SILAC analysis of compartmentalised Ras signalling

Lead Research Organisation: University of Liverpool
Department Name: Biomedical Sciences


We are interested in Ras proteins that sit near the top of many cell signalling pathways and control key cellular functions such as proliferation, migration and cell death. Cells contain three almost identical versions (isoforms) that differ only in their preferred locations within the cell. These overlapping localisations are believed to underlie the biological differences known to exist between the isoforms. This model suggests that by operating from different platforms within the cell, Ras isoforms will come into contact with distinct pools of activators and downstream signalling proteins. We plan to test this model by targetting Ras to each of the different organelles where it is known to reside and compare the signals that are produced. We will use new technology that enables the responses of networks containing thousands of proteins to be precisely analysed. Our datasets will be mined to reveal likely relationships between signalling from each compartment and functional outputs that will be confirmed using cell-based assays. Together these data will provide a comprehensive picture of compartment-specific signalling.

Technical Summary

Ras proteins are key mediators of many signalling cascades. Recently compartment-specific signalling has been proposed to underlie the biological differences between closely related isoforms. We plan to comprehensively investigate Ras-dependent signalling from multiple subcellular platforms by targetting Ras and Ras modulators to different organelles. We will screen a wide range of effector pathways using quantitative SILAC proteomic analysis of signalling generated by constitutively active Ras targetted to plasma membrane microdomains, endosomes, mitochondria, ER/Golgi and Golgi. Complementary studies in Ras null cells transfected with wild type targetted Ras will enable analysis of acute and prolonged compartment-specific signalling. Importantly, the predicted coupling of spatial signalling with specific functional outputs revealed by our datasets will be investigated in appropriate cell-based assays. Together these strategies will generate an integrated overview of compartment-specific Ras signalling pathways, the extent to which they are conserved across cell types and the physiological outputs controlled by spatially distinct signalling domains.


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Hernandez-Valladares M (2015) Comparative proteomic analysis of compartmentalised Ras signalling. in Scientific reports

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Hernandez-Valladares M (2014) Quantitative proteomic analysis of compartmentalized signaling networks. in Methods in enzymology

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Prior IA (2012) A comprehensive survey of Ras mutations in cancer. in Cancer research

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Prior IA (2012) Ras trafficking, localization and compartmentalized signalling. in Seminars in cell & developmental biology

Description We have identified the consequences of location-specific cell communication through an important protein that is frequently mutated in cancer. These observations inform our understanding of how the family of Ras proteins work and potentially helps to explain how closely related family members generate different outputs.
Exploitation Route Compartment-specific Ras signalling has emerged as an important model in Ras research in recent years. Our data provide a comprehensive overview of this phenomenon that will be of broad interest to researchers in this area. We are in the process of publishing our data which will allow researchers to access compartment-specific quantitative data on >2000 proteins and phosphosites.
Sectors Pharmaceuticals and Medical Biotechnology

Description we successfully established novel cell reagents and proteomic profiling pipelines to assay compartmentalised Ras signalling. We found that each subcellular location generates a distinct signalling output and characterised the defining features of compartment-specific Ras signalling networks. The methods also created a capacity to perform large scale cell signalling characterisation that has resulted in further successful grant funding.
First Year Of Impact 2010
Sector Pharmaceuticals and Medical Biotechnology
Description Characterising isoform-specific KRAS4A and KRAS4B signalling
Amount £121,000 (GBP)
Organisation North West Cancer Research (NWCR) 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2013 
End 01/2015
Description Why are Ras isoforms not redundant? 'Characterising isoform-specific oncogenic Ras signaling networks'
Amount £194,803 (GBP)
Organisation North West Cancer Research (NWCR) 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2015 
End 01/2018