A13
Differential phosphoproteomics of fibroblast growth factor signalling: identification of Src-mediated phosphorylation events
Debbie Cunningham1, Steve Sweet2, Helen Cooper1, John Heath1
1University of Birmingham, Birminghan, UK, 2University of Illinois at Urbana-Champaign, Urbana, IL, USA
Background
The fibroblast growth factor (FGF) family of ligands and receptors execute a wide range of biological functions in development, tissue repair, angiogenesis and cellular homeostasis. Dysregulation of the FGF signalling axis by a diversity of mechanisms is a frequent feature of a range of different tumour cell types and, as a result, the FGF pathway is the target for the development of small molecule kinase inhibitors and other forms of therapeutic intervention. However in order to better inform the development of therapeutic interventions in the FGF pathway, and analyse their molecular actions, a complete understanding of the architecture of the downstream processes activated by FGF signalling is required. In this study we apply a differential phosphoproteomics approach to the identification of phosphorylation events mediated as a consequence of activated FGFR signalling.
Method
In this study we set out to identify further direct and indirect phosphorylated protein targets for activated FGFR kinase activity using ultra-high resolution mass spectrometry techniques. From an initial survey of phosphorylation sites in FGF stimulated cells we applied SILAC combined with chemical inhibition of Src kinase activity to search for phosphorylation events that are dependent on Src kinase activity in FGF stimulated cells.
Results
This revealed a range of target proteins, including known Src substrates, downstream kinases and adaptor proteins. By iterative application of SILAC techniques specific instances of Src mediated phosphorylation events were further characterised by high coverage phosphopeptide mapping of one Src substrate protein, the multifunctional adaptor Dok1, and the further identification of Src kinase dependent Dok1 binding partners.
Conclusion
Collectively these results significantly expand the range of proteins implicated in the FGF signalling pathway, providing an inventory of phosphorylation events to monitor the activation status of the FGF pathway and reveal new targets for therapeutic intervention in FGF and Src signalling.
