title: Modulation of the chromatin phosphoproteome by the Haspin protein kinase.
abstract: Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr(3) of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date, histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg(2) and Lys(4) adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys(4) of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser(137) of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser(137) resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser(137) might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing.
authors.affiliation: From the ‡Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland;
authors.forename: Alessio
authors.initials: A
authors.lastname: Maiolica
authors.affiliation: §Department of Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland;
authors.forename: Maria
authors.initials: M
authors.lastname: de Medina-Redondo
authors.affiliation: ¶Cellular Signal Integration Group (C-SIG), Center for Biological Sequence Analysis (CBS), Department of Systems Biology, Technical University of Denmark (DTU), Lyngby, Denmark;
authors.forename: Erwin M
authors.initials: EM
authors.lastname: Schoof
authors.affiliation: ‖Oxford University, Nuffield Department of Clinical Medicine, Target Discovery Institute (TDI) and Structural Genomics Consortium (SGC), Oxford OX3 7FZ, United Kingdom;
authors.forename: Apirat
authors.initials: A
authors.lastname: Chaikuad
authors.affiliation: **Department of Experimental Oncology, European Institute of Oncology, Milan, Italy;
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Villa
authors.affiliation: ‡‡Department of Pharmaceutical Sciences, University of Piemonte Orientale "A. Avogadro" Novara, Italy;
authors.forename: Marco
authors.initials: M
authors.lastname: Gatti
authors.affiliation: §§Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany;
authors.forename: Siva
authors.initials: S
authors.lastname: Jeganathan
authors.affiliation: ¶¶Yale University School of Medicine, Department of Pharmacology, New Haven, Connecticut 06520, USA;
authors.forename: Hua Jane
authors.initials: HJ
authors.lastname: Lou
authors.affiliation: From the ‡Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland;
authors.forename: Karel
authors.initials: K
authors.lastname: Novy
authors.affiliation: From the ‡Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland;
authors.forename: Simon
authors.initials: S
authors.lastname: Hauri
authors.affiliation: §Department of Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland;
authors.forename: Umut H
authors.initials: UH
authors.lastname: Toprak
authors.affiliation: ‖‖Gene Center Munich Ludwig-Maximilians-Universität München, Munich, Germany;
authors.forename: Franz
authors.initials: F
authors.lastname: Herzog
authors.affiliation: §Department of Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland;
authors.forename: Patrick
authors.initials: P
authors.lastname: Meraldi
authors.affiliation: ‡‡Department of Pharmaceutical Sciences, University of Piemonte Orientale "A. Avogadro" Novara, Italy;
authors.forename: Lorenza
authors.initials: L
authors.lastname: Penengo
authors.affiliation: ¶¶Yale University School of Medicine, Department of Pharmacology, New Haven, Connecticut 06520, USA;
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Turk
authors.affiliation: ‖Oxford University, Nuffield Department of Clinical Medicine, Target Discovery Institute (TDI) and Structural Genomics Consortium (SGC), Oxford OX3 7FZ, United Kingdom;
authors.forename: Stefan
authors.initials: S
authors.lastname: Knapp
authors.affiliation: ¶Cellular Signal Integration Group (C-SIG), Center for Biological Sequence Analysis (CBS), Department of Systems Biology, Technical University of Denmark (DTU), Lyngby, Denmark;
authors.forename: Rune
authors.initials: R
authors.lastname: Linding
authors.affiliation: From the ‡Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland; Faculty of Science, University of Zurich, Zurich, Switzerland aebersold@imsb.biol.ethz.ch.
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
mesh_terms: D000595:Amino Acid Sequence; D064107:Aurora Kinase B / Q000378:metabolism*; D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics*; D057809:HEK293 Cells; D006367:HeLa Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008745:Methylation; D008938:Mitosis / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D060066:Protein Interaction Maps / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D002843:Chromatin; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; C576921:AURKB protein, human; D064107:Aurora Kinase B; C530656:HASPIN protein, human; D017346:Protein Serine-Threonine Kinases
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