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Molecular & Cellular Proteomics 6:908-922, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Identification of Endosomal Epidermal Growth Factor Receptor Signaling Targets by Functional Organelle Proteomics ^*,S

Taras Stasyk, Natalia Schiefermeier, Sergej Skvortsov, Heinz Zwierzina, Johan Peränen^¶, Guenther K. Bonn^|| and Lukas A. Huber^,**

From the Biocenter, Division of Cell Biology, and Department of Internal Medicine, Innsbruck Medical University, A-6020 Innsbruck, Austria, ^¶ Institute of Biotechnology, University of Helsinki, Fi-00014 Finland, and ^|| Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, A-6020 Innsbruck, Austria

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 CapZß). 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.

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