src family of tyrosine kinases [pp60c-src (Src), p59fyn (Fyn) and pp62c-yes (Yes)] are ubiquitously expressed, and are thus likely to have general roles in growth control. We have previously shown that, after addition of platelet-derived growth factor (PDGF) to quiescent cells, all three kinases become activated and associated with the PDGF receptor. We have now addressed the requirements for this association. First, we have used a baculovirus expression system to show that Fyn associates with the activated PDGF receptor in vitro in the absence of other proteins, demonstrating that the association between the two molecules is direct. Second, by generating cell lines expressing chimeric molecules consisting of Fyn sequences fused to a portion of beta-galactosidase, we found that the SH2 domain of Fyn is necessary for ligand-stimulated association with the PDGF receptor in vivo. Third, those fusion proteins that associated with the PDGF receptor also became phosphorylated in vivo following PDGF treatment, and in in vitro kinase assays, suggesting that the amino-terminal half of Fyn contains the sites of PDGF-stimulated phosphorylation. Partially purified, kinase-negative Fyn also became phosphorylated in the activated PDGF receptor complex in vitro, demonstrating that the PDGF receptor phosphorylates Fyn, rather than the novel phosphorylations occurring by autophosphorylation.
Insights
Platelet-derived growth factor (PDGF) binding activates Src family kinases, including Fyn. The Fyn SH2 domain mediates direct association with the PDGF receptor, which then phosphorylates Fyn.
Area of Science:
- Cellular signaling pathways
- Protein-protein interactions
- Receptor tyrosine kinases
Background:
- Src family kinases (Src, Fyn, Yes) are ubiquitously expressed and implicated in growth control.
- Previous studies showed Fyn, Src, and Yes kinases associate with the PDGF receptor upon PDGF stimulation.
- The specific molecular requirements for this kinase-receptor association remained unclear.
Purpose of the Study:
- To elucidate the molecular requirements for the association between Fyn and the PDGF receptor.
- To determine if the Fyn-PDGF receptor interaction is direct.
- To identify the regions of Fyn involved in PDGF-stimulated phosphorylation.
Main Methods:
- Baculovirus expression system for in vitro association studies.
- Generation of Fyn-beta-galactosidase fusion proteins in cell lines.
- In vivo and in vitro kinase assays to assess phosphorylation.
Main Results:
- Fyn directly associates with the activated PDGF receptor in vitro.
- The SH2 domain of Fyn is essential for its association with the PDGF receptor in vivo.
- PDGF-stimulated phosphorylation of Fyn occurs in its amino-terminal half.
- The PDGF receptor directly phosphorylates Fyn.
Conclusions:
- Fyn directly binds the activated PDGF receptor via its SH2 domain.
- The PDGF receptor phosphorylates Fyn, contributing to its activation.
- These findings clarify the molecular mechanism of Fyn-PDGF receptor interaction in cellular signaling.