Aneuploidy occurs in cancer cells when errors in chromosome segregation happen. Mutations leading to increased chromosome missegregation in certain cancers might be predisposing factors that accelerate tumorigenesis. By this model, the kinetochore and its regulatory system, which are essential for genome stability, are crucial in protecting against cancer development. The project's goal is to identify and characterize proteins required for mitotic chromosome segregation in eukaryotes, by using Saccharomyces cerevisiae as an experimental organism. Sgt1 and the core kinetochore protein, Skp1, promote assembly of the kinetochore core complex (CBF3) by activating Ctf13. Moreover, Skp1 and Sgt1 are part of the SCF complex (an E3 ubiquitin ligase).
Specific Aim 1 is to investigate the biological function of Sgt1. This will be investigated by determining the time at which Sgt1 binds to CEN, and analyzing Sgt1's phosphorylation status. To test the hypothesis that Sgt1 and Skp1 are key components in the potential connection between kinetochore activation and ubiquitin-mediated degradation via the SCF complex, extragenic suppressor screens will be performed.
Specific Aim 2 is to isolate and characterize proteins that interact with Sgt1. Immunoprecipitation mass spectrometry will be performed to isolate Sgt1 interactors. The function(s) of the isolated proteins will be characterized in genetics and biochemistry using the mutant strains that we will construct. Signaling from defective kinetochores to the mitotic checkpoint is thought to occur but is poorly understood. The principal investigator has shown that the spindle checkpoint protein Bub1 binds to Skp1 and that Bub1 associates with CEN DNA via Skp1. Therefore, Specific Aim 3 is to characterize the molecular interaction between kinetochores and the mitotic spindle checkpoint, especially the Bub1-Skp1 interaction. The contribution of the Bub1-Skp1 complex to the G2/M delays caused by mitotic defects, Bub1's Skp1 binding and CEN-associating domains, and additional Bub1 interactors will be analyzed. This work will reveal kinetochore functions of Skp1 and Sgt1 and novel kinetochore or cell-cycle regulators that can serve as entry points for further analysis of kinetochores in chromosome transmission and cell-cycle progression Achieving these aims will further elucidate mechanisms of cancer development.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM068418-01
Application #
6599020
Study Section
Genetics Study Section (GEN)
Program Officer
Zatz, Marion M
Project Start
2003-05-01
Project End
2008-04-30
Budget Start
2003-05-01
Budget End
2004-04-30
Support Year
1
Fiscal Year
2003
Total Cost
$275,975
Indirect Cost
Name
St. Jude Children's Research Hospital
Department
Type
DUNS #
067717892
City
Memphis
State
TN
Country
United States
Zip Code
38105
Niikura, Yohei; Kitagawa, Katsumi (2016) Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins. J Vis Exp :e53732
Niikura, Yohei; Kitagawa, Risa; Kitagawa, Katsumi (2016) CENP-A Ubiquitylation Is Inherited through Dimerization between Cell Divisions. Cell Rep 15:61-76
Niikura, Yohei; Kitagawa, Risa; Ogi, Hiroo et al. (2015) CENP-A K124 Ubiquitylation Is Required for CENP-A Deposition at the Centromere. Dev Cell 32:589-603
Ohkuni, Kentaro; Abdulle, Rashid; Kitagawa, Katsumi (2014) Degradation of centromeric histone H3 variant Cse4 requires the Fpr3 peptidyl-prolyl Cis-Trans isomerase. Genetics 196:1041-5
Bian, Yang; Kitagawa, Risa; Bansal, Parmil K et al. (2014) Synthetic genetic array screen identifies PP2A as a therapeutic target in Mad2-overexpressing tumors. Proc Natl Acad Sci U S A 111:1628-33
Kikuchi, Koji; Narita, Takeo; Pham, Van T et al. (2013) Structure-specific endonucleases xpf and mus81 play overlapping but essential roles in DNA repair by homologous recombination. Cancer Res 73:4362-71
Yang, Chunying; Tang, Xi; Guo, Xiaojing et al. (2011) Aurora-B mediated ATM serine 1403 phosphorylation is required for mitotic ATM activation and the spindle checkpoint. Mol Cell 44:597-608
Ohkuni, Kentaro; Kitagawa, Katsumi (2011) Endogenous transcription at the centromere facilitates centromere activity in budding yeast. Curr Biol 21:1695-703
Goto, Greicy H; Mishra, Ashutosh; Abdulle, Rashid et al. (2011) Bub1-mediated adaptation of the spindle checkpoint. PLoS Genet 7:e1001282
Kikuchi, Koji; Niikura, Yohei; Kitagawa, Katsumi et al. (2010) Dishevelled, a Wnt signalling component, is involved in mitotic progression in cooperation with Plk1. EMBO J 29:3470-83

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