Receptor tyrosine kinases (RTKs) exist as part of a complex network of cell surface molecules co-ordinating intracellular responses to extracellular perturbations, including growth factor stimulation. RTKs initiate numerous phosphorylation-dependent signalling cascades, which are regulated differentially in response to different ligand binding to each RTK and by the spatial distribution of RTKs within a cell. Other signalling cascades are also simultaneously transmitting information throughout the cell, which also regulate RTK cellular outputs. RTK signalling ultimately gives rise to different transcriptional responses and cellular outputs, critical in development, tissue homeostasis and disease pathogenesis. Mass-spectrometry (MS)-based quantitative proteomics and phosphoproteomics allows characterisation of global RTK-mediated signalling and cellular responses and has been invaluable in improving our understanding of RTK function in diseases such as cancer. In this thesis, I apply multiple proteomics and phosphoproteomics approaches to study the spatiotemporal regulation of Fibroblast Growth Factor Receptor 2b (FGFR2b) signalling in response to the recycling stimulus Fibroblast Growth Factor 10 (FGF10). FGFR2 is amplified in 4% of triple negative breast cancers (TNBCs), and despite effort to treat FGFR2 overexpressing TNBCs with targeted therapies, limited success has been achieved so far. Given intracellular trafficking and recycling of RTKs to the cell surface is also known to regulate oncogenic RTK signalling, we firstly dissected signalling events orchestrated by FGFR recycling ligands. We used three trafficking phosphoproteomics approaches to uncover a novel âreciprocal primingâ event between FGFR2b and epidermal growth factor receptor (EGFR), which co-ordinated the trafficking of FGFR2b in response to FGF10 and promoted FGF10-mediated cell cycle entry and proliferation in breast cancer cells. We next sought to understand how RTK recycling mediated cellular responses through regulation of recycling endosome proximal signalling events. We employed two phosphoproteomics approaches, a biochemical approach to perturb RTK internalization and recycling as well as a proximity-dependent biotinylation ascorbate peroxidase 2 (APEX2) approach. Our perturbation and proximal phosphoproteomics approaches uncovered a recycling endosome regulation of mTOR signalling and autophagy, not identified using global phosphoproteomics approaches. We showed that recycling of FGFR2b in response to FGF10 suppressed autophagy and promoted survival of breast cancer cells. This response required both recycling of FGFR2b and was FGF10 specific. Lastly, to explore how FGFR2b was integrated within the network of cell surface RTKs, we explored the reciprocal regulation between FGF10-FGFR2b and epidermal growth factor (EGF)-EGFR signalling axis using a co-stimulation approach. Interactomics and global proteomics identified a novel FGFR2b-fatty acid synthase (FASN) signalling axis. Downstream of both FGFR2b and EGFR, FASN differentially co-ordinated cellular responses. While in response to co-stimulation, a proliferative response and a co-stimulation-specific migratory response were observed in TNBC cell line BT20, which were sensitive to FASN inhibition. Collectively, the work in this thesis highlights the value of using MS-based (phospho)proteomics to study RTK signalling and highlights the importance of studying RTKs within an integrated network of RTK signalling and trafficking events.
A Quantitative Proteomics Investigation of Spatiotemporal and Integrated Receptor Tyrosine Kinase Signalling
Ferguson, H. (Author). 4 Jan 2023
Student thesis: Phd