Characterisation of a novel PDE4 and the modification of PDE4 function by phosphorylation and protein-protein interaction

Wallace, Derek A. (2006) Characterisation of a novel PDE4 and the modification of PDE4 function by phosphorylation and protein-protein interaction. PhD thesis, University of Glasgow.

Abstract

cAMP participates in cell signal transduction and influences biological responses such as muscle contraction, learning and memory, inflammatory cytokine production, and cell growth, differentiation and apoptosis. The sole means of cAMP degradation is by PDE enzymes. The PDE4 family represents the major cAMP-hydrolysing activity in many cells. Four genes encode the PDE4 isoforms and over twenty have now been characterised. Each isoform differs by virtue of a unique N-terminal intra-cellular targeting region, and by differential inclusion of regulatory UCR domains via 5' mRNA splicing. The plethora of PDE4 isoforms underpins compartmentalisation of cAMP signalling, facilitating the generation of cAMP domains or gradients. Work in this thesis characterises a novel PDE4A long isoform, namely PDE4A11. It is expressed with a species-conserved, unique 81 amino acid N- terminal region encoded by a single 5' exon. Transcript analysis of human cells and tissues alludes to tissue-specific and developmental changes in expression. PDE4A11 is targeted to the perinuclear region and membrane ruffles of COS7 cells. Sequence analysis identifies putative PIP3-mediated membrane targeting. PDE4A11 is kinetically comparable to the other PDE4A long isoforms and is inhibited by conventional PDE4 inhibitors. It is insensitive to conformational changes detected by altered rolipram inhibition, a likely consequence of reduced LR2 and/or enhanced N-terminal interactions with Lyn-SH3. Soluble and particulate forms exhibit distinct rates of thermal inactivation. PDE4A11 is activated by PKA phosphorylation at Ser-119 within UCR1. N-terminal phosphorylation by ERK1/2 should be pursued. PDE4A11 couples Beta-arrestin and is the second PDE4 isoform to interact with XAP2. Phosphorylation of PDE4 isoforms confers activity and protein interaction regulation. All PDE4 long isoforms are activated by PKA phosphorylation within UCR1. Phosphorylation of the catalytic unit by ERK1/2 occurs in all PDE4 isoforms, except PDE4A, and activates short form PDE4s and inhibits long forms. Activation of the PI-3K pathway also facilitates PDE4 activation. The identification of PDE4A5 as an authentic substrate for MAPKAPK2 is described, and the functional effects are investigated. This novel phosphorylation occurs at a serine residue, distinct from the PKA site, within UCR1. The kinase and target residue is identified in C0S1 cells expressing PDE4A5, following treatment with anisomycin, the p38 MAPK inhibitor, SB203580, and siRNA-mediated knockdown of MAPKAPK2, together with N-terminal truncation of PDE4A5 and Ser-147-Ala mutation. Phosphorylation of PDE4A5 by MAPKAPK2 has no effect on catalytic activity but delays the onset and amplitude of PKA-mediated activation. The interaction of PDE4A5 with XAP2, but not b=Beta-arrestin, is attenuated by MAPKAPK2 phosphorylation. Phosphorylation does not affect PDE4A5 intracellular targeting or its susceptibility to caspase-3 cleavage. Enzyme conformational change with a Ser-147-Asp phospho-mimetic is not observed. PDE4 inhibition reduces the production of ROS in many immune and inflammatory cells, consequential of the immuno-suppressive and anti-inflammatory effects of cAMP. Oxidative stress is implicated in the pathogenesis of disease states such as COPD and cardiac hypertrophy. The final chapter of this thesis strives to unravel the role of PDE4 inhibition in the attenuation of ROS production in cardiac myocytes. Angiotensin II promotes the delivery of p47-phox, an integral component of the active NADPH oxidase enzyme, to the membrane. Elevation of intra-cellular cAMP concentrations using forskolin and rolipram prevents p47-phox translocation, an effect that is partially rescued with the PKA inhibitor, H89. PKA directly phosphorylates pA7-phox and six putative sites are identified within the C-terminal region. PDE4A4B is able to interact directly with p47-phox and p67-phox, whereas PDE4A10 only interacts with p47-phox, indicating specificity in the PDE4 control of the PKA phosphorylation state of phox proteins. In summary, the work in this thesis contributes further to our understanding of the PDE4 regulation of intra-cellular cAMP concentrations in cells, and the specific response mediated through the multitude of isoforms and modification of function by both phosphorylation and protein interactions.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Phosphorylation, cellular signal transduction.
• Subjects: Q Science > QH Natural history; Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Housley, Prof. Miles
• Date of Award: 2006
• Depositing User: Mrs Monika Milewska-Fiertek
• Unique ID: glathesis:2006-39016
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 18 Dec 2018 14:59
• Last Modified: 20 May 2021 15:48
• URI: https://theses.gla.ac.uk/id/eprint/39016

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