Anchored cAMP signaling: Onward and upward – A short history of compartmentalized cAMP signal transduction

Intracellular signal transduction pathways require a high degree of spatial and temporal resolution in order to deliver the appropriate outputs. Specific signaling mediated by the ubiquitous second messenger cAMP and its effector, the cAMP-dependent protein kinase (PKA), is governed by the spatial organization of different pathway components by A-kinase anchoring proteins (AKAPs). This review discusses the history and future of anchored cAMP signaling pathways.     

Epac and PKA: a tale of two intracellular cAMP receptors

cAMP-mediated signaling pathways regulate a multitude of important biological processes under both physiological and pathological conditions, including diabetes, heart failure and cancer. In eukaryotic cells, the effects of cAMP are mediated by two ubiquitously expressed intracellular cAMP receptors, the classic protein kinase A (PKA)/cAMP-dependent protein kinase and the recently discovered exchange protein directly activated by cAMP (Epac)/cAMP-regulated guanine nucleotide exchange factors. Like PKA, Epac contains an evolutionally conserved cAMP binding domain that acts as a molecular switch for sensing intracellular second messenger cAMP levels to control diverse biological functions. The existence of two families of cAMP effectors provides a mechanism for a more precise and integrated control of the cAMP signaling pathways in a spatial and temporal manner. Depending upon the specific cellular environments as well as their relative abundance, distribution and localization, Epac and PKA may act independently, converge synergistically or oppose each other in regulating a specific cellular function.     

PKA compartmentalization links cAMP signaling and autophagy

Autophagy is a highly regulated degradative process crucial for maintaining cell homeostasis. This important catabolic mechanism can be nonspecific, but usually occurs with fine spatial selectivity (compartmentalization), engaging only specific subcellular sites. While the molecular machines driving autophagy are well understood, the involvement of localized signaling events in this process is not well defined. Among the pathways that regulate autophagy, the cyclic AMP (cAMP)/protein kinase A (PKA) cascade can be compartmentalized in distinct functional units called microdomains. However, while it is well established that, depending on the cell type, cAMP can inhibit or promote autophagy, the role of cAMP/PKA microdomains has not been tested. Here we show not only that the effects on autophagy of the same cAMP elevation differ in different cell types, but that they depend on a highly complex sub-compartmentalization of the signaling cascade. We show in addition that, in HT-29 cells, in which autophagy is modulated by cAMP rising treatments, PKA activity is strictly regulated in space and time by phosphatases, which largely prevent the phosphorylation of soluble substrates, while membrane-bound targets are less sensitive to the action of these enzymes. Interestingly, we also found that the subcellular distribution of PKA type-II regulatory PKA subunits hinders the effect of PKA on autophagy, while displacement of type-I regulatory PKA subunits has no effect. Our data demonstrate that local PKA activity can occur independently of local cAMP concentrations and provide strong evidence for a link between localized PKA signaling events and autophagy.Subject terms: Kinases, Autophagy     

Conserved cAMP signaling cascades regulate fungal development and virulence

Two well characterized signal transduction cascades regulating fungal development and virulence are the MAP kinase and cAMP signaling cascades. Here we review the current state of knowledge on cAMP signaling cascades in fungi. While the processes regulated by cAMP signaling in fungi are as diverse as the fungi themselves, the components involved in signal transduction are remarkably conserved. Fungal cAMP signaling cascades are also quite versatile, which is apparent from the differential regulation of similar biological processes. In this review we compare and contrast cAMP signaling pathways that regulate development in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and differentiation and virulence in the human pathogen Cryptococcus neoformans and the plant pathogen Ustilago maydis. We also present examples of interaction between the cAMP and MAP kinase signaling cascades in the regulation of fungal development and virulence.     

Cyclic adenosine monophosphate (cAMP) signaling in melanocyte pigmentation and melanomagenesis

The second messenger cyclic adenosine monophosphate (cAMP) regulates numerous functions in both benign melanocytes and melanoma cells. cAMP is generated from two distinct sources, transmembrane and soluble adenylyl cyclases (tmAC and sAC, respectively), and is degraded by a family of proteins called phosphodiesterases (PDEs). cAMP signaling can be regulated in many different ways and can lead to varied effects in melanocytes. It was recently revealed that distinct cAMP signaling pathways regulate pigmentation by either altering pigment gene expression or the pH of melanosomes. In the context of melanoma, many studies report seemingly contradictory roles for cAMP in tumorigenesis. For example, cAMP signaling has been implicated in both cancer promotion and suppression, as well as both therapy resistance and sensitization. This conundrum in the field may be explained by the fact that cAMP signals in discrete microdomains and each microdomain can mediate differential cellular functions. Here, we review the role of cAMP signaling microdomains in benign melanocyte biology, focusing on pigmentation, and in melanomagenesis.     

