title: Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein-coupled receptor kinase-dependent manner.
abstract: Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)-treated cells shows that G protein-coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.
authors.affiliation: Department of Neurology, Hematology, Metabolism, Endocrinology and Diabetology, Yamagata University Faculty of Medicine. 2-2-2 Iida-nishi, Yamagata 990-9585, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Hara
authors.forename: Shigeki
authors.initials: S
authors.lastname: Arawaka
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Sato
authors.forename: Youhei
authors.initials: Y
authors.lastname: Machiya
authors.forename: Can
authors.initials: C
authors.lastname: Cui
authors.forename: Asuka
authors.initials: A
authors.lastname: Sasaki
authors.forename: Shingo
authors.initials: S
authors.lastname: Koyama
authors.forename: Takeo
authors.initials: T
authors.lastname: Kato
mesh_terms: D001692:Biological Transport; D047390:Casein Kinase II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D054770:G-Protein-Coupled Receptor Kinase 3 / Q000235:genetics / Q000378:metabolism; D054774:G-Protein-Coupled Receptor Kinase 5 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D054768:G-Protein-Coupled Receptor Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction; D051844:alpha-Synuclein / Q000235:genetics* / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D051844:alpha-Synuclein; D012694:Serine; D047390:Casein Kinase II; D054770:G-Protein-Coupled Receptor Kinase 3; C517848:GRK3 protein, human; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; C517898:GRK5 protein, human; D004298:Dopamine