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Protein kinases are dynamic molecular switches that sample multiple conformational states. The regulatory subunit of PKA harbors two cAMP-binding domains [cyclic nucleotide-binding (CNB) domains] that oscillate between inactive and active conformations dependent on cAMP binding. The cooperative binding of cAMP to the CNB domains activates an allosteric interaction network that enables PKA to progress from the inactive to active conformation, unleashing the activity of the catalytic subunit. Despite its importance in the regulation of many biological processes, the molecular mechanism responsible for the observed cooperativity during the activation of PKA remains unclear. Here, we use optical tweezers to probe the folding cooperativity and energetics of domain communication between the cAMP-binding domains in the apo state and bound to the catalytic subunit. Our study provides direct evidence of a switch in the folding-energy landscape of the two CNB domains from energetically independent in the apo state to highly cooperative and energetically coupled in the presence of the catalytic subunit. Moreover, we show that destabilizing mutational effects in one CNB domain efficiently propagate to the other and decrease the folding cooperativity between them. Taken together, our results provide a thermodynamic foundation for the conformational plasticity that enables protein kinases to adapt and respond to signaling molecules.

Cyanobacteria are phototrophic prokaryotes that evolved oxygenic photosynthesis ∼2.7 billion y ago and are presently responsible for ∼10% of total global photosynthetic production. To cope with the evolutionary pressure of dropping ambient CO2 concentrations, they evolved a CO2-concentrating mechanism (CCM) to augment intracellular inorganic carbon (Ci) levels for efficient CO2 fixation. However, how cyanobacteria sense the fluctuation in Ci is poorly understood. Here we present biochemical, structural, and physiological insights into SbtB, a unique PII-like signaling protein, which provides new insights into Ci sensing. SbtB is highly conserved in cyanobacteria and is coexpressed with CCM genes. The SbtB protein from the cyanobacterium Synechocystis sp. PCC 6803 bound a variety of adenosine nucleotides, including the second messenger cAMP. Cocrystal structures unraveled the individual binding modes of trimeric SbtB with AMP and cAMP. The nucleotide-binding pocket is located between the subunit clefts of SbtB, perfectly matching the structure of canonical PII proteins. This clearly indicates that proteins of the PII superfamily arose from a common ancestor, whose structurally conserved nucleotide-binding pocket has evolved to sense different adenyl nucleotides for various signaling functions. Moreover, we provide physiological and biochemical evidence for the involvement of SbtB in Ci acclimation. Collectively, our results suggest that SbtB acts as a Ci sensor protein via cAMP binding, highlighting an evolutionarily conserved role for cAMP in signaling the cellular carbon status.

Epac1 is a cAMP-regulated guanine nucleotide exchange factor for the small G protein Rap. Upon cAMP binding, Epac1 undergoes a conformational change that results in its release from autoinhibition. In addition, cAMP induces the translocation of Epac1 from the cytosol to the plasma membrane. This relocalization of Epac1 is required for efficient activation of plasma membrane-located Rap and for cAMP-induced cell adhesion. This translocation requires the Dishevelled, Egl-10, Pleckstrin (DEP) domain, but the molecular entity that serves as the plasma membrane anchor and the possible mechanism of regulated binding remains elusive. Here we show that Epac1 binds directly to phosphatidic acid. Similar to the cAMP-induced Epac1 translocation, this binding is regulated by cAMP and requires the DEP domain. Furthermore, depletion of phosphatidic acid by inhibition of phospholipase D1 prevents cAMP-induced translocation of Epac1 as well as the subsequent activation of Rap at the plasma membrane. Finally, mutation of a single basic residue within a polybasic stretch of the DEP domain, which abolishes translocation, also prevents binding to phosphatidic acid. From these results we conclude that cAMP induces a conformational change in Epac1 that enables DEP domain-mediated binding to phosphatidic acid, resulting in the tethering of Epac1 at the plasma membrane and subsequent activation of Rap.

