Long non-coding RNA TDRG1 aggravates colorectal cancer stemness by binding with miR-873-5p to upregulate PRKAR2.

The effects of long non-coding RNA TDRG1 have been established in several tumors; however, its roles in colorectal cancer (CRC) progression are never been found. Here, we found that TDRG1 level was upregulated in CRC cells compared to that in normal colon epithelial cells. Additionally, TDRG1 level was remarkably upregulated in 3D non-adherent spheres derived from the parental CRC cells. Further in vitro and in vivo revealed that TDRG1 knockdown suppressed the stemness of CRC cells. What's more, combined with bioinformatics analysis, luciferase reporter and RNA pull down experiments showed that TDRG1 could bind to miR-873-5p, downregulated its level and thus increase the expression of PRKAR2. Finally, it was shown that TDRG1 functioned through the miR-873-5p/PRKAR2 axis. This study demonstrated a novel TDRG1/miR-873-5p/PRKAR2 signaling in CRC progression.

Depolarization induces nociceptor sensitization by CaV1.2-mediated PKA-II activation.

Depolarization drives neuronal plasticity. However, whether depolarization drives sensitization of peripheral nociceptive neurons remains elusive. By high-content screening (HCS) microscopy, we revealed that depolarization of cultured sensory neurons rapidly activates protein kinase A type II (PKA-II) in nociceptors by calcium influx through CaV1.2 channels. This effect was modulated by calpains but insensitive to inhibitors of cAMP formation, including opioids. In turn, PKA-II phosphorylated Ser1928 in the distal C terminus of CaV1.2, thereby increasing channel gating, whereas dephosphorylation of Ser1928 involved the phosphatase calcineurin. Patch-clamp and behavioral experiments confirmed that depolarization leads to calcium- and PKA-dependent sensitization of calcium currents ex vivo and local peripheral hyperalgesia in the skin in vivo. Our data suggest a local activity-driven feed-forward mechanism that selectively translates strong depolarization into further activity and thereby facilitates hypersensitivity of nociceptor terminals by a mechanism inaccessible to opioids.

Scorpion toxin MeuNaTxα-1 sensitizes primary nociceptors by selective modulation of voltage-gated sodium channels.

Venoms are a rich source of highly specific toxins, which allow the identification of novel therapeutic targets. We have now applied high content screening (HCS) microscopy to identify toxins that modulate pain sensitization signaling in primary sensory neurons of rat and elucidated the underlying mechanism. A set of venoms and fractions thereof were analyzed for their ability to activate type II protein kinase A (PKA-II) and extracellular signal-regulated kinases (ERK1/2). We identified MeuNaTxα-1, a sodium channel-selective scorpion α-toxin from Mesobuthus eupeus, which affected both PKA-II and ERK1/2. Recombinant MeuNaTxα-1 showed identical activity to the native toxin on mammalian voltage-gated sodium channels expressed in Xenopus laevis oocytes and induced thermal hyperalgesia in adult mice. The effect of MeuNaTxα-1 on sensory neurons was dose-dependent and tetrodotoxin-sensitive. Application of inhibitors and toxin mutants with altered sodium channel selectivity demonstrated that signaling activation in sensory neurons depends on NaV 1.2 isoform. Accordingly, the toxin was more potent in neurons from newborn rats, where NaV 1.2 is expressed at a higher level. Our results demonstrate that HCS microscopy-based monitoring of intracellular signaling is a novel and powerful tool to identify and characterize venoms and their toxins affecting sensory neurons.

Protein Kinase A Catalytic and Regulatory Subunits Interact Differently in Various Areas of Mouse Brain.