Collagen stimulation of platelets induces a rapid spatial response of cAMP and cGMP signaling scaffolds

Intracellular communication is tightly regulated in both space and time. Spatiotemporal control is important to achieve a high level of specificity in both dimensions. For instance, cAMP-dependent kinase (PKA) attains spatial resolution by interacting with distinct members of the family of A-kinase anchoring proteins (AKAPs) that position PKA at specific loci within the cell. To control the cAMP induced signal in time, distinct signal terminators such as phosphodiesterases and phosphatases are often co-localized at the AKAP scaffold. In platelets, high levels of cAMP/cGMP maintain the resting state to allow free circulation. Exposure to collagen, for instance when the vessel is damaged, triggers platelet activation through initiation of the GPVI (glycoprotein VI)/FcRγ-chain forming the onset of a plethora of signaling pathways. Consequently overall intra-platelet cAMP and cGMP levels drop, however detail on how PKA, but also cGMP-dependent protein kinase (PKG) respond in relation to their localized signaling scaffolds is currently missing. To investigate this, we employed a quantitative chemical proteomics approach in activated human platelets enabling the specific enrichment of cAMP/cGMP signaling nodes. Our data reveal that within a few minutes several specific PKA and PKG signaling nodes respond significantly to the activating signal, whereas others do not, suggesting a rapid adaption of specific localized cAMP and cGMP pools to the stimulus. Using protein phosphorylation data gathered we touch upon the potential cross-talk between protein phosphorylation and signaling scaffold function as a general theme in platelet spatiotemporal control.     

Dynamic regulation, desensitization, and cross-talk in discrete subcellular microdomains during beta2-adrenoceptor and prostanoid receptor cAMP signaling

Dynamic and localized actions of cAMP are central to the generation of discrete cellular events in response to a range of G(s)-coupled receptor agonists. In the present study we have employed a cyclic nucleotide-gated channel sensor to report acute changes in cAMP in the restricted cellular microdomains adjacent to two different G(s)-coupled receptor pathways, beta(2)-adrenoceptors and prostanoid receptors that are expressed endogenously in HEK293 cells. We probed by either selective small interference RNA-mediated knockdown or dominant negative overexpression the contribution of key signaling components in the rapid attenuation of the local cAMP signaling and subsequent desensitization of each of these G-protein-coupled receptor signaling pathways immediately following receptor activation. Direct measurements of cAMP changes just beneath the plasma membrane of single HEK293 cells reveal novel insights into key regulatory roles provided by protein kinase A-RII, beta-arrestin2, cAMP phosphodiesterase-4D3, and cAMP phosphodiesterase-4D5. We provide new evidence for distinct modes of cAMP down-regulation in these two G(s)-linked pathways and show that these distinct G-protein-coupled receptor signaling systems are subject to unidirectional, heterologous desensitization that allows for limited cross-talk between distinct, dynamically regulated pools of cAMP.     

cAMP signaling in skeletal muscle adaptation: hypertrophy, metabolism, and regeneration

Among organ systems, skeletal muscle is perhaps the most structurally specialized. The remarkable subcellular architecture of this tissue allows it to empower movement with instructions from motor neurons. Despite this high degree of specialization, skeletal muscle also has intrinsic signaling mechanisms that allow adaptation to long-term changes in demand and regeneration after acute damage. The second messenger adenosine 3',5'-monophosphate (cAMP) not only elicits acute changes within myofibers during exercise but also contributes to myofiber size and metabolic phenotype in the long term. Strikingly, sustained activation of cAMP signaling leads to pronounced hypertrophic responses in skeletal myofibers through largely elusive molecular mechanisms. These pathways can promote hypertrophy and combat atrophy in animal models of disorders including muscular dystrophy, age-related atrophy, denervation injury, disuse atrophy, cancer cachexia, and sepsis. cAMP also participates in muscle development and regeneration mediated by muscle precursor cells; thus, downstream signaling pathways may potentially be harnessed to promote muscle regeneration in patients with acute damage or muscular dystrophy. In this review, we summarize studies implicating cAMP signaling in skeletal muscle adaptation. We also highlight ligands that induce cAMP signaling and downstream effectors that are promising pharmacological targets.     

