title: Quantitative phospho-proteomics to investigate the polo-like kinase 1-dependent phospho-proteome.
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.
authors.affiliation: Research Institute of Molecular Pathology, 1030 Vienna, Austria.
authors.forename: Karin
authors.initials: K
authors.lastname: Grosstessner-Hain
authors.forename: Björn
authors.initials: B
authors.lastname: Hegemann
authors.forename: Maria
authors.initials: M
authors.lastname: Novatchkova
authors.forename: Jonathan
authors.initials: J
authors.lastname: Rameseder
authors.forename: Brian A
authors.initials: BA
authors.lastname: Joughin
authors.forename: Otto
authors.initials: O
authors.lastname: Hudecz
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Roitinger
authors.forename: Peter
authors.initials: P
authors.lastname: Pichler
authors.forename: Norbert
authors.initials: N
authors.lastname: Kraut
authors.forename: Michael B
authors.initials: MB
authors.lastname: Yaffe
authors.forename: Jan-Michael
authors.initials: JM
authors.lastname: Peters
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019295:Computational Biology; D016384:Consensus Sequence; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000145:classification / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020543:Proteome / Q000145:classification / Q000378:metabolism*; D040901:Proteomics; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000097869:Polo-Like Kinase 1
chemical_list: D018797:Cell Cycle Proteins; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D020543:Proteome; D011518:Proto-Oncogene Proteins; D000097869:Polo-Like Kinase 1