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

Lead Research Organisation: University College London
Department Name: Cell and Developmental Biology


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In a series of experiments (unpublished) we can demonstrate an unusual and unexpected link between a major G-protein signaling system - the Arf family G-proteins that control vesicular trafficking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 forms
ii) 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 IP
iv) 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.


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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M009513/1 30/09/2015 31/03/2024
1907426 Studentship BB/M009513/1 30/09/2017 02/03/2022 Lewis Tanner
Description Proteins in a range of states created as part of the PhD have been used to test and develop capacity of separation at the industrial partner's High Performance Capillary Electrophoresis unit. Presentation of results to investors had led to increased funding and improved equipment design which hope to be used for future relevant testing services (such as ELISA) in the future of the company.
First Year Of Impact 2019
Sector Manufacturing, including Industrial Biotechology
Impact Types Economic

Description Benchmarking protein CGE at DeltaDot Ltd. 
Organisation deltaDOT Ltd
Country United Kingdom 
Sector Private 
PI Contribution A plan was devised for a collaborative benchmarking of proteins and separation improvement working with DeltaDOT technology platform in Camden. Much work was put into creating in kind funding against a UCL business partnership award providing a section of the funds to cover this work. This collaboration was not awarded funding and is currently on hold posing successful outcome in the future.
Collaborator Contribution I was tasked with parley between the two parties and devising the plan for 3-5 months of work along with costings. I wrote and collaborated on finding a funding source for this work and in creating all of the application materials required. I created protein samples to be carried out with this work which may be benchmarked for use at a later date pending successful future repeat funding.
Impact Greater links between DeltaDOT and UCL have been forged and are likely to form the basis of successful, academic-private collaboration in the future.
Start Year 2019
Description Process development at GMD 
Organisation Genetic Microdevices
Country United Kingdom 
Sector Private 
PI Contribution Ongoing development of GMD trademark Highly sensitive Capillery Gel Electrophoresis platform for the separation of novel protein forms. This has involved helping alter product design and developing methods specific to protein analysis.
Collaborator Contribution Use of equipment, expertise and consumables for sample analysis.
Impact Cannot disclose.
Start Year 2018
Description Organising committee for Breakthroughs in Healthcare Conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact I was part of organising committee finding small and medium sized healthcare technology businesses to take part in talks and bring career stands. Overall 6-7 industrial partners were in attendance ranging from an AI based small London based startup to multinationals such as AstraZeneca and Medtronic. This was positive in introducing researchers and industrial partners that may make for future partnerships and for showcasing careers for students interested in Industry with relevant careers contacts available.
Year(s) Of Engagement Activity 2018
Description Performed in Science comedy showcase 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact I learnt aspects of public engagement and comedy supervised under Dr Steve Cross. For this we had to write, rehearse and perform a comedy routine based around our research as part of an evening performance at Bloomsbury Theatre. The evening was very successful with a full house and gave me the ability to perform science comedy to diverse audiences in the future.
Year(s) Of Engagement Activity 2019