title: Enhanced ubiquitylation and accelerated degradation of the dopamine transporter mediated by protein kinase C.
abstract: Dopamine transporter (DAT) localization in dopaminergic neurons plays an important role in regulating dopamine signaling. However, the mechanisms of DAT trafficking that control DAT localization are still poorly understood. To gain insight into these mechanisms, human DAT was purified in large amounts using a two-step affinity chromatography procedure from untreated HeLa cells or cells treated with phorbol 12-myristate 13-acetate (PMA). Mass spectrometric analysis of purified DAT complexes revealed the presence of several proteins, among which ubiquitin was particularly abundant in the PMA-treated sample. Western blotting of highly purified DAT protein confirmed constitutive ubiquitylation of DAT and a dramatic increase in DAT ubiquitylation in cells treated with PMA. This increase was blocked by pretreatment with the protein kinase C (PKC) inhibitor bis-indolylmaleimide. DAT ubiquitylation by ectopically expressed ubiquitin was demonstrated in cells transiently transfected with yellow fluorescent protein-tagged ubiquitin. In addition, fluorescence resonance energy transfer was detected between cyan fluorescent protein-tagged DAT and yellow fluorescent protein-tagged ubiquitin, indicative of DAT-ubiquitin conjugation. Interestingly, the largest fluorescence resonance energy transfer signals were observed in endosomes. Ubiquitylated DAT was detected in the plasma membrane using cell surface biotinylation as well as in intracellular compartments, suggesting that ubiquitylation begins at the plasma membrane and is maintained in endosomes. In both porcine aortic endothelial and HeLa cells, where PKC-dependent DAT ubiquitylation was observed, PKC activation resulted in rapid degradation of DAT (t12 = 1-2 h). Altogether, these data suggest that PKC-induced DAT ubiquitylation may target DAT to lysosomal degradation.
authors.affiliation: Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, Colorado 80045, USA.
authors.forename: Manuel
authors.initials: M
authors.lastname: Miranda
authors.forename: Christine C
authors.initials: CC
authors.lastname: Wu
authors.forename: Tatiana
authors.initials: T
authors.lastname: Sorkina
authors.forename: Davin R
authors.initials: DR
authors.lastname: Korstjens
authors.forename: Alexander
authors.initials: A
authors.lastname: Sorkin
mesh_terms: D053674:Aggrecans; D000818:Animals; D001011:Aorta / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D050483:Dopamine Plasma Membrane Transport Proteins / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D037181:Lectins, C-Type / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry; D008301:Maleimides / Q000494:pharmacology; D013058:Mass Spectrometry; D009474:Neurons / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D011509:Proteoglycans / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013552:Swine; D013755:Tetradecanoylphorbol Acetate; D014162:Transfection; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D053674:Aggrecans; D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D050483:Dopamine Plasma Membrane Transport Proteins; D016326:Extracellular Matrix Proteins; D007211:Indoles; D037181:Lectins, C-Type; D008164:Luminescent Proteins; D008301:Maleimides; D011509:Proteoglycans; D011994:Recombinant Proteins; D025801:Ubiquitin; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; C088060:bisindolylmaleimide; D013755:Tetradecanoylphorbol Acetate