Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies.
EGF, but not TGF alpha, efficiently induces degradation of the EGF receptor (EGFR). We show that EGFR was initially polyubiquitinated to the same extent upon incubation with EGF and TGF alpha, whereas the ubiquitination was more sustained by incubation with EGF than with TGF alpha. Consistently, the ubiquitin ligase c-Cbl was recruited to the plasma membrane upon activation of the EGFR with EGF and TGF alpha, but localized to endosomes only upon activation with EGF. EGF remains bound to the EGFR upon endocytosis, whereas TGF alpha dissociates from the EGFR. Therefore, the sustained polyubiquitination is explained by EGF securing the kinase activity of endocytosed EGFR. Overexpression of the dominant negative N-Cbl inhibited ubiquitination of the EGFR and degradation of EGF and EGFR. This demonstrates that EGF-induced ubiquitination of the EGFR as such is important for lysosomal sorting. Both lysosomal and proteasomal inhibitors blocked degradation of EGF and EGFR, and proteasomal inhibitors inhibited translocation of activated EGFR from the outer limiting membrane to inner membranes of multivesicular bodies (MVBs). Therefore, lysosomal sorting of kinase active EGFR is regulated by proteasomal activity. Immuno-EM showed the localization of intact EGFR on internal membranes of MVBs. This demonstrates that the EGFR as such is not the proteasomal target.
Longva
Karianne E
KE
Institute of Pathology, The University of Oslo, Rikshospitalet, N-0027 Oslo, Norway.
Blystad
Froydis D
FD
Stang
Espen
E
Larsen
Astrid M
AM
Johannessen
Lene E
LE
Madshus
Inger H
IH
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D022162:Cytoplasmic Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D010450:Endopeptidases / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D007976:Leupeptins / Q000494:pharmacology; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D009097:Multienzyme Complexes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D021381:Protein Transport / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000494:pharmacology; D050721:Proto-Oncogene Proteins c-cbl; D016211:Transforming Growth Factor alpha / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D011518:Proto-Oncogene Proteins; D016211:Transforming Growth Factor alpha; D014452:Ubiquitins; D000643:Ammonium Chloride; C067713:lactacystin; D004815:Epidermal Growth Factor; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C067419:CBL protein, human; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine