title: Protein kinase C-mediated functional regulation of dopamine transporter is not achieved by direct phosphorylation of the dopamine transporter protein.
abstract: Dopaminergic neurotransmission is terminated by the action of the presynaptic dopamine transporter (DAT). It mediates Na(+)/Cl(-) -dependent re-uptake of extracellular dopamine (DA) into the cell, and is regarded as a major regulatory mechanism for synaptic transmission. Previous works have documented that protein kinase C (PKC) activator or inhibitor alters DA uptake by DAT, suggesting that PKC phosphorylation plays an important regulatory mechanism in DAT function. Based on the existence of consensus amino acid sequences for PKC phosphorylation, it has been postulated that PKC regulation of DAT is mediated by the direct phosphorylation of DAT protein. In this study, we try to discover whether the functional regulation of DAT by PKC is due to direct phosphorylation of DAT. The PKC null mutant hDAT, where all putative PKC phosphorylation sites are eliminated, has been constructed by the replacement of serine/threonine residues with glycines. The mutation itself showed no effect on the functional activities of DAT. The DA uptake activity of PKC null mutant was equivalent to those of wild-type hDAT (80-110% of wild-type). Phorbol ester activation of PKC inhibited DA uptake of wild-type hDAT by 35%, and staurosphorine blocked the effect of phorbol ester on DA uptake. The same phenomena was observed in PKC null mutant DAT, although no significant phosphorylation was observed by PKC activation. Confocal microscopic analysis using EGFP-fused DAT revealed that the activation of PKC by phorbol ester elicited fluorescent DAT to be internalized into the intracellular space both in wild-type and PKC null mutant DAT in a similar way. These results suggest that PKC-mediated regulation of DAT function is achieved in an indirect manner, such as phosphorylation of a mediator protein or activation of a clathrin-mediated pathway.
authors.affiliation: Departments of Biochemistry and Physiology, College of Medicine, Hanyang University, Seoul, Korea.
authors.forename: M Y
authors.initials: MY
authors.lastname: Chang
authors.forename: S H
authors.initials: SH
authors.lastname: Lee
authors.forename: J H
authors.initials: JH
authors.lastname: Kim
authors.forename: K H
authors.initials: KH
authors.lastname: Lee
authors.forename: Y S
authors.initials: YS
authors.lastname: Kim
authors.forename: H
authors.initials: H
authors.lastname: Son
authors.forename: Y S
authors.initials: YS
authors.lastname: Lee
mesh_terms: D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D010712:Phosphatidic Acids / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010712:Phosphatidic Acids; D011993:Recombinant Fusion Proteins; C493214:SLC6A3 protein, human; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D004298:Dopamine