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

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

SUBMITTER: Hara S

PROVIDER: S-EPMC3667719 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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

Hara Susumu S Arawaka Shigeki S Sato Hiroyasu H Machiya Youhei Y Cui Can C Sasaki Asuka A Koyama Shingo S Kato Takeo T

Molecular biology of the cell 20130410 11

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 ...[more]

PMID: 23576548

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Project description:Parkinson´s disease (PD) is characterized by the presence of proteinaceous inclusions called Lewy bodies that are mainly composed of α-synuclein (αSyn). Elevated levels of oxidative or nitrative stresses have been implicated in αSyn related toxicity. Phosphorylation of αSyn on serine 129 (S129) modulates autophagic clearance of inclusions and is prominently found in Lewy bodies. The neighboring tyrosine residues Y125, Y133 and Y136 are phosphorylation and nitration sites. Using a yeast model of PD, we found that Y133 is required for protective S129 phosphorylation and for S129-independent proteasome clearance. αSyn can be nitrated and form stable covalent dimers originating from covalent crosslinking of two tyrosine residues. Nitrated tyrosine residues, but not di-tyrosine-crosslinked dimers, contributed to αSyn cytotoxicity and aggregation. Analysis of tyrosine residues involved in nitration and crosslinking revealed that the C-terminus, rather than the N-terminus of αSyn, is modified by nitration and di-tyrosine formation. The nitration level of wild-type αSyn was higher compared to that of A30P mutant that is non-toxic in yeast. A30P formed more dimers than wild-type αSyn, suggesting that dimer formation represents a cellular detoxification pathway in yeast. Deletion of the yeast flavohemoglobin gene YHB1 resulted in an increase of cellular nitrative stress and cytotoxicity leading to enhanced aggregation of A30P αSyn. Yhb1 protected yeast from A30P-induced mitochondrial fragmentation and peroxynitrite-induced nitrative stress. Strikingly, overexpression of neuroglobin, the human homolog of YHB1, protected against αSyn inclusion formation in mammalian cells. In total, our data suggest that C-terminal Y133 plays a major role in αSyn aggregate clearance by supporting the protective S129 phosphorylation for autophagy and by promoting proteasome clearance. C-terminal tyrosine nitration increases pathogenicity and can only be partially detoxified by αSyn di-tyrosine dimers. Our findings uncover a complex interplay between S129 phosphorylation and C-terminal tyrosine modifications of αSyn that likely participates in PD pathology.

Dataset Information

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

Publications

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

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