PKA phosphorylation and compartmentalisation of cAMP-phosphodiesterases

McSorley, Theresa (2004) PKA phosphorylation and compartmentalisation of cAMP-phosphodiesterases. PhD thesis, University of Glasgow.

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cAMP is a ubiquitous second messenger which conveys its cellular effects mainly through the activation of protein kinase A (PKA). Within a cell, activated PKA serves to phosphoiylate, and alter the conformational change of a wide variety of proteins. Cyclic-phosphodiesterases (PDEs) play a pivotal role in the regulation of cAMP signalling by determining the breakdown of cAMP to 5'-AMP. It is now well-accepted that cAMP signalling is a compartmentalised response, with PKA being tethered to distinct subcellular regions by A-kinase anchoring proteins (AKAPs). The PDE4 family exclusively hydrolyse cAMP. I studied the PKA phosphorylation of the two sites within PDE4D3 and found that the phosphorylation of Ser54 (responsible for activation) precedes that of Ser 13. I showed, for the first time, that long form PDE4s from each subfamily can be phosphoiylated and consequentially activated by PKA in intact cells. I used a novel approach to appreciate the functional consequences of the compartmentalisation of PDE4 cAMP- phosphodiesterases. To do this, I overexpressed different catalytically inactive long form PDE4s which, I hypothesised, would alter distinct cAMP 'pools' by displacing the cognate endogenous active PDE4s from their anchor sites. By detecting the phosphorylation of the inactive PDE4 by PKA, the catalytically inactive PDE4 itself acted as readout of localised PKA activity. Through this approach, I found that different PDE4 isoforms are capable of controlling distinct pools of cAMP generated by different Gs-coupled receptors. Specifically I showed that the PDE4D3 and PDE4C2 may be held in close proximity to, and play a role in the regulation of discrete sub-populations of PKA under basal levels of cAMP generation. Through microarray analysis, I studied the effect of altering compartmentalised cAMP signalling on gene expression. This was again done by overexpressing catalytically inactive PDE4s in HEK293 cells. My experiments suggest that specific PDE4 isoforms can selectively influence gene regulation in these cells. Additionally, I analysed the function of distinct PDEs within cardiac myocytes. I showed that manipulation of cAMP levels with PDE inhibitors alone, or with PDE inhibitors and selected GPCR activation, selectively altered ability of phospholamban (PLB) and ERK 1/2 to be regulated by PKA, suggesting that PLB and ERK 1/2 can be differentially controlled by specific PDE families.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Houslay, Prof. M.
Date of Award: 2004
Depositing User: Mrs Monika Milewska-Fiertek
Unique ID: glathesis:2004-30902
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 12 Oct 2018 15:43
Last Modified: 14 Jul 2021 15:36
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