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

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 (K_D ∼ 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.     

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 (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.     

“cAMP Sponge”: A Buffer for Cyclic Adenosine 3′, 5′-Monophosphate

Background

While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP).

Methods/Principal Findings

Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIβ of protein kinase A (PKA). Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named “cAMP sponge”) was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets.

Conclusions

This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.     

Bacillus bombysepticus α-Toxin Binding to G Protein-Coupled Receptor Kinase 2 Regulates cAMP/PKA Signaling Pathway to Induce Host Death

Bacterial pathogens and their toxins target host receptors, leading to aberrant behavior or host death by changing signaling events through subversion of host intracellular cAMP level. This is an efficient and widespread mechanism of microbial pathogenesis. Previous studies describe toxins that increase cAMP in host cells, resulting in death through G protein-coupled receptor (GPCR) signaling pathways by influencing adenylyl cyclase or G protein activity. G protein-coupled receptor kinase 2 (GRK2) has a central role in regulation of GPCR desensitization. However, little information is available about the pathogenic mechanisms of toxins associated with GRK2. Here, we reported a new bacterial toxin-Bacillus bombysepticus (Bb) α-toxin that was lethal to host. We showed that Bb α-toxin interacted with BmGRK2. The data demonstrated that Bb α-toxin directly bound to BmGRK2 to promote death by affecting GPCR signaling pathways. This mechanism involved stimulation of G_αs, increase level of cAMP and activation of protein kinase A (PKA). Activated cAMP/PKA signal transduction altered downstream effectors that affected homeostasis and fundamental biological processes, disturbing the structural and functional integrity of cells, resulting in death. Preventing cAMP/PKA signaling transduction by inhibitions (NF449 or H-89) substantially reduced the pathogenicity of Bb α-toxin. The discovery of a toxin-induced host death specifically linked to GRK2 mediated signaling pathway suggested a new model for bacterial toxin action. Characterization of host genes whose expression and function are regulated by Bb α-toxin and GRK2 will offer a deeper understanding of the pathogenesis of infectious diseases caused by pathogens that elevate cAMP.     

Dynamics of phosphodiesterase-induced cAMP dissociation from protein kinase A: Capturing transient ternary complexes by HDXMS

cAMP signaling is a fundamental cellular process necessary for mediating responses to hormonal stimuli. In contrast to cAMP-dependent activation of protein kinase A (PKA), an important cellular target, far less is known on termination in cAMP signaling, specifically how phosphodiesterases (PDEs) facilitate dissociation and hydrolysis of bound cAMP. In this study, we have probed the dynamics of a ternary complex of PKA and a PDE–RegA with an excess of a PDE-nonhydrolyzable cAMP analog, Sp-cAMPS by amide hydrogen/deuterium exchange mass spectrometry (HDXMS). Our results highlight how HDXMS can be used to monitor reactions together with mapping conformational dynamics of transient signaling complexes. Our results confirm a two-state model for active RegA-mediated dissociation of bound cAMP. Further, our results reveal that Sp-cAMPS and RegA mediate mutually exclusive interactions with the same region of PKA and at specific concentrations of Sp-cAMPS, RegA is capable of blocking Sp-cAMPS reassociation to PKA. This provides a molecular basis for how PDEs modulate levels of intracellular cAMP so that PKA is better suited to responding to fluxes rather than constant levels of cAMP. This study underscores how HDXMS can be a powerful tool for monitoring reactions together with mapping conformational dynamics in signaling proteins. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Protein Kinase A Opposes the Phosphorylation-dependent Recruitment of Glycogen Synthase Kinase 3β to A-kinase Anchoring Protein 220

The proximity of an enzyme to its substrate can influence rate and magnitude of catalysis. A-kinase anchoring protein 220 (AKAP220) is a multivalent anchoring protein that can sequester a variety of signal transduction enzymes. These include protein kinase A (PKA) and glycogen synthase kinase 3β (GSK3β). Using a combination of molecular and cellular approaches we show that GSK3β phosphorylation of Thr-1132 on AKAP220 initiates recruitment of this kinase into the enzyme scaffold. We also find that AKAP220 anchors GSK3β and its substrate β-catenin in membrane ruffles. Interestingly, GSK3β can be released from the multienzyme complex in response to PKA phosphorylation on serine 9, which suppresses GSK3β activity. The signaling scaffold may enhance this regulatory mechanism, as AKAP220 has the capacity to anchor two PKA holoenzymes. Site 1 on AKAP220 (residues 610–623) preferentially interacts with RII, whereas site 2 (residues 1633–1646) exhibits a dual specificity for RI and RII. In vitro affinity measurements revealed that site 2 on AKAP220 binds RII with ∼10-fold higher affinity than site 1. Occupancy of both R subunit binding sites on AKAP220 could provide a mechanism to amplify local cAMP responses and enable cross-talk between PKA and GSK3β.     

Mapping intersubunit interactions of the regulatory subunit (RIalpha) in the type I holoenzyme of protein kinase A by amide hydrogen/deuterium exchange mass spectrometry (DXMS)

Protein kinase A (PKA), a central locus for cAMP signaling in the cell, is composed of regulatory (R) and catalytic (C) subunits. The C-subunits are maintained in an inactive state by binding to the R-subunit dimer in a tetrameric holoenzyme complex (R(2)C(2)). PKA is activated by cAMP binding to the R-subunits which induces a conformational change leading to release of the active C-subunit. Enzymatic activity of the C-subunit is thus regulated by cAMP via the R-subunit, which toggles between cAMP and C-subunit bound states. The R-subunit is composed of a dimerization/docking (D/D) domain connected to two cAMP-binding domains (cAMP:A and cAMP:B). While crystal structures of the free C-subunit and cAMP-bound states of a deletion mutant of the R-subunit are known, there is no structure of the holoenzyme complex or of the cAMP-free state of the R-subunit. An important step in understanding the cAMP-dependent activation of PKA is to map the R-C interface and characterize the mutually exclusive interactions of the R-subunit with cAMP and C-subunit. Amide hydrogen/deuterium exchange mass spectrometry is a suitable method that has provided insights into the different states of the R-subunit in solution, thereby allowing mapping of the effects of cAMP and C-subunit on different regions of the R-subunit. Our study has localized interactions with the C-subunit to a small contiguous surface on the cAMP:A domain and the linker region. In addition, C-subunit binding causes increased amide hydrogen exchange within both cAMP-domains, suggesting that these regions become more flexible in the holoenzyme and are primed to bind cAMP. Furthermore, the difference in the protection patterns between RIalpha and the previously studied RIIbeta upon cAMP-binding suggests isoform-specific differences in cAMP-dependent regulation of PKA activity.     

Protein Kinase A Modulates Transforming Growth Factor-β Signaling through a Direct Interaction with Smad4 Protein

Transforming growth factor β (TGFβ) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFβ signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFβ activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290–300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281–285 and 320–329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFβ-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFβ-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo.     

Mapping the Free Energy Landscape of PKA Inhibition and Activation: A Double-Conformational Selection Model for the Tandem cAMP-Binding Domains of PKA RIα

Protein Kinase A (PKA) is the major receptor for the cyclic adenosine monophosphate (cAMP) secondary messenger in eukaryotes. cAMP binds to two tandem cAMP-binding domains (CBD-A and -B) within the regulatory subunit of PKA (R), unleashing the activity of the catalytic subunit (C). While CBD-A in RIα is required for PKA inhibition and activation, CBD-B functions as a “gatekeeper” domain that modulates the control exerted by CBD-A. Preliminary evidence suggests that CBD-B dynamics are critical for its gatekeeper function. To test this hypothesis, here we investigate by Nuclear Magnetic Resonance (NMR) the two-domain construct RIα (91–379) in its apo, cAMP₂, and C-bound forms. Our comparative NMR analyses lead to a double conformational selection model in which each apo CBD dynamically samples both active and inactive states independently of the adjacent CBD within a nearly degenerate free energy landscape. Such degeneracy is critical to explain the sensitivity of CBD-B to weak interactions with C and its high affinity for cAMP. Binding of cAMP eliminates this degeneracy, as it selectively stabilizes the active conformation within each CBD and inter-CBD contacts, which require both cAMP and W260. The latter is contributed by CBD-B and mediates capping of the cAMP bound to CBD-A. The inter-CBD interface is dispensable for intra-CBD conformational selection, but is indispensable for full activation of PKA as it occludes C-subunit recognition sites within CBD-A. In addition, the two structurally homologous cAMP-bound CBDs exhibit marked differences in their residual dynamics profiles, supporting the notion that conservation of structure does not necessarily imply conservation of dynamics.     

