miR-34c-3p Regulates Protein Kinase A Activity Independent of cAMP by Dicing prkar2b Transcripts in Theileria annulata-Infected Leukocytes.

MicroRNAs (miRNAs) are small noncoding RNAs that can play critical roles in regulating various cellular processes, including during many parasitic infections. Here, we report a regulatory role for miR-34c-3p in cAMP-independent regulation of host cell protein kinase A (PKA) activity in Theileria annulata-infected bovine leukocytes. We identified prkar2b (cAMP-dependent protein kinase A type II-beta regulatory subunit) as a novel miR-34c-3p target gene and demonstrate how infection-induced upregulation of miR-34c-3p repressed PRKAR2B expression to increase PKA activity. As a result, the disseminating tumorlike phenotype of T. annulata-transformed macrophages is enhanced. Finally, we extend our observations to Plasmodium falciparum-parasitized red blood cells, where infection-induced augmentation in miR-34c-3p levels led to a drop in the amount of prkar2b mRNA and increased PKA activity. Collectively, our findings represent a novel cAMP-independent way of regulating host cell PKA activity in infections by Theileria and Plasmodium parasites. IMPORTANCE Small microRNA levels are altered in many diseases, including those caused by parasites. Here, we describe how infection by two important animal and human parasites, Theileria annulata and Plasmodium falciparum, induce changes in infected host cell miR-34c-3p levels to regulate host cell PKA kinase activity by targeting mammalian prkar2b. Infection-induced changes in miR-34c-3p levels provide a novel epigenetic mechanism for regulating host cell PKA activity independent of fluxes in cAMP to both aggravate tumor dissemination and improve parasite fitness.

RIIß-PKA in GABAergic Neurons of Dorsal Median Hypothalamus Governs White Adipose Browning.

The RIIß subunit of  cAMP-dependent protein kinase A (PKA) is expressed in the brain and adipose tissue. RIIß-knockout mice show leanness and increased UCP1 in brown adipose tissue. The authors have previously reported that RIIß reexpression in hypothalamic GABAergic neurons rescues the leanness. However, whether white adipose tissue (WAT) browning contributes to the leanness and whether RIIß-PKA in these neurons governs WAT browning are unknown. Here, this work reports that RIIß-KO mice exhibit a robust WAT browning. RIIß reexpression in dorsal median hypothalamic GABAergic neurons (DMH GABAergic neurons) abrogates WAT browning. Single-cell sequencing, transcriptome sequencing, and electrophysiological studies show increased GABAergic activity in DMH GABAergic neurons of RIIß-KO mice. Activation of DMH GABAergic neurons or inhibition of PKA in these neurons elicits WAT browning and thus lowers body weight. These findings reveal that RIIß-PKA in DMH GABAergic neurons regulates WAT browning. Targeting RIIß-PKA in DMH GABAergic neurons may offer a clinically useful way to promote WAT browning for treating obesity and other metabolic disorders.

PKA-RIIß autophosphorylation modulates PKA activity and seizure phenotypes in mice.

Temporal lobe epilepsy (TLE) is one of the most common and intractable neurological disorders in adults. Dysfunctional PKA signaling is causally linked to the TLE. However, the mechanism underlying PKA involves in epileptogenesis is still poorly understood. In the present study, we found the autophosphorylation level at serine 114 site (serine 112 site in mice) of PKA-RIIß subunit was robustly decreased in the epileptic foci obtained from both surgical specimens of TLE patients and seizure model mice. The p-RIIß level was negatively correlated with the activities of PKA. Notably, by using a P-site mutant that cannot be autophosphorylated and thus results in the released catalytic subunit to exert persistent phosphorylation, an increase in PKA activities through transduction with AAV-RIIß-S112A in hippocampal DG granule cells decreased mIPSC frequency but not mEPSC, enhanced neuronal intrinsic excitability and seizure susceptibility. In contrast, a reduction of PKA activities by RIIß knockout led to an increased mIPSC frequency, a reduction in neuronal excitability, and mice less prone to experimental seizure onset. Collectively, our data demonstrated that the autophosphorylation of RIIß subunit plays a critical role in controlling neuronal and network excitabilities by regulating the activities of PKA, providing a potential therapeutic target for TLE.

Structural analyses of the PKA RIIß holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma.

When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIß, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIß:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIß2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIß holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIß holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.

