title: Tyrosine phosphorylation of NEDD4 activates its ubiquitin ligase activity.
abstract: Ligand binding to the receptor tyrosine kinase fibroblast growth factor (FGF) receptor 1 (FGFR1) causes dimerization and activation by transphosphorylation of tyrosine residues in the kinase domain. FGFR1 is ubiquitylated by the E3 ligase NEDD4 (also known as NEDD4-1), which promotes FGFR1 internalization and degradation. Although phosphorylation of FGFR1 is required for NEDD4-dependent endocytosis, NEDD4 directly binds to a nonphosphorylated region of FGFR1. We found that activation of FGFR1 led to activation of c-Src kinase-dependent tyrosine phosphorylation of NEDD4, enhancing the ubiquitin ligase activity of NEDD4. Using mass spectrometry, we identified several FGF-dependent phosphorylated tyrosines in NEDD4, including Tyr(43) in the C2 domain and Tyr(585) in the HECT domain. Mutating these tyrosines to phenylalanine to prevent phosphorylation inhibited FGF-dependent NEDD4 activity and FGFR1 endocytosis and enhanced cell proliferation. Mutating the tyrosines to glutamic acid to mimic phosphorylation enhanced NEDD4 activity. Moreover, the NEDD4 C2 domain bound the HECT domain, and the presence of phosphomimetic mutations inhibited this interaction, suggesting that phosphorylation of NEDD4 relieves an inhibitory intra- or intermolecular interaction. Accordingly, activation of FGFR1 was not required for activation of NEDD4 that lacked its C2 domain. Activation of c-Src by epidermal growth factor (EGF) also promoted tyrosine phosphorylation and enhanced the activity of NEDD4. Thus, we identified a feedback mechanism by which receptor tyrosine kinases promote catalytic activation of NEDD4 and that may represent a mechanism of receptor crosstalk.
authors.affiliation: Cell Biology Program, The Hospital for Sick Children, and Biochemistry Department, University of Toronto, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada.
authors.forename: Avinash
authors.initials: A
authors.lastname: Persaud
authors.affiliation: Cell Biology Program, The Hospital for Sick Children, and Biochemistry Department, University of Toronto, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada.
authors.forename: Philipp
authors.initials: P
authors.lastname: Alberts
authors.affiliation: IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Via Adamello 16, Milan 20139, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Mari
authors.affiliation: Molecular Structure and Function Program, The Hospital for Sick Children, and Molecular Genetics Department, University of Toronto, Toronto, Ontario M5G 0A4, Canada.
authors.forename: Jiefei
authors.initials: J
authors.lastname: Tong
authors.affiliation: Cell Biology Program, The Hospital for Sick Children, and Biochemistry Department, University of Toronto, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada.
authors.forename: Ryan
authors.initials: R
authors.lastname: Murchie
authors.affiliation: IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Via Adamello 16, Milan 20139, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Maspero
authors.affiliation: Cell Biology Program, The Hospital for Sick Children, and Biochemistry Department, University of Toronto, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada.
authors.forename: Frozan
authors.initials: F
authors.lastname: Safi
authors.affiliation: Molecular Structure and Function Program, The Hospital for Sick Children, and Molecular Genetics Department, University of Toronto, Toronto, Ontario M5G 0A4, Canada.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Moran
authors.affiliation: IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Via Adamello 16, Milan 20139, Italy. Dipartimento Scienze della Salute, Universita degli Studi di Milano, Via di Rudini 8, Milan 20142, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Polo
authors.affiliation: Cell Biology Program, The Hospital for Sick Children, and Biochemistry Department, University of Toronto, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada. drotin@sickkids.ca.
authors.forename: Daniela
authors.initials: D
authors.lastname: Rotin
mesh_terms: D018076:DNA, Complementary / Q000235:genetics; D004705:Endocytosis / Q000502:physiology; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D000075702:Nedd4 Ubiquitin Protein Ligases; D010766:Phosphorylation; D059748:Proteolysis; D060888:Real-Time Polymerase Chain Reaction; D020239:Receptor Cross-Talk / Q000502:physiology*; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D018076:DNA, Complementary; D056827:Endosomal Sorting Complexes Required for Transport; D014443:Tyrosine; D004815:Epidermal Growth Factor; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases; C496348:FGFR1 protein, human; D051496:Receptor, Fibroblast Growth Factor, Type 1