title: Protein interaction domain mapping of human kinetochore protein Blinkin reveals a consensus motif for binding of spindle assembly checkpoint proteins Bub1 and BubR1.
abstract: The kinetochore is a supramolecular structure essential for microtubule attachment and the mitotic checkpoint. Human blinkin/human Spc105 (hSpc105)/hKNL1 was identified originally as a mixed-lineage leukemia (MLL) fusion partner and later as a kinetochore component. Blinkin directly binds to several structural and regulatory proteins, but the precise binding sites have not been defined. Here, we report distinct and essential binding domains for Bub1 and BubR1 (here designated Bubs) at the N terminus of blinkin and for Zwint-1 and hMis14/hNsl1 at the C terminus. The minimal binding sites for Bub1 and BubR1 are separate but contain a consensus KI motif, KI(D/N)XXXF(L/I)XXLK. RNA interference (RNAi)-mediated replacement with mutant blinkin reveals that the Bubs-binding domain is functionally important for chromosome alignment and segregation. We also provide evidence that hMis14 mediates hNdc80 binding to blinkin at the kinetochore. The C-terminal fragment of blinkin locates at kinetochores in a dominant-negative fashion by displacing endogenous blinkin from kinetochores. This negative dominance is relieved by mutations of the hMis14 binding PPSS motif on the C terminus of blinkin or by fusion of the N sequence that binds to Bub1 and BubR1. Taken together, these results indicate that blinkin functions to connect Bub1 and BubR1 with the hMis12, Ndc80, and Zwint-1 complexes, and disruption of this connection may lead to tumorigenesis.
authors.affiliation: Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan. tomomi.kiyomitsu@gmail.com
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kiyomitsu
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Murakami
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Yanagida
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism; D020090:Chromosome Segregation; D003598:Cytoskeletal Proteins; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D018386:Kinetochores / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; C472643:Knl1 protein, human; D008869:Microtubule-Associated Proteins; C120529:NDC80 protein, human; C574033:NSL1 protein, human; D009687:Nuclear Proteins; C413695:ZWINT protein, human; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases