Structure-function studies of the cAMP -dependent protein kinase in vitro and in intact cells

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, Calpha and Cgamma, which share 83% identity in primary structure but differ in function. The first approach sought to identify differences in primary structure between Cgamma and Calpha, which may define functional differences between them. To this end chimeras were generated, swapping the carboxyl and amino termini between Calpha and Cgamma and were evaluated for functionality through CREB-mediated reporter assays. Wild type Calpha and Cgamma induced CREB-mediated transcriptional activation, but the chimeras failed to exhibit any activity. The second approach sought to characterize phosphorylation differences between purified PKA-Cgamma and PKA-Calpha that defines their physiological function. Two novel phosphorylation sites were identified on both isoforms by tandem mass spectrometry analysis (Cgamma S14 and Calpha/Cgamma S259). It was also determined that Cgamma expressed in Sf9 insect cells, like Calpha 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-Cgamma through determination of the rate for the reaction-limiting step, which was found to be 9-times slower than that of Calpha. The final approach sought to identify Cgamma expression in the cell through the use of a new Cgamma-specific antibody. Cgamma expression was identified following differentiation of U-937 cells suggesting a novel function for Cgamma in the cell.