Quantitative Phospho-proteomics to Investigate the Polo-like Kinase 1-Dependent Phospho-proteome*

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.

[1]  J. Peters,et al.  The Small-Molecule Inhibitor BI 2536 Reveals Novel Insights into Mitotic Roles of Polo-like Kinase 1 , 2007, Current Biology.

[2]  Jan-Michael Peters,et al.  Polo-like kinase 1 triggers the initiation of cytokinesis in human cells by promoting recruitment of the RhoGEF Ect2 to the central spindle. , 2007, Developmental cell.

[3]  P. O’Farrell,et al.  Triggering the all-or-nothing switch into mitosis. , 2001, Trends in cell biology.

[4]  Zu-Wen Sun,et al.  Mitotic Phosphorylation of Histone H3 Is Governed by Ipl1/aurora Kinase and Glc7/PP1 Phosphatase in Budding Yeast and Nematodes , 2000, Cell.

[5]  Fa Liu,et al.  Structural and functional analyses of minimal phosphopeptides targeting the polo-box domain of polo-like kinase 1 , 2009, Nature Structural &Molecular Biology.

[6]  Karl Mechtler,et al.  Dissociation of Cohesin from Chromosome Arms and Loss of Arm Cohesion during Early Mitosis Depends on Phosphorylation of SA2 , 2005, PLoS biology.

[7]  I. Sumara,et al.  The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase. , 2002, Molecular cell.

[8]  Jennifer L. Snead,et al.  A coupled chemical-genetic and bioinformatic approach to Polo-like kinase pathway exploration. , 2007, Chemistry & biology.

[9]  Andrea Musacchio,et al.  Crystal structure of the tetrameric Mad1–Mad2 core complex: implications of a ‘safety belt’ binding mechanism for the spindle checkpoint , 2002, The EMBO journal.

[10]  Stephen S. Taylor,et al.  Polo and Aurora kinases: lessons derived from chemical biology. , 2008, Current opinion in cell biology.

[11]  K. Mechtler,et al.  QIKS – Quantitative identification of kinase substrates , 2010, Proteomics.

[12]  E. Nishida,et al.  Identification of a Consensus Motif for Plk (Polo-like Kinase) Phosphorylation Reveals Myt1 as a Plk1 Substrate* , 2003, Journal of Biological Chemistry.

[13]  M. Yaffe,et al.  A motif-based profile scanning approach for genome-wide prediction of signaling pathways , 2001, Nature Biotechnology.

[14]  Karl Mechtler,et al.  BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals , 2008, Nature Methods.

[15]  Otto Hudecz,et al.  Spatial Exclusivity Combined with Positive and Negative Selection of Phosphorylation Motifs Is the Basis for Context-Dependent Mitotic Signaling , 2011, Science Signaling.

[16]  Péter Lénárt,et al.  Polo on the Rise-from Mitotic Entry to Cytokinesis with Plk1. , 2008, Developmental cell.

[17]  J. Shabanowitz,et al.  A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. , 2004, Analytical chemistry.

[18]  Jan Ellenberg,et al.  Roles of Polo-like Kinase 1 in the Assembly of Functional Mitotic Spindles , 2004, Current Biology.

[19]  J. Ellenberg,et al.  Regulation of Sister Chromatid Cohesion between Chromosome Arms , 2004, Current Biology.

[20]  G. Crooks,et al.  WebLogo: a sequence logo generator. , 2004, Genome research.

[21]  Xingzhi Xu,et al.  Cytokinesis and cancer: Polo loves ROCK'n' Rho(A). , 2010, Journal of genetics and genomics = Yi chuan xue bao.

[22]  S. Carr,et al.  A Mitotic Phosphorylation Feedback Network Connects Cdk1, Plk1, 53BP1, and Chk2 to Inactivate the G2/M DNA Damage Checkpoint , 2010, PLoS biology.

