Cyclic adenosine monophosphate (cAMP) signaling through cAMP-dependent protein kinase (PKA) is a ubiquitous mammalian signaling pathway involved in metabolism, cell proliferation, and cell death. While the PKA catalytic (C) subunit has served as a prototype for the protein kinase superfamily, the regulatory (R) subunit defines the mechanism whereby cAMP translates an extracellular signal into an intracellular biological response.

This dissertation investigates three major areas of PKA research: (1) defining the molecular features that govern RIα:C complex formation as a means to understand the allosteric regulation of cAMP-induced PKA activation; (2) elucidating the molecular rules that govern substrate recognition; and (3) understanding the molecular basis for isoform-specific activation by cAMP derivatives.

A structure of a PKA RIα:C holoenzyme was solved with a RIα deletion mutant that contains both cAMP binding domains. This structure revealed the extraordinary conformational range that the R-subunit can adopt as it toggles between binding the C-subunit and cAMP. Mutational analysis explains how Domain B is a "gate-keeper" for Domain A. A critical salt bridge links the two hydrophobic capping residues that stack against cAMP (for Domains A and B), such that binding of cAMP to Domain B can release the capping residue for Domain A. Small angle X-ray analysis of various mutant RIα-subunits revealed that Domain B is also highly dynamic. Dissection of the inhibitor sites from both cAMP binding domains shows that binding is only preserved for RII subunits. The differences observed between the RI and RII subunits suggest why the RII subunits, but not RI, can bind to the C-subunit in the absence of ATP.

A crystal structure of a complex between PKA and a specific substrate, phospholamban, was solved, revealing an overall common docking mode as the protein inhibitors at this site. Peptide array methods, site-directed mutagenesis, and biochemical analysis combined defines a unique consensus substrate recognition motif for PKA substrates as R-X_y-R-R-X-S/T-ϕ, where y is 0-4 residues.

Finally, the crystal structure of RIα bound to a cAMP analog, HE-33, was solved, revealing the structural basis of how cAMP analogs result in selective activation of Type Iα versus Type IIβ isoforms.