Phosphodiesterases and subcellular compartmentalized cAMP signaling in the cardiovascular system

Phosphodiesterases are key enzymes in the cAMP signaling cascade. They convert cAMP in its inactive form 5'-AMP and critically regulate the intensity and the duration of cAMP-mediated signals. Multiple isoforms exist that possess different intracellular distributions, different affinities for cAMP, and different catalytic and regulatory properties. This complex repertoire of enzymes provides a multiplicity of ways to modulate cAMP levels, to integrate more signaling pathways, and to respond to the specific needs of the cell within distinct subcellular domains. In this review we summarize key findings on phosphodiesterase compartmentalization in the cardiovascular system.     

cAMP and Ca signaling in secretory epithelia: Crosstalk and synergism

The Ca²⁺ and cAMP/PKA pathways are the primary signaling systems in secretory epithelia that control virtually all secretory gland functions. Interaction and crosstalk in Ca²⁺ and cAMP signaling occur at multiple levels to control and tune the activity of each other. Physiologically, Ca²⁺ and cAMP signaling operate at 5–10% of maximal strength, but synergize to generate the maximal response. Although synergistic action of the Ca²⁺ and cAMP signaling is the common mode of signaling and has been known for many years, we know very little of the molecular mechanism and mediators of the synergism. In this review, we discuss crosstalk between the Ca²⁺ and cAMP signaling and the function of IRBIT (IP₃ receptors binding protein release with IP₃) as a third messenger that mediates the synergistic action of the Ca²⁺ and cAMP signaling.     

Regulation of aldosterone production from zona glomerulosa cells by ANG II and cAMP: evidence for PKA-independent activation of CaMK by cAMP

Aldosterone production in zona glomerulosa (ZG) cells of adrenal glands is regulated by various extracellular stimuli (K(+), ANG II, ACTH) that all converge on two major intracellular signaling pathways: an increase in cAMP production and calcium (Ca(2+)) mobilization. However, molecular events downstream of the increase in intracellular cAMP and Ca(2+) content are controversial and far from being completely resolved. Here, we found that Ca(2+)/calmodulin-dependent protein kinases (CaMKs) play a predominant role in the regulation of aldosterone production stimulated by ANG II, ACTH, and cAMP. The specific CaMK inhibitor KN93 strongly reduced ANG II-, ACTH-, and cAMP-stimulated aldosterone production. In in vitro kinase assays and intact cells, we could show that cAMP-induced activation of CaMK, using the adenylate cyclase activator forskolin or the cAMP-analog Sp-5,6-DCI-cBIMPS (cBIMPS), was not mediated by PKA. Activation of the recently identified cAMP target protein Epac (exchange protein directly activated by cAMP) by 8-pCPT-2'-O-Me-cAMP had no effect on CaMK activity and aldosterone production. Furthermore, we provide evidence that cAMP effects in ZG cells do not involve Ca(2+) or MAPK signaling. Our results suggest that ZG cells, in addition to PKA and Epac/Rap proteins, contain other as yet unidentified cAMP mediator(s) involved in regulating CaMK activity and aldosterone secretion.     

Role of cAMP and phosphodiesterase signaling in liver health and disease

Liver disease is a significant health problem worldwide with mortality reaching around 2 million deaths a year. Non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) are the major causes of chronic liver disease. Pathologically, NAFLD and ALD share similar patterns of hepatic disorders ranging from simple steatosis to steatohepatitis, fibrosis and cirrhosis. It is becoming increasingly important to identify new pharmacological targets, given that there is no FDA-approved therapy yet for either NAFLD or ALD. Since the evolution of liver diseases is a multifactorial process, several mechanisms involving parenchymal and non-parenchymal hepatic cells contribute to the initiation and progression of liver pathologies. Moreover, certain protective molecular pathways become repressed during liver injury including signaling pathways such as the cyclic adenosine monophosphate (cAMP) pathway. cAMP, a key second messenger molecule, regulates various cellular functions including lipid metabolism, inflammation, cell differentiation and injury by affecting gene/protein expression and function. This review addresses the current understanding of the role of cAMP metabolism and consequent cAMP signaling pathway(s) in the context of liver health and disease. The cAMP pathway is extremely sophisticated and complex with specific cellular functions dictated by numerous factors such abundance, localization and degradation by phosphodiesterases (PDEs). Furthermore, because of the distinct yet divergent roles of both of its effector molecules, the cAMP pathway is extensively targeted in liver injury to modify its role from physiological to therapeutic, depending on the hepatic condition. This review also examines the behavior of the cAMP-dependent pathway in NAFLD, ALD and in other liver diseases and focuses on PDE inhibition as an excellent therapeutic target in these conditions.     