Protein kinase A (PKA) are tetramers of two catalytic and two regulatory subunits, docked at precise intracellular sites to provide localized phosphorylating activity, triggered by cAMP binding to regulatory subunits and subsequent dissociation of catalytic subunits. It is unclear whether in the brain PKA dissociated subunits may also be found. PKA catalytic subunit was examined in various mouse brain areas using immunofluorescence, equilibrium binding and western blot, to reveal its location in comparison to regulatory subunits type RI and RII. In the cerebral cortex, catalytic subunits colocalized with clusters of RI, yet not all RI clusters were bound to catalytic subunits. In stria terminalis, catalytic subunits were in proximity to RI but separated from them. Catalytic subunits clusters were also present in the corpus striatum, where RII clusters were detected, whereas RI clusters were absent. Upon cAMP addition, the distribution of regulatory subunits did not change, while catalytic subunits were completely released from regulatory subunits. Unpredictably, catalytic subunits were not solubilized; instead, they re-targeted to other binding sites within the tissue, suggesting local macromolecular reorganization. Hence, the interactions between catalytic and regulatory subunits of protein kinase A consistently vary in different brain areas, supporting the idea of multiple interaction patterns.

Roles of phosphodiesterases in the regulation of the cardiac cyclic nucleotide cross-talk signaling network.

The balanced signaling between the two cyclic nucleotides (cNs) cAMP and cGMP plays a critical role in regulating cardiac contractility. Their degradation is controlled by distinctly regulated phosphodiesterase isoenzymes (PDEs), which in turn are also regulated by these cNs. As a result, PDEs facilitate communication between the ß-adrenergic and Nitric Oxide (NO)/cGMP/Protein Kinase G (PKG) signaling pathways, which regulate the synthesis of cAMP and cGMP respectively. The phenomena in which the cAMP and cGMP pathways influence the dynamics of each other are collectively referred to as cN cross-talk. However, the cross-talk response and the individual roles of each PDE isoenzyme in shaping this response remain to be fully characterized. We have developed a computational model of the cN cross-talk network that mechanistically integrates the ß-adrenergic and NO/cGMP/PKG pathways via regulation of PDEs by both cNs. The individual model components and the integrated network model replicate experimentally observed activation-response relationships and temporal dynamics. The model predicts that, due to compensatory interactions between PDEs, NO stimulation in the presence of sub-maximal ß-adrenergic stimulation results in an increase in cytosolic cAMP accumulation and corresponding increases in PKA-I and PKA-II activation; however, the potentiation is small in magnitude compared to that of NO activation of the NO/cGMP/PKG pathway. In a reciprocal manner, ß-adrenergic stimulation in the presence of sub-maximal NO stimulation results in modest cGMP elevation and corresponding increase in PKG activation. In addition, we demonstrate that PDE2 hydrolyzes increasing amounts of cAMP with increasing levels of ß-adrenergic stimulation, and hydrolyzes increasing amounts of cGMP with decreasing levels of NO stimulation. Finally, we show that PDE2 compensates for inhibition of PDE5 both in terms of cGMP and cAMP dynamics, leading to cGMP elevation and increased PKG activation, while maintaining whole-cell ß-adrenergic responses similar to that prior to PDE5 inhibition. By defining and quantifying reactions comprising cN cross-talk, the model characterizes the cross-talk response and reveals the underlying mechanisms of PDEs in this non-linear, tightly-coupled reaction system.

cAMP-dependent Protein Kinase (PKA) Signaling Is Impaired in the Diabetic Heart.

Diabetes mellitus causes cardiac dysfunction and heart failure that is associated with metabolic abnormalities and autonomic impairment. Autonomic control of ventricular function occurs through regulation of cAMP-dependent protein kinase (PKA). The diabetic heart has suppressed ß-adrenergic responsiveness, partly attributable to receptor changes, yet little is known about how PKA signaling is directly affected. Control and streptozotocin-induced diabetic mice were therefore administered 8-bromo-cAMP (8Br-cAMP) acutely to activate PKA in a receptor-independent manner, and cardiac hemodynamic function and PKA signaling were evaluated. In response to 8Br-cAMP treatment, diabetic mice had impaired inotropic and lusitropic responses, thus demonstrating postreceptor defects. This impaired signaling was mediated by reduced PKA activity and PKA catalytic subunit content in the cytoplasm and myofilaments. Compartment-specific loss of PKA was reflected by reduced phosphorylation of discrete substrates. In response to 8Br-cAMP treatment, the glycolytic activator PFK-2 was robustly phosphorylated in control animals but not diabetics. Control adult cardiomyocytes cultured in lipid-supplemented media developed similar changes in PKA signaling, suggesting that lipotoxicity is a contributor to diabetes-induced ß-adrenergic signaling dysfunction. This work demonstrates that PKA signaling is impaired in diabetes and suggests that treating hyperlipidemia is vital for proper cardiac signaling and function.

