cAMP-dependent Protein Kinase Activity Research Articles

Effects of Different Na+ Concentrations on cAMP-Dependent Protein Kinase Activity in Postmortem Meat.

cAMP-dependent protein kinase (PKA) activity regulates protein phosphorylation, with Na+ playing a crucial role in PKA activity. The aim of this study was to investigate the effects of different Na+ concentrations on PKA activity and protein phosphorylation level in postmortem muscle. The study consisted of two experiments: (1) NaCl of 0, 20, 100, 200 and 400 mM was added to a muscle homogenate incubation model to analyze the effect of Na+ concentration on PKA activity, and (2) the same concentrations were added to pure PKA in vitro incubation models at 4 °C to verify the effect of Na+ on PKA activity. The PKA activity of the muscle homogenate model increased with storage time in groups with different Na+ concentrations. High concentrations of Na+ inhibited sarcoplasmic protein phosphorylation. The PKA activity at 24 h of storage and the sarcoplasmic protein phosphorylation level at 12 h of storage in the group with 200 mM Na+ was lower than that of the other groups. After 1 h incubation, the PKA activity of samples in the 200 mM Na+ group was inhibited and lower than that in the other Na+ groups in the in vitro incubation model. These results suggest that the Na+ concentration at 200 mM could better inhibit PKA activity. This study provided valuable insights for enhancing curing efficiency and improving meat quality.

Persistent PKA activation redistributes NaV1.5 to the cell surface of adult rat ventricular myocytes.

During chronic stress, persistent activation of cAMP-dependent protein kinase (PKA) occurs, which can contribute to protective or maladaptive changes in the heart. We sought to understand the effect of persistent PKA activation on NaV1.5 channel distribution and function in cardiomyocytes using adult rat ventricular myocytes as the main model. PKA activation with 8CPT-cAMP and okadaic acid (phosphatase inhibitor) caused an increase in Na+ current amplitude without altering the total NaV1.5 protein level, suggesting a redistribution of NaV1.5 to the myocytes' surface. Biotinylation experiments in HEK293 cells showed that inhibiting protein trafficking from intracellular compartments to the plasma membrane prevented the PKA-induced increase in cell surface NaV1.5. Additionally, PKA activation induced a time-dependent increase in microtubule plus-end binding protein 1 (EB1) and clustering of EB1 at myocytes' peripheral surface and intercalated discs (ICDs). This was accompanied by a decrease in stable interfibrillar microtubules but an increase in dynamic microtubules along the myocyte surface. Imaging and coimmunoprecipitation experiments revealed that NaV1.5 interacted with EB1 and β-tubulin, and both interactions were enhanced by PKA activation. We propose that persistent PKA activation promotes NaV1.5 trafficking to the peripheral surface of myocytes and ICDs by providing dynamic microtubule tracks and enhanced guidance by EB1. Our proposal is consistent with an increase in the correlative distribution of NaV1.5, EB1, and β-tubulin at these subcellular domains in PKA-activated myocytes. Our study suggests that persistent PKA activation, at least during the initial phase, can protect impulse propagation in a chronically stressed heart by increasing NaV1.5 at ICDs.

Identification of the susceptible genes and mechanism underlying the comorbid presence of coronary artery disease and rheumatoid arthritis: a network modularization analysis

