SILAC analysis of compartmentalised Ras signalling

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.

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.