title: Zwint-1 is a novel Aurora B substrate required for the assembly of a dynein-binding platform on kinetochores.
abstract: Aurora B (AurB) is a mitotic kinase responsible for multiple aspects of mitotic progression, including assembly of the outer kinetochore. Cytoplasmic dynein is an abundant kinetochore protein whose recruitment to kinetochores requires phosphorylation. To assess whether AurB regulates recruitment of dynein to kinetochores, we inhibited AurB using ZM447439 or a kinase-dead AurB construct. Inhibition of AurB reduced accumulation of dynein at kinetochores substantially; however, this reflected a loss of dynein-associated proteins rather than a defect in dynein phosphorylation. We determined that AurB inhibition affected recruitment of the ROD, ZW10, zwilch (RZZ) complex to kinetochores but not zwint-1 or more-proximal kinetochore proteins. AurB phosphorylated zwint-1 but not ZW10 in vitro, and three novel phosphorylation sites were identified by tandem mass spectrometry analysis. Expression of a triple-Ala zwint-1 mutant blocked kinetochore assembly of RZZ-dependent proteins and induced defects in chromosome movement during prometaphase. Expression of a triple-Glu zwint-1 mutant rendered cells resistant to AurB inhibition during prometaphase. However, cells expressing the triple-Glu mutant failed to satisfy the spindle assembly checkpoint (SAC) at metaphase because poleward streaming of dynein/dynactin/RZZ was inhibited. These studies identify zwint-1 as a novel AurB substrate required for kinetochore assembly and for proper SAC silencing at metaphase.
authors.affiliation: Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Kasuboski
authors.forename: Jason R
authors.initials: JR
authors.lastname: Bader
authors.forename: Patricia S
authors.initials: PS
authors.lastname: Vaughan
authors.forename: Sinji B F
authors.initials: SB
authors.lastname: Tauhata
authors.forename: Michael
authors.initials: M
authors.lastname: Winding
authors.forename: Meghan A
authors.initials: MA
authors.lastname: Morrissey
authors.forename: Michelle V
authors.initials: MV
authors.lastname: Joyce
authors.forename: William
authors.initials: W
authors.lastname: Boggess
authors.forename: Larissa
authors.initials: L
authors.lastname: Vos
authors.forename: Gordon K
authors.initials: GK
authors.lastname: Chan
authors.forename: Edward H
authors.initials: EH
authors.lastname: Hinchcliffe
authors.forename: Kevin T
authors.initials: KT
authors.lastname: Vaughan
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D064107:Aurora Kinase B; D064127:Aurora Kinases; D001549:Benzamides / Q000494:pharmacology; D056445:Cytoplasmic Dyneins / Q000378:metabolism*; D000072159:Dynactin Complex; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D018386:Kinetochores / Q000378:metabolism*; D059566:M Phase Cell Cycle Checkpoints; D008677:Metaphase; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011799:Quinazolines / Q000494:pharmacology; D051381:Rats; D059010:Single-Cell Analysis; D059008:Time-Lapse Imaging
chemical_list: C474722:4-(4-(N-benzoylamino)anilino)-6-methoxy-7-(3-(1-morpholino)propoxy)quinazoline; D001549:Benzamides; D000072159:Dynactin Complex; D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D011799:Quinazolines; C413695:ZWINT protein, human; C576921:AURKB protein, human; C576925:Aurkb protein, rat; D064107:Aurora Kinase B; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D056445:Cytoplasmic Dyneins