ObjectiveThe comorbidities of coronary artery disease (CAD) and rheumatoid arthritis (RA) are mutual risk factors, which lead to higher mortality, but the biological mechanisms connecting the two remain unclear. Here, we aimed to identify the risk genes for the comorbid presence of these two complex diseases using a network modularization approach, to offer insights into clinical therapy and drug development for these diseases.MethodThe expression profile data of patients CAD with and without RA were obtained from the GEO database (GSE110008). Based on the differentially expressed genes (DEGs), weighted gene co-expression network analysis (WGCNA) was used to construct a gene network, detect co-expression modules, and explore their relation to clinical traits. The Zsummary index, gene significance (GS), and module membership (MM) were utilized to screen the important differentiated modules and hub genes. The GO and KEGG pathway enrichment analysis were applied to analyze potential mechanisms.ResultBased on the 278 DEGs obtained, 41 modules were identified, of which 17 and 24 modules were positively and negatively correlated with the comorbid occurrence of CAD and RA (CAD&RA), respectively. Thirteen modules with Zsummary < 2 were found to be the underlying modules, which may be related to CAD&RA. With GS ≥ 0.5 and MM ≥ 0.8, 49 hub genes were identified, such as ADO, ABCA11P, POT1, ZNF141, GPATCH8, ATF6 and MIA3, etc. The area under the curve values of the representative seven hub genes under the three models (LR, KNN, SVM) were greater than 0.88. Enrichment analysis revealed that the biological functions of the targeted modules were mainly involved in cAMP-dependent protein kinase activity, demethylase activity, regulation of calcium ion import, positive regulation of tyrosine, phosphorylation of STAT protein, and tissue migration, etc.ConclusionThirteen characteristic modules and 49 susceptibility hub genes were identified, and their corresponding molecular functions may reflect the underlying mechanism of CAD&RA, hence providing insights into the development of clinical therapies against these diseases.

β3 Receptor Signaling in Pregnant Human Myometrium Suggests a Role for β3 Agonists as Tocolytics.

Preterm labor leading to preterm birth is the leading cause of infant morbidity and mortality. At the present time, nothing can reliably halt labor once it begins. The knowledge that agonists of the β2 adrenergic receptor relax airway smooth muscle and are effective in the treatment of asthma led to the notion that β2 mimetics would prevent preterm birth by relaxing uterine smooth muscle. The activation of cAMP-dependent protein kinase by β2 receptors is unable to provide meaningful tocolysis. The failure of β2 agonists such as ritodrine and terbutaline to prevent preterm birth suggests that the regulation of uterine smooth muscle is disparate from that of airway. Other smooth muscle quiescent-mediating molecules, such as nitric oxide, relax vascular smooth muscle in a cGMP-protein kinase G-dependent manner; however, nitric oxide activation of protein kinase G fails to explain the relaxation of the myometrium to nitric oxide. Moreover, nitric oxide-mediated relaxation is blunted in preterm labor, and thus, for this reason and because of the fall in maternal blood pressure, nitric oxide cannot be employed as a tocolytic. The β3 adrenergic receptor-mediated relaxation of the human myometrium is claimed to be cAMP-dependent protein kinase-dependent. This is scientifically displeasing given the failure of β2 agonists as tocolytics and suggests a non-canonical signaling role for β3AR in myometrium. The addition of the β3 agonist mirabegron to pregnant human myometrial strips in the tissue bath relaxes oxytocin-induced contractions. Mirabegron stimulates nitric oxide production in myometrial microvascular endothelial cells, and the relaxation of uterine tissue in vitro is partially blocked by the addition of the endothelial nitric oxide synthase blocker Nω-Nitro-L-arginine. Recent data suggest that both endothelial and smooth muscle cells respond to β3 stimulation and contribute to relaxation through disparate signaling pathways. The repurposing of approved medications such as mirabegron (Mybetriq™) tested in human myometrium as uterine tocolytics can advance the prevention of preterm birth.

Multifaceted Roles of GLP-1 and Its Analogs: A Review on Molecular Mechanisms with a Cardiotherapeutic Perspective.

Diabetes is one of the chronic metabolic disorders which poses a multitude of life-debilitating challenges, including cardiac muscle impairment, which eventually results in heart failure. The incretin hormone glucagon-like peptide-1 (GLP-1) has gained distinct recognition in reinstating glucose homeostasis in diabetes, while it is now largely accepted that it has an array of biological effects in the body. Several lines of evidence have revealed that GLP-1 and its analogs possess cardioprotective effects by various mechanisms related to cardiac contractility, myocardial glucose uptake, cardiac oxidative stress and ischemia/reperfusion injury, and mitochondrial homeostasis. Upon binding to GLP-1 receptor (GLP-1R), GLP-1 and its analogs exert their effects via adenylyl cyclase-mediated cAMP elevation and subsequent activation of cAMP-dependent protein kinase(s) which stimulates the insulin release in conjunction with enhanced Ca2+ and ATP levels. Recent findings have suggested additional downstream molecular pathways stirred by long-term exposure of GLP-1 analogs, which pave the way for the development of potential therapeutic molecules with longer lasting beneficial effects against diabetic cardiomyopathies. This review provides a comprehensive overview of the recent advances in the understanding of the GLP-1R-dependent and -independent actions of GLP-1 and its analogs in the protection against cardiomyopathies.