Localization of cystic fibrosis transmembrane conductance regulator signaling complexes in human salivary gland striated duct cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent protein kinase (PKA)-regulated Cl(-) channel, crucial for epithelial cell regulation of salt and water transport. Previous studies showed that ezrin, an actin binding and A-kinase anchoring protein (AKAP), facilitates association of PKA with CFTR. We used immunohistochemistry and immunogold transmission electron microscopy to localize CFTR, ezrin, and PKA type II regulatory (RII) and catalytic (C) subunits in striated duct cells of human parotid and submandibular glands. Immunohistochemistry localized the four proteins mainly to the apical membrane and the apical cytoplasm of striated duct cells. In acinar cells, ezrin localized to the luminal membrane, and PKA RII subunits were present in secretory granules, as previously described. Immunogold labeling showed that CFTR and PKA RII and C subunits were localized to the luminal membrane and associated with apical granules and vesicles of striated duct cells. Ezrin was present along the luminal membrane, on microvilli and along the junctional complexes between cells. Double labeling showed specific protein associations with apical granules and vesicles and along the luminal membrane. Ezrin, CFTR, and PKA RII and C subunits are co-localized in striated duct cells, suggesting the presence of signaling complexes that serve to regulate CFTR activity.

PKA-type I selective constrained peptide disruptors of AKAP complexes.

A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotemporal modulators of cAMP-dependent protein kinase activity in cells. Although AKAPs organize a plethora of diverse pathways, their cellular roles are often elusive due to the dynamic nature of these signaling complexes. AKAPs can interact with the type I or type II PKA holoenzymes by virtue of high-affinity interactions with the R-subunits. As a means to delineate AKAP-mediated PKA signaling in cells, we sought to develop isoform-selective disruptors of AKAP signaling. Here, we report the development of conformationally constrained peptides named RI-STapled Anchoring Disruptors (RI-STADs) that target the docking/dimerization domain of the type 1 regulatory subunit of PKA. These high-affinity peptides are isoform-selective for the RI isoforms, can outcompete binding by the classical AKAP disruptor Ht31, and can selectively displace RIα, but not RIIα, from binding the dual-specific AKAP149 complex. Importantly, these peptides are cell-permeable and disrupt Type I PKA-mediated phosphorylation events in the context of live cells. Hence, RI-STAD peptides are versatile cellular tools to selectively probe anchored type I PKA signaling events.

3α-diol administration decreases hippocampal PKA (II) mRNA expression and impairs Morris water maze performance in adult male rats.

The effect of testosterone and its metabolites on learning and memory has been the subject of many studies. This study used the Morris water maze task to investigate the effect of intra-hippocampal injection of 3α-diol (one of the metabolites of testosterone) on acquisition stage of spatial memory in adult male rats. During the experiment we observed that 3α-diol, significantly impaired Morris water maze performance in treated rat's compared with controls. Because signaling event mediated by protein kinase A (PKA) especially PKA (II) are critical for many neuronal functions such as learning and memory, the hippocampus was analyzed for mRNA expression of PKA (II) using TaqMan real time RT-PCR. The results indicated that the transcription levels of PKA (II) were significantly decreased in animals treated with 3α-diol compared with controls. Thus, the findings suggest that administration of 3α-diol in hippocampus of adult male rats impairs memory function, possibly via down-regulation of PKA.

Microtubule-associated type II protein kinase A is important for neurite elongation.

