Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein-coupled receptor kinase-dependent manner

Most a-synuclein (alpha-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 alpha-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 alpha-syn, whereas cytosolic alpha-syn is phosphorylated exclusively by CK2. Expression of wild-type alpha-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into alpha-syn. However, wild-type and S129A alpha-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 alpha-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that alpha-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.