Control of βAR- and N-methyl-D-aspartate (NMDA) Receptor-Dependent cAMP Dynamics in Hippocampal Neurons

Norepinephrine, a neuromodulator that activates β-adrenergic receptors (βARs), facilitates learning and memory as well as the induction of synaptic plasticity in the hippocampus. Several forms of long-term potentiation (LTP) at the Schaffer collateral CA1 synapse require stimulation of both βARs and N-methyl-D-aspartate receptors (NMDARs). To understand the mechanisms mediating the interactions between βAR and NMDAR signaling pathways, we combined FRET imaging of cAMP in hippocampal neuron cultures with spatial mechanistic modeling of signaling pathways in the CA1 pyramidal neuron. Previous work implied that cAMP is synergistically produced in the presence of the βAR agonist isoproterenol and intracellular calcium. In contrast, we show that when application of isoproterenol precedes application of NMDA by several minutes, as is typical of βAR-facilitated LTP experiments, the average amplitude of the cAMP response to NMDA is attenuated compared with the response to NMDA alone. Models simulations suggest that, although the negative feedback loop formed by cAMP, cAMP-dependent protein kinase (PKA), and type 4 phosphodiesterase may be involved in attenuating the cAMP response to NMDA, it is insufficient to explain the range of experimental observations. Instead, attenuation of the cAMP response requires mechanisms upstream of adenylyl cyclase. Our model demonstrates that Gs-to-Gi switching due to PKA phosphorylation of βARs as well as Gi inhibition of type 1 adenylyl cyclase may underlie the experimental observations. This suggests that signaling by β-adrenergic receptors depends on temporal pattern of stimulation, and that switching may represent a novel mechanism for recruiting kinases involved in synaptic plasticity and memory.     

A Long Lasting β1 Adrenergic Receptor Stimulation of cAMP/Protein Kinase A (PKA) Signal in Cardiac Myocytes

Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated β₁ adrenergic receptor (β₁AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t_½ of 3.9 h) in cardiac myocytes. However, the β₁AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. β₁AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the β₁AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response.     

Single Turnover Autophosphorylation Cycle of the PKA RIIβ Holoenzyme

To provide tight spatiotemporal signaling control, the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) holoenzyme typically nucleates a macromolecular complex or a “PKA signalosome.” Using the RIIβ holoenzyme as a prototype, we show how autophosphorylation/dephosphorylation of the RIIβ subunit, as well as cAMP and metal ions, contribute to the dynamics of PKA signaling. While we showed previously that the RIIβ holoenzyme could undergo a single turnover autophosphorylation with adenosine triphosphate and magnesium (MgATP) and trap both products in the crystal lattice, we asked here whether calcium could trap an ATP:RIIβ holoenzyme since the RIIβ holoenzyme is located close to ion channels. The 2.8Å structure of an RIIβ^p ₂:C₂:(Ca₂ADP)₂ holoenzyme, supported by biochemical and biophysical data, reveals a trapped single phosphorylation event similar to MgATP. Thus, calcium can mediate a single turnover event with either ATP or adenosine-5''-(β,γ-imido)triphosphate (AMP-PNP), even though it cannot support steady-state catalysis efficiently. The holoenzyme serves as a “product trap” because of the slow off-rate of the pRIIβ subunit, which is controlled by cAMP, not by phosphorylation of the inhibitor site. By quantitatively defining the RIIβ signaling cycle, we show that release of pRIIβ in the presence of cAMP is reduced by calcium, whereas autophosphorylation at the phosphorylation site (P-site) inhibits holoenzyme reassociation with the catalytic subunit. Adding a single phosphoryl group to the preformed RIIβ holoenzyme thus creates a signaling cycle in which phosphatases become an essential partner. This previously unappreciated molecular mechanism is an integral part of PKA signaling for type II holoenzymes.     

