Abstract
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 (RIα) subunit, where RIα 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 RIα. Furthermore our results indicate that binding of RIα activates PDE catalysis several fold demonstrating a dual function of RIα, 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 RIα 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 RIα generated as an end product remains bound to RegA. The PKA C-subunit then displaces RegA and reassociates with cAMP-free RIα 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 RIα that has important consequences for PKA regulation and cAMP signal termination.
Highlights
Because we identified the phosphate binding cassette (PBC) as one of the regions mediating interactions with RegA, we set out to test if the catalytic domain of RegA could hydrolyze cAMP bound to RI␣
In order to study interactions between PDEs and PKA we have chosen the elements of a simple eukaryotic model- Dictyostelium discoideum, which presents a simple model for cAMP-PKA signaling with single isoforms of the PKA R (RI␣) and Csubunit (C␣) that both show high homology to mammalian PKA subunits [18]
All of the residues involved in cAMP binding and allostery are invariant between D. discoideum RI␣ and mammalian (B. taurus) RI␣ (Supplemental Fig. 5) [18]
Summary
Ferences in subcellular localization, tissue specificity, and function [7]. Of these, RI␣ and C␣ are the two isoforms that are distributed across all cell-types [8, 9]. Of the mammalian RI␣ sequence, including the N-terminal dimerization/docking domain, whereas the sequences of the cAMP-binding domains (CNB:A and CNB:B) are highly invariant among RI␣ across species [18] These indicate that the PDE-RI␣ interactions must involve the cAMP-binding domains and would be independent of AKAPs. Earlier studies showed that RegA was capable of interacting with RI␣ from D. discoideum and with the monomeric N-terminal truncation mutant of mammalian (Bos taurus) RI␣ (RI␣ [91–379]) [17]. It has been assumed until now that reassociation of the PKA holoenzyme occurs through a reversal of the activation process in which C-subunit binding to cAMP-bound RI␣ facilitates dissociation of cAMP leading to regeneration of the holoenzyme [19] This is supported by evidence showing affinity of cAMP for the holoenzyme being significantly lower than that for free RI␣ [20]. By catalyzing hydrolysis of cAMP-bound to the RI␣, PDEs actively prime RI␣ for reassociation with the C-subunit and are critical for signal termination in cAMP signaling
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