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Title: Redox-dependent signalling via Protein Kinase A type I within distinct subcellular compartments
Author: Lomas, Oliver
ISNI:       0000 0004 6499 6927
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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Background and Aims : Numerous proteins, important for cell signalling, are modified by the balance of oxidative or reducing stimuli within the cell - the redox state. The regulatory subunit Iα (RIα) of Protein Kinase A (PKA) contains conserved cysteine residues that are readily oxidised to form a disulphide bond via the thiol group of other cysteines. It has been suggested that the formation of the disulphide bond between RIα subunits of the PKA holoenzyme increases the activity of the enzyme. The aims of the project were to examine whether the intrinsic activity of the enzyme or its functional localisation were increased by redox modification. Methods : Live-cell fluorescent imaging of Fluoresence Resonance Energy Transfer (FRET) has been used to investigate real-time changes in PKA activity and Fluorescence Resonance After Photo-Bleaching (FRAP) has been used to investigate changes in sub-ceullar localisation of PKA regulatory subunits. Results : The addition of the oxidant, hydrogen peroxide, to cells expressing FRET-based reporters of PKA activity was not associated with an increase in PKA activity compared to cells that expressed PKA-RIα that were incapable of undergoing disulphide bond formation. Mutation of the cysteine residue in PKA-RIα was, however, associated with subtle differences in sub-cellular diffusibility of the protein, which supports the hypthesiss that such redox modification appears to influence the mobility and hence anchoring of the protein within the cell. Conclusions : In summary, the data presented in this thesis do not support the notion that PKA can be activated directly through oxidation the RIα subunit. However, modulation of classical cAMP-PKA signalling by the redox state of the cell may be brought about by subtle changes in the anchoring and hence localisation of PKA-RIα as implied by changes in diffusibility within the cytoplasm.
Supervisor: Zaccolo, Manuela ; Casadei, Barbara Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available