Bidirectional crosstalk of the cAMP/ROS-dependent signaling pathways in inflammatory macrophage: An activation of formononetin

Bacterial lipopolysaccharide (LPS) is a toxic stimulant to macrophage inflammation. Inflammation intersects cell metabolism and often directs host immunopathogenesis stress. We aim here at pharmacological discovering of formononetin (FMN) action, to which anti-inflammatory signaling spans across immune membrane receptors and second messenger metabolites. In ANA-1 macrophage stimulated by LPS, and simultaneous treatment with FMN, results show the Toll-like receptor 4 (TLR4) and estrogen receptor (ER) signals, in concert with reactive oxygen species (ROS) and cyclic adenosine monophosphate (cAMP), respectively. LPS stimulates inactivation of the ROS-dependent nuclear factor erythroid 2-related factor 2 (Nrf2) by upregulating TLR4, but it does not affect cAMP. However, FMN treatment not only activates Nrf2 signaling by TLR4 inhibition, but also it activates cAMP-dependent protein kinase activities by upregulating ER. The cAMP activity gives rise to phosphorylation (p-) of protein kinase A, liver kinase B1 and 5’-AMP activated protein kinase (AMPK). Moreover, bidirectional signal crosstalk is amplified between p-AMPK and ROS, as FMN combinational validation with AMPK activator/inhibitor/target small-interfering RNA or ROS scavenger. The signal crosstalk is well positioned serving as the ˈplug-inˈ knot for rather long signaling axis, and the immune-to-metabolic circuit via ER/TLR4 signal transduction. Collectively, convergence of the FMN-activated signals drives significant reduction of cyclooxygenase-2, interleukin-6 and NLR family pyrin domain-containing protein 3, in LPS-stimulated cell. Although anti-inflammatory signaling is specifically related to the immune-type macrophage, the p-AMPK antagonizing effect arises from FMN combination with ROS scavenger H-bond donors. Information of our work assists in predictive traits against macrophage inflammatory challenges, using phytoestrogen discoveries.

Adenylyl cyclase 6 plays a minor role in the mouse inner ear and retina

Adenylyl cyclase 6 (AC6) synthesizes second messenger cAMP in G protein-coupled receptor (GPCR) signaling. In cochlear hair cells, AC6 distribution relies on an adhesion GPCR, ADGRV1, which is associated with Usher syndrome (USH), a condition of combined hearing and vision loss. ADGRV1 is a component of the USH type 2 (USH2) protein complex in hair cells and photoreceptors. However, the role of AC6 in the inner ear and retina has not been explored. Here, we found that AC6 distribution in hair cells depends on the USH2 protein complex integrity. Several known AC6 regulators and effectors, which were previously reported to participate in ADGRV1 signaling in vitro, are localized to the stereociliary compartments that overlap with AC6 distribution in hair cells. In young AC6 knockout (Adcy6−/−) mice, the activity of cAMP-dependent protein kinase, but not Akt kinase, is altered in cochleas, while both kinases are normal in vestibular organs. Adult Adcy6−/− mice however exhibit normal hearing function. AC6 is expressed in mouse retinas but rarely in photoreceptors. Adcy6−/− mice have slightly enhanced photopic but normal scotopic vision. Therefore, AC6 may participate in the ADGRV1 signaling in hair cells but AC6 is not essential for cochlear and retinal development and maintenance.

