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Title: Biochemical analysis of human cancer-associated pseudokinases
Author: Bailey, Fiona P.
ISNI:       0000 0004 5356 706X
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2014
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Mutation or aberrant expression of protein kinase domains can lead to the development of a variety of cancers, which is why these proteins have become the focus of many clinical therapies. ~10% of the human kinome have been classified as pseudokinases, because they lack at least one of the highly conserved motifs that were originally used to help define the protein kinase superfamily. Due to these deviations from the normal situation, pseudokinases were originally predicted to be catalytically inactive, often based solely upon bioinformatic sequence alignment evidence. However, the discovery of pseudokinases that can hydrolyze or bind to ATP either at, or below, the nucleotide concentrations typically found in cells, suggests that each pseudokinase requires a thorough and individual biochemical analysis using a combination of techniques in order to reveal its biological function. Trib2 and NOK are two pseudokinases that contain a highly atypical 'DFG' metal ion binding motif, when compared to canonical kinase domains, and both are implicated in a variety of human cancers. Trib2 is predicted to be a Ser/Thr pseudokinase and is very closely related to the Trib1 and Trib3 pseudokinases in humans, and to the Tribbles pseudokinase that was originally characterized in the model fruit fly Drosophila Melanogaster. Along with an E3-ligase, Trib2 is required to target the tumor-suppressing transcription factor C/EBP? for degradation in cells. The results presented in this thesis include the first demonstration of a Mg2+ ionindependent ATP binding and autophosphorylation function for Trib2 in vitro. NOK is a pseudokinase that exhibits similarity to the RPTK sub-family, however it does not have a detectable ligand-binding domain, but does contain a putative transmembrane domain that lies adjacent to the pseudokinase domain. Overexpression of NOK mRNA has been observed in a variety of cancers, but its mode of action is still unclear. I describe in this thesis that NOK localizes to the nuclear membrane of cells and contains a localization sequence that resembles the EGFR Nuclear Localization Sequence, which translocates to the nucleus via the Inner Nuclear Membrane. Work described in this thesis also describes the development of tools to pave the way for future cellular analysis of both Trib2 and NOK, which will reveal whether these pseudokinases could be exciting, and novel drug targets in human diseases.
Supervisor: Eyers, P. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available