Receptor tyrosine kinase (RTK) signalling through extracellular-signal-regulated kinases (ERKs) has pivotal roles during metazoan development, underlying processes as diverse as fate determination, differentiation, proliferation, survival, migration and growth. Abnormal RTK/ERK signalling has been extensively documented to contribute to developmental disorders and disease, most notably in oncogenic transformation by mutant RTKs1 or downstream pathway components such as Ras and Raf2. Although the core RTK/ERK signalling cassette has been characterized by decades of research using mammalian cell culture and forward genetic screens in model organisms, signal propagation through this pathway is probably regulated by a larger network of moderate, context-specific proteins. The genes encoding these proteins may not have been discovered through traditional screens owing, in particular, to the requirement for visible phenotypes. To obtain a global view of RTK/ERK signalling, we performed an unbiased, RNA interference (RNAi), genome-wide, high-throughput screen in Drosophila cells using a novel, quantitative, cellular assay monitoring ERK activation. Here we show that ERK pathway output integrates a wide array of conserved cellular processes. Further analysis of selected components—in multiple cell types with different RTK ligands and oncogenic stimuli—validates and classifies 331 pathway regulators. The relevance of these genes is highlighted by our isolation of a Ste20-like kinase and a PPM-family phosphatase that seem to regulate RTK/ERK signalling in vivo and in mammalian cells. Novel regulators that modulate specific pathway outputs may be selective targets for drug discovery.
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We thank A. Philippakis and P. Hong for helpful discussions of statistical approaches, M. Melnick for dpERK antibody assistance, L. Kockel for pioneering the phospho-specific antibody high-throughput screen approach in our laboratory and help with in vivo analysis, C. Micchelli and R. Binari for assistance with genetic manipulations and interpretation, and other current and former members of the Perrimon lab for reagents and discussions. We thank L. Kockel, C. Micchelli, M. Kulkarni, B. Neel, R. Dasgupta, R. Binari and B. Mathey-Prevot for critical manuscript review. A.F. is a recipient of the Medical Scientist Training Program (MSTP) grant. N.P. is an investigator of the Howard Hughes Medical Institute.
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Algorithms for Molecular Biology (2015)