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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

In silico analysis of substrate binding zone of PLK2.A. Hydrophobic surface calculation of acidophilic kinases PLK2, PLK1, CK2α, CK1δ. In yellow the hydrophobic areas. Kinase active sites have been indicated by an arrow. B. Interaction between PLK2 and the phosphopeptide EAIAELDtLNEESYK (P31946). −3 and +1 leucine residues are shown in yellow, threonine in blue. ATP is shown in spheres.
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pone-0111018-g004: In silico analysis of substrate binding zone of PLK2.A. Hydrophobic surface calculation of acidophilic kinases PLK2, PLK1, CK2α, CK1δ. In yellow the hydrophobic areas. Kinase active sites have been indicated by an arrow. B. Interaction between PLK2 and the phosphopeptide EAIAELDtLNEESYK (P31946). −3 and +1 leucine residues are shown in yellow, threonine in blue. ATP is shown in spheres.

Mentions: Of special interest is the enrichment in hydrophobic residues close to the PLK2 target residue, at −2 (the above-mentioned leucine) and at +1 position. The preference for hydrophobic residues is uncommon among acidophilic kinases even if this feature is shared with PLK1 [29]. Therefore we decided to further investigate this aspect. To provide a structural basis for this enrichment in hydrophobic residues at −2 and +1 position, an in silico analysis of the substrate binding zone of PLK2 was performed. Analyzing the hydrophobic amino acid distribution of PLK2 (Figure 4A) it is possible to observe the presence of hydrophobic regions in the active site (yellow areas). These hydrophobic regions, albeit less pronounced, are also present in the active site of PLK1 that also displays a preference for hydrophobic residues at −3 and +1 position (Figure 3C). By sharp contrast, these two hydrophobic regions are absent in the acidophilic kinases CK2 and CK1δ active sites (Figure 4A) consistent with the aminoacid preference observed in Figure 3.


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

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

In silico analysis of substrate binding zone of PLK2.A. Hydrophobic surface calculation of acidophilic kinases PLK2, PLK1, CK2α, CK1δ. In yellow the hydrophobic areas. Kinase active sites have been indicated by an arrow. B. Interaction between PLK2 and the phosphopeptide EAIAELDtLNEESYK (P31946). −3 and +1 leucine residues are shown in yellow, threonine in blue. ATP is shown in spheres.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111018-g004: In silico analysis of substrate binding zone of PLK2.A. Hydrophobic surface calculation of acidophilic kinases PLK2, PLK1, CK2α, CK1δ. In yellow the hydrophobic areas. Kinase active sites have been indicated by an arrow. B. Interaction between PLK2 and the phosphopeptide EAIAELDtLNEESYK (P31946). −3 and +1 leucine residues are shown in yellow, threonine in blue. ATP is shown in spheres.
Mentions: Of special interest is the enrichment in hydrophobic residues close to the PLK2 target residue, at −2 (the above-mentioned leucine) and at +1 position. The preference for hydrophobic residues is uncommon among acidophilic kinases even if this feature is shared with PLK1 [29]. Therefore we decided to further investigate this aspect. To provide a structural basis for this enrichment in hydrophobic residues at −2 and +1 position, an in silico analysis of the substrate binding zone of PLK2 was performed. Analyzing the hydrophobic amino acid distribution of PLK2 (Figure 4A) it is possible to observe the presence of hydrophobic regions in the active site (yellow areas). These hydrophobic regions, albeit less pronounced, are also present in the active site of PLK1 that also displays a preference for hydrophobic residues at −3 and +1 position (Figure 3C). By sharp contrast, these two hydrophobic regions are absent in the acidophilic kinases CK2 and CK1δ active sites (Figure 4A) consistent with the aminoacid preference observed in Figure 3.

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