Interplay of Ca2+ and cAMP signaling in the insulin-secreting MIN6 beta-cell line

Ca2+ and cAMP are important second messengers that regulate multiple cellular processes. Although previous studies have suggested direct interactions between Ca2+ and cAMP signaling pathways, the underlying mechanisms remain unresolved. In particular, direct evidence for Ca2+-regulated cAMP production in living cells is incomplete. Genetically encoded fluorescence resonance energy transfer-based biosensors have made possible real-time imaging of spatial and temporal gradients of intracellular cAMP concentration in single living cells. Here, we used confocal microscopy, fluorescence resonance energy transfer, and insulin-secreting MIN6 cells expressing Epac1-camps, a biosynthetic unimolecular cAMP indicator, to better understand the role of intracellular Ca2+ in cAMP production. We report that depolarization with high external K+, tolbutamide, or glucose caused a rapid increase in cAMP that was dependent on extracellular Ca2+ and inhibited by nitrendipine, a Ca2+ channel blocker, or 2',5'-dideoxyadenosine, a P-site antagonist of transmembrane adenylate cyclases. Stimulation of MIN6 cells with glucose in the presence of tetraethylammonium chloride generated concomitant Ca2+ and cAMP oscillations that were abolished in the absence of extracellular Ca2+ and blocked by 2',5'-dideoxyadenosine or 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase. Simultaneous measurements of Ca2+ and cAMP concentrations with Fura-2 and Epac1-camps, respectively, revealed a close temporal and causal interrelationship between the increases in cytoplasmic Ca2+ and cAMP levels following membrane depolarization. These findings indicate highly coordinated interplay between Ca2+ and cAMP signaling in electrically excitable endocrine cells and suggest that Ca2+-dependent cAMP oscillations are derived from an increase in adenylate cyclase activity and periodic activation and inactivation of cAMP-hydrolyzing phosphodiesterase.     

cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages

Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways of their mammalian hosts during infection. In this study, cAMP levels associated with Mycobacterium tuberculosis and Mycobacterium bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased c . 50-fold during infection of J774.16 macrophages, relative to the cAMP levels within bacteria incubated in tissue culture media alone. cAMP levels also increased within the macrophage cytoplasm upon uptake of live, but not dead, mycobacteria. The presence of albumin in the absence of oleic acid significantly decreased cAMP secretion and production by both M. tuberculosis and M. bovis BCG. These results suggest that cAMP signaling plays a role in the interaction of tuberculosis-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.     

Protein kinase A-mediated gating of neuregulin-dependent ErbB2-ErbB3 activation underlies the synergistic action of cAMP on Schwann cell proliferation

In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) enhances the action of neuregulin, the most potent known mitogen for SCs, by synergistically increasing the activation of two crucial signaling pathways: ERK and Akt. However, the underlying mechanism of cross-talk between neuregulin and cAMP signaling remains mostly undefined. Here, we report that the activation of protein kinase A (PKA), but not that of exchange protein activated by cAMP (EPAC), enhances S-phase entry of SCs by synergistically enhancing the ligand-dependent tyrosine phosphorylation/activation of the neuregulin co-receptor, ErbB2-ErbB3. The role of PKA in neuregulin-ErbB signaling was confirmed using PKA inhibitors, pathway-selective cAMP analogs, and natural ligands stimulating PKA activity in SCs, such as adenosine and epinephrine. Two basic observations defined the synergistic action of PKA as "gating" for neuregulin-ErbB signaling: 1) the activation of PKA was not sufficient to induce S-phase entry or the activation of either ErbB2 or ErbB3; and 2) the presence of neuregulin was strictly required to ignite ErbB activation and thereby ERK and Akt signaling. However, PKA directly phosphorylated ErbB2 on Thr-686, a highly conserved intracellular regulatory site that was required for the PKA-mediated synergistic enhancement of neuregulin-induced ErbB2-ErbB3 activation and proliferation in SCs. The gating action of PKA on neuregulin-induced ErbB2-ErbB3 activation has important biological significance, because it insures signal amplification into the ERK and Akt pathways without compromising either the neuregulin dependence or the high specificity of ErbB signaling pathways.     

