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. 2011 Nov;10(11):M111.008540.
doi: 10.1074/mcp.M111.008540. Epub 2011 Aug 21.

Quantitative Phospho-Proteomics to Investigate the Polo-Like Kinase 1-dependent Phospho-Proteome

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Free PMC article

Quantitative Phospho-Proteomics to Investigate the Polo-Like Kinase 1-dependent Phospho-Proteome

Karin Grosstessner-Hain et al. Mol Cell Proteomics. .
Free PMC article

Abstract

Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells. We compared phosphorylation sites in HeLa cells that were or were not treated with the PLK1-inhibitor BI 4834, by labeling peptides via methyl esterification, fractionation of peptides by strong cation exchange chromatography, and phosphopeptide enrichment via immobilized metal affinity chromatography. Analysis by quantitative mass spectrometry identified 4070 unique mitotic phosphorylation sites on 2069 proteins. Of these, 401 proteins contained one or multiple phosphorylation sites whose abundance was decreased by PLK1 inhibition. These include proteins implicated in PLK1-regulated processes such as DNA damage, mitotic spindle formation, spindle assembly checkpoint signaling, and chromosome segregation, but also numerous proteins that were not suspected to be regulated by PLK1. Analysis of amino acid sequence motifs among phosphorylation sites down-regulated under PLK1 inhibition in this data set identified two potential novel variants of the PLK1 consensus motif.

Figures

Fig. 1.
Fig. 1.
Sample generation and control experiments. A, Flow scheme summarizing the experimental strategy to identify PLK1-inhibitor-sensitive phosphorylation sites (numbers in bold reflect singly and multiply phosphorylated peptides, all other numbers are based on singly phosphorylated peptides). B, Time line to generate highly homogeneous mitotic cell cultures. Five different BI 4834 incubation times in the 3 h nocodazole arrest were tested. C, To identify the time of BI 4834 treatment required for full PLK1 inhibition with smallest indirect effect, samples were generated as described in B and analyzed for the mitotic status using phosphorylation of Histone H3 on serine 10 (H3 S10 ph) and phospho-dependent CDC27 shift, for CDK1 activity by probing CDK1 inhibitory phosphorylation on threonine 14 and tyrosine 15 (CDK1 T14,Y15 ph) and for PLK1 activity by probing STAG2 phosphorylation on serine 1224 (STAG2 S1224 ph).
Fig. 2.
Fig. 2.
Relative (phospho-)peptide frequency distribution and evaluation of MS data. A, Distribution of log2 ratios of all detected phosphopeptides and nonphosphopeptides. In total 9313 phosphopeptides and 12635 unphosphorylated peptides were detected. B, Validation of PLK1 targets by Western blot analysis with phospho-specific antibodies against CENPF, WAPAL, and EIF4B. Bar diagrams indicate the corresponding ratios found by MS quantification.
Fig. 3.
Fig. 3.
Bioinformatic analysis of the PLK1-dependent phospho-proteome and functional annotation of PLK1-dependent proteins. A, Extracted phosphorylation site-centered 15-mers of all unique phosphorylation sites were compared with Scansite predictions for 27 kinases (only five are shown, see supplementary Fig. S3 for full analysis). Only top-scoring kinase hits were accepted for each 15-mer to avoid redundancy. Predicted PLK1 targets are significantly enriched in the BI 4834-sensitive data set compared with the nonsensitive, which is indicated by the Fisher's Exact test p value of 3.905E-08 (*). B, Kinase consensus logos significantly enriched in either of the BI 4834-regulated data sets. The enrichment of the logo in comparison to the not-regulated (N-R) phosphopeptides is shown as bar diagram (S for sensitive, I for induced). Selected logos from the BI 4834-sensitive phosphopeptides (c-e) resemble the reported PLK1 consensus sequence whereas logos f and g represent a newly discovered potential PLK1 motif. C, Selected phospho-proteins showing BI 4834-dependent regulation according to subcellular localization and biological function are listed according to their regulatory ratio.

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