Neuritogenesis is a process through which neurons generate their widespread axon and dendrites. The microtubule cytoskeleton plays crucial roles throughout neuritogenesis. Our previous study indicated that the amount of type II protein kinase A (PKA) on microtubules significantly increased upon neuronal differentiation and neuritogenesis. While the overall pool of PKA has been shown to participate in various neuronal processes, the function of microtubule-associated PKA during neuritogenesis remains largely unknown. First, we showed that PKA localized to microtubule-based region in different neurons. Since PKA is essential for various cellular functions, globally inhibiting PKA activity will causes a wide variety of phenotypes in neurons. To examine the function of microtubule-associated PKA without changing the total PKA level, we utilized the neuron-specific PKA anchoring protein MAP2. Overexpressing the dominant negative MAP2 construct that binds to type II PKA but cannot bind to the microtubule cytoskeleton in dissociated hippocampal neurons removed PKA from microtubules and resulted in compromised neurite elongation. In addition, we demonstrated that the association of PKA with microtubules can also enhance cell protrusion using the non-neuronal P19 cells. Overexpressing a MAP2 deletion construct which does not target PKA to the microtubule cytoskeleton caused non-neuronal cells to generate shorter cell protrusions than control cells overexpressing wild-type MAP2 that anchors PKA to microtubules. Finally, we demonstrated that the ability of microtubule-associated PKA to promote protrusion elongation was independent of MAP2 phosphorylation. This suggests other proteins in close proximity to the microtubule cytoskeleton are involved in this process.

Non-steroidal anti-inflammatory drugs activate NADPH oxidase in adipocytes and raise the H2O2 pool to prevent cAMP-stimulated protein kinase a activation and inhibit lipolysis.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) -aspirin, naproxen, nimesulide, and piroxicam- lowered activation of type II cAMP-dependent protein kinase A (PKA-II) in isolated rat adipocytes, decreasing adrenaline- and dibutyryl cAMP (Bt2cAMP)-stimulated lipolysis. The molecular bases of insulin-like actions of NSAID were studied. RESULTS: Based on the reported inhibition of lipolysis by H2O2, catalase was successfully used to block NSAID inhibitory action on Bt2cAMP-stimulated lipolysis. NSAID, at (sub)micromolar range, induced an H2O2 burst in rat adipocyte plasma membranes and in whole adipocytes. NSAID-mediated rise of H2O2 was abrogated in adipocyte plasma membranes by: diphenyleneiodonium, an inhibitor of NADPH oxidase (NOX); the NOX4 antibody; and cytochrome c, trapping the NOX-formed superoxide. These three compounds prevented the inhibition of Bt2cAMP-stimulated lipolysis by NSAIDs. Inhibition of aquaporin-mediated H2O2 transport with AgNO3 in adipocytes allowed NOX activation but prevented the lipolysis inhibition promoted by NSAID: i.e., once synthesized, H2O2 must reach the lipolytic machinery. Since insulin inhibits adrenaline-stimulated lipolysis, the effect of aspirin on isoproterenol-stimulated lipolysis in rat adipocytes was studied. As expected, isoproterenol-mediated lipolysis was blunted by both insulin and aspirin. CONCLUSIONS: NSAIDs activate NOX4 in adipocytes to produce H2O2, which impairs cAMP-dependent PKA-II activation, thus preventing isoproterenol-activated lipolysis. H2O2 signaling in adipocytes is a novel and important cyclooxygenase-independent effect of NSAID.

Activation of both protein kinase A (PKA) type I and PKA type II isozymes is required for retinoid-induced maturation of acute promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) is characterized by granulopoietic differentiation arrest at the promyelocytic stage. In most cases, this defect can be overcome by treatment with all-trans-retinoic acid (ATRA), leading to complete clinical remission. Cyclic AMP signaling has a key role in retinoid treatment efficacy: it enhances ATRA-induced maturation in ATRA-sensitive APL cells (including NB4 cells) and restores it in some ATRA-resistant cells (including NB4-LR1 cells). We show that the two cell types express identical levels of the Cα catalytic subunit and comparable global cAMP-dependent protein kinase A (PKA) enzyme activity. However, the maturation-resistant NB4-LR1 cells have a PKA isozyme switch: compared with the NB4 cells, they have decreased content of the juxtanuclearly located PKA regulatory subunit IIα and PKA regulatory subunit IIß, and a compensatory increase of the generally cytoplasmically distributed PKA-RIα. Furthermore, the PKA regulatory subunit II exists mainly in the less cAMP-responsive nonautophosphorylated state in the NB4-LR1 cells. By the use of isozyme-specific cAMP analog pairs, we show that both PKA-I and PKA-II must be activated to achieve maturation in NB4-LR1 as well as NB4 cells. Therefore, special attention should be paid to activating not only PKA-I but also PKA-II in attempts to enhance ATRA-induced APL maturation in a clinical setting.