Activity of cAMP-Dependent Protein Kinase is Reduced in Protein-Energy Malnourished Rats

Glucagon decreases glutathione synthesis in hepatocytes from well-nourished rats. However, in hepatocytes from malnourished rats, glucagon does not inhibit glutathione synthesis, suggesting a desensitization of cAMP-mediated signal transduction. We investigated the mechanism for this desensitization of cAMP-mediated responsiveness in malnourished rats by comparing the signal transduction pathways in rats fed very low protein diets (0.5 g protein/100 g diet) with those of rats fed diets adequate in protein (15 g protein/100 g diet) for 2 wk. Glucagon receptor and forskolin-stimulated cAMP production were greater in hepatocytes from malnourished rats. Stimulation of adenylyl cyclase with forskolin, guanine nucleotides or manganese in hepatic membranes was also enhanced after malnutrition. Moreover, quantity of the stimulatory guanine nucleotide regulatory protein was 70–80% greater in hepatocytes from malnourished rats but the inhibitory guanine nucleotide regulatory protein was not different. These results suggested that desensitization of cAMP-mediated signal transduction after malnutrition occurred at a site distal to cAMP production. Maximal activity of cAMP-dependent protein kinase was 60% lower in liver homogenates from malnourished rats compared with controls. This difference in activity was confined to the cytosolic compartment, with no difference in activity observed in the particulate fraction. Lower activity of cAMP-dependent protein kinase in the cytosol of malnourished rats was associated with a 43% reduction in the quantity of regulatory subunit type I, with no difference in the regulatory subunit type II. These data indicate that desensitization of cAMP signal transduction in rat liver after malnutrition is due to a decrease in the quantity and activity of cAMP-dependent protein kinase.

Comparative and integrative analysis of transcriptomic and epigenomic-wide DNA methylation changes in African American prostate cancer

ABSTRACT African American (AA) men have the highest incidence and mortality rate from Prostate cancer (PCa) than any other racial/ethnic group. To date, PCa genomic studies have largely under-represented tumour samples from AA men. We measured genome-wide DNA methylation in benign and tumor prostate tissues from AA men using the Illumina Infunium 850 K EPIC array. mRNA expression database from a subset of the AA biospecimen were used to assess correlation of transcriptome and methylation datasets. Genome-wide methylation analysis identified 11,460 probes that were significant (p < 0.01) and differentially methylated in AA PCa compared to normal prostate tissues and showed significant (p < 0.01) inverse-correlation with mRNA expression. Ingenuity pathway analysis and Gene Ontology analysis in our AA dataset compared with TCGA dataset showed similarities in methylation patterns: top candidate genes with significant hypermethylation and corresponding down-regulated gene expression were associated with biological pathways in hemidesmosome assembly, mammary gland development, epidermis development, hormone biosynthesis, and cell communication. In addition, top candidate genes with significant hypomethylation and corresponding up-regulated gene expression were associated with biological pathways in macrophage differentiation, cAMP-dependent protein kinase activity, protein destabilization, transcription co-repression, and fatty acid biosynthesis. In contrast, differences in genome-wide methylation in our AA dataset compared with TCGA dataset were enriched for genes in steroid signalling, immune signalling, chromatin structure remodelling and RNA processing. Overall, differential methylation of AMIGO3, IER3, UPB1, GRM7, TFAP2C, TOX2, PLSCR2, ZNF292, ESR2, MIXL1, BOLL, and FGF6 were significant and uniquely associated with PCa progression in our AA cohort.

Very Low Protein Diets Induce a Rapid Decrease in Hepatic cAMP-Dependent Protein Kinase Followed by a Slower Increase in Adenylyl Cyclase Activity in Rats

Recent evidence indicates that cAMP-mediated responses are desensitized in liver during malnutrition. While receptor-stimulated production of cAMP is increased in hepatocytes from rats fed very low protein diets for 14 d, activity of cAMP-dependent protein kinase (PKA) is decreased in liver cytosol. The present study investigated the time course for this desensitization. Weanling rats were fed either a 0.5 (malnourished) or 15% protein (control) diet for 1, 3, 7 or 14 d. Total PKA activity decreased after only 3 d of feeding the low protein diet. This decrease was confined to the cytosolic compartment and was associated with a lower quantity of immunoreactive RI regulatory subunit of PKA, with no difference in the quantity of immunoreactive RII regulatory subunit. In contrast, basal-, MnCl2- and guanine nucleotide regulatory protein-stimulated adenylyl cyclase activities were not greater in liver membranes of malnourished rats than in those of the control rats until the 2nd wk of feeding. Greater activity was paralleled by an increase in the quantity of the stimulatory guanine nucleotide regulatory protein at d 14. The inhibitory guanine nucleotide regulatory protein quantity did not differ between dietary groups. Greater cAMP production was not mediated by changes in PKA phosphorylation of adenylyl cyclase because preincubation of membranes with purified PKA catalytic subunit decreased MnCl2-stimulated cAMP production equally in liver membranes of both control and malnourished rats. Similarly, treatment with alkaline phosphatase decreased adenylyl cyclase activity but did not eliminate the difference in adenylyl cyclase activity between control and malnourished rats. These data demonstrate that loss of PKA activity is an early response to a low protein diet and that, subsequently, a number of molecular adaptations occur which increase cAMP production. These changes may be adaptive responses to malnutrition that maintain essential cAMP-dependent functions.