Voltage gating of hyperpolarization-activated cation (HCN) channels is potentiated by direct binding of cAMP to a cytoplasmic cAMP-sensing domain (CSD). When unliganded, the CSD inhibits hyperpolarization-dependent opening of the HCN channel gate; cAMP binding relieves this autoinhibition so that opening becomes more favorable thermodynamically. This autoinhibition-relief mechanism is conserved with that of several other cyclic nucleotide receptors using the same ligand-binding fold. Besides its thermodynamic effect, cAMP also modulates the depolarization-dependent deactivation rate by kinetically trapping channels in an open state. Here we report studies of strong open-state trapping in an HCN channel showing that the well-established autoinhibition-relief model is insufficient. Whereas deletion of the CSD mimics the thermodynamic open-state stabilization usually associated with cAMP binding, CSD deletion removes rather than mimics the kinetic effect of strong open-state trapping. Substitution of different CSD sequences leads to variation of the degree of open-state trapping in the liganded channel but not in the unliganded channel. CSD-dependent open-state trapping is observed during a voltage-dependent deactivation pathway, specific to the secondary open state that is formed by mode shift after prolonged hyperpolarization activation. This hysteretic activation–deactivation cycle is preserved by CSD substitution, but the change in deactivation kinetics of the liganded channel resulting from CSD substitution is not correlated with the change in autoinhibition properties. Thus the liganded and the unliganded forms of the CSD respectively provide the structural determinants for open-state trapping and autoinhibition, such that two distinct mechanisms for cAMP regulation can operate in one receptor.

In the heart, cAMP is a key regulator of excitation–contraction coupling and its biological effects are mainly associated with the activity of protein kinase A (PKA). The aim of this study was to investigate the contribution of the cAMP-binding protein Epac (Exchange protein directly activated by cAMP) in the regulation of the contractile properties of rat ventricular cardiac myocytes. We report that both PKA and Epac increased cardiac sarcomere contraction but through opposite mechanisms. Differently from PKA, selective Epac activation by the cAMP analog 8-(4-chlorophenylthio)-2′- O -methyl-cAMP (8-pCPT) reduced Ca 2+ transient amplitude and increased cell shortening in intact cardiomyocytes and myofilament Ca 2+ sensitivity in permeabilized cardiomyocytes. Moreover, ventricular myocytes, which were infected in vivo with a constitutively active form of Epac, showed enhanced myofilament Ca 2+ sensitivity compared to control cells infected with green fluorescent protein (GFP) alone. At the molecular level, Epac increased phosphorylation of 2 key sarcomeric proteins, cardiac Troponin I (cTnI) and cardiac Myosin Binding Protein-C (cMyBP-C). The effects of Epac activation on myofilament Ca 2+ sensitivity and on cTnI and cMyBP-C phosphorylation were independent of PKA and were blocked by protein kinase C (PKC) and Ca 2+ calmodulin kinase II (CaMKII) inhibitors. Altogether these findings identify Epac as a new regulator of myofilament function.

The cAMP-mediated allosteric transition in the catabolite activator protein (CAP; also known as the cAMP receptor protein, CRP) is a textbook example of modulation of DNA-binding activity by small-molecule binding. Here we report the structure of CAP in the absence of cAMP, which, together with structures of CAP in the presence of cAMP, defines atomic details of the cAMP-mediated allosteric transition. The structural changes, and their relationship to cAMP binding and DNA binding, are remarkably clear and simple. Binding of cAMP results in a coil-to-helix transition that extends the coiled-coil dimerization interface of CAP by 3 turns of helix and concomitantly causes rotation, by approximately 60 degrees , and translation, by approximately 7 A, of the DNA-binding domains (DBDs) of CAP, positioning the recognition helices in the DBDs in the correct orientation to interact with DNA. The allosteric transition is stabilized further by expulsion of an aromatic residue from the cAMP-binding pocket upon cAMP binding. The results define the structural mechanisms that underlie allosteric control of this prototypic transcriptional regulatory factor and provide an illustrative example of how effector-mediated structural changes can control the activity of regulatory proteins.

