title: Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests.
abstract: The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1(Mph1) kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1(Mph1) kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors in vitro, demonstrating that Mad3p modification can directly influence Cdc20(Slp1)-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1(Mph1) kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Judith
authors.initials: J
authors.lastname: Zich
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Karen
authors.initials: K
authors.lastname: May
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Paraskevopoulos
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Onur
authors.initials: O
authors.lastname: Sen
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Heather M
authors.initials: HM
authors.lastname: Syred
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Sjaak
authors.initials: S
authors.lastname: van der Sar
authors.affiliation: Edinburgh Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Hitesh
authors.initials: H
authors.lastname: Patel
authors.affiliation: Scripps Research Institute, La Jolla, California, United States of America.
authors.forename: James J
authors.initials: JJ
authors.lastname: Moresco
authors.affiliation: Scripps Research Institute, La Jolla, California, United States of America.
authors.forename: Ali
authors.initials: A
authors.lastname: Sarkeshik
authors.affiliation: Scripps Research Institute, La Jolla, California, United States of America.
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.|Department of Bioanalytics, Institute of Biotechnology, Technische Universitat Berlin, Berlin, Germany.
authors.forename: Juri
authors.initials: J
authors.lastname: Rappsilber
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Hardwick
mesh_terms: D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome / Q000378:metabolism*; D064199:Cdc20 Proteins / Q000378:metabolism*; D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000166:cytology / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism*
chemical_list: D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D029702:Schizosaccharomyces pombe Proteins; C512748:mad3 protein, S pombe; C577504:slp1 protein, S pombe; D064173:Anaphase-Promoting Complex-Cyclosome; C570446:Mph1 protein, S pombe; D011494:Protein Kinases