title: Ligand-induced lysosomal epidermal growth factor receptor (EGFR) degradation is preceded by proteasome-dependent EGFR de-ubiquitination.
abstract: Studies on the differential routing of internalized epidermal growth factor receptors (EGFRs) induced by EGF, TGF alpha, and the superagonist EGF-TGF alpha chimera E4T suggested a correlation between receptor recycling and their mitogenic potency. EGFR sorting to lysosomes depends on its kinase domain and its ubiquitination by Cbl proteins. Proteasomes have also been proposed to regulate EGFR degradation, but the underlying mechanism remains obscure. Here we evaluated EGFR activation, Cbl recruitment, EGFR ubiquitination and degradation in response to EGF, TGF alpha, and E4T. We also determined the fate of activated EGFRs and Cbl proteins by using v-ATPase (bafilomycin A1) and proteasome (lactacystin) inhibitors. Our results demonstrate that E4T and TGF alpha provoke decreased Cbl recruitment, EGFR ubiquitination and EGFR degradation compared with EGF. Furthermore, bafilomycin treatment blocks EGFR but not c-Cbl degradation. In contrast, lactacystin treatment blocks EGF-induced c-Cbl degradation but does not block EGFR degradation, even though lactacystin causes a minor delay in EGFR degradation. Surprisingly, even though bafilomycin completely blocks EGFR degradation, it does not prevent EGFR de-ubiquitination upon prolonged EGF stimulation. Strikingly, when combined with bafilomycin, lactacystin treatment stabilizes the ubiquitinated EGFR and prevents its de-ubiquitination. We conclude that the enhanced EGFR recycling that has been observed in HER-14 cells following TGF alpha or E4T stimulation correlates with decreased EGFR ubiquitination and EGFR degradation, and that proteasomal activity is required for de-ubiquitination of the EGFR prior to its lysosomal degradation.
authors.affiliation: Department of Cell Biology, University of Nijmegen, 6525 ED Nijmegen, The Netherlands.
authors.forename: Husam A J
authors.initials: HA
authors.lastname: Alwan
authors.forename: Everardus J J
authors.initials: EJ
authors.lastname: van Zoelen
authors.forename: Jeroen E M
authors.initials: JE
authors.lastname: van Leeuwen
mesh_terms: D016475:3T3 Cells; D000818:Animals; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D008024:Ligands; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016211:Transforming Growth Factor alpha; D025801:Ubiquitin; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex