Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.746532
Title: Investigation of mechanisms for restricting the activity of cyclic-AMP dependent protein kinase
Author: Walker-Gray, R. A.
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2017
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Abstract:
Cyclic AMP (cAMP) is an ancient second messenger that is essential for many cellular processes including synaptic plasticity and control of heart rate and contractility. Cyclic AMP-dependent protein kinase (PKA) is the major intracellular receptor for cAMP. PKA consists of dimeric regulatory (R) subunits that bind and inhibit catalytic (C) subunits. PKA is activated upon binding of cAMP to the R subunits, which leads to the release of C subunits, and phosphorylation of intracellular protein substrates. An enduring challenge in cAMP research is to understand how PKA activity is directed to specific substrates, as the C subunits exhibit only limited substrate specificity in vitro. Elevations of cAMP are controlled in both space and time in the cell. This is achieved by the co-localization of enzymes for both the synthesis (cyclases) and breakdown (phosphodiesterases) of cAMP. Anchoring proteins are also essential for directing PKA to substrates in their immediate vicinity. However, a mechanism is yet to be established to explain how the activity of the C subunit of PKA is restrained following its dissociation from R subunits. This thesis details three parallel investigations that apply novel approaches with the shared aim of understanding how C subunit restraint is achieved. First, using quantitative immunoblotting in conjunction with purified PKA subunits, I investigated PKA subunit stoichiometry, finding that PKA R subunits typically outnumber C subunits by ~15-fold. Second, I developed a novel approach for monitoring R subunit isoform-specific association with C subunits in cells, with temporal precision. Comparative experiments using this approach and measurements with a fluorescent reporter of PKA activity show that only a small portion of C subunits need be dissociated to achieve high PKA activity. Third, I applied and developed a novel cross-linking coupled to mass spectrometry (XL-MS) protocol for analysis of the structure of PKA complexes. Insights include the likely orientation of PKA complexes that contain type II R (RII) subunits towards the membrane, and identification of a possible conformational change in PKA upon binding an anchoring protein. Together these experiments illuminate several aspects of PKA to show how the activity of this critical signalling enzyme is restrained within cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.746532  DOI: Not available
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