TRAFFIC PROTEOMICS · Proteomic analysis of signaling induced by receptor tyrosine kinase endocytosis
FP7 — People (Marie Curie Actions)
- Duration
- 2010-03-01 → 2012-02-29
- EU contribution
- €206,129
- Participants
- 1
- Scheme
- MC-IEF
Lines connect the coordinator with its partners.
Results in brief
Proteomic analysis of signalling induced by receptor tyrosine kinase endocytosis
Receptor tyrosine kinase (RTK)-dependent signalling controls many cellular processes. Tight regulation of signalling propagation and specificity is often lost in various diseases such as cancer. Endocytosis, the process by which cells sort RTKs for either degradation or recycling, is a potent regulator of signalling specificity and duration, resulting in different cellular outcomes. However, the molecular bases of endocytosis-dependent signalling are not fully understood. Here, we focused on epidermal growth factor receptor (EGFR) and fibroblast growth factor receptors 1 and 2 (FGFRs 1 and 2) as RTKs model systems to define the signalling cascades that are specifically regulated by the different endocytic pathways. Our study was based on an unbiased approach consisting of mass spectrometry (MS)-driven quantitative proteomics (in particular phosphoproteomics), followed by the use of appropriate functional assay to validate the results. Overview of the work: (a) establishment and optimisation of a workflow for the enrichment and the analysis of phosphopeptides (phosphoproteomics); (b) establishment of a new protocol for the enrichment of tyrosine-phosphorylated containing peptides; (c) generation and analysis of biological replicates of phosphoproteomes of the three mentioned RTKs taken at different time points. We characterised the dynamic activation of signalling cascades focusing on very early (upon 1 minute stimulation), early (upon 8 minutes) and late (upon 40 minutes) events; (d) employment of a large-scale quantitative strategy based on pull-down assays using either phosphopeptides or SH2 domains as baits to further characterise RTKs-dependent signaling cascades; (e) use of small-interfering ribonucleic acid (siRNA) and confocal microscopy to validate the results obtained in the phosphoproteomics screening. Results and conclusions: (a) The stimulation of each RTK with ligands that induce either receptor degradation or recycling results in the activation of RTK-specific signalling cascades. We have not found a common signature associated to each endocytic route. (b) For each RTK we found specific signalling events and cellular processes associated to one or the other ligand: both ligand specificity and timing are required for the activation of the right intracellular cascade resulting in the right cellular response. (c) Already upon 1 minute stimulation (very early events), we observed significant differences in both RTKs phosphorylation and signalling activation. (d) In particular, a very early molecular switch controls the fate of FGFR2, one of the RTKs considered in this project. (e) The combination of advanced MS-based functional proteomics (phosphoproteomics, phosphopeptides pull-down assays) with more classical biochemical (i.e. siRNA screening) and cellular (i.e. microscopy) techniques allowed us to perform not only a system-wide analysis of RTKs entire cascades but also to further characterise some of their previously unknown individual components. (f) We believed that such a system biology approach shed light on some previously uncharacterised molecular aspects of RTK signalling and regulation. (g) Finally, since aberrant RTK signalling and trafficking are hallmarks of various diseases, including many cancers, the implications of our studies extend beyond basic research, paving the way to better define the pathogenesis of RTK-driven diseases, with the potential to identify novel therapeutic targets.
Data: CORDIS, © European Union
Project objective
Stimulated Receptor Tyrosine Kinases (RTKs) regulate several key cellular processes, such as cell proliferation, differentiation, migration and transformation, by activating specific intracellular signaling pathways. Endocytosis, the process by which cells internalize and sort RTKs for either degradation or recycling, is a potent regulator of signal transduction specificity and cellular outcomes. Several RTKs have been shown to escape from the canonical degradative route under certain conditions, resulting in signaling amplification - which often results in tumorigenesis. In particular, EGFR and FGFRs follow opposite endocytic routes upon stimulation by different ligands. This application focuses on EGFR and FGFRs as model systems to define signaling cascades that are specifically regulated by the different endocytic pathways. We will integrate an unbiased data-driven approach consisting of quantitative proteomics, based on high-resolution mass-spectrometry in combination with proteome-wide metabolic incorporation of stable isotopes, followed by proper functional assays to validate the results. This system biology approach will shed light on some previously uncharacterized aspects of RTK signaling regulation at the whole-cell level, gaining insights into the molecular mechanisms underlying endocytosis-driven cellular responses. Since aberrant RTK signaling is a hallmark of various diseases, such as many cancers, the implications of our studies extend beyond basic research, offering the opportunity to identify novel therapeutic targets, e.g. for the development of anti-cancer drugs. These objectives are part of European research’s efforts to elucidate physiological processes, search for pathogenic events and novel targets and contribute to the technological progress of protein research.
Original text from CORDIS.
Participants
- KOBENHAVNS UNIVERSITET · KOBENHAVNCoordinatorDenmark
Links
Data: CORDIS, © European Union