Function and regulation of cyclic nucleotide phosphodiesterases

Abstract

Cyclic AMP is an important second messenger involved in a variety of cellular responses to extracellular signals. Cyclic AMP phosphodiesterases (PDEs) play a role in signal transduction by hydrolyzing cellular cAMP thus regulating cAMP dependent protein kinase A activity. PDE7 belongs to one of the eleven PDE families identified in mammalian cells. PDE7A1 is the first family member and is characterized as a high-affinity cAMP-specific PDE.;The first chapter of my thesis analyzes the regulation of PDE7A1 and cAMP signaling in the pancreatic beta cell line betaTC3. PDE7A1 co-localizes with type II PKA in the Golgi apparatus and associates with the PKA catalytic subunit in these cells. The interaction domain of PDE7A1 and PKA C was mapped to the N-terminal region of PDE7A1 which contains two repeat sequences each bearing a PKA pseudosubstrate recognition site. In an in vitro kinase assay, PDE7A1 can inhibit PKA kinase activity. Its N-terminal region is not only necessary but also sufficient for PKA inhibition. In yeast, PDE7A1 and this N-terminal domain can reverse the phenotype resulting from PKA constitutive-activation independently of its phosphodiesterase activity. These data suggest that in addition to hydrolyzing cAMP, PDE7A1 can inhibit PKA kinase activity by interaction via the N-terminal PKA pseudosubstrate repeat sequences. This dual regulatory mechanism can make PDE7A1 a potent terminator of basal cAMP signaling in eukaryotic cells.;The second chapter of my thesis is focused on the cloning and characterization of PDE7A2 which is a muscle form of PDE7A. PDE7A2 is encoded by a smaller transcript than that of PDE7A1 and is very abundant in skeletal muscle and heart. Cloning the PDE7A2 cDNA from muscle demonstrated that PDE7A2 is a novel 5' splice variant of PDE7A1 with a new, hydrophobic N-terminus. The 456 amino acid PDE7A2 is detected on Western Blots as a band with an apparent mobility of 50 kDa. PDE7A2 distributes to particulate cell fractions and is localized on the plasma membrane of skeletal muscle and heart. Enzyme kinetic analysis shows that PDE7A2 is also a high affinity cAMP specific PDE with a Km of 0.1 muM.;The third chapter of my thesis analyzes the role cAMP PDEs play in regulation of insulin secretion from pancreatic beta cells. Cyclic nucleotide PDE activities in betaTC3 cells were identified with family and isozyme specific inhibitors. PDE1C, 4A, 4D and a cGMP specific PDE were found present in soluble fractions and PDE3 in particulate fractions of betaTC3 cells. PDE7A1 protein was also observed in betaTC3 cells, but its PDE activity was not detected. Selective inhibition of PDE1C but not of PDE4 augmented glucose-stimulated insulin secretion in both betaTC3 cells and pancreatic islets. PDE1C of betaTC3 cells is a novel isozyme possessing a Km of 0.47 muM for cAMP and 0.25 muM for cGMP. Increased responsiveness of PDE1C activity to Calcium/Calmodulin is observed upon exposure of cells to glucose. These observations suggest a glucose-dependent feedback mechanism of insulin secretion regulated by PDE1C.