P-CREB and p-DARPP-32 orchestrating the modulatory role of cAMP/PKA signaling pathway enhanced by Roflumilast in rotenone-induced Parkinson's disease in rats

Recently, phosphodiesterases (PDEs) have gained great attention due to their implication in Parkinson's disease (PD) pathogenesis. Noteworthy, the PDE4 enzyme is highly expressed in the striatum and selectively degrades cyclic adenosine monophosphate (cAMP). The cAMP was shown to play a vital role in dopamine (DA) signaling besides maintaining the plasticity of dopaminergic neurons as well as protecting them from inflammation and oxidative stress-mediated death. Thus, PDE4 inhibition could be a promising strategy for treating PD. Accordingly, the present study investigated the neuroprotective efficacy of roflumilast, a PDE4 inhibitor, in abolishing neurodegeneration in the rotenone-induced PD model. Rotenone (1.5 mg/kg, s.c) was delivered via 11 injections on matching days. Roflumilast treatment (0.5 mg/kg, p.o) was given daily after the fifth rotenone injection. Roflumilast significantly reversed rotenone's adverse effects, as it enhanced trophic factors expression and abrogated inflammation as well as oxidative stress. Thus, promoting dopaminergic neuronal plasticity and survival, as well as restoring striatal DA level and function, which resulted in enhanced motor performance. The beneficial effect of roflumilast was mediated through inhibition of striatal PDE4 with consequent activation of cAMP-dependent protein kinase A (PKA) signaling pathways, including the cAMP response element-binding protein (CREB) pathway and dopamine and cAMP-regulated phosphoprotein 32,000 (DARPP-32) pathway that is essential for maintaining dopaminergic function. Therefore, the present work sheds light on the substantial neuroprotective potential of roflumilast in treating PD through the activation of the cAMP/PKA cascade.

Glucocorticoid effects on working memory impairment require l-type calcium channel activity within prefrontal cortex

Previous findings have indicated that glucocorticoid hormones impair working memory via an interaction with the β-adrenoceptor-cAMP signaling cascade to rapidly increase cAMP-dependent protein kinase (PKA) activity within the prefrontal cortex (PFC). However, it remains elusive how such activation of PKA can affect downstream cellular mechanisms in regulating PFC cognitive function. PKA is known to activate l-type voltage-gated Ca2+ channels (LTCCs) which regulate a broad range of cellular processes, including neuronal excitability and neurotransmitter release. The present experiments examined whether LTCC activity within the PFC is required in mediating glucocorticoid and PKA effects on spatial working memory. Male Sprague Dawley rats received bilateral administration of the LTCC inhibitor diltiazem together with either the glucocorticoid receptor agonist RU 28362 or PKA activator Sp-cAMPS into the PFC before testing on a delayed alternation task in a T-maze. Both RU 28362 and Sp-cAMPS impaired working memory, whereas the LTCC inhibitor diltiazem fully blocked the working memory impairment induced by either RU 28362 or Sp-cAMPS. Conversely, bilateral administration of the LTCC agonist Bay K8644 into the PFC was sufficient to impair working memory. Thus, these findings provide support for the view that glucocorticoids, via an interaction with the β-adrenergic signaling cascade and enhanced PKA activity levels, impair working memory by increasing LTCC activity in the PFC.

Adenylate cyclase A amplification and functional diversification during Polyspondylium pallidum development

BackgroundIn Dictyostelium discoideum (Ddis), adenylate cyclase A (ACA) critically generates the cAMP oscillations that coordinate aggregation and morphogenesis. Unlike group 4 species like Ddis, other groups do not use extracellular cAMP to aggregate. However, deletion of cAMP receptors (cARs) or extracellular phosphodiesterase (PdsA) in Polyspondylium pallidum (Ppal, group 2) blocks fruiting body formation, suggesting that cAMP oscillations ancestrally control post-aggregative morphogenesis. In group 2, the acaA gene underwent several duplications. We deleted the three Ppal aca genes to identify roles for either gene and tested whether Ppal shows transient cAMP-induced cAMP accumulation, which underpins oscillatory cAMP signalling.ResultsIn contrast to Ddis, pre-aggregative Ppal cells did not produce a pulse of cAMP upon stimulation with the cAR agonist 2′H-cAMP, but acquired this ability after aggregation. Deletion of Ppal aca1, aca2 and aca3 yielded different phenotypes. aca1ˉ cells showed relatively thin stalks, aca2ˉ showed delayed secondary sorogen formation and aca3ˉ formed less aggregation centers. The aca1ˉaca2ˉ and aca1ˉaca3ˉ mutants combined individual defects, while aca2ˉaca3ˉ and aca1ˉaca3ˉaca2ˉ additionally showed > 24 h delay in aggregation, with only few aggregates with fragmenting streams being formed. The fragments developed into small fruiting bodies with stalk and spore cells. Aggregation was restored in aca2ˉaca3ˉ and aca1ˉaca3ˉaca2ˉ by 2.5 mM 8Br-cAMP, a membrane-permeant activator of cAMP-dependent protein kinase (PKA). Like Ddis, Ppal sorogens also express the adenylate cyclases ACR and ACG. We found that prior to aggregation, Ddis acaˉ/ACG cells produced a pulse of cAMP upon stimulation with 2′H-cAMP, indicating that cAMP oscillations may not be dependent on ACA alone.ConclusionsThe three Ppal replicates of acaA perform different roles in stalk morphogenesis, secondary branch formation and aggregation, but act together to enable development by activating PKA. While even an aca1ˉaca3ˉaca2ˉ mutant still forms (some) fruiting bodies, suggesting little need for ACA-induced cAMP oscillations in this process, we found that ACG also mediated transient cAMP-induced cAMP accumulation. It, therefore, remains likely that post-aggregative Ppal morphogenesis is organized by cAMP oscillations, favouring a previously proposed model, where cAR-regulated cAMP hydrolysis rather than its synthesis dominates oscillatory behaviour.

IRAS/Nischarin modulates morphine reward by glutamate receptor activation in the nucleus accumbens of mouse brain

The I1 imidazoline receptor and its candidate protein imidazoline receptor antisera-selected (IRAS)/Nischarin are linked to μ opioid receptor (MOR) functions associated with MOR trafficking. We previously demonstrated that IRAS may play an important role in the development of morphine tolerance and physical dependence in vivo. However, the effects of IRAS on morphine psychological dependence are not fully understood. To extend these studies, we investigated the impact of IRAS on morphine dependence in conditioned place preference (CPP) experiments and explored the underlying mechanisms. Knockout of IRAS enhanced the acquisition and reinstatement of morphine-induced CPP. Conditional-knockout of IRAS in the nucleus accumbens (NAc) reproduced higher CPP, and overexpression of IRAS in the NAc rescued the increased morphine-induced CPP in IRAS-/- mice. IRAS-/- mice showed dramatic cAMP-dependent protein kinase (PKA) activation, upregulation of the phosphorylation of the AMPA receptor GluR1-S845 and NMDA receptor NR1-S897 in the NAc after CPP experiment. Moreover, knockout of IRAS induced an increase in spontaneous excitatory postsynaptic current (sEPSC) frequency and a decrease in the AMPA/NMDA ratio in the NAc after chronic morphine treatment. The selective AMPA receptor antagonist NBQX could inhibit morphine CPP in WT mice, while its effect was significantly reduced in IRAS-/- mice. Together, our results demonstrate that IRAS contributes to the regulation of morphine dependence and that the alteration of glutamatergic transmission in the NAc may participate in the effect of IRAS.

Mitochondrial a Kinase Anchor Proteins in Cardiovascular Health and Disease: A Review Article on Behalf of the Working Group on Cellular and Molecular Biology of the Heart of the Italian Society of Cardiology

Second messenger cyclic adenosine monophosphate (cAMP) has been found to regulate multiple mitochondrial functions, including respiration, dynamics, reactive oxygen species production, cell survival and death through the activation of cAMP-dependent protein kinase A (PKA) and other effectors. Several members of the large family of A kinase anchor proteins (AKAPs) have been previously shown to locally amplify cAMP/PKA signaling to mitochondria, promoting the assembly of signalosomes, regulating multiple cardiac functions under both physiological and pathological conditions. In this review, we will discuss roles and regulation of major mitochondria-targeted AKAPs, along with opportunities and challenges to modulate their functions for translational purposes in the cardiovascular system.

Development of a phos-tag-based fluorescent biosensor for sensitive detection of protein kinase in cancer cells.

Protein kinase can catalyze the phosphorylation of peptides/proteins, and it is closely associated with various human diseases such as cancer, immune deficiencies, and Alzheimer's disease. Sensitive monitoring of protein kinase activity is significant for biochemical research and drug discovery. Herein, we develop a phos-tag-based fluorescent biosensor for sensitive detection of cAMP-dependent protein kinase (PKA) activity in cancer cells. We design a peptide-DNA conjugate and a signal probe for PKA activity assay, and employ a biotinylated phos-tag (a selective phosphate-binding agent) to recognize and capture the phosphorylated peptide-DNA substrate. The peptide-DNA conjugates as the catalytic substrates can translate the peptide signal to a nucleotide signal for the initiation of the RNase HII-driven cycling signal amplification. The magnetic nanobeads (MBs) as the carriers can separate and enrich the phosphorylated peptide in complex matrices. Meanwhile, the combination of MBs with the phos-tag-mediated phosphate group recognition can effectively eliminate the interference from the complex matrix, and the introduction of single-molecule detection endows this assay with high sensitivity. This biosensor can achieve a detection limit of 1.98 × 10-8 U μL-1 and a wide dynamic range from 1 × 10-7 to 1 × 10-2 U μL-1. Moreover, this biosensor can be applied for the screening of PKA inhibitor and the measurement of cellular PKA activity, holding great potential in biomedical research and clinical diagnosis.

Engineered synaptic tools reveal localized cAMP signaling in synapse assembly.

The physiological mechanisms driving synapse formation are elusive. Although numerous signals are known to regulate synapses, it remains unclear which signaling mechanisms organize initial synapse assembly. Here, we describe new tools, referred to as "SynTAMs" for synaptic targeting molecules, that enable localized perturbations of cAMP signaling in developing postsynaptic specializations. We show that locally restricted suppression of postsynaptic cAMP levels or of cAMP-dependent protein-kinase activity severely impairs excitatory synapse formation without affecting neuronal maturation, dendritic arborization, or inhibitory synapse formation. In vivo, suppression of postsynaptic cAMP signaling in CA1 neurons prevented formation of both Schaffer-collateral and entorhinal-CA1/temporoammonic-path synapses, suggesting a general principle. Retrograde trans-synaptic rabies virus tracing revealed that postsynaptic cAMP signaling is required for continuous replacement of synapses throughout life. Given that postsynaptic latrophilin adhesion-GPCRs drive synapse formation and produce cAMP, we suggest that spatially restricted postsynaptic cAMP signals organize assembly of postsynaptic specializations during synapse formation.

The cAMP Inducers Modify N-Acetylaspartate Metabolism in Wistar Rat Brain.

