title: Identification of endosomal epidermal growth factor receptor signaling targets by functional organelle proteomics.
abstract: Epidermal growth factor (EGF) receptor (EGFR) signal transduction is organized by scaffold and adaptor proteins, which have specific subcellular distribution. On a way from the plasma membrane to the lysosome EGFRs are still in their active state and can signal from distinct subcellular locations. To identify organelle-specific targets of EGF receptor signaling on endosomes a combination of subcellular fractionation, two-dimensional DIGE, fluorescence labeling of phosphoproteins, and MALDI-TOF/TOF mass spectrometry was applied. All together 23 EGF-regulated (phospho)proteins were identified as being differentially associated with endosomal fractions by functional organelle proteomics; among them were proteins known to be involved in endosomal trafficking and cytoskeleton rearrangement (Alix, myosin-9, myosin regulatory light chain, Trap1, moesin, cytokeratin 8, septins 2 and 11, and CapZbeta). Interestingly R-Ras, a small GTPase of the Ras family that regulates cell survival and integrin activity, was associated with endosomes in a ligand-dependent manner. EGF-dependent association of R-Ras with late endosomes was confirmed by confocal laser scanning immunofluorescence microscopy and Western blotting of endosomal fractions. EGFR tyrosine kinase inhibitor gefitinib was used to confirm EGF-dependent regulation of all identified proteins. EGF-dependent association of signaling molecules, such as R-Ras, with late endosomes suggests signaling specification through intracellular organelles.
authors.affiliation: Biocenter, Division of Cell Biology, Innsbruck Medical University, A-6020 Innsbruck, Austria.
authors.forename: Taras
authors.initials: T
authors.lastname: Stasyk
authors.forename: Natalia
authors.initials: N
authors.lastname: Schiefermeier
authors.forename: Sergej
authors.initials: S
authors.lastname: Skvortsov
authors.forename: Heinz
authors.initials: H
authors.lastname: Zwierzina
authors.forename: Johan
authors.initials: J
authors.lastname: Peränen
authors.forename: Guenther K
authors.initials: GK
authors.lastname: Bonn
authors.forename: Lukas A
authors.initials: LA
authors.lastname: Huber
mesh_terms: D018929:Cell Culture Techniques; D015180:Electrophoresis, Gel, Two-Dimensional; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D000077156:Gefitinib; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics; D011799:Quinazolines / Q000494:pharmacology; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011506:Proteins; D011799:Quinazolines; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D048049:Extracellular Signal-Regulated MAP Kinases; D000077156:Gefitinib