Cyclic AMP can promote APL progression and protect myeloid leukemia cells against anthracycline-induced apoptosis.

We show that cyclic AMP (cAMP) elevating agents protect blasts from patients with acute promyelocytic leukemia (APL) against death induced by first-line anti-leukemic anthracyclines like daunorubicin (DNR). The cAMP effect was reproduced in NB4 APL cells, and shown to depend on activation of the generally cytoplasmic cAMP-kinase type I (PKA-I) rather than the perinuclear PKA-II. The protection of both NB4 cells and APL blasts was associated with (inactivating) phosphorylation of PKA site Ser118 of pro-apoptotic Bad and (activating) phosphorylation of PKA site Ser133 of the AML oncogene CREB. Either event would be expected to protect broadly against cell death, and we found cAMP elevation to protect also against 2-deoxyglucose, rotenone, proteasome inhibitor and a BH3-only mimetic. The in vitro findings were mirrored by the findings in NSG mice with orthotopic NB4 cell leukemia. The mice showed more rapid disease progression when given cAMP-increasing agents (prostaglandin E2 analog and theophylline), both with and without DNR chemotherapy. The all-trans retinoic acid (ATRA)-induced terminal APL cell differentiation is a cornerstone in current APL treatment and is enhanced by cAMP. We show also that ATRA-resistant APL cells, believed to be responsible for treatment failure with current ATRA-based treatment protocols, were protected by cAMP against death. This suggests that the beneficial pro-differentiating and non-beneficial pro-survival APL cell effects of cAMP should be weighed against each other. The results suggest also general awareness toward drugs that can affect bone marrow cAMP levels in leukemia patients.

PTEN directly activates the actin depolymerization factor cofilin-1 during PGE2-mediated inhibition of phagocytosis of fungi.

Macrophage ingestion of the yeast Candida albicans requires its recognition by multiple receptors and the activation of diverse signaling programs. Synthesis of the lipid mediator prostaglandin E(2) (PGE(2)) and generation of cyclic adenosine monophosphate (cAMP) also accompany this process. Here, we characterized the mechanisms underlying PGE(2)-mediated inhibition of phagocytosis and filamentous actin (F-actin) polymerization in response to ingestion of C. albicans by alveolar macrophages. PGE(2) suppressed phagocytosis and F-actin formation through the PGE(2) receptors EP2 and EP4, cAMP, and activation of types I and II protein kinase A. Dephosphorylation and activation of the actin depolymerizing factor cofilin-1 were necessary for these inhibitory effects of PGE(2). PGE(2)-dependent activation of cofilin-1 was mediated by the protein phosphatase activity of PTEN (phosphatase and tensin homolog deleted on chromosome 10), with which it directly associated. Because enhanced production of PGE(2) accompanies many immunosuppressed states, the PTEN-dependent pathway described here may contribute to impaired antifungal defenses.

Cyclic AMP induces IPC leukemia cell apoptosis via CRE-and CDK-dependent Bim transcription.

