title: PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination.
abstract: Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1). Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the N-and C-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40-50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications.
authors.affiliation: Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX, 79968, United States of America.
authors.forename: Susana P
authors.initials: SP
authors.lastname: Barrera
authors.affiliation: Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX, 79968, United States of America.
authors.forename: Vicente
authors.initials: V
authors.lastname: Castrejon-Tellez
authors.affiliation: Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX, 79968, United States of America.
authors.forename: Margarita
authors.initials: M
authors.lastname: Trinidad
authors.affiliation: Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX, 79968, United States of America.
authors.forename: Elisa
authors.initials: E
authors.lastname: Robles-Escajeda
authors.affiliation: Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX, 79968, United States of America.
authors.forename: Javier
authors.initials: J
authors.lastname: Vargas-Medrano
authors.affiliation: Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX, 79968, United States of America.
authors.forename: Armando
authors.initials: A
authors.lastname: Varela-Ramirez
authors.affiliation: Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX, 79968, United States of America.
authors.forename: Manuel
authors.initials: M
authors.lastname: Miranda
mesh_terms: D000818:Animals; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D050487:Glycine Plasma Membrane Transport Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction; D013552:Swine; D054875:Ubiquitination
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D050487:Glycine Plasma Membrane Transport Proteins; D010703:Phorbol Esters; C493267:Slc6a9 protein, mouse; D011493:Protein Kinase C