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Title: An investigation into Cdc28 phosphorylation of Ipl1 and the role of Haspin-like kinases in yeast cell cycle control
Author: Corbishley, Stephen J.
Awarding Body: University of Dundee
Current Institution: University of Dundee
Date of Award: 2013
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The chromosomal passenger complex (CPC) is a conserved, essential protein complex in eukaryotes, consisting of Ipl1/Aurora B kinase and three regulatory subunits: Sli15/INCENP, Bir1/Survivin and Nbl1/Borealin. The CPC has numerous cell cycle roles, the best characterised of which is the regulation of kinetochore-microtubule attachments during metaphase to promote chromosome bi-orientation on the mitotic spindle. To efficiently perform these functions, the CPC must be properly regulated and localised. The original defining characteristic of 'chromosomal passenger' proteins was their dynamic localisation: associated with chromatin and centromeres during early mitosis followed by translocation to the spindle midzone during anaphase. However, exactly how the CPC is properly localised and regulated is not fully understood. Recent studies demonstrated that phosphorylation of the CPC regulatory subunits by cyclin-dependent kinase (CDK) regulates localisation of the complex. For example, in budding yeast and human cells the dephosphorylation of CDKphosphorylated sites in Sli15/INCENP is required for the efficient translocation of the CPC from centromeres in metaphase to the spindle in anaphase. Many Ipl1/Aurora B homologues also contain CDK phosphorylation motifs but whether modification of these sites is required for proper CPC localisation and function is not known - here this was investigated in budding yeast. Preventing phosphorylation of CDK consensus sites in the N-terminus of Ipl1 was found to result in its premature localisation to the spindle in metaphase; this was dependent on the yeast microtubule binding EB1 homologue Bim1. However, preventing or mimicking phosphorylation at these sites had no effect on cell viability or chromosome bi-orientation. During the course of this study, several groups demonstrated that the conserved Haspin kinase in fission yeast, Xenopus and human cells contributes to CPC localisation at the centromere during metaphase by phosphorylating histone H3 on Threonine 3 (H3- T3). In light of these findings, two budding yeast Haspin kinase homologues (Alk1 and Alk2) were investigated. Although roles for Alk1 and Alk2 in histone H3 phosphorylation and CPC recruitment could not be conclusively determined, unusual phenotypes were observed in cells lacking both Alk1 and Alk2. In these cells, the spindle was frequently confined to the bud and incorrectly aligned. If these cells were arrested in metaphase, the bud became significantly larger than the mother cell. When anaphase was eventually triggered, the spindle elongated and then broke down entirely within the bud. These abnormalities were independent of histone H3-T3 phosphorylation, since mutation of H3-T3 to non-phosphorylatable alanine did not result in these phenotypes. Thus Alk1 and Alk2 share novel and redundant roles in spindle positioning, cell polarity and bud growth that are particularly important during a metaphase arrest.
Supervisor: Stark, Michael Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available