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.