title: Kinase activity of fission yeast Mph1 is required for Mad2 and Mad3 to stably bind the anaphase promoting complex.
abstract: Defects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1, 2]. The spindle checkpoint delays anaphase onset until all chromosomes are attached to spindle microtubules in a bipolar fashion [3, 4]. Mad2 is a key checkpoint component that undergoes conformational activation, catalyzed by a Mad1-Mad2 template enriched at unattached kinetochores [5]. Mad2 and Mad3 (BubR1) then bind and inhibit Cdc20 to form the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C). Checkpoint kinases (Aurora, Bub1, and Mps1) are critical for checkpoint signaling, yet they have poorly defined roles and few substrates have been identified [6-8]. Here we demonstrate that a kinase-dead allele of the fission yeast MPS1 homolog (Mph1) is checkpoint defective and that levels of APC/C-associated Mad2 and Mad3 are dramatically reduced in this mutant. Thus, MCC binding to fission yeast APC/C is dependent on Mph1 kinase activity. We map and mutate several phosphorylation sites in Mad2, producing mutants that display reduced Cdc20-APC/C binding and an inability to maintain checkpoint arrest. We conclude that Mph1 kinase regulates the association of Mad2 with its binding partners and thereby mitotic arrest.
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, EH9 3JR, UK.
authors.forename: Judith
authors.initials: J
authors.lastname: Zich
authors.forename: Alicja M
authors.initials: AM
authors.lastname: Sochaj
authors.forename: Heather M
authors.initials: HM
authors.lastname: Syred
authors.forename: Laura
authors.initials: L
authors.lastname: Milne
authors.forename: Atlanta G
authors.initials: AG
authors.lastname: Cook
authors.forename: Hiro
authors.initials: H
authors.lastname: Ohkura
authors.forename: Juri
authors.initials: J
authors.lastname: Rappsilber
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Hardwick
mesh_terms: D000705:Anaphase; D064173:Anaphase-Promoting Complex-Cyclosome; D064127:Aurora Kinases; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D018386:Kinetochores / Q000378:metabolism; D059566:M Phase Cell Cycle Checkpoints; D064246:Mad2 Proteins; D013058:Mass Spectrometry; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012568:Schizosaccharomyces / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics / Q000378:metabolism*
chemical_list: D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D029702:Schizosaccharomyces pombe Proteins; C577696:mad2 protein, S pombe; C512748:mad3 protein, S pombe; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; C570446:Mph1 protein, S pombe; D011494:Protein Kinases; D064127:Aurora Kinases; C495594:Bub1 protein, S pombe; D017346:Protein Serine-Threonine Kinases