Forskolin-inducible cAMP Pathway Negatively Regulates T-cell Proliferation by Uncoupling the Interleukin-2 Receptor Complex

Cytokine-mediated regulation of T-cell activity involves a complex interplay between key signal transduction pathways. Determining how these signaling pathways cross-talk is essential to understanding T-cell function and dysfunction. In this work, we provide evidence that cross-talk exists between at least two signaling pathways: the Jak3/Stat5 and cAMP-mediated cascades. The adenylate cyclase activator forskolin (Fsk) significantly increased intracellular cAMP levels and reduced proliferation of the human T-cells via inhibition of cell cycle regulatory genes but did not induce apoptosis. To determine this inhibitory mechanism, effects of Fsk on IL-2 signaling was investigated. Fsk treatment of MT-2 and Kit 225 T-cells inhibited IL-2-induced Stat5a/b tyrosine and serine phosphorylation, nuclear translocation, and DNA binding activity. Fsk treatment also uncoupled IL-2 induced association of the IL-2Rβ and γ_c chain, consequently blocking Jak3 activation. Interestingly, phosphoamino acid analysis revealed that Fsk-treated cells resulted in elevated serine phosphorylation of Jak3 but not Stat5, suggesting that Fsk can negatively regulate Jak3 activity possibly mediated through PKA. Indeed, in vitro kinase assays and small molecule inhibition studies indicated that PKA can directly serine phosphorylate and functionally inactivate Jak3. Taken together, these findings suggest that Fsk activation of adenylate cyclase and PKA can negatively regulate IL-2 signaling at multiple levels that include IL-2R complex formation and Jak3/Stat5 activation.     

EP2 Receptor Signaling Pathways Regulate Classical Activation of Microglia

Activation of EP2 receptors by prostaglandin E2 (PGE₂) promotes brain inflammation in neurodegenerative diseases, but the pathways responsible are unclear. EP2 receptors couple to Gα_s and increase cAMP, which associates with protein kinase A (PKA) and cAMP-regulated guanine nucleotide exchange factors (Epacs). Here, we studied EP2 function and its signaling pathways in rat microglia in their resting state or undergoing classical activation in vitro following treatment with low concentrations of lipopolysaccharide and interferon-γ. Real time PCR showed that PGE₂ had no effect on expression of CXCL10, TGF-β1, and IL-11 and exacerbated the rapid up-regulation of mRNAs encoding cyclooxygenase-2, inducible NOS, IL-6, and IL-1β but blunted the production of mRNAs encoding TNF-α, IL-10, CCL3, and CCL4. These effects were mimicked fully by the EP2 agonist butaprost but only weakly by the EP1/EP3 agonist 17-phenyl trinor PGE₂ or the EP4 agonist CAY10598 and not at all by the EP3/EP1 agonist sulprostone and confirmed by protein measurements of cyclooxygenase-2, IL-6, IL-10, and TNF-α. In resting microglia, butaprost induced cAMP formation and altered the mRNA expression of inflammatory mediators, but protein expression was unchanged. The PKA inhibitor H89 had little or no effect on inflammatory mediators modulated by EP2, whereas the Epac activator 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate acetoxymethyl ester mimicked all butaprost effects. These results indicate that EP2 activation plays a complex immune regulatory role during classical activation of microglia and that Epac pathways are prominent in this role.     

Evolutionary Paths of the cAMP-Dependent Protein Kinase (PKA) Catalytic Subunits

3′,5′-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Cα and Cβ, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Cα and Cβ arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Cα/Cβ kinase family is exceptionally high, a small set of signature residues defining Cα and Cβ subfamilies were identified. These conserved residues might be important for functions that are unique to the Cα or Cβ clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods.