Search

Primary pigmented nodular adrenocortical disease (PPNAD) and bilateral macronodular adrenocortical disease (BMAD) are 2 forms of adrenocortical nodular diseases causing Cushing's syndrome but are 2 very distinct conditions. PPNAD, affecting mostly young patients with an almost constant severe Cushing's syndrome, is characterized by pigmented micronodules, usually less than 1 cm, not always visible on imaging. On the contrary, BMAD is predominantly diagnosed in the fifth and sixth decades, with highly variable degrees of cortisol excess, from mild autonomous cortisol secretion to overt Cushing's syndrome. BMAD presents as large bilateral adrenal macronodules, easily observed on imaging. Both diseases are often genetically determined: frequently PPNAD is observed in a multiple neoplasia syndrome, Carney complex, and a germline genetic defect is identified in around 80% of index cases, always affecting key actors of the cAMP/protein kinase A (PKA) pathway: mostly PRKAR1A, encoding the PKA 1-alpha regulatory subunit. On the other hand, BMAD appears mostly isolated, and 2 predisposing genes are known at present: ARMC5, accounting for around 20% of index cases, and the recently identified KDM1A, causing the rare presentation with food-dependent Cushing's syndrome, mediated by the ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in adrenal nodules. GIPR was the first demonstrated receptor to illegitimately regulate cortisol secretion in nodular adrenocortical diseases, and a myriad of other receptors and paracrine signals were discovered afterward. The last 30 years were pivotal in the understanding of the genetics and pathophysiology of bilateral adrenocortical nodular diseases, leading to a personalized approach of these fascinating conditions., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

Protein kinase A (PKA) is a ubiquitous cAMP-dependent enzyme in mammalian tissues. The inactive PKA holoenzyme disassociates into a homodimer of regulatory (R) subunits and two active catalytic (C) subunits upon cAMP binding to two tandem domains (termed CBD-A and CBD-B) in R subunits. The release of cAMP facilitates reassociation of R and C subunits, resetting PKA to its basal state. The cAMP-mediated structural changes in the activation-termination cycle remain partially understood. The multimeric states of PKA complicate the issue and are particularly less studied. Therefore, we computationally investigated the conformational dynamics of the PKA RIα homodimer in different cAMP-bound states. The absence of cAMP in two CBDs differently affects the conformational dynamics of protomers. Moreover, such disparate responses are extended to the dimer interface constituted by the N-terminal helical sub-domains termed N3A motifs. The removal of cAMP from CBD-A induces large-scale structural changes of individual R subunits towards the holoenzyme state, consistent with previous simulations of a single R subunit. Meanwhile it keeps the structural heterogeneity of the N3A-N3A' dimer interface observed in the fully bound state. By contrast, the removal of cAMP from CBD-B does not affect individual R subunits but alters the conformational space of the N3A-N3A' dimer interface. The cAMP-coupled structural changes of each protomer and conserved conformational space of the N3A-N3A' dimer interface are essential for the transition between the fully cAMP-bound R 2 homodimer and the R 2 C 2 holoenzyme as suggested by their crystal structures. Our work provides structural insights into the regulatory mechanism of cAMP in PKA signaling.

Spatiotemporal regulation of intracellular signaling molecules, such as the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), ensures proper cellular function. Liquid-liquid phase separation (LLPS) of the ubiquitous PKA regulatory subunit RIα promotes cAMP compartmentation and signaling specificity. However, the molecular determinants of RIα LLPS remain unclear. Here, we reveal that two separate dimerization interfaces, combined with the cAMP-induced unleashing of the PKA catalytic subunit (PKA-C) from the pseudosubstrate inhibitory sequence, drive RIα condensate formation in the cytosol of mammalian cells, which is antagonized by docking to A-kinase anchoring proteins. Strikingly, we find that the RIα pseudosubstrate region is critically involved in forming a non-canonical R:C complex, which recruits active PKA-C to RIα condensates to maintain low basal PKA activity in the cytosol. Our results suggest that RIα LLPS not only facilitates cAMP compartmentation but also spatially restrains active PKA-C, thus highlighting the functional versatility of biomolecular condensates in driving signaling specificity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The integration of innovative medical technologies and interdisciplinary collaboration could improve the treatment of cancer, a globally prevalent and often deadly disease. Despite recent advancements, current cancer therapies fail to specifically address recurrence and target cancer stem cells (CSCs), which contribute to relapse. In this study, we utilized three types of cancer cells, from which three types of CSCs were further derived, to conduct a proteomic analysis. Additionally, shared cell surface biomarkers were identified as potential targets for a comprehensive treatment strategy. The selected biomarkers were evaluated through short hairpin RNA treatment, which revealed contrasting functions in cancer cells and CSCs. Knockdown of the identified proteins revealed that they regulate the epithelial-mesenchymal transition (EMT) and stemness via the ERK signaling pathway. Resistance to anticancer agents was consequently reduced, ultimately enhancing the overall anticancer effects of the treatment. Additionally, the significance of these biomarkers in clinical patient outcomes was confirmed using bioinformatics. Our study suggests a novel cancer treatment strategy that addresses the limitations of current anticancer therapies.

The cAMP/PKA and mitogen-activated protein kinase (MAPK) signaling cascade control many cellular processes and are highly regulated for optimal cellular responses upon external stimuli. Phosphodiesterase 8A (PDE8A) is an important regulator that inhibits signaling via cAMP-dependent PKA by hydrolyzing intracellular cAMP pool. Conversely, PDE8A activates the MAPK pathway by protecting CRAF/Raf1 kinase from PKA-mediated inhibitory phosphorylation at Ser259 residue, a binding site of scaffold protein 14-3-3. It still remains enigmatic as to how the cross-talk involving PDE8A regulation influences cAMP/PKA and MAPK signaling pathways. Here, we report that PDE8A interacts with 14-3-3ζ in both yeast and mammalian system, and this interaction is enhanced upon the activation of PKA, which phosphorylates PDE8A's Ser359 residue. Biophysical characterization of phospho-Ser359 peptide with 14-3-3ζ protein further supports their interaction. Strikingly, 14-3-3ζ reduces the catalytic activity of PDE8A, which upregulates the cAMP/PKA pathway while the MAPK pathway is downregulated. Moreover, 14-3-3ζ in complex with PDE8A and cAMP-bound regulatory subunit of PKA, RIα, delays the deactivation of PKA signaling. Our results define 14-3-3ζ as a molecular switch that operates signaling between cAMP/PKA and MAPK by associating with PDE8A., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cardiac myxoma (CM) is the most common benign cardiac tumor, and most CMs are left atrial myxomas (LAMs). Six variations of KIF1C, c.899 A > T, c.772 T > G, c.352 A > T, c.2895 C > T, c.3049 G > A, and c.*442_*443dup in left atrial myxoma tissues are identified by whole-exome sequencing (WES) and Sanger sequencing. RNA-seq and function experiments show the reduction of the expression of KIF1C and PRKAR1A caused by rare variations of KIF1C. KIF1C is observed to be located in the nucleus, bind to the promoter region of PRKAR1A, and regulate its transcription. Reduction of KIF1C decreases PRKAR1A expression and activates the PKA, which causes an increase in ERK1/2 phosphorylation and SRC-mediated STAT3 activation, a reduction of CDH1, TP53, CDKN1A, and BAX, and eventually promotes tumor formation both in vitro and in vivo. The results suggest that inhibition of KIF1C promotes the pathogenesis of LAM through positive feedback formed by the crosstalk between KIF1C and PRKAR1A., (© 2023. The Author(s).)

The Carney complex (CNC) is an autosomal dominant disorder characterized by endocrine and nonendocrine tumors. Loss-of-function variants of protein kinase A regulatory subunit 1 alpha (PRKAR1A) are common causes of CNC. Here, we present the case of a patient with CNC with a novel PRKAR1A missense variant. A 21-year-old woman was diagnosed with CNC secondary to acromegaly and adrenal Cushing syndrome. Genetic analysis revealed a novel missense heterozygous variant of PRKAR1A (c.176A>T). Her relatives, suspected of having CNC, also carried the same variant. RNA analysis revealed that this variant led to nonsense-mediated mRNA decay. In vitro functional analysis of the variant confirmed its role in increasing protein kinase A activity and cyclic adenosine monophosphate levels. This study broadens our understanding of the genetic spectrum of CNC. We suggest that PRKAR1A genetic testing and counseling be recommended for patients with CNC and their families.

In the heart, protein kinase A (PKA) is critical for activating calcium handling and sarcomeric proteins in response to beta-adrenergic stimulation leading to increased myocardial contractility and performance. The catalytic activity of PKA is tightly regulated by regulatory subunits that inhibit the catalytic subunit until released by cAMP binding. Phosphorylation of type II regulatory subunits promotes PKA activation; however, the role of phosphorylation in type I regulatory subunits remain uncertain. Here, we utilize human induced pluripotent stem cell cardiomyocytes (iPSC-CMs) to identify STK25 as a kinase of the type Iα regulatory subunit PRKAR1A. Phosphorylation of PRKAR1A leads to inhibition of PKA kinase activity and increased binding to the catalytic subunit in the presence of cAMP. Stk25 knockout in mice diminishes Prkar1a phosphorylation, increases Pka activity, and augments contractile response to beta-adrenergic stimulation. Together, these data support STK25 as a negative regulator of PKA signaling through phosphorylation of PRKAR1A., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Protein kinase A (PKA) is a holoenzyme consisting of a regulatory (R)-subunit dimer and two catalytic (C)-subunits. There are two major families of C-subunits, C α and C β , and four functionally nonredundant R-subunits (RI α , RI β , RII α , RII β ). In addition to binding to and being regulated by the R-subunits, the C-subunits are regulated by two tail regions that each wrap around the N- and C-lobes of the kinase core. Although the C-terminal (Ct-) tail is classified as an intrinsically disordered region (IDR), the N-terminal (Nt-) tail is dominated by a strong helix that is flanked by short IDRs. In contrast to the Ct-tail, which is a conserved and highly regulated feature of all PKA, PKG, and protein kinase C protein kinase group (AGC) kinases, the Nt-tail has evolved more recently and is highly variable in vertebrates. Surprisingly and in contrast to the kinase core and the Ct-tail, the entire Nt-tail is not conserved in nonmammalian PKAs. In particular, in humans, C β actually represents a large family of C-subunits that are highly variable in their Nt-tail and also expressed in a highly tissue-specific manner. Although we know so much about the C α 1-subunit, we know almost nothing about these C β isoforms wherein C β 2 is highly expressed in lymphocytes, and C β 3 and C β 4 isoforms account for ∼50% of PKA signaling in brain. Based on recent disease mutations, the C β proteins appear to be functionally important and nonredundant with the C α isoforms. Imaging in retina also supports nonredundant roles for C β as well as isoform-specific localization to mitochondria. This represents a new frontier in PKA signaling. SIGNIFICANCE STATEMENT: How tails and adjacent domains regulate each protein kinase is a fundamental challenge for the biological community. Here we highlight how the N- and C-terminal tails of PKA (Nt-tails/Ct-tails) affect the structure and regulate the function of the kinase core and show the combinatorial variations that are introduced into the Nt-tail of the C α - and C β -subunits in contrast to the Ct-tail, which is conserved across the entire AGC subfamily of protein kinases., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Hexokinase 2 (HK2), which catalyzes the first committed step in glucose metabolism, is induced in cancer cells. HK2's role in tumorigenesis has been attributed to its glucose kinase activity. Here, we describe a kinase independent HK2 activity, which contributes to metastasis. HK2 binds and sequesters glycogen synthase kinase 3 (GSK3) and acts as a scaffold forming a ternary complex with the regulatory subunit of protein kinase A (PRKAR1a) and GSK3β to facilitate GSK3β phosphorylation and inhibition by PKA. Thus, HK2 functions as an A-kinase anchoring protein (AKAP). Phosphorylation by GSK3β targets proteins for degradation. Consistently, HK2 increases the level and stability of GSK3 targets, MCL1, NRF2, and particularly SNAIL. In addition to GSK3 inhibition, HK2 kinase activity mediates SNAIL glycosylation, which prohibits its phosphorylation by GSK3. Finally, in mouse models of breast cancer metastasis, HK2 deficiency decreases SNAIL protein levels and inhibits SNAIL-mediated epithelial mesenchymal transition and metastasis., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Large-cell calcifying Sertoli cell tumors (LCCSCTs) are among the most frequent lesions occurring in male Carney complex (CNC) patients. Although they constitute a key diagnostic criterion for this rare multiple neoplasia syndrome resulting from inactivating mutations of the tumor suppressor PRKAR1A, leading to unrepressed PKA activity, LCCSCT pathogenesis and origin remain elusive. Mouse models targeting Prkar1a inactivation in all somatic populations or separately in each cell type were generated to decipher the molecular and paracrine networks involved in the induction of CNC testis lesions. We demonstrate that the Prkar1a mutation was required in both stromal and Sertoli cells for the occurrence of LCCSCTs. Integrative analyses comparing transcriptomic, immunohistological data and phenotype of mutant mouse combinations led to the understanding of human LCCSCT pathogenesis and demonstrated PKA-induced paracrine molecular circuits in which the aberrant WNT4 signal production is a limiting step in shaping intratubular lesions and tumor expansion both in a mouse model and in human CNC testes.

cAMP-dependent protein kinase (PKA) signaling plays various roles during mammalian spermatogenesis, ranging from the regulation of gene expression to the modulation of sperm motility. However, the molecular mechanisms that govern the multifaceted functions of PKA during spermatogenesis remain largely unclear. We previously found that PKA regulatory subunit I α (RIα) and catalytic subunit α (Cα) co-sediment with polyribosomal fractions of mouse testis lysate on sucrose gradient and the stimulation of PKA activity facilitates protein synthesis in post-meiotic elongating spermatids, indicating that type I PKA is intricately associated with protein translation machinery and regulates protein synthesis during mouse spermiogenesis. Since PKA activity is often regulated by interacting proteins that form complexes with its regulatory subunits, the identification of PKA-RIα interacting proteins in post-meiotic spermatogenic cells will facilitate our understanding of its regulatory roles in protein synthesis and spermiogenesis. In the present study, we applied a yeast two-hybrid screen to identify PKA-Riα-binding proteins using a cDNA library generated from mouse round and elongating spermatids. Numerous proteins were found to potentially interact with PKA-RIα, including proteostasis modulators, metabolic enzymes, cytoskeletal regulators, and mitochondrial proteins, many of which are specifically expressed in testes. Consistently, the examination of MENA (mouse ENA/VASP homolog) in developing mouse testes suggested that post-meiotic spermatogenic cells express a short isoform of MENA that interacts with PKA-RIα in yeast two-hybrid assay. The identification of PKA-RIα interacting proteins provides us solid basis to further explore how PKA signaling regulates protein synthesis and cellular morphogenesis during mouse spermatogenesis.

Protein kinase A (PKA) is the main receptor for the universal cAMP second messenger. PKA is a tetramer with two catalytic (C) and two regulatory (R) subunits, each including two tandem cAMP-binding domains, i.e. CBD-A and -B. Activation of the complex occurs with cAMP binding first to CBD-B, followed by a second molecule of cAMP binding to CBD-A, which causes the release of the active C-subunit. Unlike previous constructs for eukaryotic cAMP-binding domains (CBDs), the 29.5 kDa construct analyzed here [i.e. RIα (119-379)] spans the CBDs in full and provides insight into inter-domain communication. In this note we report the 1 H, 13 C, and 15  N backbone assignments of cAMP-bound RIα (119-379) CBDs (BMRB No. 50920)., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function., Competing Interests: The authors declare no competing interest.

Background: Kinase oxidation is a critical signaling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, PKARIα (type-1 protein kinase A) can be reversibly oxidized, forming interprotein disulfide bonds in the holoenzyme complex. However, the effect of PKARIα disulfide formation on downstream signaling in the heart, particularly under states of oxidative stress such as ischemia and reperfusion (I/R), remains unexplored., Methods: Atrial tissue obtained from patients before and after cardiopulmonary bypass and reperfusion and left ventricular (LV) tissue from mice subjected to I/R or sham surgery were used to assess PKARIα disulfide formation by immunoblot. To determine the effect of disulfide formation on PKARIα catalytic activity and subcellular localization, live-cell fluorescence imaging and stimulated emission depletion super-resolution microscopy were performed in prkar1 knock-out mouse embryonic fibroblasts, neonatal myocytes, or adult LV myocytes isolated from "redox dead" (Cys17Ser) PKARIα knock-in mice and their wild-type littermates. Comparison of intracellular calcium dynamics between genotypes was assessed in fura2-loaded LV myocytes, whereas I/R-injury was assessed ex vivo., Results: In both humans and mice, myocardial PKARIα disulfide formation was found to be significantly increased (2-fold in humans, P =0.023; 2.4-fold in mice, P <0.001) in response to I/R in vivo. In mouse LV cardiomyocytes, disulfide-containing PKARIα was not found to impact catalytic activity, but instead led to enhanced AKAP (A-kinase anchoring protein) binding with preferential localization of the holoenzyme to the lysosome. Redox-dependent regulation of lysosomal two-pore channels by PKARIα was sufficient to prevent global calcium release from the sarcoplasmic reticulum in LV myocytes, without affecting intrinsic ryanodine receptor leak or phosphorylation. Absence of I/R-induced PKARIα disulfide formation in "redox dead" knock-in mouse hearts resulted in larger infarcts (2-fold, P <0.001) and a concomitant reduction in LV contractile recovery (1.6-fold, P <0.001), which was prevented by administering the lysosomal two-pore channel inhibitor Ned-19 at the time of reperfusion., Conclusions: Disulfide modification targets PKARIα to the lysosome, where it acts as a gatekeeper for two-pore channel-mediated triggering of global calcium release. In the postischemic heart, this regulatory mechanism is critical for protection from extensive injury and offers a novel target for the design of cardioprotective therapeutics.

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 RIIβ, 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 RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ2C2 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 RIIβ holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIβ 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., Competing Interests: The authors have declared that no competing interests exist.

Reactive oxygen species (ROS) have been shown to prolong cardiac action potential duration resulting in afterdepolarizations, the cellular basis of triggered arrhythmias. As previously shown, protein kinase A type I (PKA I) is readily activated by oxidation of its regulatory subunits. However, the relevance of this mechanism of activation for cardiac pathophysiology is still elusive. In this study, we investigated the effects of oxidation-activated PKA I on cardiac electrophysiology. Ventricular cardiomyocytes were isolated from redox-dead PKA-RI Cys17Ser knock-in (KI) and wild-type (WT) mice and exposed to H 2 O 2 (200 µmol/L) or vehicle (Veh) solution. In WT myocytes, exposure to H 2 O 2 significantly increased oxidation of the regulatory subunit I (RI) and thus its dimerization (threefold increase in PKA RI dimer). Whole cell current clamp and voltage clamp were used to measure cardiac action potentials (APs), transient outward potassium current ( I to ) and inward rectifying potassium current ( I K1 ), respectively. In WT myocytes, H 2 O 2 exposure significantly prolonged AP duration due to significantly decreased I to and I K1 resulting in frequent early afterdepolarizations (EADs). Preincubation with the PKA-specific inhibitor Rp-8-Br-cAMPS (10 µmol/L) completely abolished the H 2 O 2 -dependent decrease in I to and I K1 in WT myocytes. Intriguingly, H 2 O 2 exposure did not prolong AP duration, nor did it decrease I to , and only slightly enhanced EAD frequency in KI myocytes. Treatment of WT and KI cardiomyocytes with the late I Na inhibitor TTX (1 µmol/L) completely abolished EAD formation. Our results suggest that redox-activated PKA may be important for H 2 O 2 -dependent arrhythmias and could be important for the development of specific antiarrhythmic drugs. NEW & NOTEWORTHY Oxidation-activated PKA type I inhibits transient outward potassium current ( I to ) and inward rectifying potassium current ( I K1 ) and contributes to ROS-induced APD prolongation as well as generation of early afterdepolarizations in murine ventricular cardiomyocytes.

Protein kinase A (PKA) regulatory subunit type 1A (PRKAR1A) defects lead to primary pigmented nodular adrenocortical disease (PPNAD). The KIT protooncogene (c-KIT) is not known to be expressed in the normal adrenal cortex (AC). In this study, we investigated the expression of c-KIT and its ligand, stem cell factor (SCF), in PPNAD and other cortisol-producing tumors of the adrenal cortex. mRNA and protein expression, by qRT-PCR, immunohistochemistry (IHC) and immunoblotting (IB), respectively, were studied. We then tested c-KIT and SCF responses to PRKAR1A introduction and PKA stimulation in adrenocortical cell lines CAR47 and H295R, which were also treated with the KIT inhibitor, imatinib mesylate (IM). Mice xenografted with H295R cells were treated with IM. There was increased c-KIT mRNA expression in PPNAD; IHC showed KIT and SCF immunoreactivity within certain nodular areas in PPNAD. IB data was consistent with IHC and mRNA data. PRKAR1A-deficient CAR47 cells expressed c-KIT; this was enhanced by forskolin and lowered by PRKAR1A reintroduction. Knockdown of PKA's catalytic subunit (PRKACA) by siRNA reduced c-KIT levels. Treatment of the CAR47 cells with IM resulted in reduced cell viability, growth arrest, and apoptosis. Treatment with IM of mice xenografted with H295 cells inhibited further tumor growth. We conclude that c-KIT is expressed in PPNAD, an expression that appears to be dependent on PRKAR1A and/or PKA activity. In a human adrenocortical cell line and its xenografts in mice, c-KIT inhibition decreased growth, suggesting that c-KIT inhibitors may be a reasonable alternative therapy to be tested in PPNAD, when other treatments are not optimal.

The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mutations in the protein kinase A (PKA) regulatory subunit type 1A (PRKAR1A) and armadillo repeat-containing 5 (ARMC5) genes cause Cushing's syndrome (CS) due to primary pigmented nodular adrenocortical disease (PPNAD) and primary bilateral macronodular adrenocortical hyperplasia (PBMAH), respectively. Between the two genes, ARMC5 is highly polymorphic with several variants in the population, whereas PRKAR1A has very little, if any, non-pathogenic variation in its coding sequence. We tested the hypothesis that ARMC5 variants may affect the clinical presentation of PPNAD and CS among patients with PRKAR1A mutations. In this study, 91 patients with PPNAD due to PRKAR1A mutations were tested for abnormal cortisol secretion or CS and for ARMC5 sequence variants. Abnormal cortisol secretion was present in 71 of 74 patients with ARMC5 variants, whereas 11 of 17 patients negative for ARMC5 variants did not have hypercortisolemia. The presence of ARMC5 variants was a statistically strong predictor of CS among patients with PRKAR1A mutations (P < 0.001). Among patients with CS due to PPNAD, ARMC5 variants were associated with lower cortisol levels at baseline (P = 0.04) and after high dose dexamethasone administration (P = 0.02). The ARMC5 p.I170V variant increased ARMC5 protein accumulation in vitro and decreased viability of NCI-H295 cells (but not HEK 293T cells). PPNAD tissues with ARMC5 variants showed stronger ARMC5 protein expression than those that carried a normal ARMC5 sequence. Taken together, our results suggest that ARMC5 variants among patients with PPNAD due to PRKAR1A defects may play the role of a genetic modifier for the presence and severity of hypercortisolemia.

In cAMP-Protein Kinase A (PKA) signaling, A-kinase anchoring protein scaffolds assemble PKA in close proximity to phosphodiesterases (PDE), kinase-substrates to form signaling islands or 'signalosomes'. In its basal state, inactive PKA holoenzyme (R2:C2) is activated by binding of cAMP to regulatory (R)-subunits leading to dissociation of active catalytic (C)-subunits. PDEs hydrolyze cAMP-bound to the R-subunits to generate 5'-AMP for termination and resetting the cAMP signaling. Mechanistic basis for cAMP signaling has been derived primarily by focusing on the proteins in isolation. Here, we set out to simulate cAMP signaling activation-termination cycles in a signalosome-like environment with PDEs and PKA subunits in close proximity to each other. Using a combination of fluorescence polarization and amide hydrogen exchange mass spectrometry with regulatory (RIα), C-subunit (Cα) and PDE8 catalytic domain, we have tracked movement of cAMP through activation-termination cycles. cAMP signaling operates as a continuum of four phases: (1) Activation and dissociation of PKA into R- and C-subunits by cAMP and facilitated by substrate (2) PDE recruitment to R-subunits (3) Hydrolysis of cAMP to 5'-AMP (4) Reassociation of C-subunit to 5'-AMP-bound-RIα in the presence of excess ATP to reset cAMP signaling to form the inactive PKA holoenzyme. Our results demonstrate that 5'-AMP is not merely a passive hydrolysis end-product of PDE action. A 'ligand-free' state R subunit does not exist in signalosomes as previously assumed. Instead the R-subunit toggles between cAMP- or 5'-AMP bound forms. This highlights, for the first time, the importance of 5'-AMP in promoting adaptation and uncovers adenylate control in cAMP signaling., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

There is a significant unmet need for a safe and effective therapy for the treatment of children with congenital hyperinsulinism. We hypothesized that amplification of the glucagon signaling pathway could ameliorate hyperinsulinism associated hypoglycemia. In order to test this we evaluated the effects of loss of Prkar1a, a negative regulator of Protein Kinase A in the context of hyperinsulinemic conditions. With reduction of Prkar1a expression, we observed a significant upregulation of hepatic gluconeogenic genes. In wild type mice receiving a continuous infusion of insulin by mini-osmotic pump, we observed a 2-fold increase in the level of circulating ketones and a more than 40-fold increase in Kiss1 expression with reduction of Prkar1a. Loss of Prkar1a in the Sur1-/- mouse model of KATP hyperinsulinism significantly attenuated fasting induced hypoglycemia, decreased the insulin/glucose ratio, and also increased the hepatic expression of Kiss1 by more than 10-fold. Together these data demonstrate that amplification of the hepatic glucagon signaling pathway is able to rescue hypoglycemia caused by hyperinsulinism., Competing Interests: MMS, PAH and KF work for Alnylam Pharamaceuticals; MMS, PAH and KF hold shares in Alnylam Pharmaceuticals stock; DDDL received funding from Alnylam Pharmaceuticals to conduct this study. We confirm that this does not alter our adherence to the PLOS ONE policies on sharing data and materials and have updated our Competing Interests statement.

Adrenocortical tumors (ACTs) frequently cause steroid excess and present cell-cycle dysregulation. cAMP/PKA signaling is involved in steroid synthesis and play a role in cell-cycle regulation. We investigated, by cell synchronization in the different phases of the cell-cycle, the control of steroidogenesis and the contribution of PKA in adrenocortical cells (H295R and culture of primary pigmented nodular adrenocortical disease cells). Cells showed increased steroidogenesis and a maximal PKA activity at G2 phase, and a reduction at G1 phase. PRKACA overexpression, or cAMP stimulation, enhanced PKA activity and induced steroidogenesis in all synchronized groups but is not sufficient to drive cell-cycle progression. PRKAR1A inactivation enhanced PKA activity and induced STAR gene expression, only in cells in G1, and triggered cell-cycle progression in all groups. These findings provide evidence for a tight association between steroidogenesis and cell-cycle in ACTs. Moreover, PRKAR1A is essential for mediating the function of PKA activity on both steroidogenesis and cell-cycle progression in adrenocortical cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Type I PKA regulatory α-subunit (RIα; encoded by the Prkar1a gene) serves as the predominant inhibitor protein of the catalytic subunit of cAMP-dependent protein kinase (PKAc). However, recent evidence suggests that PKA signaling can be initiated by cAMP-independent events, especially within the context of cellular oxidative stress such as ischemia-reperfusion (I/R) injury. We determined whether RIα is actively involved in the regulation of PKA activity via reactive oxygen species (ROS)-dependent mechanisms during I/R stress in the heart. Induction of ex vivo global I/R injury in mouse hearts selectively downregulated RIα protein expression, whereas RII subunit expression appears to remain unaltered. Cardiac myocyte cell culture models were used to determine that oxidant stimulus (i.e., H 2 O 2 ) alone is sufficient to induce RIα protein downregulation. Transient increase of RIα expression (via adenoviral overexpression) negatively affects cell survival and function upon oxidative stress as measured by increased induction of apoptosis and decreased mitochondrial respiration. Furthermore, analysis of mitochondrial subcellular fractions in heart tissue showed that PKA-associated proteins are enriched in subsarcolemmal mitochondria (SSM) fractions and that loss of RIα is most pronounced at SSM upon I/R injury. These data were supported via electron microscopy in A-kinase anchoring protein 1 (AKAP1)-knockout mice, where loss of AKAP1 expression leads to aberrant mitochondrial morphology manifested in SSM but not interfibrillar mitochondria. Thus, we conclude that modification of RIα via ROS-dependent mechanisms induced by I/R injury has the potential to sensitize PKA signaling in the cell without the direct use of the canonical cAMP-dependent activation pathway. NEW & NOTEWORTHY We uncovered a previously undescribed phenomenon involving oxidation-induced activation of PKA signaling in the progression of cardiac ischemia-reperfusion injury. Type I PKA regulatory subunit RIα, but not type II PKA regulatory subunits, is dynamically regulated by oxidative stress to trigger the activation of the catalytic subunit of PKA in cardiac myocytes. This effect may play a critical role in the regulation of subsarcolemmal mitochondria function upon the induction of ischemic injury in the heart.

Purpose: To evaluate the clinical and pathologic characteristics of conjunctival myxoid lesions, with specific focus on PRKAR1A studies, in order to distinguish neoplastic conjunctival myxoma from other myxoid conjunctival lesions., Methods: A retrospective, interventional, multicenter study of all patients with conjunctival myxoma, conjunctival stromal tumor, or reactive fibromyxoid proliferation diagnosed during 1988-2018. Patient and family medical histories and clinical and pathologic characteristics of excised lesions were assessed., Results: There were 28 patients with conjunctival myxoid lesions diagnosed as myxoma (16/28), conjunctival stromal tumor (10/28), or reactive fibromyxoid proliferation (2/28). The patients with abundant myxoid matrix lesions (14/28, 50%) were younger (mean 49 [range 23-68] years) than those with scant-to-moderate myxoid matrix lesions (14/28, mean 61 [range 18-82] years; P = .04). Abundant myxoid matrix lesions more likely contained predominantly stellate cells (6/14 [43%] vs 0/14 [0%]; P = .05) and fibrillar collagen (13/14 [93%] vs 2/14 [14%]; P < .0001), conforming to the standard morphologic definition of myxoma. Absence of PRKAR1A protein expression was found in 2 lesions with morphologic features of myxoma (2/14, 14%), 1 of which demonstrated a pathogenic mutation in the PRKAR1A gene. There was no difference between the lesions with respect to other clinical and pathologic parameters., Conclusions: PRKAR1A plays a role in the development of a subset of conjunctival myxomas, particularly in tumors fulfilling stringent morphologic criteria for myxoma. With the exception of PRKAR1A studies, current immunohistochemical panels cannot reliably distinguish between neoplastic conjunctival myxomas and other myxoid lesions, underscoring the importance of morphology in establishing accurate diagnosis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Myeloid-derived suppressor cells (MDSCs) play a pivotal role in mediating the formation of an immunosuppressive environment and assisting tumors in evading the host immune response. However, the mechanism through which tumors manipulate the differentiation and function of MDSCs remains unclear. Here, we report that hypoxia-induced glioma cells can stimulate the differentiation of functional MDSCs by transferring exosomal miR-29a and miR-92a to MDSCs. Our results showed that glioma-derived exosomes (GEXs) can enhance the differentiation of functional MDSCs both in vitro and in vivo, and hypoxia-induced GEXs (H-GEXs) demonstrated a stronger MDSCs induction ability than did normoxia-induced GEXs (N-GEXs). A subsequent miRNA sequencing analysis of N-GEXs and H-GEXs revealed that hypoxia-induced exosomal miR-29a and miR-92a expression induced the propagation of MDSCs. miR-29a and miR-92a activated the proliferation and function of MDSCs by targeting high-mobility group box transcription factor 1 (Hbp1) and protein kinase cAMP-dependent type I regulatory subunit alpha (Prkar1a), respectively. Altogether, the results of our study provide new insights into the role of glioma exosomal miRNAs in mediating the formation of immunosuppressive microenvironments in tumors and elucidate the underlying exosomal miR-29a/miR-92a-based regulatory mechanism responsible for the modulation of functional MDSC induction., (© 2018 UICC.)

Protein kinase A (PKA), the main effector of cAMP in eukaryotes, is a paradigm for the mechanisms of ligand-dependent and allosteric regulation in signalling. Here we report the orthologous but cAMP-independent PKA of the protozoan Trypanosoma and identify 7-deaza-nucleosides as potent activators (EC 50  ≥ 6.5 nM) and high affinity ligands (K D  ≥ 8 nM). A co-crystal structure of trypanosome PKA with 7-cyano-7-deazainosine and molecular docking show how substitution of key amino acids in both CNB domains of the regulatory subunit and its unique C-terminal αD helix account for this ligand swap between trypanosome PKA and canonical cAMP-dependent PKAs. We propose nucleoside-related endogenous activators of Trypanosoma brucei PKA (TbPKA). The existence of eukaryotic CNB domains not associated with binding of cyclic nucleotides suggests that orphan CNB domains in other eukaryotes may bind undiscovered signalling molecules. Phosphoproteome analysis validates 7-cyano-7-deazainosine as powerful cell-permeable inducer to explore cAMP-independent PKA signalling in medically important neglected pathogens.

Objective: To analyze the biological network regulation and key proteins of gene expression microarray in human normal prostate epithelial cells after treated with low-dose cadmium, and provide some new theoretical evidence for the pathogenesis of cadmium-related prostate cancer., Methods: We downloaded 19 copies of gene chip data from the Gene Expression Omnibus (GEO), involving 9 samples of prostate epithelial cells exposed to low-dose cadmium and 10 cases of normal control. Using the Gene-Cloud of Biotechnology Informs platform, GenClip2.0 and Sytoscape 3.5.1, we screened differentially expressed genes, explored their protein interaction networks and biological pathways and, from the perspective of transcriptome, analyzed the changes in the genetic network of normal human prostate epithelial cells and their possible molecular biological functions after low-dose cadmium treatment., Results: Totally, 1 050 (1.92%) differentially expressed genes were found in the prostate epithelial cells treated with low-dose cadmium, involved in such biological functions as the cell physiological process, MAPK regulation, regulation of intracellular signal transduction, and immunological effect. The HSP90AB1, BUB3 and PRKAR1A genes were the core nodes of the protein network, which showed statistically significant differences in their expressions and a correlation with the malignant transformation of normal cells., Conclusions: Low-dose cadmium can cause genetic changes in normal human prostate epithelial cells and the differentially expressed genes are mainly involved in such biological functions as the cell physiological process, MAPK regulation, regulation of intracellular signal transduction, and immunological effect.

Microcystin-leucine arginine (MC-LR) causes serum testosterone declines and male reproductive disorders. However, the molecular mechanisms underlying the pathological changes are still unclear. In the present study, we aimed to investigate the toxic effects of MC-LR on gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus. Our results demonstrated that MC-LR could enter GnRH neurons and inhibit GnRH synthesis, resulting in the decrease of serum GnRH and testosterone levels. The inhibitory effects of MC-LR on GnRH synthesis were identified to be associated with activation of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element-binding protein (CREB)/c-Fos signaling pathway. With miRNA microarray analyses, we found that miR-329-3p was down-regulated most dramatically in MC-LR-treated GT1-7 cells. We then further identified that miR-329-3p regulated PRKAR1A and PRKACB expression and thus influenced GnRH synthesis. This is the first study to explore the molecular mechanism underlying the inhibitory effects of MC-LR on GnRH synthesis in the hypothalamus. Our data have provided a new perspective in the development of diagnosis and treatment strategies for male infertility as a result of dysfunction of the hypothalamic-pituitary-gonadal axis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cholangiocarcinoma (CCA) is a cancer of the bile ducts that is associated with poor prognosis and poor treatment outcome. Approximately one-third of CCA patients can undergo surgery, but the recurrence rate is high and chemotherapy often cannot satisfactorily prolong survival. Cellular immunotherapy based on adoptive T-cell transfer is a potential treatment for CCA; however, the development of this technology and the search for an appropriate tumor-associated antigen are still ongoing. To enhance the cytotoxic activity of effector T cells against CCA, we developed self-differentiated monocyte-derived dendritic cells (SD-DC) presenting cAMP-dependent protein kinase type I-alpha regulatory subunit (PRKAR1A), which is an overexpressed protein that plays a role in the regulation of tumor growth to activate T cells for CCA cell killing. Dendritic cells (DCs) transduced with lentivirus harboring tri-cistronic cDNA sequences (SD-DC-PR) could produce granulocyte-macrophage colony-stimulating factor, interleukin-4, and PRKAR1A. SD-DC showed similar phenotypes to those of DCs derived by conventional method. Autologous effector T cells (CD3+, CD8+) activated by SD-DC-PR exhibited greater cytotoxic activity against CCA than those activated by conventionally-derived DCs. Effector T cells activated by SD-DC-PR killed 60% of CCA cells at an effector-to-target ratio of 15:1, which is approximately twofold greater than the cell killing performance of those stimulated with control DC. The cytotoxic activities of effector T cells activated by SD-DC-PR against CCA cells were significantly associated with the expression levels of PRKR1A in CCA cells. This finding that SD-DC-PR effectively stimulated autologous effector T cells to kill CCA cells may help to accelerate the development of novel therapies for treating CCA.

Spermatogenesis is a tightly regulated process involving germ cell-specific and germ cell-predominant genes. Here we investigate a novel germ cell-specific gene, Spatc1l (spermatogenesis and centriole associated 1 like). Expression analyses show that SPATC1L is expressed in mouse and human testes. We find that mouse SPATC1L localizes to the neck region in testicular sperm. Moreover, SPATC1L associates with the regulatory subunit of protein kinase A (PKA). Using CRISPR/Cas9-mediated genome engineering, we generate mice lacking SPATC1L. Disruption of Spatc1l in mice leads to male sterility owing to separation of sperm heads from tails. The lack of SPATC1L is associated with a reduction in PKA activity in testicular sperm, and we identify capping protein muscle Z-line beta as a candidate target of phosphorylation by PKA in testis. Taken together, our results implicate the SPATC1L-PKA complex in maintaining the stability of the sperm head-tail junction, thereby revealing a new molecular basis for sperm head-tail integrity., (© 2018 Gwangju Institute of Science and Technology.)

Anxiety disorders are associated with abnormalities in fear-learning and bias to threat; early life experiences are influential to the development of an anxiety-like phenotype in adulthood. We recently reported that adult mice (Prkar1a+/-) with haploinsufficiency for the main regulatory subunit of the protein kinase A (PKA) exhibit an anxiety-like phenotype associated with increased PKA activity in the amygdala. PKA is the main effector of cyclic adenosine mono-phosphate signaling, a key pathway involved in the regulation of fear learning. Since anxiety has developmental and genetic components, we sought to examine the interaction of a genetic defect associated with anxiety phenotype and early life experiences. We investigated the effects of neonatal maternal separation or tactile stimulation on measures of behavior typical to adolescence as well as developmental changes in the behavioral phenotype between adolescent and adult wild-type (WT) and Prkar1a+/- mice. Our results showed developmental differences in assays of anxiety and novelty behavior for both genotypes. Adolescent mice showed increased exploratory and novelty seeking behavior compared to adult counterparts. However, early life experiences modulated behavior in adolescent WT differently than in adolescent Prkar1a+/- mice. Adolescent WT mice exposed to early life tactile stimulation showed attenuation of anxiety-like behavior, whereas an increase in exploratory behavior was found in Prkar1a+/- adolescent mice. The finding of behavioral differences that are apparent during adolescence in Prkar1a +/- mice suggests that long-term exposure of the brain to increased PKA activity during critical developmental periods contributes to the anxiety-like phenotype noted in the adult animals with increased PKA activity., (Published by Elsevier B.V.)

Expression of the intracellular form amastigote specific genes in the Leishmania donovani parasite plays a major role in parasite replication in the macrophage. In the current work, we have characterized a novel hypothetical gene, Ld30b that is specifically transcribed in the intracellular stage of the parasite. The recombinant Ld30b protein exists as a pentamer in solution as identified by native-PAGE and size exclusion gel chromatography. Structural analysis using circular dichroism and molecular modeling indicate that Ld30b belongs to family of cAMP-dependent protein kinase type I-alpha regulatory subunit. Co-localization immunofluorescence microscopy and western blot analyses (using anti-Ld30b antibody and anti-hypoxanthine-guanine phosphoribosyl transferase, a glycosome marker) on the isolated parasite glycosome organelle fractions show that Ld30b is localized in glycosome, though lacked a glycosome targeting PTS1/2 signal in the protein sequence. Episomal expression of Ld30b in the parasite caused the arrest of promastigotes and amastigotes growth in vitro. Cell cycle analysis using flow cytometry indicates that these parasites are arrested in 'sub G0/G1' phase of the cell cycle. Single allele knockout of Ld30b in the parasite similarly attenuated its growth by accumulation of cells in the S phase of cell cycle, thus confirming the probable importance of appropriate level of protein in the cells. Studying such intracellular stage expressing genes might unravel novel regulatory pathways for the development of drugs or vaccine candidates against leishmaniasis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene cause mostly somatotropinomas and/or prolactinomas in a subset of familial isolated pituitary adenomas (FIPA). AIP has been shown to interact with phosphodiesterases (PDEs) and G proteins, suggesting a link to the cyclic AMP (cAMP)-dependent protein kinase (PKA) pathway. Upregulation of PKA is seen in sporadic somatotropinomas that carry GNAS mutations, and those in Carney complex that are due to PRKAR1A mutations. To elucidate the mechanism of AIP-dependent pituitary tumorigenesis, we studied potential functional and physical interactions of AIP with PKA's main subunits PRKAR1A (R1α) and PRKACA (Cα). We found that AIP physically interacts with both R1α and Cα; this interaction is enhanced when all three components are present, but maintained during Cα-R1α dissociation by PKA activation, indicating that AIP binds Cα/R1α both in complex and separately. The interaction between AIP and R1α/Cα is reduced when the frequent AIP pathogenic mutation p.R304* is present. AIP protein levels are regulated both by translation and the ubiquitin/proteasome pathway and Cα stabilizes both AIP and R1α protein levels. AIP reduction by siRNA leads to an increase of PKA activity, which is disproportionately enhanced during PDE4-inhibition. We show that AIP interacts with the PKA pathway on multiple levels, including a physical interaction with both the main regulatory (R1α) and catalytic (Cα) PKA subunits and a functional interaction with PDE4-dependent PKA activation. These findings provide novel insights on the mechanisms of AIP-dependent pituitary tumorigenesis., (Published by Oxford University Press 2018. This work is written by US Government employees and is in the public domain in the US.)

cAMP-dependent protein kinase (PKAc) is a pivotal signaling protein in eukaryotic cells. PKAc has two well-characterized regulatory subunit proteins, RI and RII (each having α and β isoforms), which keep the PKAc catalytic subunit in a catalytically inactive state until activation by cAMP. Previous reports showed that the RIα regulatory subunit is phosphorylated by cGMP-dependent protein kinase (PKG) in vitro , whereupon phosphorylated RIα no longer inhibits PKAc at normal (1:1) stoichiometric ratios. However, the significance of this phosphorylation as a mechanism for activating type I PKA holoenzymes has not been fully explored, especially in cellular systems. In this study, we further examined the potential of RIα phosphorylation to regulate physiologically relevant "desensitization" of PKAc activity. First, the serine 101 site of RIα was validated as a target of PKGIα phosphorylation both in vitro and in cells. Analysis of a phosphomimetic substitution in RIα (S101E) showed that modification of this site increases PKAc activity in vitro and in cells, even without cAMP stimulation. Numerous techniques were used to show that although Ser 101 variants of RIα can bind PKAc, the modified linker region of the S101E mutant has a significantly reduced affinity for the PKAc active site. These findings suggest that RIα phosphorylation may be a novel mechanism to circumvent the requirement of cAMP stimulus to activate type I PKA in cells. We have thus proposed a model to explain how PKG phosphorylation of RIα creates a "sensitized intermediate" state that is in effect primed to trigger PKAc activity.

The adrenal cortex undergoes remodeling during fetal and postnatal life. How zona reticularis emerges in the postnatal gland to support adrenarche, a process whereby higher primates increase prepubertal androgen secretion, is unknown. Using cell-fate mapping and gene deletion studies in mice, we show that activation of PKA has no effect on the fetal cortex, while it accelerates regeneration of the adult cortex, triggers zona fasciculata differentiation that is subsequently converted into a functional reticularis-like zone, and drives hypersecretion syndromes. Remarkably, PKA effects are influenced by sex. Indeed, testicular androgens increase WNT signaling that antagonizes PKA, leading to slower adrenocortical cell turnover and delayed phenotype whereas gonadectomy sensitizes males to hypercorticism and reticularis-like formation. Thus, reticularis results from ultimate centripetal conversion of adult cortex under the combined effects of PKA and cell turnover that dictate organ size. We show that PKA-induced progenitor recruitment is sexually dimorphic and may provide a paradigm for overrepresentation of women in adrenal diseases.

