title: Early signaling dynamics of the epidermal growth factor receptor.
abstract: 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.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139.
authors.forename: Raven J
authors.identifier: http://orcid.org/0000-0002-3856-7454
authors.initials: RJ
authors.lastname: Reddy
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139.
authors.forename: Aaron S
authors.initials: AS
authors.lastname: Gajadhar
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Swenson
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Rothenberg
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139.
authors.forename: Timothy G
authors.initials: TG
authors.lastname: Curran
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 fwhite@mit.edu.
authors.forename: Forest M
authors.initials: FM
authors.lastname: White
mesh_terms: D001940:Breast / Q000166:cytology / Q000201:enzymology*; D002455:Cell Division; D002460:Cell Line; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D066246:ErbB Receptors / Q000819:agonists / Q000502:physiology*; D005260:Female; D006801:Humans; D046912:Multiprotein Complexes; D010749:Phosphoprotein Phosphatases / Q000502:physiology; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors; D019061:src-Family Kinases / Q000502:physiology
chemical_list: D016895:Culture Media, Serum-Free; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D004815:Epidermal Growth Factor; C512478:EGFR protein, human; D066246:ErbB Receptors; D019061:src-Family Kinases; D010749:Phosphoprotein Phosphatases
keywords: epidermal growth factor receptor; mass spectrometry; signal transduction; tyrosine phosphorylation