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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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Related in: MedlinePlus

Workflow for PLK2 peptide substrate identification.
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pone-0111018-g001: Workflow for PLK2 peptide substrate identification.

Mentions: Samples were labeled according to the dimethyl labeling method described in [23] and following the scheme reported in Figure 1. 400 µg of each peptide solution (control sample and PLK2 phosphorylated sample) was diluted to 500 µl of 5% formic acid. Each sample was then divided into two identical aliquots of 250 µl to perform a “forward” and a “reverse” experiment. Two isotopic forms of formaldehyde were used: the “light” form (CH2O) and the “medium” form (CD2O). Labeling was performed on-column using SepPak Vac 1cc C18 Cartridges, as described in [23]. Samples were mixed in a 1∶1 ratio as described in Figure 1 and dried under vacuum.


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

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

Workflow for PLK2 peptide substrate identification.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111018-g001: Workflow for PLK2 peptide substrate identification.
Mentions: Samples were labeled according to the dimethyl labeling method described in [23] and following the scheme reported in Figure 1. 400 µg of each peptide solution (control sample and PLK2 phosphorylated sample) was diluted to 500 µl of 5% formic acid. Each sample was then divided into two identical aliquots of 250 µl to perform a “forward” and a “reverse” experiment. Two isotopic forms of formaldehyde were used: the “light” form (CH2O) and the “medium” form (CD2O). Labeling was performed on-column using SepPak Vac 1cc C18 Cartridges, as described in [23]. Samples were mixed in a 1∶1 ratio as described in Figure 1 and dried under vacuum.

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