Date of Award
Doctor of Philosophy (PhD)
Stephen J. Beebe
There are 518 protein kinase genes in the human genome; this constitutes about 1.7% of all human genes. The cAMP-dependent protein kinase (PKA) serves as the prototypic model for the study of kinases because it contains a conserved catalytic core shared with all eukaryotic kinases, it is the simplest kinase, and it is one of the best-characterized serine/threonine kinases. PKA is ubiquitous in mammals and regulates multiple physiological mechanisms such as the cell cycle, apoptosis, cell motility, energy metabolism, and gene transcription through a well-defined intracellular signaling pathway. While PKA clearly has a central physiological role it is still unclear how PKA mediates multiple physiological mechanisms at the cellular level. Four approaches were used to explore this question using two PKA catalytic subunits, Cα and Cγ, which share 83% identity in primary structure but differ in function. The first approach sought to identify differences in primary structure between Cγ and Cα, which may define functional differences between them. To this end chimeras were generated, swapping the carboxyl and amino termini between Cα and Cγ and were evaluated for functionality through CREB-mediated reporter assays. Wild type Cα and Cγ induced CREB-mediated transcriptional activation, but the chimeras failed to exhibit any activity. The second approach sought to characterize phosphorylation differences between purified PKA-Cγ and PKA-Cα that defines their physiological function. Two novel phosphorylation sites were identified on both isoforms by tandem mass spectrometry analysis (Cγ S14 and Cα/Cγ S259). It was also determined that Cγ expressed in Sf9 insect cells, like Cα expressed in mammalian cells, is phosphorylated at T197 and S338 and the modification at T197 is important to the function of both isoforms. The third approach sought to characterize the kinetic mechanism of PKA-Cγ through determination of the rate for the reaction-limiting step, which was found to be 9-times slower than that of Cα. The final approach sought to identify Cγ expression in the cell through the use of a new Cγ-specific antibody. Cγ expression was identified following differentiation of U-937 cells suggesting a novel function for Cγ in the cell.
Morris, Gary Z..
"Structure-Function Studies of the cAMP-Dependent Protein Kinase In Vitro and in Intact Cells"
(2006). Doctor of Philosophy (PhD), dissertation, , Old Dominion University, DOI: 10.25777/ptf4-s531