cAMP and the cAMP binding domain (CBD) constitute a ubiquitous regulatory switch that translates an extracellular signal into a biological response. The CBD contains α- and β-subdomains with cAMP binding to a phosphate binding cassette (PBC) in the β-sandwich. The major receptors for cAMP in mammalian cells are the regulatory subunits (R-subunits) of PKA where cAMP and the catalytic subunit compete for the same CBD. The R-subunits inhibit kinase activity, whereas cAMP releases that inhibition. Here, we use NMR to map at residue resolution the cAMP-dependent interaction network of the CBD-A domain of isoform Iα of the R-subunit of PKA. Based on H/D, H/H, and N z exchange data, we propose a molecular model for the allosteric regulation of PKA by cAMP. According to our model, cAMP binding causes long-range perturbations that propagate well beyond the immediate surroundings of the PBC and involve two key relay sites located at the C terminus of β 2 (I163) and N terminus of β 3 (D170). The I163 site functions as one of the key triggers of global unfolding, whereas the D170 locus acts as an electrostatic switch that mediates the communication between the PBC and the B-helix. Removal of cAMP not only disrupts the cap for the B′ helix within the PBC, but also breaks the circuitry of cooperative interactions stemming from the PBC, thereby uncoupling the α- and β-subdomains. The proposed model defines a signaling mechanism, conserved in every genome, where allosteric binding of a small ligand disrupts a large protein–protein interface.

cAMP-binding domains from several different proteins were analyzed to determine the properties and interactions of this recognition motif. Systematic computational analyses, including structure-based sequence comparison, surface matching, affinity grid analysis, and analyses of the ligand protein interactions were carried out. These analyses show distinctive roles of the sugar phosphate and the adenine in the cAMP-binding module. We propose that the cAMP-binding regulatory proteins function by providing an allosteric system in which the presence or absence of cAMP produces a substantial structural change through the loss of hydrophobic interactions with the adenine ring and consequent repositioning of the C helix. The modified positioning of the helix in turn is recognized by a protein-binding event, completing the allostery.

Second messenger cAMP regulates many cellular functions through its effectors, such as cAMP-dependent protein kinase (PKA) and Epac (exchange proteins directly activated by cAMP). Spatial and temporal control of cAMP signaling is crucial to differential regulation of cellular targets involved in various signaling cascades. To investigate the compartmentalized cAMP signaling, we constructed fluorescent indicators that report intracellular cAMP dynamics and Epac activation by sandwiching the full-length Epac1 between cyan and yellow mutants of GFP. Elevations of cAMP decreased FRET and increased the ratio of cyan-to-yellow emissions by 10–30% in living mammalian cells. This response can be reversed by removing cAMP-elevating agents and abolished by mutating the critical residue responsible for cAMP binding. Targeting of the reporter to the plasma membrane, where cAMP is produced in response to the activation of β-adrenergic receptor, revealed a faster cAMP response at the membrane than in the cytoplasm and mitochondria. Simultaneous imaging with targeted cAMP indicator and PKA activity reporter allowed the detection of a much delayed PKA response in the nucleus after the rapid accumulation of cAMP at the plasma membrane of the same cell, despite the immediate presence of a pool of cAMP in the nucleus. Thus, cAMP dynamics and the activation of its effectors are precisely controlled spatiotemporally in vivo .

cAMP, through the activation of cAMP-dependent protein kinase (PKA), is involved in transcriptional regulation. In eukaryotic cells, cAMP is not considered to alter the binding affinity of CREB/ATF to cAMP-responsive element (CRE) but to induce serine phosphorylation and consequent increase in transcriptional activity. In contrast, in prokaryotic cells, cAMP enhances the DNA binding of the catabolite repressor protein to regulate the transcription of several operons. The structural similarity of the cAMP binding sites in catabolite repressor protein and regulatory subunit of PKA type II (RII) suggested the possibility of a similar role for RII in eukaryotic gene regulation. Herein we report that RIIβ subunit of PKA is a transcription factor capable of interacting physically and functionally with a CRE. In contrast to CREB/ATF, the binding of RIIβ to a CRE was enhanced by cAMP, and in addition, RIIβ exhibited transcriptional activity as a Gal4-RIIβ fusion protein. These experiments identify RIIβ as a component of an alternative pathway for regulation of CRE-directed transcription in eukaryotic cells.