Neuronal N-acetylaspartate production appears in the presence of aspartate N-acetyltransferase (NAT8L) and binds acetyl groups from acetyl-CoA with aspartic acid. Further N-acetylaspartate pathways are still being elucidated, although they seem to involve neuron-glia crosstalk. Together with N-acetylaspartate, NAT8L takes part in oligoglia and astroglia cell maturation, myelin production, and dopamine-dependent brain signaling. Therefore, understanding N-acetylaspartate metabolism is an emergent task in neurobiology. This project used in in vitro and in vivo approaches in order to establish the impact of maturation factors and glial cells on N-acetylaspartate metabolism. Embryonic rat neural stem cells and primary neurons were maturated with either nerve growth factor, trans-retinoic acid or activators of cAMP-dependent protein kinase A (dibutyryl-cAMP, forskolin, theophylline). For in vivo, adult male Wistar rats were injected with theophylline (20 mg/kg b.w.) daily for two or eight weeks. Our studies showed that the N-acetylaspartate metabolism differs between primary neurons and neural stem cell cultures. The presence of glia cells protected N-acetylaspartate metabolism from dramatic changes within the maturation processes, which was impossible in the case of pure primary neuron cultures. In the case of differentiation processes, our data points to dibutyryl-cAMP as the most prominent regulator of N-acetylaspartate metabolism.

Expanded Supercooling Capacity With No Cryoprotectant Accumulation Underlies Cold Tolerance of the European Grapevine Moth

The European grapevine moth, Lobesia botrana (Denis et Schiffermuller), is a serious invasive pest that causes significant losses to the flowers and fruits of grapes in most of the world. This multivoltine pest passes the winter as the third-generation diapausing pupa. The current study was designed to compare nondiapausing (first and second generations) and diapausing pupae (third generation) and to investigate the relationship among cold tolerance, the supercooling point (SCP), and diapause development of the third-generation diapausing pupae. The lethal temperatures (LTs) for the three generations were determined using 24-h exposure at subzero temperatures. The mean SCP of the pupae was estimated at approx. -22.6°C, the lowest level of which (-23.7°C) was recorded in the well-developed diapausing pupae in February. The highest level of cold tolerance was also recorded in February. There were no significant differences among the temperatures required to kill 30, 50, and 90% of the pupae. The temperatures significantly decreased from October onward and reached the lowest levels in February during which the lowest SCP and the highest cold tolerance were observed in the diapausing pupae. No significant differences were found in the cryoprotectant levels, among the diapausing and nondiapausing pupae, and the diapause development. The highest activity of cAMP-dependent protein kinase (AMPK) was recorded in the late diapause in February. The findings suggested a relationship among SCP depression, cold tolerance enhancement, and diapause development. A bimodal cold-tolerance strategy (freeze-intolerant and freeze-tolerant) was found to be a feature of the pupae.

Identification of Novel Substrates for cGMP Dependent Protein Kinase (PKG) through Kinase Activity Profiling to Understand Its Putative Role in Inherited Retinal Degeneration.

Inherited retinal degenerative diseases (IRDs), which ultimately lead to photoreceptor cell death, are characterized by high genetic heterogeneity. Many IRD-associated genetic defects affect 3′,5′-cyclic guanosine monophosphate (cGMP) levels. cGMP-dependent protein kinases (PKGI and PKGII) have emerged as novel targets, and their inhibition has shown functional protection in IRDs. The development of such novel neuroprotective compounds warrants a better understanding of the pathways downstream of PKGs that lead to photoreceptor degeneration. Here, we used human recombinant PKGs in combination with PKG activity modulators (cGMP, 3′,5′-cyclic adenosine monophosphate (cAMP), PKG activator, and PKG inhibitors) on a multiplex peptide microarray to identify substrates for PKGI and PKGII. In addition, we applied this technology in combination with PKG modulators to monitor kinase activity in a complex cell system, i.e. the retinal cell line 661W, which is used as a model system for IRDs. The high-throughput method allowed quick identification of bona fide substrates for PKGI and PKGII. The response to PKG modulators helped us to identify, in addition to ten known substrates, about 50 novel substrates for PKGI and/or PKGII which are either specific for one enzyme or common to both. Interestingly, both PKGs are able to phosphorylate the regulatory subunit of PKA, whereas only PKGII can phosphorylate the catalytic subunit of PKA. In 661W cells, the results suggest that PKG activators cause minor activation of PKG, but a prominent increase in the activity of cAMP-dependent protein kinase (PKA). However, the literature suggests an important role for PKG in IRDs. This conflicting information could be reconciled by cross-talk between PKG and PKA in the retinal cells. This must be explored further to elucidate the role of PKGs in IRDs.