Accurate chromosome segregation is critical as unequal distribution of the genomic DNA results in impaired cell function or cell death. Kinetochores, the multi-protein structures assembled on centromeric DNA, drive chromosome segregation. Chromosome segregation is under supervision of mitotic spindle checkpoint. The mitotic spindle checkpoint is a surveillance mechanism ensuring that cells enter anaphase with all kinetochores properly attached to spindle microtubules and thereby preventing missegregation. Some checkpoint proteins are localised at kinetochore where they generate and enhance the checkpoint signal. Mps1 (Mph1 in S. pombe) and Aurora B (Ark1 in S. pombe) kinases are required for precise chromosome segregation and mitotic spindle checkpoint in fission yeast. In this study we investigate the roles of Mph1 and Ark1 in regulating the S. pombe kinetochore protein Spc7, which is the homologue of human Blinkin/KNL1. We demonstrated that both kinases target the N-terminus of Spc7. Loss of phosphorylation on the candidate phosphosites results in sensitivity to microtubule depolymerizing drugs indicating mitotic defects. As Blinkin has been proposed to be a docking platform for checkpoint proteins, we tested the possibility that Mph1 kinase is involved in kinetochore targeting of checkpoint proteins, Bub1 and Bub3. Our results demonstrate that Mph1-dependent phosphorylation of Spc7 at conserved MELT motifs is required for Bub1 and Bub3 kinetochore localisation. We were able to reconstitute the interaction between Spc7 and the Bub proteins in vitro demonstrating that the Spc7 phosphorylation is sufficient for Bub1 and Bub3 association with Spc7, most likely with Bub3 making the Spc7 contact. Mimicking phosphorylation at the MELT motifs leads to constitutive Bub1 localisation at kinetochores. We also showed that the N-terminus of Spc7 has microtubule binding activity regulated by Ark1 kinase. Mimicking phosphorylation at Ark1 sites results in reduced amount of recombinant Spc7 co-precipitating with microtubules in microtubule binding assays. Moreover, two stretches of basic residues, that contribute to Spc7 microtubule binding activity, have been mapped in the extreme Nterminus of Spc7. Spc7 also interacts with PP1 phosphatase, Dis2 in S. pombe, which is required for checkpoint silencing, but the mechanism of this interactions remains to be determined. These findings allow us to speculate on Spc7 role(s) in coupling microtubule binding with spindle checkpoint activation and silencing.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:578484 |
| Date | January 2013 |
| Creators | Sochaj, Alicja Maria |
| Contributors | Hardwick, Kevin; Harrington, Lea |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/7763 |
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