title: Cbl and Itch binding sites in ERBB4 CYT-1 and CYT-2 mediate K48- and K63-polyubiquitination, respectively.
abstract: ERBB receptors have an important function in mammalian development and normal physiology, but overexpression and poor downregulation of ERBB receptors have been associated with malignant growth. Ligand-induced ERBB receptor signaling is terminated by clathrin-dependent receptor endocytosis, followed by incorporation of activated receptor complexes into multi-vesicular bodies and subsequent degradation in lysosomes. In the case of ERBB1, also known as the EGF receptor, it has been shown that ubiquitination serves as a signal to facilitate internalization and subsequent endosomal sorting, but little is known about the role of ubiquitination of other ERBB receptors. In the present study we investigated the regulation of ubiquitination and deubiquitination of the ERBB4 CYT-1 and CYT-2 isoforms in the context of chimeric EGFR-ERBB4 receptors. We demonstrate that EGFR-ERBB4 CYT-2 chimera shows decreased ligand-induced downregulation and EGF-degradation, as well as enhanced EGF recycling, when compared to EGFR-ERBB4 CYT-1. Moreover we show that the mutation Y1103F in the binding site for Cbl which is present in both CYT-1 and CYT-2, does not influence ERBB4 endosomal trafficking. We further demonstrate that total ligand-induced ubiquitination of CYT-1 is higher than that of CYT-2, whereby CYT-1 ubiquitination is mainly dependent on the PPXY(1056) Itch binding site for the E3-ligase Itch which is only present in CYT-1, while that of CYT-2 is dependent on the Y1103 Cbl binding site. The E3-ligase c-Cbl is more efficiently phosphorylated upon EGF stimulation of the CYT-2 than the CYT-1 isoform. Moreover our data show that the pY1103 Cbl binding site is required for K48-polyubiquitination of both CYT-1 and CYT-2, whereas the PPXY(1056) Itch binding site is required for K63-polyubiquitination of CYT-1. We further demonstrate that EGF stimulation of EGFR-ERBB4 CYT-1 and CYT-2 does not result in efficient binding to and tyrosine phosphorylation of the ESCRT-0 subunit Hrs. Finally, even though CYT-1 shows ligand-induced K63-polyubiquitination, it is not subjected to deubiquitination by the K63 polyubiquitin-specific AMSH deubiquitinating enzyme, while CYT-1 is slightly deubiquitinated by USP8. We conclude that Cbl and Itch binding sites in ERBB4 CYT-1 and CYT-2 mediate K48- and K63-polyubiquitination, respectively.
authors.affiliation: Department of Cell and Applied Biology, Faculty of Science, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.
authors.forename: Inez M J
authors.initials: IM
authors.lastname: Meijer
authors.forename: Walter
authors.initials: W
authors.lastname: van Rotterdam
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: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D015536:Down-Regulation; D011992:Endosomes / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism*; D066247:Receptor, ErbB-4; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins / Q000378:metabolism*; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D004815:Epidermal Growth Factor; C432966:ITCH protein, human; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C587689:ERBB4 protein, human; D066246:ErbB Receptors; C587690:Erbb4 protein, mouse; D066247:Receptor, ErbB-4; D008239:Lysine