The investigation of two novel PDE4A enzymes

Johnston, Lee Ann (2002) The investigation of two novel PDE4A enzymes. PhD thesis, University of Glasgow.

Abstract

As the central function of PDEs within cells is to control the levels of the cyclic nucleotides, it was surprising to find that the PDE4A gene encoded a splice variant, called PDE4A7 (2EL) that is catalytically inactive. This protein was investigated further by firstly determining its intracellular localisation using the methods of subcellular fractionation and Laser Scanning Confocal Microscopy (LSCM). Interestingly, PDE4A7 when over-expressed in COS-1 cells was found exclusively within the nuclear compartment. This was in contrast to that of the catalytically active long isoform, PDE4A4B, which was found to be extranuclear. Subcellular fractionation studies revealed that the major fraction of PDE4A4B was localised within the cytosolic (S2) fraction, whereas PDE4A7 was localised exclusively within the low speed particulate (PI) fraction. Therefore, despite sharing identical core regions, both PDE4A4B and PDE4A7 were found in completely different compartments. Using chimeric constructs, I firstly demonstrated that the central core was exclusively located to the particulate (PI) fraction even in the absence of the PDE4A7 N-terminal region. Secondly, I demonstrated that replacing the C- terminal of PDE4A7 with 19 amino acids from the C-terminal region that is common to all the active PDE4As was enough to cause re-distribution of a fraction of the PDE4A7 central core to the cytosolic (S2) fraction. These analyses established that the cytosolic targeting observed with the active PDE4A enzymes can be attributed to a small portion of sequence located within their C-terminal regions. In Chapter 4, the yeast two hybrid screening procedure was used to try and identify binding partners for PDE4A7. Proteins identified as potential binding partners for PDE4A7 included; CREB binding protein (CBP), RanBPM9 and niRNA from the KIAA0160 gene. However, upon further analysis I demonstrated that both CBP and RanBPM9 were false positives generated from the yeast two-hybrid screen. The interaction between PDE4A7 and the mRNA from the KIAA0160 gene has yet to be reevaluated. In Chapter 5, I used the methods of gene microarray and RT PCR to address whether the functional role of PDE4A7 within the nucleus was to control the expression levels of other genes. Gene microarray results demonstrated that the expression levels of a large number of genes changed in response to the over-expression of PDE4A7. Next, RT- PCR was used in an attempt to verify a small number of these changes. However, RT-PCR analysis did not support the changes observed with gene microarray. It was concluded that microarray had generated false positive data for these particular genes. As yet the basis for the nuclear targeting of PDE4A7 and the function of this protein within this compartment remains to be elucidated. Finally, in Chapter 6, I performed partial characterisation studies on a novel PDE4A isoform, called HSPDE4A11. Transient expression of HSPDE4A11 in COS-7 cells allowed the detection of a ll8kDa protein in both the soluble and particulate fractions. The soluble and particulate forms of HSPDE4A11 exhibited similar Km values for cAMP hydrolysis (~2.5muM) and Vmax values.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Houslay, Professor M.
• Date of Award: 2002
• Depositing User: Enlighten Team
• Unique ID: glathesis:2002-71061
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 May 2019 10:49
• Last Modified: 08 Aug 2022 10:25
• Thesis DOI: 10.5525/gla.thesis.71061
• URI: https://theses.gla.ac.uk/id/eprint/71061