Structural and Dynamic Effects of Phosphorylation of Protein Kinase a Catalytic Subunit

Abstract

Protein Kinase A (PKA) is an enzyme involved in many physiological processes due to its role in signaling pathways, including regulation of metabolism and memory formation. Mutations in PKA are associated with cancer, cardiac disorders, and neurodegenerative disorders. PKA is a tetramer comprised of two regulatory (R) and two catalytic (c) subunits, forming the holoenzyme complex. My work focuses on the C-subunit. Phosphorylation of different residues in the PKA C-subunit lead to distinct phosphorylation states (phospho-states) with roles that are not well understood. I have studied and compared the structures and dynamics of two phospho-states using protein crystallography. Using room temperature X-ray diffraction data collected at the Berkeley Advanced Light Source and a published starting model, I solved the phases for the electron densities and refined the atomic structure for each dataset. Each dataset, comprised of diffraction data from several crystals per complex, were scaled and merged for final ensemble refinement. Ensemble refinement of each complex found differences in dynamics between phospho-states in several important secondary structures in the protein: the A-Helix, the Hydrophobic Motif, and the H-Helix. This could lead to functional differences of the two phospho-states in the cell. To further study functional differences, I used fluorescence polarization biochemical assays to compare the cyclic AMP-dependent activation of the holoenzyme complex formed with each phospho-state of the C-subunit. Studying the effects of phosphorylation on the structure and dynamics of PKA will lead to a better understanding of how these differences affect their function in the cell. The differential dynamics observed in the ensemble refinements of each phospho-state of the C-subunit could lead to insights on the binding of inhibitors and development of functionally specific therapeutics.