PRKAR2B-HIF-1α loop promotes aerobic glycolysis and tumour growth in prostate cancer.

OBJECTIVES: Reprogramming of cellular metabolism is profoundly implicated in tumorigenesis and can be exploited to cancer treatment. Cancer cells are known for their propensity to use glucose-dependent glycolytic pathway instead of mitochondrial oxidative phosphorylation for energy generation even in the presence of oxygen, a phenomenon known as Warburg effect. The type II beta regulatory subunit of protein kinase A (PKA), PRKAR2B, is highly expressed in castration-resistant prostate cancer (CRPC) and contributes to tumour growth and metastasis. However, whether PRKAR2B regulates glucose metabolism in prostate cancer remains largely unknown. MATERIALS AND METHODS: Loss-of-function and gain-of-function studies were used to investigate the regulatory role of PRKAR2B in aerobic glycolysis. Real-time qPCR, Western blotting, luciferase reporter assay and chromatin immunoprecipitation were employed to determine the underlying mechanisms. RESULTS: PRKAR2B was sufficient to enhance the Warburg effect as demonstrated by glucose consumption, lactate production and extracellular acidification rate. Mechanistically, loss-of-function and gain-of-function studies showed that PRKAR2B was critically involved in the tumour growth of prostate cancer. PRKAR2B was able to increase the expression level of hypoxia-inducible factor 1α (HIF-1α), which is a key mediator of the Warburg effect. Moreover, we uncovered that HIF-1α is a key transcription factor responsible for inducing PRKAR2B expression in prostate cancer. Importantly, inhibition of glycolysis by the glycolytic inhibitor 2-deoxy-d-glucose (2-DG) or replacement of glucose in the culture medium with galactose (which has a much lower rate than glucose entry into glycolysis) largely compromised PRKAR2B-mediated tumour-promoting effect. Similar phenomenon was noticed by genetic silencing of HIF-1α. CONCLUSIONS: Our study identified that PRKAR2B-HIF-1α loop enhances the Warburg effect to enable growth advantage in prostate cancer.

Hypoxia and the hypoxia inducible factor 1α activate protein kinase A by repressing RII beta subunit transcription.

Overactivation of the cAMP signal transduction pathway plays a central role in the pathogenesis of endocrine tumors. Genetic aberrations leading to increased intracellular cAMP or directly affecting PKA subunit expression have been identified in inherited and sporadic endocrine tumors, but are rare indicating the presence of nongenomic pathological PKA activation. In the present study, we examined the impact of hypoxia on PKA activation using human growth hormone (GH)-secreting pituitary tumors as a model of an endocrine disease displaying PKA-CREB overactivation. We show that hypoxia activates PKA and enhances CREB transcriptional activity and subsequently GH oversecretion. This is due to a previously uncharacterized ability of HIF-1α to suppress the transcription of the PKA regulatory subunit 2B (PRKAR2B) by sequestering Sp1 from the PRKAR2B promoter. The present study reveals a novel mechanism through which the transcription factor HIF-1α transduces environmental signals directly onto PKA activity, without affecting intracellular cAMP concentrations. By identifying a point of interaction between the cellular microenvironment and intracellular enzyme activation, neoplastic, and nonneoplastic diseases involving overactivated PKA pathway may be more efficiently targeted.

Transcriptional regulation of PRKAR2B by miR-200b-3p/200c-3p and XBP1 in human prostate cancer.

The cyclic adenosine monophosphate (cAMP)-activated protein kinase A (PKA) pathway is profoundly implicated in Prostate cancer (PCa) progression. Previously, we showed that PRKAR2B, the type II-beta regulatory subunit of PKA, is highly expressed in castration-resistant prostate cancer (CRPC) and can induce epithelial-mesenchymal transition by activating Wnt/ß-catenin signaling in PCa cells. However, the molecular mechanism of dysregulated PRKAR2B expression pattern is still largely unknown. In this study, we found that the mutation, copy number alteration, and methylation status of PRKAR2B gene have no correlation with its expression level in PCa. Then, we identified two microRNAs (miR-200b-3p and miR-200c-3p) to be critical regulators of PRKAR2B expression in PCa. Notably, miR-200b-3p and miR-200c-3p expression were significantly downregulated in metastatic CRPC and negatively correlated with the expression level of PRKAR2B in PCa tissues. Moreover, we characterized X-Box Binding Protein 1 (XBP1) as a key transcription factor responsible for PRKAR2B expression in PCa. Importantly, miR-200b-3p/200c-3p or XBP1 knockdown inhibited PCa cell proliferation and promoted cell apoptosis and these inhibitory roles could be largely restored by PRKAR2B, suggesting that PRKAR2B is a functional mediator of miR-200b-3p, miR-200c-3p, and XBP1 in PCa. Collectively, our study firstly identified miR-200b-3p/200c-3p and XBP1 as the critical upstream regulators of PRKAR2B in PCa and provided novel insights to PRKAR2B-driven PCa progression.

Two PKA RIα holoenzyme states define ATP as an isoform-specific orthosteric inhibitor that competes with the allosteric activator, cAMP.

Protein kinase A (PKA) holoenzyme, comprised of a cAMP-binding regulatory (R)-subunit dimer and 2 catalytic (C)-subunits, is the master switch for cAMP-mediated signaling. Of the 4 R-subunits (RIα, RIß, RIIα, RIIß), RIα is most essential for regulating PKA activity in cells. Our 2 RIα2C2 holoenzyme states, which show different conformations with and without ATP, reveal how ATP/Mg2+ functions as a negative orthosteric modulator. Biochemical studies demonstrate how the removal of ATP primes the holoenzyme for cAMP-mediated activation. The opposing competition between ATP/cAMP is unique to RIα. In RIIß, ATP serves as a substrate and facilitates cAMP-activation. The isoform-specific RI-holoenzyme dimer interface mediated by N3A-N3A' motifs defines multidomain cross-talk and an allosteric network that creates competing roles for ATP and cAMP. Comparisons to the RIIß holoenzyme demonstrate isoform-specific holoenzyme interfaces and highlights distinct allosteric mechanisms for activation in addition to the structural diversity of the isoforms.

Epigenetic regulation of diabetogenic adipose morphology.

OBJECTIVE: Hypertrophic white adipose tissue (WAT) morphology is associated with insulin resistance and type 2 diabetes. The mechanisms governing hyperplastic versus hypertrophic WAT expansion are poorly understood. We assessed if epigenetic modifications in adipocytes are associated with hypertrophic adipose morphology. A subset of genes with differentially methylated CpG-sites (DMS) in the promoters was taken forward for functional evaluation. METHODS: The study included 126 women who underwent abdominal subcutaneous biopsy to determine adipose morphology. Global transcriptome profiling was performed on WAT from 113 of the women, and CpG methylome profiling on isolated adipocytes from 78 women. Small interfering RNAs (siRNA) knockdown in human mesenchymal stem cells (hMSCs) was used to assess influence of specific genes on lipid storage. RESULTS: A higher proportion of CpG-sites were methylated in hypertrophic compared to hyperplastic WAT. Methylation at 35,138 CpG-sites was found to correlate to adipose morphology. 2,102 of these CpG-sites were also differentially methylated in T2D; 98% showed directionally consistent change in methylation in WAT hypertrophy and T2D. We identified 2,508 DMS in 638 adipose morphology-associated genes where methylation correlated with gene expression. These genes were over-represented in gene sets relevant to WAT hypertrophy, such as insulin resistance, lipolysis, extracellular matrix organization, and innate immunity. siRNA knockdown of ADH1B, AZGP1, C14orf180, GYG2, HADH, PRKAR2B, PFKFB3, and AQP7 influenced lipid storage and metabolism. CONCLUSION: CpG methylation could be influential in determining adipose morphology and thereby constitute a novel antidiabetic target. We identified C14orf180 as a novel regulator of adipocyte lipid storage and possibly differentiation.

A Genome-Wide Association Study for Susceptibility to Visual Experience-Induced Myopia.

Purpose: The rapid rise in prevalence over recent decades and high heritability of myopia suggest a role for gene-environment (G × E) interactions in myopia susceptibility. Few such G × E interactions have been discovered to date. We aimed to test the hypothesis that genetic analysis of susceptibility to visual experience-induced myopia in an animal model would identify novel G × E interaction loci. Methods: Chicks aged 7 days (n = 987) were monocularly deprived of form vision for 4 days. A genome-wide association study (GWAS) was carried out in the 20% of chicks most susceptible and least susceptible to form deprivation (n = 380). There were 304,963 genetic markers tested for association with the degree of induced axial elongation in treated versus control eyes (A-scan ultrasonography). A GWAS candidate region was examined in the following three human cohorts: CREAM consortium (n = 44,192), UK Biobank (n = 95,505), and Avon Longitudinal Study of Parents and Children (ALSPAC; n = 4989). Results: A locus encompassing the genes PIK3CG and PRKAR2B was genome-wide significantly associated with myopia susceptibility in chicks (lead variant rs317386235, P = 9.54e-08). In CREAM and UK Biobank GWAS datasets, PIK3CG and PRKAR2B were enriched for strongly-associated markers (meta-analysis lead variant rs117909394, P = 1.7e-07). In ALSPAC participants, rs117909394 had an age-dependent association with refractive error (-0.22 diopters [D] change over 8 years, P = 5.2e-04) and nearby variant rs17153745 showed evidence of a G × E interaction with time spent reading (effect size -0.23 D, P = 0.022). Conclusions: This work identified the PIK3CG-PRKAR2B locus as a mediator of susceptibility to visually induced myopia in chicks and suggests a role for this locus in conferring susceptibility to myopia in human cohorts.

FOXG1 Regulates PRKAR2B Transcriptionally and Posttranscriptionally via miR200 in the Adult Hippocampus.

Rett syndrome is a complex neurodevelopmental disorder that is mainly caused by mutations in MECP2. However, mutations in FOXG1 cause a less frequent form of atypical Rett syndrome, called FOXG1 syndrome. FOXG1 is a key transcription factor crucial for forebrain development, where it maintains the balance between progenitor proliferation and neuronal differentiation. Using genome-wide small RNA sequencing and quantitative proteomics, we identified that FOXG1 affects the biogenesis of miR200b/a/429 and interacts with the ATP-dependent RNA helicase, DDX5/p68. Both FOXG1 and DDX5 associate with the microprocessor complex, whereby DDX5 recruits FOXG1 to DROSHA. RNA-Seq analyses of Foxg1cre/+ hippocampi and N2a cells overexpressing miR200 family members identified cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) as a target of miR200 in neural cells. PRKAR2B inhibits postsynaptic functions by attenuating protein kinase A (PKA) activity; thus, increased PRKAR2B levels may contribute to neuronal dysfunctions in FOXG1 syndrome. Our data suggest that FOXG1 regulates PRKAR2B expression both on transcriptional and posttranscriptional levels.

PRKAR2B promotes prostate cancer metastasis by activating Wnt/ß-catenin and inducing epithelial-mesenchymal transition.

Castration-resistant prostate cancers (CRPC) that occur after the failure of androgen-blocking therapies cause most of the deaths in prostate cancer (PCa) patients. In a previous study we identified that PRKAR2B expression is upregulated in CRPC and possesses potentials to develop CRPC. Here we further investigated the underlying mechanism of PRKAR2B in regulating prostate cancer metastasis. We established an androgen-independent LNCaPcell line (LNCaP-AI), and investigated the function of PRKAR2B on regulating cell invasion in vitro and in vivo. We found that PRKAR2B expression was markedly increased in LNCaP-AI cells and metastatic CRPC (mCRPC) tissues compared to LNCaP cells and primary PCa specimens, respectively. PRKAR2B level was significantly correlated with the Gleason score and lymph nodes metastasis in PCa. In vitro, PRKAR2B overexpression promoted cell invasion, whereas knockdown of PRKAR2B in CRPC cells inhibited cell invasion. PRKAR2B overexpression also promoted tumor metastasis in vivo. PRKAR2B resulted in a decreased expression of E-cadherin and an increased expression of Vimentin, N-cadherin, Fibronectin, indicating that PRKAR2B induced epithelial-mesenchymal transition (EMT). PRKAR2B activated Wnt/ß-catenin signaling in CRPC cells. More important, inhibition of Wnt/ß-catenin attenuated PRKAR2B-induced EMT and cancer cells invasion. Our results provided novel insights to PRKAR2B-driven CRPC cell invasion and indicated that PRKAR2B might be served as a potential target for CRPC therapy.

Knockdown of PRKAR2B Results in the Failure of Oocyte Maturation.

BACKGROUND/AIMS: Cyclic adenosine monophosphate (cAMP)-dependent type 2 regulatory subunit beta (Prkar2b) is a regulatory isoform of cAMP-dependent protein kinase (PKA), which is the primary target for cAMP actions. In oocytes, PKA and the pentose phosphate pathway (PPP) have important roles during the germinal vesicle (GV) stage arrest of development. Although the roles of the PKA signal pathway have been studied in the development of oocyte, there has been no report on the function of PRKAR2B, a key regulator of PKA. METHODS: Using reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR), immunohistochemistry, and immunofluorescence, we determined the relative expression of Prkar2b in various tissues, including ovarian follicles, during oocyte maturation. Prkar2b-interfering RNA (RNAi) microinjection was conducted to confirm the effect of Prkar2b knockdown, and immunofluorescence, qRT-PCR, and time-lapse video microscopy were used to analyze Prkar2b-deficient oocytes. RESULTS: Prkar2b is strongly expressed in the ovarian tissues, particularly in the growing follicle. During oocyte maturation, the highest expression of Prkar2b was during metaphase I (MI), with a significant decrease at metaphase II (MII). RNAi-mediated Prkar2b suppression resulted in MI-stage arrest during oocyte development, and these oocytes exhibited abnormal spindle formation and chromosome aggregation. Expression of other members of the PKA family (except for Prkaca) were decreased, and the majority of the PPP factors were also reduced in Prkar2b-deficient oocytes. CONCLUSION: These results suggest that Prkar2b is closely involved in the maturation of oocytes by controlling spindle formation and PPP-mediated metabolism.

Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches.

Protein phosphorylation by cyclic AMP-dependent protein kinase (PKA) underlies key cellular processes, including sympathetic stimulation of heart cells, and potentiation of synaptic strength in neurons. Unrestrained PKA activity is pathological, and an enduring challenge is to understand how the activity of PKA catalytic subunits is directed in cells. We developed a light-activated cross-linking approach to monitor PKA subunit interactions with temporal precision in living cells. This enabled us to refute the recently proposed theory that PKA catalytic subunits remain tethered to regulatory subunits during cAMP elevation. Instead, we have identified other features of PKA signaling for reducing catalytic subunit diffusion and increasing recapture rate. Comprehensive quantitative immunoblotting of protein extracts from human embryonic kidney cells and rat organs reveals that regulatory subunits are always in large molar excess of catalytic subunits (average ∼17-fold). In the majority of organs tested, type II regulatory (RII) subunits were found to be the predominant PKA subunit. We also examined the architecture of PKA complexes containing RII subunits using cross-linking coupled to mass spectrometry. Quantitative comparison of cross-linking within a complex of RIIß and Cß, with or without the prototypical anchoring protein AKAP18α, revealed that the dimerization and docking domain of RIIß is between its second cAMP binding domains. This architecture is compatible with anchored RII subunits directing the myristylated N terminus of catalytic subunits toward the membrane for release and recapture within the plane of the membrane.

Distinct PKA regulatory subunits mediate PGE2 inhibition of TGFß-1-stimulated collagen I translation and myofibroblast differentiation.

Prostaglandin E2 (PGE2), via cAMP signaling, inhibits a variety of fibroblast functions relevant to fibrogenesis. Among these are their translation of collagen I protein and their differentiation to myofibroblasts. PKA is central to these actions, with cAMP binding to regulatory (R) subunits leading to the release of catalytic subunits. Here we examined the role of specific PKAR subunit isoforms in these inhibitory actions in transforming growth factor ß-1 (TGFß-1)-stimulated human lung fibroblasts (HLFs). HLFs expressed all four R subunit isoforms. siRNA-mediated knockdown of subunits PKARIα and PKARIIα had no effect on PGE2 inhibition of either process. However, knockdown of PKARIß selectively attenuated PGE2 inhibition of collagen I protein expression, whereas knockdown of PKARIIß selectively attenuated PGE2 inhibition of expression of the myofibroblast differentiation marker, α-smooth muscle actin (α-SMA). cAMP analogs that selectively activate either PKARIß or PKARIIß exclusively inhibited collagen I synthesis or differentiation, respectively. In parallel, the PKARIß agonist (but not a PKARIIß agonist) reduced phosphorylation of two proteins involved in protein translation, protein kinase B (AKT) and mammalian target of rapamycin (mTOR). By contrast, the PKARIIß agonist (but not a PKARIß agonist) reduced levels of the differentiation-associated phosphorylated focal adhesion kinase (p-FAK) as well as the relative mRNA and protein expression of serum response factor (SRF), a transcription factor necessary for myofibroblast differentiation. Our results demonstrate that cAMP inhibition of collagen I translation and myofibroblast differentiation reflects the actions of distinct PKAR subunits.

Crosstalk from cAMP to ERK1/2 emerges during postnatal maturation of nociceptive neurons and is maintained during aging.

Maturation of nociceptive neurons depends on changes in transcription factors, ion channels and neuropeptides. Mature nociceptors initiate pain in part by drastically reducing the activation threshold via intracellular sensitization signaling. Whether sensitization signaling also changes during development and aging remains so far unknown. Using a novel automated microscopy approach, we quantified changes in intracellular signaling protein expression and in their signaling dynamics, as well as changes in intracellular signaling cascade wiring, in sensory neurons from newborn to senescent (24 months of age) rats. We found that nociceptive subgroups defined by the signaling components protein kinase A (PKA)-RIIß (also known as PRKAR2B) and CaMKIIα (also known as CAMK2A) developed at around postnatal day 10, the time of nociceptor maturation. The integrative nociceptor marker, PKA-RIIß, allowed subgroup segregation earlier than could be achieved by assessing the classical markers TRPV1 and Nav1.8 (also known as SCN10A). Signaling kinetics remained constant over lifetime despite in part strong changes in the expression levels. Strikingly, we found a mechanism important for neuronal memory - i.e. the crosstalk from cAMP and PKA to ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) - to emerge postnatally. Thus, maturation of nociceptors is closely accompanied by altered expression, activation and connectivity of signaling pathways known to be central for pain sensitization and neuronal memory formation.

The Activation of Protein Kinase A by the Calcium-Binding Protein S100A1 Is Independent of Cyclic AMP.

Biochemical and structural studies demonstrate that S100A1 is involved in a Ca2+-dependent interaction with the type 2α and type 2ß regulatory subunits of protein kinase A (PKA) (RIIα and RIIß) to activate holo-PKA. The interaction was specific for S100A1 because other calcium-binding proteins (i.e., S100B and calmodulin) had no effect. Likewise, a role for S100A1 in PKA-dependent signaling was established because the PKA-dependent subcellular redistribution of HDAC4 was abolished in cells derived from S100A1 knockout mice. Thus, the Ca2+-dependent interaction between S100A1 and the type 2 regulatory subunits represents a novel mechanism that provides a link between Ca2+ and PKA signaling, which is important for the regulation of gene expression in skeletal muscle via HDAC4 cytosolic-nuclear trafficking.

FRET biosensor uncovers cAMP nano-domains at ß-adrenergic targets that dictate precise tuning of cardiac contractility.

Compartmentalized cAMP/PKA signalling is now recognized as important for physiology and pathophysiology, yet a detailed understanding of the properties, regulation and function of local cAMP/PKA signals is lacking. Here we present a fluorescence resonance energy transfer (FRET)-based sensor, CUTie, which detects compartmentalized cAMP with unprecedented accuracy. CUTie, targeted to specific multiprotein complexes at discrete plasmalemmal, sarcoplasmic reticular and myofilament sites, reveals differential kinetics and amplitudes of localized cAMP signals. This nanoscopic heterogeneity of cAMP signals is necessary to optimize cardiac contractility upon adrenergic activation. At low adrenergic levels, and those mimicking heart failure, differential local cAMP responses are exacerbated, with near abolition of cAMP signalling at certain locations. This work provides tools and fundamental mechanistic insights into subcellular adrenergic signalling in normal and pathological cardiac function.

Differential expression of the protein kinase A subunits in normal adrenal glands and adrenocortical adenomas.

Somatic mutations in protein kinase A catalytic α subunit (PRKACA) were found to be causative for 30-40% of cortisol-producing adenomas (CPA) of the adrenal gland, rendering PKA signalling constitutively active. In its resting state, PKA is a stable and inactive heterotetramer, consisting of two catalytic and two regulatory subunits with the latter inhibiting PKA activity. The human genome encodes three different PKA catalytic subunits and four different regulatory subunits that are preferentially expressed in different organs. In normal adrenal glands all regulatory subunits are expressed, while CPA exhibit reduced protein levels of the regulatory subunit IIß. In this study, we linked for the first time the loss of RIIß protein levels to the PRKACA mutation status and found the down-regulation of RIIß to arise post-transcriptionally. We further found the PKA subunit expression pattern of different tumours is also present in the zones of the normal adrenal cortex and demonstrate that the different PKA subunits have a differential expression pattern in each zone of the normal adrenal gland, indicating potential specific roles of these subunits in the regulation of different hormones secretion.