Characterization of Kinases Involved in the Phosphorylation of Aggregated α-Synuclein

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