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Building mechanistic models of kinase-driven signaling pathways requires quantitative measurements of protein phosphorylation across physiologically relevant conditions, but this is rarely done because of the insensitivity of traditional technologies. By using a multiplexed deep phosphoproteome profiling workflow, we were able to generate a deep phosphoproteomics dataset of the EGFR-MAPK pathway in non-transformed MCF10A cells across physiological ligand concentrations with a time resolution of <12 min and in the presence and absence of multiple kinase inhibitors. An improved phosphosite mapping technique allowed us to reliably identify >46,000 phosphorylation sites on >6600 proteins, of which >4500 sites from 2110 proteins displayed a >2-fold increase in phosphorylation in response to EGF. This data was then placed into a cellular context by linking it to 15 previously published protein databases. We found that our results were consistent with much, but not all previously reported data regarding the activation and negative feedback phosphorylation of core EGFR-ERK pathway proteins. We also found that EGFR signaling is biphasic with substrates downstream of RAS/MAPK activation showing a maximum response at <3ng/ml EGF while direct substrates, such as HGS and STAT5B, showing no saturation. We found that RAS activation is mediated by at least 3 parallel pathways, two of which depend on PTPN11. There appears to be an approximately 4-minute delay in pathway activation at the step between RAS and RAF, but subsequent pathway phosphorylation was extremely rapid. Approximately 80 proteins showed a >2-fold increase in phosphorylation across all experiments and these proteins had a significantly higher median number of phosphorylation sites (~18) relative to total cellular phosphoproteins (~4). Over 60% of EGF-stimulated phosphoproteins were downstream of MAPK and included mediators of cellular processes such as gene transcription, transport, signal transduction and cytoskeletal arrangement. Their phosphorylation was either linear with respect to MAPK activation or biphasic, corresponding to the biphasic signaling seen at the level of the EGFR. This deep, integrated phosphoproteomics data resource should be useful in building mechanistic models of EGFR and MAPK signaling and for understanding how downstream responses are regulated.
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Discovering the dynamic world of the EGFR-MAPK phosphoproteome: Feng, Sanford and colleagues developed a multiplexed deep phosphoproteome profiling workflow unveiling 4500 protein sites exhibiting increased phosphorylation upon EGF stimulation.