Identification of the PLK2-Dependent Phosphopeptidome by Quantitative Proteomics

Polo-like kinase 2 (PLK2) has been recently recognized as the major enzyme responsible for phosphorylation of a-synuclein
at S129 in vitro and in vivo, suggesting that this kinase may play a key role in the pathogenesis of Parkinson's disease and
other synucleinopathies. Moreover PLK2 seems to be implicated in cell division, oncogenesis, and synaptic regulation of the
brain. However little is known about the phosphoproteome generated by PLK2 and, consequently the overall impact of
PLK2 on cellular signaling. To fill this gap we exploited an approach based on in vitro kinase assay and quantitative
phosphoproteomics. A proteome-derived peptide library obtained by digestion of undifferentiated human neuroblastoma
cell line was exhaustively dephosphorylated by lambda phosphatase followed by incubation with or without PLK2
recombinant kinase. Stable isotope labeling based quantitative phosphoproteomics was applied to identify the
phosphosites generated by PLK2. A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified
by LC-MS/MS. Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase
specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated
database of in cell/vivo phosphorylation sites