Selective inhibition of histamine-evoked Ca2+ signals by compartmentalized cAMP in human bronchial airway smooth muscle cells

Intracellular Ca²⁺ and cAMP typically cause opposing effects on airway smooth muscle contraction. Receptors that stimulate these pathways are therapeutic targets in asthma and chronic obstructive pulmonary disease. However, the interactions between different G protein-coupled receptors (GPCRs) that evoke cAMP and Ca²⁺ signals in human bronchial airway smooth muscle cells (hBASMCs) are poorly understood. We measured Ca²⁺ signals in cultures of fluo-4-loaded hBASMCs alongside measurements of intracellular cAMP using mass spectrometry or [³H]-adenine labeling. Interactions between the signaling pathways were examined using selective ligands of GPCRs, and inhibitors of Ca²⁺ and cAMP signaling pathways. Histamine stimulated Ca²⁺ release through inositol 1,4,5-trisphosphate (IP₃) receptors in hBASMCs. β₂-adrenoceptors, through cAMP and protein kinase A (PKA), substantially inhibited histamine-evoked Ca²⁺ signals. Responses to other Ca²⁺-mobilizing stimuli were unaffected by cAMP (carbachol and bradykinin) or minimally affected (lysophosphatidic acid). Prostaglandin E₂ (PGE₂), through EP₂ and EP₄ receptors, stimulated formation of cAMP and inhibited histamine-evoked Ca²⁺ signals. There was no consistent relationship between the inhibition of Ca²⁺ signals and the amounts of intracellular cAMP produced by different stimuli. We conclude that β-adrenoceptors, EP₂ and EP₄ receptors, through cAMP and PKA, selectively inhibit Ca²⁺ signals evoked by histamine in hBASMCs, suggesting that PKA inhibits an early step in H₁ receptor signaling. Local delivery of cAMP within hyperactive signaling junctions mediates the inhibition.     

Regulation of cAMP responses by the G12/13 pathway converges on adenylyl cyclase VII

Regulation of intracellular cAMP by multiple pathways enables differential function of this ubiquitous second messenger in a context-dependent manner. Modulation of G(s)-stimulated intracellular cAMP has long been known to be modulated by the G(i) and G(q)/Ca(2+) pathways. Recently, the G(13) pathway was also shown to facilitate cAMP responses in murine macrophage cells. We report here that this synergistic regulation of cAMP synthesis by the G(s) and G(13) pathways is mediated by a specific isoform of adenylyl cyclase, AC7. Furthermore, this signaling paradigm exists in several hematopoietic lineages and can be recapitulated by exogenous expression of AC7 in HEK 293 cells. Mechanistic characterization of this synergistic interaction indicates that it occurs downstream of receptor activation and it can be mediated by the alpha subunit of either G(12) or G(13). Our results demonstrate that AC7 is a specific downstream effector of the G(12/13) pathway.     

cAMP binding protein assay for widespread use in cell signaling studies

cAMP plays a critical role in intracellular signaling pathways that regulate proliferation or differentiation. The cAMP binding protein assay, using a naturally derived cAMP binding protein, is one of the most widely used methods for cAMP determination. The major steps of this binding assay include purification of the binding protein, cAMP extraction from samples, and quantification of the cAMP Most purification methods of the cAMP binding protein were published before 1975, and many of the materials and methods are outdated. Here we describe an updated method of purification of cAMP binding protein from bovine skeletal muscle with the advantages of simplicity, low cost, and high yield The isolation procedures can be completed in two days using commercially available materials and equipment. The cAMP binding properties of the isolated protein can be utilizedfor more than two years. Binding protein isolatedfrom 1 kg bovine muscle is sufficientfor at least 3 x10(4) assay tubes. Furthemore, we describe the techniques of cAMP extraction and quantification that have been used successfully in studying parathyroid hormone signaling as an example of a G protein-linked seven transmembrane domain receptor that signals through the protein kinase A pathway.