Phosphodiesterases Catalyze Hydrolysis of cAMP-bound to Regulatory Subunit of Protein Kinase A and Mediate Signal Termination

Although extensive structural and biochemical studies have provided molecular insights into the mechanism of cAMP-dependent activation of protein kinase A (PKA), little is known about signal termination and the role of phosphodiesterases (PDEs) in regulatory feedback. In this study we describe a novel mode of protein kinase A-anchoring protein (AKAP)-independent feedback regulation between a specific PDE, RegA and the PKA regulatory (RI alpha) subunit, where RI alpha functions as an activator of PDE catalysis. Our results indicate that RegA, in addition to its well-known role as a PDE for bulk cAMP in solution, is also capable of hydrolyzing cAMP-bound to RI alpha. Furthermore our results indicate that binding of RI alpha activates PDE catalysis several fold demonstrating a dual function of RI alpha, both as an inhibitor of the PKA catalytic ( C) subunit and as an activator for PDEs. Deletion mutagenesis has localized the sites of interaction to one of the cAMP-binding domains of RI alpha and the catalytic PDE domain of RegA whereas amide hydrogen/deuterium exchange mass spectrometry has revealed that the cAMP-binding site ( phosphate binding cassette) along with proximal regions important for relaying allosteric changes mediated by cAMP, are important for interactions with the PDE catalytic domain of RegA. These sites of interactions together with measurements of cAMP dissociation rates demonstrate that binding of RegA facilitates dissociation of cAMP followed by hydrolysis of the released cAMP to 5'AMP. cAMP-free RI alpha generated as an end product remains bound to RegA. The PKA C-subunit then displaces RegA and reassociates with cAMP-free RI alpha to regenerate the inactive PKA holoenzyme thereby completing the termination step of cAMP signaling. These results reveal a novel mode of regulatory feedback between PDEs and RI alpha that has important consequences for PKA regulation and cAMP signal termination. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.002295, 1-15, 2011.