title: The Mps1 kinase modulates the recruitment and activity of Cnn1(CENP-T) at Saccharomyces cerevisiae kinetochores.
abstract: Kinetochores are conserved protein complexes that bind the replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we dissected the phospho-regulated dynamic interaction between conserved kinetochore protein Cnn1(CENP-T), the centromere region, and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization are unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain (HFD) that associates with the centromere region and a N-terminal Spc24/Spc25 interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that the established Ndc80 binding site in the N terminus of Cnn1, Cnn1(60-84), should be extended with flanking residues, Cnn1(25-91), to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1-S74, based on in vitro experiments demonstrating the Cnn1-Ndc80 complex interaction. We demonstrate that from G1 through metaphase, Cnn1 localizes via both its HFD and N-terminal Spc24/Spc25 interaction sequence, and deletion or mutation of either region results in anomalous Cnn1 kinetochore levels. At anaphase onset (when Mps1 activity decreases) Cnn1 becomes enriched mainly via the N-terminal Spc24/Spc25 interaction sequence. In sum, we provide the first in vivo evidence of Cnn1 preanaphase linkages with the kinetochore and enrichment of the linkages during anaphase.
authors.affiliation: Purdue University, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University Center for Cancer Research, West Lafayette, Indiana 47907-2091.
authors.forename: Kriti Shrestha
authors.initials: KS
authors.lastname: Thapa
authors.affiliation: Istituto FIRC (Fondazione Italiana per la Ricerca sul Cancro) di Oncologia Molecolare, 20139 Milan, Italy.
authors.forename: Amanda
authors.initials: A
authors.lastname: Oldani
authors.affiliation: European Institute of Oncology, Department of Experimental Oncology, 20139 Milan, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Pagliuca
authors.affiliation: European Institute of Oncology, Department of Experimental Oncology, 20139 Milan, Italy.
authors.forename: Peter
authors.identifier: 0000-0001-9772-5881
authors.initials: P
authors.lastname: De Wulf
authors.affiliation: Purdue University, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University Center for Cancer Research, West Lafayette, Indiana 47907-2091 thazbun@purdue.edu.
authors.forename: Tony R
authors.identifier: 0000-0003-0675-8093
authors.initials: TR
authors.lastname: Hazbun
mesh_terms: D000595:Amino Acid Sequence; D000705:Anaphase; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D018386:Kinetochores / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; C574223:Cnn1 protein, S cerevisiae; C112472:NDC80 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C500717:Spc24 protein, S cerevisiae; C500718:Spc25 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C093416:MPS1 protein, S cerevisiae
keywords: CENP-T; Cnn1; Mps1; centromere; kinetochore