Abstract
Polo-like kinase 2 (PLK2) has been recently recognized as the major enzyme responsible for phosphorylation of α-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.
Highlights
The Polo like-kinase 2 (PLK2) is a serine/threonine kinase belonging to the POLO like kinase family playing a role in cell cycle progression, mitosis, cytokinesis, and DNA damage response
PLK2 is involved in cell cycle regulation, is required for centriole duplication in mammalian cells [4], regulates mitotic spindle in the mammary gland [5], and is a direct transcriptional target of p53 activating G2-M checkpoint, which prevents mitotic catastrophe following spindle damage [6]
In this regard PLK2 can bind and phosphorylate the mutant p53, inducing an oncogenic feedback loop in cancer cells [8], or may promote Mcl1 stabilization, providing resistance to cell death induced by TRAIL in Cholangiocarcinoma [9]
Summary
The Polo like-kinase 2 (PLK2) is a serine/threonine kinase belonging to the POLO like kinase family playing a role in cell cycle progression, mitosis, cytokinesis, and DNA damage response. To increase the number of identified phosphopeptides, each sample was analyzed three times with the same chromatographic conditions but using different fragmentation methods as described in [24].
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