title: Role of Intrinsic and Extrinsic Factors in the Regulation of the Mitotic Checkpoint Kinase Bub1.
abstract: The spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores to ensure accurate sister chromatid segregation during mitosis. The SAC members Bub1 and BubR1 are paralogs that underwent significant functional specializations during evolution. We report an in-depth characterization of the kinase domains of Bub1 and BubR1. BubR1 kinase domain binds nucleotides but is unable to deliver catalytic activity in vitro. Conversely, Bub1 is an active kinase regulated by intra-molecular phosphorylation at the P+1 loop. The crystal structure of the phosphorylated Bub1 kinase domain illustrates a hitherto unknown conformation of the P+1 loop docked into the active site of the Bub1 kinase. Both Bub1 and BubR1 bind Bub3 constitutively. A hydrodynamic characterization of Bub1:Bub3 and BubR1:Bub3 demonstrates both complexes to have 1:1 stoichiometry, with no additional oligomerization. Conversely, Bub1:Bub3 and BubR1:Bub3 combine to form a heterotetramer. Neither BubR1:Bub3 nor Knl1, the kinetochore receptor of Bub1:Bub3, modulate the kinase activity of Bub1 in vitro, suggesting autonomous regulation of the Bub1 kinase domain. We complement our study with an analysis of the Bub1 substrates. Our results contribute to the mechanistic characterization of a crucial cell cycle checkpoint.
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227, Dortmund, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Breit
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227, Dortmund, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Bange
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227, Dortmund, Germany.
authors.forename: Arsen
authors.initials: A
authors.lastname: Petrovic
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227, Dortmund, Germany.
authors.forename: John R
authors.initials: JR
authors.lastname: Weir
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227, Dortmund, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Müller
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227, Dortmund, Germany.
authors.forename: Doro
authors.initials: D
authors.lastname: Vogt
authors.affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227, Dortmund, Germany.|Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitätsstrasse, 45141, Essen, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Musacchio
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018360:Crystallography, X-Ray; D006801:Humans; D059566:M Phase Cell Cycle Checkpoints; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000378:metabolism; D010766:Phosphorylation; D000075223:Poly-ADP-Ribose Binding Proteins; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D013379:Substrate Specificity
chemical_list: C489444:BUB3 protein, human; D018797:Cell Cycle Proteins; D009707:Nucleosomes; D000075223:Poly-ADP-Ribose Binding Proteins; D012694:Serine; C584277:BUB1 protein, human; D017346:Protein Serine-Threonine Kinases