PMID- 26929352 OWN - NLM STAT- MEDLINE DCOM- 20160811 LR - 20181202 IS - 1091-6490 (Electronic) IS - 0027-8424 (Print) IS - 0027-8424 (Linking) VI - 113 IP - 11 DP - 2016 Mar 15 TI - Early signaling dynamics of the epidermal growth factor receptor. PG - 3114-9 LID - 10.1073/pnas.1521288113 [doi] AB - 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. FAU - Reddy, Raven J AU - Reddy RJ AUID- ORCID: 0000-0002-3856-7454 AD - Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139. FAU - Gajadhar, Aaron S AU - Gajadhar AS AD - Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139. FAU - Swenson, Eric J AU - Swenson EJ AD - Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139. FAU - Rothenberg, Daniel A AU - Rothenberg DA AD - Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139. FAU - Curran, Timothy G AU - Curran TG AD - Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139. FAU - White, Forest M AU - White FM AD - 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. LA - eng GR - R01CA096504/CA/NCI NIH HHS/United States GR - P30 CA014051/CA/NCI NIH HHS/United States GR - T32GM008334/GM/NIGMS NIH HHS/United States GR - U01 CA154966/CA/NCI NIH HHS/United States GR - U54 CA112967/CA/NCI NIH HHS/United States GR - T32 GM008334/GM/NIGMS NIH HHS/United States GR - U54CA112967/CA/NCI NIH HHS/United States GR - R01 CA118705/CA/NCI NIH HHS/United States GR - R01CA118705/CA/NCI NIH HHS/United States GR - R01 CA096504/CA/NCI NIH HHS/United States PT - Comparative Study PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20160229 PL - United States TA - Proc Natl Acad Sci U S A JT - Proceedings of the National Academy of Sciences of the United States of America JID - 7505876 RN - 0 (Culture Media, Serum-Free) RN - 0 (Multiprotein Complexes) RN - 0 (Phosphoproteins) RN - 62229-50-9 (Epidermal Growth Factor) RN - EC 2.7.10.1 (EGFR protein, human) RN - EC 2.7.10.1 (ErbB Receptors) RN - EC 2.7.10.2 (src-Family Kinases) RN - EC 3.1.3.16 (Phosphoprotein Phosphatases) SB - IM MH - Breast/cytology/*enzymology MH - Cell Division MH - Cell Line MH - Culture Media, Serum-Free/pharmacology MH - Enzyme Activation/drug effects MH - Epidermal Growth Factor/pharmacology MH - Epithelial Cells/drug effects/enzymology MH - ErbB Receptors/agonists/*physiology MH - Female MH - Humans MH - Multiprotein Complexes MH - Phosphoprotein Phosphatases/physiology MH - Phosphoproteins/analysis MH - Phosphorylation/drug effects MH - Protein Processing, Post-Translational/drug effects MH - Signal Transduction/*physiology MH - Time Factors MH - src-Family Kinases/physiology PMC - PMC4801278 OTO - NOTNLM OT - epidermal growth factor receptor OT - mass spectrometry OT - signal transduction OT - tyrosine phosphorylation COIS- The authors declare no conflict of interest. EDAT- 2016/03/02 06:00 MHDA- 2016/08/12 06:00 PMCR- 2016/09/15 CRDT- 2016/03/02 06:00 PHST- 2016/03/02 06:00 [entrez] PHST- 2016/03/02 06:00 [pubmed] PHST- 2016/08/12 06:00 [medline] PHST- 2016/09/15 00:00 [pmc-release] AID - 1521288113 [pii] AID - 201521288 [pii] AID - 10.1073/pnas.1521288113 [doi] PST - ppublish SO - Proc Natl Acad Sci U S A. 2016 Mar 15;113(11):3114-9. doi: 10.1073/pnas.1521288113. Epub 2016 Feb 29.