UC San Diego

cAMP-dependent protein kinase (PKA) is one of the most ubiquitous kinases in cells that relies on four functionally non-redundant cAMP sensing Regulatory (R). Largely influenced by the signaling state of the cell, the catalytic subunits of PKA exist in either the unbound active state or as part of phosphorylation-silent R- subunit nucleated macromolecular assemblies. The guiding theme of this thesis was to gain insights into RI[alpha]- mediated PKA signaling complexes utilizing X-ray crystallography, small-angle X-ray scattering (SAXS), as well as complementary biochemical techniques. Specifically, this study targeted the elucidation of the RI[alpha] homodimer complex structure, a small membrane A- kinase anchoring protein (smAKAP) -bound RI[alpha] dimerization domain (D/D) structure, the RI[alpha] holoenzyme (RI[alpha]:C)₂ complex, and the RI-mediated interaction with P-Rex1. At the start of this project, only monomeric R structures where known, which fell short in explaining cAMP binding for the full-length protein as compared to the truncated R subunits. With X-ray crystallography, the full-length RI[alpha] homodimer structure was solved and revealed a novel, isoform- specific interface between the protomers. Next, the crystal structure of smAKAP's A-kinase binding domain (smAKAP-AKB) in complex with the dimerization/docking domain of PKA-RI[alpha] (PKA-RI[alpha]D/D) was solved, which revealed a unique interaction surface with a large number of contact residues to drive smAKAP's high specificity. Furthermore, we present insights that have been gained for the assembly and function of the RI[alpha] holoenzyme with the description of an intriguing model and a low-resolution structure. Additionally, a focus on disease mutations of the RI[alpha] gene uncovers explanations for the downstream molecular effects that result in the severe clinical outcomes of Carney Complex disease and acrodysostosis-1. Finally, the RI-subunits' C- termini potential as a scaffolding module was tested. Based on our experiments we propose a binding model for the PDZ domains of P-Rex1 to the RI[alpha]/[Beta]CNB-B domain. In summary, this thesis emphasizes RI[alpha] as an essential regulator and partner for PKA cell signaling assemblies