Characterisation of new cellular signalling systems using novel, rapid, high resolution protein electrophoresis

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A very large fraction of the human genome, and the genomes of other metazoans, encodes proteins that respond rapidly to changes in external and internal cellular environments. For example about 5% is given over to signaling systems that operate through GTP binding proteins and about 2.5% accounts for protein kinases. In addition, many hundreds more genes code proteins that support or respond to these two systems. In a series of experiments (unpublished) we can demonstrate an unusual and unexpected link between a major G-protein signaling system - the Arf family G-proteins that control vesicular trafficking and intracellular transport, cytoskeletal dynamics and phosphoinositide metabolism - and the Src family of non-receptor tyrosine kinases that contains major cellular oncogenes with roles in cell growth, differentiation, cell shape, migration and survival, and specialised cell signals. We can show that Arfs are tyrosine phosphorylated by Src kinases in a manner dependent on the Arf activation state - in the GDP bound "inactive" cytosolic form Arfs are poor substrates and in the GTP bound, membrane associated "active" form they are excellent substrates for the the kinases. Proofs in place (and relevant molecular biological tools and methods available) include: i) in vitro phosphorylation of pure recombinant GTP-Arf isoforms by pure recombinant Src and Lck enzymes but not GDP Arf formsii) demonstration of Arf protein tyrosine phosphorylation using transfection and immunoprecipitation (IP) of HA-tagged GTP-Arf1 and GTP-Arf6 but not GDP forms.iii) mass spectrometric identification of the phosphorylation site on Arf1 and Arf6 following IPiv) IP of native, wild-type Arfs and demonstration of tyrosine phosphorylation.v) differences in membrane association dynamics of wild-type Arf GFP fusion proteins and those with YF mutants (phosphorylation site eliminated) using FRAP confocal microscopy. We propose to use newly developed advanced, rapid protein separation and quantification techniques to fully investigate the importance of combined kinase-G protein signalling in the new coupled Src-Arf system. The project will have a manageable remit and a realistic prospect for success within the very tight 4-year timeframe. This project is technically, conceptually and theoretically very demanding. For example the dynamic properties of the GDP/GTP, GEF/GAP switch of G-proteins is (in reality) poorly understood (mathematical proofs and models in Stanley and Thomas 2016) compared to the kinase/phosphatase phosphorylation/dephosphorylaton cycle. As a result the dynamics of the combined system are not intuitive since the four-way interconversion of GDP-Arf, GTP-Arf, GDP-phospho Arf and GTP-phospho Arf forms is entirely novel in the cell signalling field. We therefore need methods to simultaneously isolate and quantify all four states of the Arf proteins at sufficient speed and very high species resolution (Appendix 1) to allow dynamics to be studied over highly sampled experimental time courses. Time series data will underpin mathematical models completely describing the dynamics of the coupled system that will supplement the cell biological and biochemical data described and allow a complete systems biological understanding of this unique combined cell signalling motif. The mathematical and computational approach will allow the complete description of the system for transparent and unambiguous communication to other researchers.