title: A chimeric pre-ubiquitinated EGF receptor is constitutively endocytosed in a clathrin-dependent, but kinase-independent manner.
abstract: The roles of EGF receptor (EGFR) kinase activity and ubiquitination in EGFR endocytosis have been controversial. The adaptor protein and ubiquitin ligase Cbl has reportedly been required. Consistently, we now report that siRNA-mediated knock-down of c-Cbl and Cbl-b significantly slowed clathrin-dependent internalization of activated wild-type (wt) EGFR by inhibiting recruitment of the EGFR to clathrin-coated pits. However, a chimeric protein consisting of wt-EGFR, a C-terminal linker and four linearly connected ubiquitins was found to interact with Eps15 and epsin 1 and to be constitutively endocytosed in a clathrin-dependent manner. Interestingly, endocytosis of this fusion protein did not require binding of EGF. Nor was kinase activity required, and the fusion protein was endocytosed in the presence of an EGFR kinase inhibitor, which efficiently counteracted tyrosine phosphorylation. This demonstrates that ubiquitination over-rides the requirement for kinase activity in recruitment of the EGFR to clathrin-coated pits.
authors.affiliation: Institute of Pathology, Faculty of Medicine, University of Oslo, 0027 Oslo, Norway.
authors.forename: Vibeke
authors.initials: V
authors.lastname: Bertelsen
authors.forename: Malgorzata Magdalena
authors.initials: MM
authors.lastname: Sak
authors.forename: Kamilla
authors.initials: K
authors.lastname: Breen
authors.forename: Marianne S
authors.initials: MS
authors.lastname: Rødland
authors.forename: Lene E
authors.initials: LE
authors.lastname: Johannessen
authors.forename: Linton M
authors.initials: LM
authors.lastname: Traub
authors.forename: Espen
authors.initials: E
authors.lastname: Stang
authors.forename: Inger Helene
authors.initials: IH
authors.lastname: Madshus
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D050721:Proto-Oncogene Proteins c-cbl / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; C486204:EPN2 protein, human; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; C114154:epsin; D004815:Epidermal Growth Factor; D050721:Proto-Oncogene Proteins c-cbl; D066246:ErbB Receptors