title: Protein kinase C (PKC)-promoted endocytosis of glutamate transporter GLT-1 requires ubiquitin ligase Nedd4-2-dependent ubiquitination but not phosphorylation.
abstract: Glutamate transporter-1 (GLT-1) is the main glutamate transporter in the central nervous system, and its concentration severely decreases in neurodegenerative diseases. The number of transporters in the plasma membrane reflects the balance between their insertion and removal, and it has been reported that the regulated endocytosis of GLT-1 depends on its ubiquitination triggered by protein kinase C (PKC) activation. Here, we identified serine 520 of GLT-1 as the primary target for PKC-dependent phosphorylation, although elimination of this serine did not impair either GLT-1 ubiquitination or endocytosis in response to phorbol esters. In fact, we present evidence indicating that the ubiquitin ligase Nedd4-2 mediates the PKC-dependent ubiquitination and down-regulation of GLT-1. Overexpression of Nedd4-2 increased the ubiquitination of the transporter and promoted its degradation. Moreover, phorbol myristate acetate enhanced Nedd4-2 phosphorylation and the formation of GLT-1·Nedd4-2 complexes, whereas siRNA knockdown of Nedd4-2 prevented ubiquitination, endocytosis, and the concomitant decrease in GLT-1 activity triggered by PKC activation. These results indicate that GLT-1 endocytosis is independent of its phosphorylation and that Nedd4-2 mediates PKC-dependent down-regulation of the transporter.
authors.affiliation: Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain.
authors.forename: Noemí
authors.initials: N
authors.lastname: García-Tardón
authors.forename: Inmaculada M
authors.initials: IM
authors.lastname: González-González
authors.forename: Jaime
authors.initials: J
authors.lastname: Martínez-Villarreal
authors.forename: Enrique
authors.initials: E
authors.lastname: Fernández-Sánchez
authors.forename: Cecilio
authors.initials: C
authors.lastname: Giménez
authors.forename: Francisco
authors.initials: F
authors.lastname: Zafra
mesh_terms: D000818:Animals; D019556:COS Cells; D002273:Carcinogens / Q000494:pharmacology; D002522:Chlorocebus aethiops; D004285:Dogs; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D027342:Excitatory Amino Acid Transporter 2; D050491:Glutamate Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000075702:Nedd4 Ubiquitin Protein Ligases; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects / Q000502:physiology*; D029867:Xenopus Proteins; D014982:Xenopus laevis
chemical_list: D002273:Carcinogens; D056827:Endosomal Sorting Complexes Required for Transport; D027342:Excitatory Amino Acid Transporter 2; D050491:Glutamate Plasma Membrane Transport Proteins; C493397:SLC1A2 protein, human; D029867:Xenopus Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618460:nedd4l protein, Xenopus; D044767:Ubiquitin-Protein Ligases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate