Omics approaches to explore cellular signaling networks

Receptor Tyrosine Kinases (RTKs) are fundamental molecular sensors that enable cells to interpret their environment and to decide whether to grow, move, differentiate, or die. RTKs reside at the plasma membrane, but can also enter into the cell, accumulate in specific cytoplasmic areas named endosomes and go back to the cell surface. This process is called receptor trafficking. Trafficking from and to the plasma membrane alters the way in which RTKs transmit the environmental signals that allow cells to interpret their surroundings. In particular, receptor accumulation into endosomes (internalization) followed by recycling to the cell surface regulates intracellular signaling cascades leading to the activation of specific long-term cellular responses. Importantly, such imbalances in receptor trafficking have been associated with human diseases (i.e. breast cancer), making this process a key focal point for study in biosciences. Using a highly multidisciplinary approach this project aims to uncover the key regulators of RTK trafficking and their influence on signaling specificity and cellular decisions.
Focusing on Fibroblast Growth Factor Receptor 2b (FGFR2b), which plays major roles during development and cancer progression, we have identified the kinase RSK2 as a novel regulator of FGFR2b trafficking and outputs upon stimulation with certain ligands. 'Functional proteomics', which integrates quantitative Mass Spectrometry (MS)-based proteomics, bioinformatics, functional assays, and imaging techniques, and RNA-seq will be used to generate RSK2-dependent signatures to be validated in FGFR2b trafficking and cellular assays in 2D and 3D cell culture. The specific aims of this project are:

1) To dissect the role of RSK2 in FGFR2b trafficking using immunofluorescence- and biochemical-based assays in epithelial cells where the expression level of RSK2 will be manipulated (i.e. CRISPR/Cas9 technology).
2) To generate a comprehensive RSK2-dependent signature in epithelial cells performing MS-based phosphoproteomics upon early time points stimulation, and quantitative proteomics and RNA-seq upon late time points stimulation, followed by bioinformatics analysis to integrate all these data sets. Selected candidates will be validated using trafficking and functional assays.
3) To explore how RSK2 affects cellular outputs, i.e. cell migration, in cells depleted of RSK2.

By combining cutting-edge -omics technology, bioinformatics, and functional assays, the student will make a significant contribution to our understanding of how recycling-dependent outputs could be manipulated to re-direct cellular decisions.

This highly multidisciplinary project integrates cutting-edge mass spectrometry (MS)-based (phospho) proteomics and transcriptomics with bioinformatics, and functional assays, addressing BBSRC remits for `systems approaches to the biosciences' and 'data driven biology' in bioscience. Omics investigation of proteins involved in trafficking of receptor tyrosine kinases will generate complex datasets to be analyzed with advanced bioinformatics tools followed by validation in a variety of cell-based assays. Through this 'new way of working', this project will exploit how a predictive systems biology approach enables advancements in scientific discoveries when integrated with defined biological endpoints. Furthermore, training will be provided in quantitative proteomics, transcriptomics, bioinformatics, and core research disciplines (molecular and cell biology), thus forming the next generation of protein scientists.