In our recent paper, we conclude that Dictyostelium Ric8 is as a nonreceptor guanine nucleotide exchange factor (GEF) for the Gα protein important for amplification of G protein–coupled receptor (GPCR) signaling (1). In their letter, Tall et al. dispute this conclusion and suggest that a function of Dictyostelium Ric8 in folding of the Gα protein might better explain the phenotype than its function as GEF (2). However, we show that WT and ric8-null cells have similar steady-state expression levels of Gα. To test the functionality of the Gα2 protein, we analyzed the interaction between Gα2 and the cAR1 receptor. In vitro, this was measured as the inhibition of cAMP binding induced by full activation of Gα2 with nonhydrolysable GTPγS. The reduction in cAMP binding in ric8 null is similar to that in WT, indicating that a similar amount of Gα is functionally coupled to the receptor. Second, dissociation of Gα2βγ was monitored in vivo by measuring the cAMP-induced FRET change between the FRET pair Gα2-Cerulean and Gβ-Venus. The FRET signal before stimulation is similar in ric8-null cells compared with WT cells, indicating the presence of similar amounts of Gα2βγ complex. Furthermore, we show that Ric8 is not essential for the initial dissociation of heterotrimeric G proteins and subsequent activation of Ras by uniform chemoattractant. Consistent with its function as GEF, recombinant Ric8 stimulates nucleotide exchange of Gα2 in vitro, and the in vivo FRET assays showed that prolonged dissociation of Gα2βγ on uniform cAMP stimulation requires Ric8. Together, we conclude in the paper that these results provide no evidence that Ric8 could play a role as chaperone for Gα2 folding but clearly show that Ric8 functions as a GEF and amplifier of Gα2 signaling.

Historical perspectives and the underlying mechanisms of allostery have been recently reviewed (1–4), increasingly highlighting conformation selection and population shift (5). A classical example is transcription factor (TF) catabolite activator protein (CAP) activation by cAMP in response to extracellular stimuli (6). cAMP–CAP–DNA complex was the first TF with 3D structure (7), and it was postulated that cAMP binding causes structural rearrangements allowing CAP to bind DNA (6, 8). Twenty-eight years later, NMR structures have confirmed the allosteric transition paradigm; as reported in this issue of PNAS, Popovych et al. (9) found that without cAMP binding, the CAP apo form loses 3 helical turns and the DNA binding domain rotates, shifting from its DNA binding-ready state. Consistent with theoretical expectation, some mutations shifted the ensemble toward a DNA-compatible active state even without cAMP binding (9). Coiled-coil order-disorder transition coupled with cAMP binding permits efficient allo steric control; yet, it is not necessarily used in other structurally similar cAMP binding domains (10). Protein kinase A undergoes a kink transition sufficient to break the inactive protein kinase A (PKA) tetramer complex on cAMP binding (11). In the CAP/fumarate and nitrate reduction regulator (FNR) superfamily, the CooA (effector: CO) does not involve an order–disorder transition but dissimilative nitrate respiration regulator (DNR) (effector: NO) does (9).

We have isolated and sequenced cDNA clones representing portions of the polyadenylylated transcripts of the dunce+ gene. These define an open reading frame of 1086 bases and some of the 5'- and 3'-untranslated regions of the transcripts. The deduced amino acid sequence is strikingly homologous to the amino acid sequence of a Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase isolated from bovine brain and more weakly related to the predicted amino acid sequence of a yeast cAMP phosphodiesterase. These homologies, together with prior genetic and biochemical studies, provide unambiguous evidence that dunce+ codes for a phosphodiesterase. In addition, the dunce+ gene product shares a seven-amino acid sequence with a regulatory subunit of cAMP-dependent protein kinase that is predicted to be part of the cAMP binding site. We also identify a weak homology between a region of the dunce+ gene product and the egg-laying hormone precursor of Aplysia californica. The open reading frame is divided in the genome by four introns.

