Investigation of the mechanisms of the G2/M phase transition in human cells – the role of Greatwall kinase

Vesely, Clare (2013) Investigation of the mechanisms of the G2/M phase transition in human cells – the role of Greatwall kinase. Doctoral thesis (PhD), University of Sussex.

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Abstract

Understanding the mechanisms and factors that govern cell cycle control is key
to developing more effective treatments for human diseases, such as cancer. The human
MASTL gene encodes an AGC family kinase, Greatwall kinase, that is conserved
among higher eukaryotes. The protein contains an unusual bifurcated kinase domain
separated by a stretch of nonconserved amino acids. In Drosophila, mutations in
Greatwall cause the failure of chromosomes to condense resulting in a delayed entry in
to and progression through mitosis. In mitotic Xenopus egg extracts, immunodepletion
of Greatwall results in exit from M phase, characterised by the decondensation of the
chromosomes and the reforming of the nuclear envelope. The addition of purified
Greatwall to egg extracts immunodepleted for Greatwall causes precocious
phosphorylation of Cdc25 and premature entry into mitosis. These reports indicate that
Greatwall plays an important role in the control of mitosis but little is known about the
function of Greatwall kinase in human cells, its structure, or control of its activity.

This project aimed to elucidate the role of this novel kinase in human cells. To
this end, the gene has been cloned and antibodies generated to allow the study of human
Greatwall kinase. RNAi-mediated knockdown of Greatwall in HeLa cells caused
aberrant mitotic progression and apoptosis. To gain further insight into the mechanism
of Greatwall activation, the Greatwall kinase structure was modelled and key motifs of
the kinase fold identified. In particular, a key activating phosphorylation was identified,
and a specific antibody raised to this site, allowing investigation of the regulation of
Greatwall activity at mitotic entry and exit. The use of chemical genetics to attempt to
specifically inhibit the kinase in human cells is described. Finally, evidence is presented
that Greatwall kinase may represent a promising new biomarker and drug target for
cancer therapy.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Subjects: Q Science > QH Natural history > QH0301 Biology > QH0426 Genetics
Q Science > QH Natural history > QH0301 Biology > QH0426 Genetics > QH0447 Genes. Alleles. Genome
Q Science > QH Natural history > QH0301 Biology > QH0573 Cytology
Depositing User: Library Cataloguing
Date Deposited: 01 Nov 2013 07:34
Last Modified: 12 Oct 2015 11:55
URI: http://sro.sussex.ac.uk/id/eprint/46829

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