Early signaling dynamics of the epidermal growth factor receptor.
Despite extensive study of the EGF receptor (EGFR) signaling network, the immediate posttranslational changes that occur in response to growth factor stimulation remain poorly characterized; as a result, the biological mechanisms underlying signaling initiation remain obscured. To address this deficiency, we have used a mass spectrometry-based approach to measure system-wide phosphorylation changes throughout the network with 10-s resolution in the 80 s after stimulation in response to a range of eight growth factor concentrations. Significant changes were observed on proteins far downstream in the network as early as 10 s after stimulation, indicating a system capable of transmitting information quickly. Meanwhile, canonical members of the EGFR signaling network fall into clusters with distinct activation patterns. Src homology 2 domain containing transforming protein (Shc) and phosphoinositol 3-kinase (PI3K) phosphorylation levels increase rapidly, but equilibrate within 20 s, whereas proteins such as Grb2-associated binder-1 (Gab1) and SH2-containing tyrosine phosphatase (SHP2) show slower, sustained increases. Proximity ligation assays reveal that Shc and Gab1 phosphorylation patterns are representative of separate timescales for physical association with the receptor. Inhibition of phosphatases with vanadate reveals site-specific regulatory mechanisms and also uncovers primed activating components in the network, including Src family kinases, whose inhibition affects only a subset of proteins within the network. The results presented highlight the complexity of signaling initiation and provide a window into exploring mechanistic hypotheses about receptor tyrosine kinase (RTK) biology.
['Reddy|Raven J|RJ|http://orcid.org/0000-0002-3856-7454', 'Gajadhar|Aaron S|AS|', 'Swenson|Eric J|EJ|', 'Rothenberg|Daniel A|DA|', 'Curran|Timothy G|TG|', 'White|Forest M|FM|']
D001940:Breast D002455:Cell Division D002460:Cell Line D016895:Culture Media, Serum-Free D004789:Enzyme Activation D004815:Epidermal Growth Factor D004847:Epithelial Cells D066246:ErbB Receptors D005260:Female D006801:Humans D046912:Multiprotein Complexes D010749:Phosphoprotein Phosphatases D010750:Phosphoproteins D010766:Phosphorylation D011499:Protein Processing, Post-Translational D015398:Signal Transduction D013997:Time Factors D019061:src-Family Kinases
epidermal growth factor receptor mass spectrometry signal transduction tyrosine phosphorylation