UC San Diego

The catalytic (C) subunit of cAMP-dependent protein kinase (PKA) is a member of the AGC group of protein kinases. PKA has served as a structural and biochemical prototype for understanding the protein kinase superfamily. In cells, PKA is regulated primarily by three different mechanisms : cAMP binding to the regulatory subunits, PKI, and phosphorylation on the turn motif and activation loop. The main focus of this dissertation was to investigate the role of turn motif and activation loop phosphorylation on the regulation of PKA structure and function. The molecular basis for activation loop (Thr197) phosphorylation was explored using biochemical and structural methods. An X-ray crystal structure of the unphosphorylated C-subunit revealed that the N- and C- lobes of the kinase were separated and there was a dramatic loss in hydrogen bonding at the active site. These changes were correlated with a decrease in activity, loss of stability, and increased dynamics of the enzyme as measured by hydrogen/deuterium exchange. Next, the role of turn motif (Ser338) phosphorylation in the C-terminal tail of PKA was investigated by structural and biochemical methods. Even though the structure of the unphosphorylated protein was similar to wild type PKA, our studies showed that the phosphorylation at the turn motif was a necessary precursor for activation loop phosphorylation, and is essential for maintaining normal PKA activity in mammalian cells. Finally, the phosphoinositide-dependent protein kinase (PDK1) is known to phosphorylate the activation loops of numerous AGC kinases. We sought to determine the molecular basis for substrate recognition and activation loop phosphorylation by PDK1. An internal Glu-Arg salt bridge was found to be a critical nonlinear recognition motif in all PDK1 substrates tested. This salt bridge is highly conserved and disease-associated SNPs at the Glu and Arg positions have been found in numerous protein kinases indicating that this may be a common recognition motif in the protein kinase superfamily