Dudka, Anna Agnieszka
(2010).
Identification of novel components in fibroblast growth factor signalling.
University of Birmingham.
Ph.D.
Abstract
Fibroblast growth factor receptors play many roles in development, cell proliferation and differentiation. They possess intrinsic tyrosine kinase activity which enables activation of other signalling proteins, formation of multiprotein signalling complexes and activation of downstream cascades. The goal of this project was to identify novel FGFR1 interacting proteins. The strategy employed for mapping novel partners on the basis of peptide-protein interaction was peptide pull-down. Using synthetic pairs of phosphorylated and unphosphorylated peptides, pull down experiments were performed to enrich phospho-specific binding partners which then were identified by mass spectrometry. Experiments carried out using FGFR1 peptides revealed novel proteins associated with receptor. Signal transducer and activator of transcription 3 (STAT3) was identified as a phospho-dependent partner for Tyr677 of FGFR1. Mutation of this tyrosine to phenylalanine eliminated the binding of STAT3 to FGFR1. Furthermore, it was presented that STAT3 tyrosine phosphorylation required over-expression of FGFRs, as shown in the breast cancer cell line, SUM-52PE. The inhibition of Src and Janus non receptor tyrosine kinases decreased FGF1-induced tyrosine STAT3 phosphorylation. The findings suggested that FGFR kinase activity was mandatory for physical association between FGFR and STAT3 and its subsequent tyrosine activation by Src and Jak kinases. Moreover, Src and Jak2 were demonstrated to form a complex with kinase active FGFR1. Finally, STAT3 was serine phosphorylated by JNK and ERK kinases, which were activated by FGF1 stimulation. Since over-expression of FGFRs is correlated with tumour development and STAT3 is a well known oncogene, it is possible that the FGFRSTAT3 signalling pathway is up regulated in cancer cells and therefore merits consideration as a therapeutic target.
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