[23]  Hongtao Yu,et al.  Phosphorylation- and polo-box-dependent binding of Plk1 to Bub1 is required for the kinetochore localization of Plk1. , 2006, Molecular biology of the cell.

[24]  J. Peters,et al.  The cohesin complex and its roles in chromosome biology. , 2008, Genes & development.

[25]  T. Hirano,et al.  Cohesin release is required for sister chromatid resolution, but not for condensin-mediated compaction, at the onset of mitosis. , 2002, Genes & development.

[26]  D. Hunt,et al.  Use of differential isotopic labeling and mass spectrometry to analyze capacitation-associated changes in the phosphorylation status of mouse sperm proteins. , 2009, Journal of proteome research.

[27]  Kyung S. Lee,et al.  Polo-box domain: a versatile mediator of polo-like kinase function , 2010, Cellular and Molecular Life Sciences.

[28]  C. Sunkel,et al.  polo, a mitotic mutant of Drosophila displaying abnormal spindle poles. , 1988, Journal of cell science.

[29]  T. Veenstra,et al.  Self-regulated Plk1 recruitment to kinetochores by the Plk1-PBIP1 interaction is critical for proper chromosome segregation. , 2006, Molecular cell.

[30]  A. Desai,et al.  A Combined Approach for the Localization and Tandem Affinity Purification of Protein Complexes from Metazoans , 2005, Science's STKE.

[31]  Steven P Gygi,et al.  Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations , 2005, Nature Methods.

[32]  K. Hofmann,et al.  PICH, a Centromere-Associated SNF2 Family ATPase, Is Regulated by Plk1 and Required for the Spindle Checkpoint , 2007, Cell.

[33]  J. Kornhauser,et al.  PhosphoSite: A bioinformatics resource dedicated to physiological protein phosphorylation , 2004, Proteomics.

[34]  Michael B. Yaffe,et al.  Scansite 2.0: proteome-wide prediction of cell signaling interactions using short sequence motifs , 2003, Nucleic Acids Res..

[35]  M. Mann,et al.  Parts per Million Mass Accuracy on an Orbitrap Mass Spectrometer via Lock Mass Injection into a C-trap*S , 2005, Molecular & Cellular Proteomics.

[36]  Karl Mechtler,et al.  Mitotic regulation of the human anaphase‐promoting complex by phosphorylation , 2003, The EMBO journal.

[37]  M. Mann,et al.  Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks , 2006, Cell.

[38]  J. Peters,et al.  Wapl Controls the Dynamic Association of Cohesin with Chromatin , 2006, Cell.

[39]  H. Lane,et al.  Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes , 1996, The Journal of cell biology.

[40]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[41]  R. Wolthuis,et al.  Polo-like Kinase-1 Is Required for Bipolar Spindle Formation but Is Dispensable for Anaphase Promoting Complex/Cdc20 Activation and Initiation of Cytokinesis* , 2004, Journal of Biological Chemistry.

[42]  R. Durbin,et al.  Systematic Analysis of Human Protein Complexes Identifies Chromosome Segregation Proteins , 2010, Science.

[43]  S. Carr,et al.  Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells , 2009, PLoS biology.

[44]  Feng Zhang,et al.  The Plk1-dependent Phosphoproteome of the Early Mitotic Spindle* , 2010, Molecular & Cellular Proteomics.

[45]  Lennart Martens,et al.  A guide to the Proteomics Identifications Database proteomics data repository , 2009, Proteomics.

[46]  Michael B Yaffe,et al.  Structure and function of Polo-like kinases , 2005, Oncogene.

[47]  Kristen M. Naegle,et al.  An integrated comparative phosphoproteomic and bioinformatic approach reveals a novel class of MPM-2 motifs upregulated in EGFRvIII-expressing glioblastoma cells. , 2008, Molecular bioSystems.

[48]  A. Ullrich,et al.  Targeting polo-like kinase 1 for cancer therapy , 2006, Nature Reviews Cancer.