title: A study of protein-protein interactions involving type 4 phosphodiesterase
Creators: name: Beard, Matthew Brian
Publisher: name: University of Glasgow
Year: 1999
Subjects: subject: Molecular biology.

description: The cyclic adenosine monophosphate (cAMP) signalling pathway is centrally involved in the regulation of cell function by many extracellular messengers. Signalling through the cAMP pathway is involved in the acute regulation of numerous metabolic processes including glucose and lipid metabolism, neurotransmission and membrane trafficking. The cAMP pathway also functions to affect gene expression and can thereby mediate the long term control of processes such as cell growth and differentiation. Every stage in the cAMP signalling pathway is mediated by a family of enzymes. This provides opportunities for cell type and developmental stage specific variations in the control of the cAMP signalling, dependent upon the particular isoforms of each of the components of the cAMP pathway that are expressed in a given cell. In addition, each stage in the cAMP pathway is able to interact with components of other intracellular signalling pathways and this allows for a cell to modulate its response to any one stimuli depending upon the other stimulatory inputs that it receives. An important aspect of intracellular signalling is the spatial organisation of the components of the various pathways within the 3 dimensional cell interior. The paradigm for the spatial organisation of signalling proteins as a regulatory factor arose with the identification of protein domains that mediated the rapid clustering of tyrosine kinases around activated insulin receptors. It has since become apparent that the regulated targeting of a wide variety of signalling proteins to specific subcellular locations is an essential feature of much of the signalling that occurs within eukaryotic cells. In chapter 3 of this thesis I have shown that the type 4 phosphodiesterase (PDE4) splice variant PDE4D4 can interact specifically with Src homology 3 (SH3) domains in vitro. HSPDE4D4 showed a selectivity for the SH3 domains that it could interact with and bound most strongly to the SH3 domains of the tyrosyl kinases Src, Lyn, Fyn and Abl and also the SH3 domain of the cytoskeletal protein fodrin. In contrast it did not bind to a number of other SH3 domains that were tested. In chapter 4 of this thesis I have shown for the first time that it is possible to express both full length HSPDE4D4 and also the unique, alternatively spliced, amino terminal 166 amino acids of HSPDE4D4 in E.coli as fusion proteins with maltose binding protein (MBP) and to purify these species to near homogeneity by affinity chromatography. I used these purified fusion proteins to demonstrate a direct protein-protein interaction between the alternatively spliced, amino terminal region of HSPDE4D4 and the SH3 domain of Lyn kinase. By engineering a recognition site for a high fidelity viral protease (TEV) into the MBP-HSPDE4D4 fusion protein, I was able to separate proteolytically the MBP and the HSPDE4D4 moieties and, for the first time, to perform a limited characterisation of highly purified, full length HSPDE4D4. This species migrated on SDS-PAGE with and apparent Mw of 121kDa and catalysed the hydrolysis of cAMP with a Km of 2.3muM and an apparent Vmax of 3490mumol/min/mg. In chapter 5 of this thesis I have mapped sub-regions within the amino terminal of RNPDE4A5 that are involved in the interaction with SH3 domains, in the subcellular targeting and in the regulation of the catalytic activity of this isoform. My data suggest that the extreme amino terminal region, residues 1-10, of RNPDE4A5 are important in the interaction of this isoform with the SH3 domain of Lyn kinase. The region between residues 218-259 of RNPDE4A5 appears to be involved in both the subcellular targeting and the regulation of the catalytic activity of this enzyme. In chapter 6 of this thesis I have shown that 2 highly conserved regions of sequence called Upstream Conserved Regions 1 and 2 (UCRl and UCR2) that characterise the amino terminal regions of all long PDE4 isoforms can bind to one another. I have shown that this interaction is stabilised by electrostatic bonds between specific residues located in the carboxyl terminal portion of UCRl and the amino terminal portion of UCR2. This interaction can be regulated by the phosphoiylation of a serine residue that lies within the amino terminal region of UCRl and forms a consensus recognition site for protein kinase A (PKA).