Byrne, Ashleigh Maria (2014) Functional characterisation of phosphodiesterase 4D7 in prostate cancer. PhD thesis. http://theses.gla.ac.uk/5275/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Functional Characterisation of Phosphodiesterase 4D7 in Prostate Cancer A Thesis Presented by Ashleigh Maria Byrne To the In accordance with the requirements for the degree of Doctor of Philosophy In Integrated Biology Institute of Cardiovascular and Medical Sciences College of Medical, Veterinary and Life Sciences 2014 Certavi et vici Table of Contents Abstract ...................................................................................... i Author’s Declaration ..................................................................... iii Acknowledgements ........................................................................ iv List of Figures .............................................................................. v List of Tables ............................................................................... ix Abbreviations ............................................................................... x Publications ............................................................................... xiii Abstracts and Posters ................................................................... xiii 1 cAMP Signalling, Phosphodiesterases and Prostate Cancer ................. 14 1.1 G protein Coupled Receptors and Heterotrimeric G proteins. ............ 17 1.2 Adenylyl Cyclases ................................................................ 20 1.3 Guanylyl Cyclases ................................................................ 22 1.4 Phosphodiesterases .............................................................. 22 1.4.1 Overview of PDE Families ................................................. 23 1.4.2 Nomenclature ............................................................... 26 1.4.3 PDE Structure ............................................................... 27 1.4.4 PDE Oligomerisation ........................................................ 30 1.4.5 PDEI family .................................................................. 31 1.4.6 The Phosphodiesterase 4 Family ......................................... 39 1.4.7 PDE4 Regulation ............................................................ 54 1.5 cAMP Effector Proteins ......................................................... 66 1.5.1 PKA ........................................................................... 66 1.5.2 Exchange Proteins Directly Activated by cAMP (EPAC) ................ 70 1.5.3 Cyclic Nucleotide gated Ion Channels ................................... 73 1.6 The Human Male Prostate Gland-a Brief Synopsis of Biology and Structure 74 1.6.1 Function of the Male Human Prostate ................................... 74 1.6.2 Structure of the Male Human Prostate .................................. 74 1.7 Signalling in the Prostate ....................................................... 76 1.7.1 Androgens and the AR ...................................................... 76 1.8 Prostate Pathogenesis- Benign Disease and Prostate Cancer ............. 83 1.8.1 Benign Prostatic Hyperplasia (BPH) ...................................... 83 1.8.2 Prostate Stem Cells and Senescence..................................... 84 1.8.3 The Intra-prostatic Immune System; Prostatitis and Inflammation . 84 1.9 Prostate Cancer .................................................................. 86 ii 1.9.1 Steroid Signalling in PC and BPH ......................................... 87 1.9.2 Other Genetic Abnormalities in Prostate Cancer ...................... 91 1.9.3 Androgen-Independent Prostate Cancer ................................ 93 1.9.4 Suggested Mechanisms for Progression into Androgen Insensitive Prostate Cancer ...................................................................... 94 1.9.5 Prostate Cancer Diagnosis and Treatment .............................. 97 1.10 cAMP Signalling in the Prostate and Prostate Cancer ................... 105 1.10.1 cAMP PDEs in Prostate Cancer ......................................... 105 1.10.2 Protein Kinase A and Prostate Cancer ............................... 106 1.10.3 EPAC and Prostate Cancer ............................................. 108 1.10.4 Neuroendocrine Differentiation in Prostate Cancer ............... 110 Aims of Research ....................................................................... 112 2 Materials and Methods ........................................................... 113 2.1 Molecular Biology ............................................................... 113 2.1.1 Cloning and PCR ........................................................... 113 2.1.2 PCR Product Clean-up ..................................................... 114 2.1.3 Restriction Endonuclease Digestion of DNA ............................ 114 2.1.4 Agarose Gel Electrophoresis ............................................. 115 2.1.5 Ligation ..................................................................... 115 2.1.6 Transformation of Competent Cells .................................... 115 2.1.7 Sequencing ................................................................. 116 2.1.8 Storage of Plasmid DNA ................................................... 116 2.1.9 Isolation of plasmid DNA .................................................. 117 2.1.10 Bioinformatic Analysis .................................................. 117 2.1.11 RNA Extraction and Purification ...................................... 117 2.1.12 Nucleic acid Quantification ............................................ 118 2.1.13 cDNA Synthesis .......................................................... 118 2.1.14 Quantitative Real Time PCR ........................................... 119 2.2 Protein Chemistry .............................................................. 121 2.2.1 Peptide Array Technology ................................................ 121 2.2.2 Phosphorylation Assays ................................................... 123 2.2.3 PDE4 Activity Assay ........................................................ 123 2.2.4 Protein-Protein Interactions ............................................. 126 2.2.5 Expression and Purification of Recombinant Proteins ................ 129 2.2.6 Protein Concentration Assay ............................................. 130 2.3 Protein Analysis ................................................................. 130 2.3.1 SDS-PAGE ................................................................... 130 iii 2.3.2 Coomassie staining ........................................................ 131 2.3.3 Western Immunoblotting ................................................. 131 2.4 Mammalian Cell Culture ....................................................... 134 2.4.1 Culture of Human Cell Lines ............................................. 134 2.4.2 Transfection of Cell Lines. ............................................... 135 2.4.3 siRNA Mediated Gene Knockdown in VCaP Cells ...................... 136 2.4.4 xCelligence Measurement of Cell Proliferation and Migration ...... 137 2.5 PLA Probe Staining and Confocal Microscopy ............................... 140 2.6 Statistical Analysis .............................................................. 141 3 PDE4D7 Signalling is Altered as Prostate Cancer Progresses ............. 142 3.1 Introduction ..................................................................... 142 3.2 Results ........................................................................... 144 3.2.1 Phosphodiesterase 4D7 is downregulated as prostate cancer becomes androgen-insensitive ................................................................ 144 3.2.2 Validation of PDE4D7 protein expression in AS and AI PC cell lines 148 3.2.3 PDE4D7 Mediates Prostate Cancer Cell Proliferation ................. 156 3.2.4 PDE4D7 may Mediate Prostate Cancer Cell Migration. ............... 164 3.2.5 Loss of PDE4D7 during the transition from AS to AI PC may be due to an Altered Epigenome .............................................................. 167 3.3 Discussion ........................................................................ 172 3.4 Chapter Summary ............................................................... 179 4 Unique N-terminal Phosphorylation of PDE4D7 ............................. 180 4.1 Introduction ..................................................................... 180 4.2 Results ........................................................................... 183 4.2.1 The Unique N-terminal Region of PDE4D7 Contains a PKA Consensus Site 183 4.2.2 PDE4D7-Ser42 is Phosphorylated by PKA in vitro ..................... 185 4.2.3 PDE4D7 Ser42 is Phosphorylated by PKA ex vivo-Overexpression System 190 4.2.4 PDE4D7 Ser42 Phosphorylation Negatively Regulates Enzyme Activity 193 4.2.5 PDE4D7 Ser42 is Phosphorylated by PKA ex vivo-Endogenous