Carney complex is a multiple neoplasia syndrome having endocrine and non-endocrine manifestations. Diagnostic criteria include myxoma, lentigines, and primary pigmented nodular adrenocortical disease, amongst other signs/symptoms. In most cases it is an autosomal dominant disease, and diagnosis therefore requires study and follow-up of the family members. Inactivating mutations of the PRKAR1A gene were identified as the main cause of the disease, although since 2015 other disease-related genes, including PRKACA and PRKACB activating mutations, have also been related with Carney complex. This review will address the genetic aspects related to Carney complex., (Copyright © 2017 SEEN y SED. Publicado por Elsevier España, S.L.U. All rights reserved.)

BACKGROUND The aim of this study was to explore the role of intermedin and its mechanism in cholesterol efflux of macrophage THP-1 and RAW264.7 cell lines in atherosclerosis (AS). MATERIAL AND METHODS ApoE-/- mice were fed with a high-fat diet, and the concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured. The lipidoses of the aortic sinus were analyzed by hematoxylin and eosin staining, and the cAMP level was detected by enzyme-linked immunosorbent assay (ELISA). The expressions of ATP-binding cassette transporter (ABCA1) were tested by real-time PCR and Western blot analysis. RESULTS IMD decreased serum TC and LDL-C, and increased serum HDL-C level in apoE-/- mice and attenuated AS plaque areas. In vitro, IMD increased intracellular cAMP concentration in a dose-dependent manner in THP-1 and RAW264.7 cell lines, which enhanced the expression of ABCA1 and increased cholesterol efflux rate. However, this effect was inhibited by PKAI in the RAW 264.7 cell line but not in the THP-1 cell line. CONCLUSIONS IMD can ameliorate the development of AS in ApoE-/- mice and regulate cholesterol balance in the RAW264.7 cell line through the cAMP-PKA pathway.

Toxoplasma gondii encodes three protein kinase A catalytic (PKAc1-3) and one regulatory (PKAr) subunits to integrate cAMP-dependent signals. Here, we show that inactive PKAc1 is maintained at the parasite pellicle by interacting with acylated PKAr. Either a conditional knockdown of PKAr or the overexpression of PKAc1 blocks parasite division. Conversely, down-regulation of PKAc1 or stabilisation of a dominant-negative PKAr isoform that does not bind cAMP triggers premature parasite egress from infected cells followed by serial invasion attempts leading to host cell lysis. This untimely egress depends on host cell acidification. A phosphoproteome analysis suggested the interplay between cAMP and cGMP signalling as PKAc1 inactivation changes the phosphorylation profile of a putative cGMP-phosphodiesterase. Concordantly, inhibition of the cGMP-dependent protein kinase G (PKG) blocks egress induced by PKAc1 inactivation or environmental acidification, while a cGMP-phosphodiesterase inhibitor circumvents egress repression by PKAc1 or pH neutralisation. This indicates that pH and PKAc1 act as balancing regulators of cGMP metabolism to control egress. These results reveal a crosstalk between PKA and PKG pathways to govern egress in T. gondii ., (© 2017 The Authors.)

The failure of the polycystins (PCs) to function in primary cilia is thought to be responsible for autosomal dominant polycystic kidney disease (ADPKD). Primary cilia integrate multiple cellular signaling pathways, including calcium, cAMP, Wnt, and Hedgehog, which control cell proliferation and differentiation. It has been proposed that mutated PCs result in reduced intracellular calcium, which in turn upregulates cAMP, protein kinase A (PKA) signaling, and subsequently other proliferative signaling pathways. However, the role of PKA in ADPKD has not been directly ascertained in vivo, although the expression of the main regulatory subunit of PKA in cilia and other compartments (PKA-RIα, encoded by PRKAR1A ) is increased in a mouse model orthologous to ADPKD. Therefore, we generated a kidney-specific knockout of Prkar1a to examine the consequences of constitutive upregulation of PKA on wild-type and Pkd1 hypomorphic ( Pkd1 RC ) backgrounds. Kidney-specific loss of Prkar1a induced renal cystic disease and markedly aggravated cystogenesis in the Pkd1 RC models. In both settings, it was accompanied by upregulation of Src, Ras, MAPK/ERK, mTOR, CREB, STAT3, Pax2 and Wnt signaling. On the other hand, Gli3 repressor activity was enhanced, possibly contributing to hydronephrosis and impaired glomerulogenesis in some animals. To assess the relevance of these observations in humans we looked for and found evidence for kidney and liver cystic phenotypes in the Carney complex, a tumoral syndrome caused by mutations in PRKAR1A These observations expand our understanding of the pathogenesis of ADPKD and demonstrate the importance of PRKAR1A highlighting PKA as a therapeutic target in ADPKD., (Copyright © 2017 the American Physiological Society.)

We identify a previously unresolved, unrecognized, and highly stable conformation of the protein kinase A (PKA) regulatory subunit RIα. This conformation, which we term the "Flipback" structure, bridges conflicting characteristics in crystallographic structures and solution experiments of the PKA RIα heterotetramer. Our simulations reveal a hinge residue, G235, in the B/C helix that is conserved through all isoforms of RI. Brownian dynamics simulations suggest that the Flipback conformation plays a role in cAMP association to the A domain of the R subunit.

Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide selectivity is still not fully understood. Analysis of isolated cyclic nucleotide-binding (CNB) domains of PKA regulatory subunit type Iα (RIα) reveals that the C-terminal CNB-B has a higher cAMP affinity and selectivity than the N-terminal CNB-A. Here, we show that introducing cGMP-specific residues using site-directed mutagenesis reduces the selectivity of CNB-B, while the combination of two mutations (G316R/A336T) results in a cGMP-selective binding domain. Furthermore, introducing the corresponding mutations (T192R/A212T) into the PKA RIα CNB-A turns this domain into a highly cGMP-selective domain, underlining the importance of these contacts for achieving cGMP specificity. Binding data with the generic purine nucleotide 3',5'-cyclic inosine monophosphate (cIMP) reveal that introduced arginine residues interact with the position 6 oxygen of the nucleobase. Co-crystal structures of an isolated CNB-B G316R/A336T double mutant with either cAMP or cGMP reveal that the introduced threonine and arginine residues maintain their conserved contacts as seen in PKG I CNB-B. These results improve our understanding of cyclic nucleotide binding and the molecular basis of cyclic nucleotide specificity., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

The tumor microenvironment (TME) has an impact on breast cancer progression by creating a pro-inflammatory milieu within the tumor. However, little is known about the roles of miRNAs in cells of the TME during this process. We identified six putative oncomiRs in a breast cancer dataset, all strongly correlating with poor overall patient survival. Out of the six candidates, miR-1246 was upregulated in aggressive breast cancer subtypes and expressed at highest levels in mesenchymal stem/stroma cells (MSCs). Functionally, miR-1246 led to a p65-dependent increase in transcription and release of pro-inflammatory mediators IL-6, CCL2 and CCL5 in MSCs, and increased NF-κB activity. The pro-inflammatory phenotype of miR-1246 in MSCs was independent of TNFα stimulations and mediated by direct targeting of the tumor-suppressors PRKAR1A and PPP2CB. In vitro recapitulation of the TME revealed increased Stat3 phosphorylation in breast epithelial (MCF10A) and cancer cells (SK-BR-3, MCF7, T47D) upon incubation with conditioned medium (CM) of MSCs overexpressing miR-1246. Additionally, this stimulation enhanced proliferation of MCF10A cells, increased migration of MDA-MB-231 cells and induced attraction of THP-1 monocytic cells. Our data shows that miR-1246 acts as both key-enhancer of pro-inflammatory responses in MSCs and putative oncomiR in breast cancer, suggesting its influence on cancer-related inflammation and breast cancer progression.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved heterotrimeric kinase complex consisting of a catalytic subunit, α, and two regulatory subunits, β and γ. Previously, we demonstrated that Snf1, the Saccharomyces cerevisiae ortholog of AMPK, negatively regulates the unfolded protein response (UPR) pathway and the Hog1 MAP kinase pathway in ER stress response. However, it remains unclear how the alternate three β subunits, Sip1, Sip2, and Gal83, of the Snf1 complex participate in ER stress response. Here, we show that Gal83 plays a major role in Snf1-mediated downregulation of the UPR and Hog1 pathways. Gal83 is the most abundant β subunit in the normal state and further induced by ER stress. This induction is mediated via activation of the GAL83 promoter by the UPR. When expressed under the control of the GAL83 promoter, Sip2 exhibits potent functional activity equivalent to Gal83. Our results suggest that the functional significance of the β subunit of Snf1 AMPK in ER stress response is defined by modulation of the expression level through regulation of the promoter activity.

Close-range electrostatic interactions that form salt bridges are key components of protein stability. Here we investigate the role of these charged interactions in modulating the allosteric activation of protein kinase A (PKA) via computational and experimental mutational studies of a conserved basic patch located in the regulatory subunit's B/C helix. Molecular dynamics simulations evidenced the presence of an extended network of fluctuating salt bridges spanning the helix and connecting the two cAMP binding domains in its extremities. Distinct changes in the flexibility and conformational free energy landscape induced by the separate mutations of Arg239 and Arg241 suggested alteration of cAMP-induced allosteric activation and were verified through in vitro fluorescence polarization assays. These observations suggest a mechanical aspect to the allosteric transition of PKA, with Arg239 and Arg241 acting in competition to promote the transition between the two protein functional states. The simulations also provide a molecular explanation for the essential role of Arg241 in allowing cooperative activation, by evidencing the existence of a stable interdomain salt bridge with Asp267. Our integrated approach points to the role of salt bridges not only in protein stability but also in promoting conformational transition and function.

Osteochondromyxomas (OMX) in the context of Carney complex (CNC) and fibrous dysplasia (FD)-like lesions (FDLL) in mice, as well as isolated myxomas in humans may be caused by inactivation of PRKAR1A, the gene coding for the type 1a regulatory subunit (R1α) of cAMP-dependent protein kinase (PKA). OMXs and FDLL in mice lacking Prkar1a grow from abnormal proliferation of adult bone stromal cells (aBSCs). Prkar1a and Prkaca (coding for Cα) haploinsufficiency leads to COX2 activation and prostaglandin E2 (PGE2) production that, in turn, activates proliferation of aBSCs. Celecoxib is a cyclooxygenase-2 (COX2) inhibitor. We hypothesized that COX-2 inhibition may have an effect in FD and FDLL. In vitro treatment of a human cell line prepared from a FD patient with Celecoxib resulted in decreased PGE2 and cell proliferation. Treatment of mice haploinsufficient for R1α and Cα with 1500 mg/kg Celecoxib led to decreased PGE2 and proliferation and increased apoptosis, with a corresponding gene expression profile, resulting in dramatic reduction of tumor growth. Furthermore, the treatment improved the organization of cortical bone that was adjacent to the tumor. We conclude that, in vitro and in vivo, Celecoxib had an inhibitory effect on FD cell proliferation and in mouse FDLL structure, respectively. We speculate that COX-2 inhibitors offer an attractive alternative to current treatments for benign tumors such as OMX and FD that, apart from tumor suppression, may mechanically stabilize affected bones., (Published by Elsevier Ireland Ltd.)

Protein Kinase cAMP-Dependent Regulatory Type I Alpha (PRKAR1A) is a tissue-specific extinguisher that transduces a signal through phosphorylation of different target proteins. Loss of PRKAR1A was frequently observed in endocrine neoplasia and stromal cell tumors. However, a few cases were seen in epithelial tumors. Previously, we first found that PRKAR1A was downregulated in lung adenocarcinoma patients. Thus, the present study aimed to clarify its clinical implication and biological function as a tumor suppressor in lung adenocarcinoma. The low levels of PRKAR1A transcript were correlated with tumor progression and poor overall survival. The re-expression of PRKAR1A in H1299 cells suppressed the tumor cell proliferation and migration; stable knockdown (KD) of PRKAR1A in A549 cells enhanced this function both in vitro and in vivo. Moreover, KD of PRKAR1A in A549 cells promoted the statistical colonization of circulating tumor cells to the lungs in nude mice. These effects by PRKAR1A were attributed to inhibiting E-cadherin expression. Elevated E-cadherin significantly suppressed the PRKAR1A-KD induced cell proliferation and migration. Most notably, deletion of PRKAR1A inhibited E-cadherin by activating ERK/Snail signaling. In conclusion, PRKAR1A was a potent suppressor, and through the inhibition of PRKAR1A-ERK-Snail-E-cadherin axis could serve as a potential therapeutic target.

Most disease-related mutations that impair cAMP protein kinase A (PKA) signaling are present within the regulatory (R) PKA RI alpha-subunit (RIα). Although mutations in the PRKAR1A gene are linked to Carney complex (CNC) disease and, more recently, to acrodysostosis-1 (ACRDYS1), the two diseases show contrasting phenotypes. While CNC mutations cause increased PKA activity, ACRDYS1 mutations result in decreased PKA activity and cAMP resistant holoenzymes. Mapping the ACRDYS1 disease mutations reveals their localization to the second of two tandem cAMP-binding (CNB) domains (CNB-B), and here, we characterize a recurrent deletion mutant where the last 14 residues are missing. The crystal structure of a monomeric form of this mutant (RIα92-365) bound to the catalytic (C)-subunit reveals the dysfunctional regions of the RIα subunit. Beyond the missing residues, the entire capping motif is disordered (residues 357-379) and explains the disrupted cAMP binding. Moreover, the effects of the mutation extend far beyond the CNB-B domain and include the active site and N-lobe of the C-subunit, which is in a partially open conformation with the C-tail disordered. A key residue that contributes to this crosstalk, D267, is altered in our structure, and we confirmed its functional importance by mutagenesis. In particular, the D267 interaction with Arg241, a residue shown earlier to be important for allosteric regulation, is disrupted, thereby strengthening the interaction of D267 with the C-subunit residue Arg194 at the R:C interface. We see here how the switch between active (cAMP-bound) and inactive (holoenzyme) conformations is perturbed and how the dynamically controlled crosstalk between the helical domains of the two CNB domains is necessary for the functional regulation of PKA activity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Molecular inotropy refers to cardiac contractility that can be modified to affect overall heart pump performance. Here we show evidence of a new molecular pathway for positive inotropy by a cardiac-restricted microRNA (miR). We report enhanced cardiac myocyte performance by acute titration of cardiac myosin-embedded miR-208a. The observed positive effect was independent of host gene myosin effects with evidence of negative regulation of cAMP-specific 3',5'-cyclic phosphodiesterase 4D (PDE4D) and the regulatory subunit of PKA (PRKAR1α) content culminating in PKA-site dependent phosphorylation of cardiac troponin I (cTnI) and phospholamban (PLN). Further, acute inhibition of miR-208a in adult myocytes in vitro increased PDE4D expression causing reduced isoproterenol-mediated phosphorylation of cTnI and PLN. Next, rAAV-mediated miR-208a gene delivery enhanced heart contractility and relaxation parameters in vivo. Finally, acute inducible increases in cardiac miR-208a in vivo reduced PDE4D and PRKAR1α, with evidence of increased content of several complementary miRs harboring the PDE4D recognition sequence. Physiologically, this resulted in significant cardiac cTnI and PLN phosphorylation and improved heart performance in vivo. As phosphorylation of cTnI and PLN is critical to myocyte function, titration of miR-208a represents a potential new mechanism to enhance myocardial performance via the PDE4D/PRKAR1α/PKA phosphoprotein signaling pathway.