title: PKCβ-dependent phosphorylation of the glycine transporter 1.
abstract: The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the glycine transporter 1 (GlyT1) and the clearance rate for glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the years, it has been shown that PKC tightly regulates the activity of several neurotransmitter transporters. In the present work, by stably expressing three N-terminus GlyT1 isoforms in porcine aortic endothelial cells and assaying for [(32)P]-orthophosphate metabolic labeling, we demonstrated that the isoforms GlyT1a, GlyT1b, and GlyT1c were constitutively phosphorylated, and that phosphorylation was dramatically enhanced, in a time dependent fashion, after PKC activation by phorbol ester. The phosphorylation was PKC-dependent, since pre-incubation of the cells with bisindolylmaleimide I, a selective PKC inhibitor, abolished the phorbol ester-induced phosphorylation. Blotting with specific anti-phospho-tyrosine antibodies did not yield any signal that could correspond to GlyT1 tyrosine phosphorylation, suggesting that the phosphorylation occurs at serine and/or threonine residues. In addition, a 23-40%-inhibition on V(max) was obtained by incubation with phorbol ester without a significant change on the apparent Km value. Furthermore, pre-incubation of the cells with the selective PKCα/β inhibitor Gö6976 abolished the downregulation effect of phorbol ester on uptake and phosphorylation, whereas the selective PKCβ inhibitors (PKCβ inhibitor or LY333531) prevented the phosphorylation without affecting glycine uptake, defining a specific role of classical PKC on GlyT1 uptake and phosphorylation. Taken together, these data suggest that conventional PKCα/β regulates the uptake of glycine, whereas PKCβ is responsible for GlyT1 phosphorylation.
authors.affiliation: Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX 79968, USA.
authors.forename: Javier
authors.initials: J
authors.lastname: Vargas-Medrano
authors.forename: Vicente
authors.initials: V
authors.lastname: Castrejon-Tellez
authors.forename: Fernando
authors.initials: F
authors.lastname: Plenge
authors.forename: Ivan
authors.initials: I
authors.lastname: Ramirez
authors.forename: Manuel
authors.initials: M
authors.lastname: Miranda
mesh_terms: D000818:Animals; D002460:Cell Line; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D005998:Glycine / Q000378:metabolism*; D050487:Glycine Plasma Membrane Transport Proteins / Q000378:metabolism*; D007211:Indoles / Q000037:antagonists & inhibitors; D008301:Maleimides / Q000037:antagonists & inhibitors; D010766:Phosphorylation / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D050487:Glycine Plasma Membrane Transport Proteins; D007211:Indoles; D008301:Maleimides; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; C070515:bisindolylmaleimide I; D005998:Glycine