An isoform (RI beta) of the regulatory type I subunit gene of cAMP-dependent protein kinase (EC 2.7.1.37) has been characterized in mouse. The open reading frame of the RI beta cDNA is 72% identical in nucleotide sequence with the previously cloned RI gene, now referred to as RI alpha. Both genes code for a protein of 380 amino acids and their proteins are 82% identical in amino acid sequence. Sequence similarity is highest in the regions that form the pseudosubstrate-binding site of the catalytic subunit and the two cAMP binding domains. The amino-terminal portion shows the greatest dissimilarity, suggesting that the isoforms may differ in their dimerization properties or interaction with other proteins. In contrast to RI alpha, which is constitutively expressed in all tissues, RI beta is expressed in a highly tissue-specific manner. Brain and spinal cord contained significant levels of RI beta mRNA, testis RNA gave a detectable signal, and all other tissues tested were negative. Expression of a RI beta cDNA in NIH 3T3 cells resulted in the appearance of a RI subunit protein that migrated more slowly than RI alpha after NaDodSO4/PAGE. The native form of RI beta in brain could also be distinguished from RI alpha by its abnormal migration on NaDodSO4/PAGE. RI beta protein produced in 3T3 cells was shown to be functional by its ability to form a cAMP-dependent holoenzyme with the catalytic subunit.

Extracellular cAMP plays a crucial role in regulating the developmental program of Dictyostelium discoideum, functioning as a chemotactic agent, as well as a signal that regulates expression of developmentally expressed genes. These activities appear to be mediated by a cell-surface receptor for cAMP. We have studied the regulation of this receptor in cells developed in starved suspension cultures exposed to 50 nM pulses of cAMP every 6 min. cAMP-pulsed cells display roughly 10-fold higher cAMP receptor levels than cells that developed on filters or that were starved in suspension without cAMP pulses. Based on saturation binding analysis, the superinduced binding activity represents an increase in receptor number, while receptor affinity for cAMP is unaffected. Photoaffinity labeling of superinduced cells results in specific labeling of the same molecules that are labeled in starved cells. This increased cAMP binding activity was also detected in membrane preparations from cAMP-pulsed cells. These results provide evidence for an unusual mode of receptor regulation: autogenous induction of the receptor by its ligand.

Discrepancies between adenosine 3′:5′-cyclic monophosphate (cAMP) and steroid production have been frequently observed in isolated target cells stimulated by low concentrations of trophic hormone. This dissociation is particularly marked in the interstitial cells of the testis, where testosterone production is elicited by gonadotropin concentrations in the picomolar range. Because of these observations, and a disparity between steroidogenesis and protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activation in Leydig cells, the role of cAMP as a mediator of the acute steroidogenic response has been questioned. This problem has been further analyzed by assay of free and occupied cAMP-binding sites of the regulatory subunit of protein kinase in basal and hormone-stimulated cells. Free sites were measured by a [ 3 H]-cAMP-binding assay, and occupied sites were measured by radioimmunoassay of endogenous cAMP eluted from receptor protein. After stimulation of purified Leydig cells with 0.1-10 pM human chorionic gonadotropin, a dose-dependent decrease in available [ 3 H]cAMP-binding sites was observed, with no change in binding affinity. The reduction in cAMP-binding sites was equivalent to the increase in occupancy of cAMP receptors by endogenous nucleotide formed during gonadotropin action. Fractional occupancy of cAMP receptors rose progressively from basal values of 0.2-0.40 to full saturation as intracellular cAMP rose 10- to 30-fold during hormone stimulation. The testosterone dose-response curve was coincident with the initial part of the cAMP-receptor occupancy curve. These changes in endogenous cAMP binding to the regulatory subunit were accompanied by a significant increase in protein kinase activity in gonadotropin-stimulated Leydig cells. These observations provide direct evidence for the role of cAMP and protein kinase during hormonal activation of steroidogenesis in the Leydig cell by low concentrations of gonadotropin.