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Identification of the PLK2-dependent phosphopeptidome by quantitative proteomics [corrected].

Franchin C, Cesaro L, Pinna LA, Arrigoni G, Salvi M - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy; Proteomics Center of Padova University, Padova, Italy.

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.

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Two-sample logo analysis of phosphosites generated by individual kinases vs. random S/T proteome.PLK2 phosphopeptides identified in this paper (A) or bona fide CK2 (B), PLK1 (C), and CK1δ (D) substrates collected from PhosphositePlus database, have been analyzed as described in Materials and Methods.
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pone-0111018-g003: Two-sample logo analysis of phosphosites generated by individual kinases vs. random S/T proteome.PLK2 phosphopeptides identified in this paper (A) or bona fide CK2 (B), PLK1 (C), and CK1δ (D) substrates collected from PhosphositePlus database, have been analyzed as described in Materials and Methods.

Mentions: Several considerations can be made observing the Two-sample logo of Figure 3A. Foremost this analysis confirms the acidophilic nature of PLK2 (initially observed by Johnson et al.[29]), showing an enrichment of acidic residues in all positions considered. Positions upstream from the site of phosphorylation (in particular from −3 to −1) display a higher selection consistent with previous observations that the specific determinants of PLK2 are mostly located on the N-terminal side of the target residue [13], [20], [30]. Moreover the main determinants in PLK2 target selection here identified correlate well with previous observations [13], [20], [30].


Identification of the PLK2-dependent phosphopeptidome by quantitative proteomics [corrected].

Franchin C, Cesaro L, Pinna LA, Arrigoni G, Salvi M - PLoS ONE (2014)

Two-sample logo analysis of phosphosites generated by individual kinases vs. random S/T proteome.PLK2 phosphopeptides identified in this paper (A) or bona fide CK2 (B), PLK1 (C), and CK1δ (D) substrates collected from PhosphositePlus database, have been analyzed as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206460&req=5

pone-0111018-g003: Two-sample logo analysis of phosphosites generated by individual kinases vs. random S/T proteome.PLK2 phosphopeptides identified in this paper (A) or bona fide CK2 (B), PLK1 (C), and CK1δ (D) substrates collected from PhosphositePlus database, have been analyzed as described in Materials and Methods.
Mentions: Several considerations can be made observing the Two-sample logo of Figure 3A. Foremost this analysis confirms the acidophilic nature of PLK2 (initially observed by Johnson et al.[29]), showing an enrichment of acidic residues in all positions considered. Positions upstream from the site of phosphorylation (in particular from −3 to −1) display a higher selection consistent with previous observations that the specific determinants of PLK2 are mostly located on the N-terminal side of the target residue [13], [20], [30]. Moreover the main determinants in PLK2 target selection here identified correlate well with previous observations [13], [20], [30].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy; Proteomics Center of Padova University, Padova, Italy.

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.

Show MeSH
Related in: MedlinePlus