Active Site Coupling in PDE:PKA Complexes Promotes Resetting of Mammalian cAMP Signaling

Abstract

Cyclic 3'5' adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (KD ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and highlights an entirely new class of binding partners for RIα. This study also highlights applications of structural mass spectrometry combined with computational docking for mapping dynamics in transient signaling protein complexes. Together, these results present a novel and critical role for phosphodiesterases in moderating local concentrations of cAMP in microdomains and signal resetting.

Unidirectional allostery in the regulatory subunit RIα facilitates efficient deactivation of protein kinase A

“…Recently, Anand and coworkers (37,38) reported that phosphodiesterases (PDEs) can bind to and catalyze cAMP release from PKA RIα. The ligands are channeled from the tandem CBDs to the active sites of a PDE dimer and then hydrolyzed.…”

Parallel Allostery by cAMP and PDE Coordinates Activation and Termination Phases in cAMP Signaling

“…Samples were injected onto a nano-UPLC HDX sample manager (Waters, Milford, MA) as described in Wales et al (39). Online immobilized pepsin digestion and reverse phase liquid chromatography were carried out as previously described in Krishnamurthy et al (27). Peptides separated from previous liquid chromatography were subsequently sprayed onto a SYNAPT G2-Si mass spectrometer (Waters) acquiring in MS E mode.…”

“…PDEs, enzymes responsible for catalyzing hydrolysis of cAMP to 5 0 AMP, initiate the termination phase of the cAMP signaling pathway by forming direct interactions with the cyclic-nucleotide-binding (CNB) pocket domains of PKA R-subunit and hydrolyzing the bound cAMP ( Fig. 1 A) (19,27). The R-subunit of D. discoideum (henceforth referred to as R D ) differs from its mammalian homologs in being monomeric, and lacks an N-terminal dimerization domain, but contains two canonical cyclic AMP binding sites in two distinct domains, CNB domains A and B (denoted CNB:A and CNB:B) ( Fig.…”

“…The transition from holoenzyme form to the cAMP-bound form constitutes the activation phase of the cAMP signaling pathway. In the termination phase, cyclic nucleotide PDEs form direct interactions with cAMP-bound PKAR (docking model (27)) and cause the release and dissociation of bound cAMP. Free PKAR can now bind PKAC and regenerate the holoenzyme, thereby terminating the cAMP pathway.…”

“…These conformational transitions have been the focus of several studies to map the allosteric transitions in response to cAMP and C-subunit binding (33,37,38). There have been far fewer studies on how PDEs interact to hydrolyze cAMP bound to PKA R-subunit and initiate cAMP signal termination (19,27). Further, how signals in the cAMP signaling pathway converge and branch off from a target through alternate allosteric pathways is of enormous interest.…”