Characterization of kinases involved in the phosphorylation of aggregated α‐synuclein

Abstract α‐Synuclein (α‐syn) is the major component of pathological inclusions characteristic of several neurodegenerative disorders, such as Parkinson's disease. The major posttranslational modification of α‐syn is phosphorylation at S129, and previous studies estimate that approximately 90% of α‐syn in proteinaceous, pathological inclusions is phosphorylated at this site. α‐Syn can be phosphorylated by polo‐like kinases (PLKs) 1–3 and casein kinases (CK) 1 and 2; however, the kinases associated with the hyperphosphorylation of aggregated α‐syn are still under debate. Using a high‐efficiency cellular model of α‐syn aggregate formation, we found that selective inhibitors for CK2 and PLKs each partially inhibited S129 phosphorylation of soluble (nonaggregated) α‐syn, but only PLK inhibitors modestly attenuated the phosphorylation of aggregated α‐syn. In addition, none of the kinase inhibitors used had a substantial effect on the propensity of α‐syn to aggregate. Overexpression of all PLKs promoted robust phosphorylation of soluble α‐syn, but none altered the propensity of α‐syn to aggregate. Overexpression of only PLK2 increased phosphorylation of aggregated α‐syn at S129, which likely is due to increased phosphorylation of soluble α‐syn, which then was incorporated into aggregates. Overexpression of PLK1 and treatment with BI2536 resulted in a significant reduction of phosphorylated, aggregated α‐syn protein, beyond that of BI2536 treatment alone. These studies suggest that phosphorylation of α‐syn is independent of α‐syn aggregate formation, that PLK1 is involved in the phosphorylation of aggregated α‐syn at S129 in this system, and that mechanisms resulting in hyperphosphorylation of aggregated α‐syn appear to be independent of those responsible for the phosphorylation of soluble α‐syn. © 2010 Wiley‐Liss, Inc.