title: Preubiquitinated chimeric ErbB2 is constitutively endocytosed and subsequently degraded in lysosomes.
abstract: The oncoprotein ErbB2 is endocytosis-deficient, probably due to its interaction with Heat shock protein 90. We previously demonstrated that clathrin-dependent endocytosis of ErbB2 is induced upon incubation of cells with Ansamycin derivatives, such as geldanamycin and its derivative 17-AAG. Furthermore, we have previously demonstrated that a preubiquitinated chimeric EGFR (EGFR-Ub(4)) is constitutively endocytosed in a clathrin-dependent manner. We now demonstrate that also an ErbB2-Ub(4) chimera is endocytosed constitutively and clathrin-dependently. Upon expression, the ErbB2-Ub(4) was further ubiquitinated, and by Western blotting, we demonstrated the formation of both Lys48-linked and Lys63-linked polyubiquitin chains. ErbB2-Ub(4) was constitutively internalized and eventually sorted to late endosomes and lysosomes where the fusion protein was degraded. ErbB2-Ub(4) was not cleaved prior to internalization. Interestingly, over-expression of Ubiquitin Interaction Motif-containing dominant negative fragments of the clathrin adaptor proteins epsin1 and Eps15 negatively affected endocytosis of ErbB2. Altogether, this argues that ubiquitination is sufficient to induce clathrin-mediated endocytosis and lysosomal degradation of the otherwise plasma membrane localized ErbB2. Also, it appears that C-terminal cleavage is not required for endocytosis.
authors.affiliation: Institute of Clinical Medicine, University of Oslo, Rikshospitalet, 0027 Oslo, Norway.
authors.forename: Tram Thu
authors.initials: TT
authors.lastname: Vuong
authors.forename: Christian
authors.initials: C
authors.lastname: Berger
authors.forename: Vibeke
authors.initials: V
authors.lastname: Bertelsen
authors.forename: Marianne Skeie
authors.initials: MS
authors.lastname: Rødland
authors.forename: Espen
authors.initials: E
authors.lastname: Stang
authors.forename: Inger Helene
authors.initials: IH
authors.lastname: Madshus
mesh_terms: D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D055503:Protein Multimerization; D059748:Proteolysis / Q000187:drug effects; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D016227:Benzoquinones; D047029:Lactams, Macrocyclic; D047428:Protein Kinase Inhibitors; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C112765:tanespimycin