title: Sequential multisite phospho-regulation of KNL1-BUB3 interfaces at mitotic kinetochores.
abstract: Regulated recruitment of the kinase-adaptor complex BUB1/BUB3 to kinetochores is crucial for correcting faulty chromosome-spindle attachments and for spindle assembly checkpoint (SAC) signaling. BUB1/BUB3 localizes to kinetochores by binding phosphorylated MELT motifs (MELpT) in the kinetochore scaffold KNL1. Human KNL1 has 19 repeats that contain a MELT-like sequence. The repeats are, however, larger than MELT, and repeat sequences can vary significantly. Using systematic screening, we show that only a limited number of repeats is "active." Repeat activity correlates with the presence of a vertebrate-specific SHT motif C-terminal to the MELT sequence. SHT motifs are phosphorylated by MPS1 in a manner that requires prior phosphorylation of MELT. Phospho-SHT (SHpT) synergizes with MELpT in BUB3/BUB1 binding in vitro and in cells, and human BUB3 mutated in a predicted SHpT-binding surface cannot localize to kinetochores. Our data show sequential multisite regulation of the KNL1-BUB1/BUB3 interaction and provide mechanistic insight into evolution of the KNL1-BUB3 interface.
authors: Molecular Cancer Research, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands.
authors: Mathijs
authors: M
authors: Vleugel
authors: Molecular Cancer Research, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands; Department of Medical Oncology, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands.
authors: Manja
authors: M
authors: Omerzu
authors: Molecular Cancer Research, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands; Department of Medical Oncology, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands.
authors: Vincent
authors: V
authors: Groenewold
authors: Molecular Cancer Research, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands.
authors: Michael A
authors: MA
authors: Hadders
authors: Molecular Cancer Research, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands.
authors: Susanne M A
authors: SMA
authors: Lens
authors: Molecular Cancer Research, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands; Department of Medical Oncology, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands; Cancer Genomics Netherlands, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands. Electronic address: g.j.p.l.kops@umcutrecht.nl.
authors: Geert J P L
authors: GJPL
authors: Kops
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D018386:Kinetochores / Q000187:drug effects / Q000378:metabolism*; D059566:M Phase Cell Cycle Checkpoints / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008938:Mitosis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation; D000075223:Poly-ADP-Ribose Binding Proteins; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D059008:Time-Lapse Imaging; D050257:Tubulin Modulators / Q000494:pharmacology
chemical_list: C489444:BUB3 protein, human; D018797:Cell Cycle Proteins; C472643:Knl1 protein, human; D008869:Microtubule-Associated Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D050257:Tubulin Modulators; D011505:Protein-Tyrosine Kinases; C584277:BUB1 protein, human; D017346:Protein Serine-Threonine Kinases; C075840:TTK protein, human; D015739:Nocodazole