Proteomics Analysis Identifies Phosphorylation-dependent α-Synuclein Protein Interactions

Abstract

Mutations and copy number variation in theHere we used the combination of peptide pulldown assays and mass spectrometry to identify and compare protein-protein interactions of phosphorylated and nonphosphorylated ␣-synuclein. We showed that non-phosphorylated ␣-synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas ␣-synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated ␣-synuclein was highly enriched in certain cytoskeletal proteins, in vesicular trafficking proteins, and in a small number of enzymes involved in protein serine phosphorylation. This targeted comparative proteomics approach for unbiased identification of protein-protein interactions sug-

α-Synuclein interacts with the switch region of Rab8a in a Ser129 phosphorylation-dependent manner

“…4). This finding is in line with a previous observation that αS phosphorylated at S129 is found specifically in pull downs with vesicular trafficking proteins (McFarland et al, 2008). Notably, the S129D variant of αS also showed enhanced Rab8a binding (Fig.…”

“…In line with these findings, phosphorylation of αS at S129 can -dependent on the genetic background -reduce αS-induced defects in vesicle trafficking (Sancenon et al, 2012). Thus, our study provides molecular insights into the functional consequences of phosphorylation of αS at S129 and supports the role of changes in protein-protein interactions for αS pathogenicity (McFarland et al, 2008).…”

Role of Ser129 phosphorylation of α-synuclein in melanoma cells

“…These results suggest that S129 phosphorylation regulates the asyn release in melanoma cells. Indeed, McFarland et al demonstrated that S129 phosphorylation enables a-syn to interact with vesicular trafficking proteins of mouse brain (McFarland et al, 2008). Most recently, Wang et al detected the selective elevation of S129-phosphorylated a-syn in the cerebrospinal fluid of PD patients (Wang et al, 2012).…”

“…Lou et al reported that the S129 phosphorylation reduces the ability of a-syn to regulate tyrosine hydroxylase (TH) and protein phosphatase 2A (Lou et al, 2010). Recently, McFarland et al showed that the C-terminal peptide of S129-phosphorylated a-syn interacts preferentially with cytoskeletal proteins and vesicular trafficking proteins (McFarland et al, 2008), suggesting that the S129 phosphorylation likely has a profound effect on protein trafficking. Despite these observations, the physiological relevance of S129 phosphorylation is still unclear.…”

“…a-Syn localizes to synaptic vesicles on the presynaptic side and the nucleus -hence the name 'synuclein' (Maroteaux et al, 1988;Auluck et al, 2010). Although the exact function of a-syn is still unclear, substantial evidence now exists to suggest that this protein is predominantly natively unfolded in solution but can bind to phospholipid membranes by adopting an amphipathic helical conformation in its N-terminal amino acids (Bennett, 2005;McFarland et al, 2008).…”