title: Proteomic analysis of the epidermal growth factor receptor (EGFR) interactome and post-translational modifications associated with receptor endocytosis in response to EGF and stress.
abstract: Aberrant expression, activation, and stabilization of epidermal growth factor receptor (EGFR) are causally associated with several human cancers. Post-translational modifications and protein-protein interactions directly modulate the signaling and trafficking of the EGFR. Activated EGFR is internalized by endocytosis and then either recycled back to the cell surface or degraded in the lysosome. EGFR internalization and recycling also occur in response to stresses that activate p38 MAP kinase. Mass spectrometry was applied to comprehensively analyze the phosphorylation, ubiquitination, and protein-protein interactions of wild type and endocytosis-defective EGFR variants before and after internalization in response to EGF ligand and stress. Prior to internalization, EGF-stimulated EGFR accumulated ubiquitin at 7 K residues and phosphorylation at 7 Y sites and at S(1104). Following internalization, these modifications diminished and there was an accumulation of S/T phosphorylations. EGFR internalization and many but not all of the EGF-induced S/T phosphorylations were also stimulated by anisomycin-induced cell stress, which was not associated with receptor ubiquitination or elevated Y phosphorylation. EGFR protein interactions were dramatically modulated by ligand, internalization, and stress. In response to EGF, different E3 ubiquitin ligases became maximally associated with EGFR before (CBL, HUWE1, and UBR4) or after (ITCH) internalization, whereas CBLB was distinctively most highly EGFR associated following anisomycin treatment. Adaptin subunits of AP-1 and AP-2 clathrin adaptor complexes also became EGFR associated in response to EGF and anisomycin stress. Mutations preventing EGFR phosphorylation at Y(998) or in the S(1039) region abolished or greatly reduced EGFR interactions with AP-2 and AP-1, and impaired receptor trafficking. These results provide new insight into spatial, temporal, and mechanistic aspects of EGFR regulation.
authors.affiliation: From the ‡The Hospital For Sick Children, Program in Molecular Structure and Function, Princess Margaret Cancer Centre, and Department of Molecular Genetics, University of Toronto. Peter Gilgan Centre for Research and Learning, 686 Bay Street, Toronto M5G 0A4, Canada.
authors.forename: Jiefei
authors.initials: J
authors.lastname: Tong
authors.affiliation: From the ‡The Hospital For Sick Children, Program in Molecular Structure and Function, Princess Margaret Cancer Centre, and Department of Molecular Genetics, University of Toronto. Peter Gilgan Centre for Research and Learning, 686 Bay Street, Toronto M5G 0A4, Canada.
authors.forename: Paul
authors.initials: P
authors.lastname: Taylor
authors.affiliation: From the ‡The Hospital For Sick Children, Program in Molecular Structure and Function, Princess Margaret Cancer Centre, and Department of Molecular Genetics, University of Toronto. Peter Gilgan Centre for Research and Learning, 686 Bay Street, Toronto M5G 0A4, Canada m.moran@utoronto.ca.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Moran
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D000841:Anisomycin / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology*; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction; D013312:Stress, Physiological / Q000502:physiology*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D033962:Adaptor Protein Complex 2; D000900:Anti-Bacterial Agents; D018808:Transcription Factor AP-1; D004815:Epidermal Growth Factor; D000841:Anisomycin; D044767:Ubiquitin-Protein Ligases; C512478:EGFR protein, human; D066246:ErbB Receptors; D048051:p38 Mitogen-Activated Protein Kinases