Fibroblast growth factors (FGFs) are paracrine or endocrine signaling proteins that when activated by their ligands, can elicit a wide range of healthy and disease-related processes such as cell proliferation and the epithelial-to-mesenchymal transition (EMT). The detailed molecular pathway dynamics that coordinate these responses have remained unclear. To elucidate and monitor these dynamics, we stimulated MCF-7 breast cancer cells with either FGF2, 3, 4, 10, or 19. Following activation of the receptor, we quantified the kinase activity dynamics of 44 kinases using a targeted mass spectrometry assay. Our system-wide kinase activity data, supplemented with (phopsho)proteomics data, reveal ligand-dependent distinct pathway dynamics, elucidate the involvement of not earlier reported kinases such as MARK, and revise some of the pathway effects on biological outcomes. In addition, logic-based dynamic modeling of the kinome dynamics further verifies the biological goodness-of-fit of the predicted models and reveals tight regulation of the RAF kinase family.

FGF-stimulated MCF-7 cells were digested using trypsin and LysC, heavy labeled phosphopeptides were added, and the samples were desalted and enriched for phosphopeptides. Subsequently, the samples were measured on a Thermo Altis.