The IPC-81 cell line is derived from the transplantable BNML model of acute myelogenic leukemia (AML), known to be a reliable predictor of the clinical efficiency of antileukemic agents, like the first-line AML anthracycline drug daunorubicin (DNR). We show here that cAMP acted synergistically with DNR to induce IPC cell death. The DNR-induced death differed from that induced by cAMP by (1) not involving Bim induction, (2) being abrogated by GSK3ß inhibitors, (3) by being promoted by the HSP90/p23 antagonist geldanamycin and truncated p23 and (4) by being insensitive to the CRE binding protein (CREB) antagonist ICER and to cyclin-dependent protein kinase (CDK) inhibitors. In contrast, the apoptosis induced by cAMP correlated tightly with Bim protein expression. It was abrogated by Bim (BCL2L11) downregulation, whether achieved by the CREB antagonist ICER, by CDK inhibitors, by Bim-directed RNAi, or by protein synthesis inhibitor. The forced expression of BimL killed IPC-81(WT) cells rapidly, Bcl2-overexpressing cells being partially resistant. The pivotal role of CREB and CDK activity for Bim transcription is unprecedented. It is also noteworthy that newly developed cAMP analogs specifically activating PKA isozyme I (PKA-I) were able to induce IPC cell apoptosis. Our findings support the notion that AML cells may possess targetable death pathways not exploited by common anti-cancer agents.

PKA regulatory subunits mediate synergy among conserved G-protein-coupled receptor cascades.

G-protein-coupled receptors sense extracellular chemical or physical stimuli and transmit these signals to distinct trimeric G-proteins. Activated Gα-proteins route signals to interconnected effector cascades, thus regulating thresholds, amplitudes and durations of signalling. Gαs- or Gαi-coupled receptor cascades are mechanistically conserved and mediate many sensory processes, including synaptic transmission, cell proliferation and chemotaxis. Here we show that a central, conserved component of Gαs-coupled receptor cascades, the regulatory subunit type-II (RII) of protein kinase A undergoes adenosine 3'-5'-cyclic monophosphate (cAMP)-dependent binding to Gαi. Stimulation of a mammalian Gαi-coupled receptor and concomitant cAMP-RII binding to Gαi, augments the sensitivity, amplitude and duration of Gαi:ßγ activity and downstream mitogen-activated protein kinase signalling, independent of protein kinase A kinase activity. The mechanism is conserved in budding yeast, causing nutrient-dependent modulation of a pheromone response. These findings suggest a direct mechanism by which coincident activation of Gαs-coupled receptors controls the precision of adaptive responses of activated Gαi-coupled receptor cascades.

Protein kinase A activity and anchoring are required for ovarian cancer cell migration and invasion.

Epithelial ovarian cancer (EOC) is the deadliest of the gynecological malignancies, due in part to its clinically occult metastasis. Therefore, understanding the mechanisms governing EOC dissemination and invasion may provide new targets for antimetastatic therapies or new methods for detection of metastatic disease. The cAMP-dependent protein kinase (PKA) is often dysregulated in EOC. Furthermore, PKA activity and subcellular localization by A-kinase anchoring proteins (AKAPs) are important regulators of cytoskeletal dynamics and cell migration. Thus, we sought to study the role of PKA and AKAP function in both EOC cell migration and invasion. Using the plasma membrane-directed PKA biosensor, pmAKAR3, and an improved migration/invasion assay, we show that PKA is activated at the leading edge of migrating SKOV-3 EOC cells, and that inhibition of PKA activity blocks SKOV-3 cell migration. Furthermore, we show that while the PKA activity within the leading edge of these cells is mediated by anchoring of type-II regulatory PKA subunits (RII), inhibition of anchoring of either RI or RII PKA subunits blocks cell migration. Importantly, we also show--for the first time--that PKA activity is up-regulated at the leading edge of SKOV-3 cells during invasion of a three-dimensional extracellular matrix and, as seen for migration, inhibition of either PKA activity or AKAP-mediated PKA anchoring blocks matrix invasion. These data are the first to demonstrate that the invasion of extracellular matrix by cancer cells elicits activation of PKA within the invasive leading edge and that both PKA activity and anchoring are required for matrix invasion. These observations suggest a role for PKA and AKAP activity in EOC metastasis.

Estrogen-dependent activation of neutral cholesterol ester hydrolase underlying gender difference of atherogenesis in apoE-/- mice.

OBJECTIVE: Mechanisms underlying gender difference of atherogenesis were investigated focusing on direct effects of estrogen on the artery. METHODS: First, male and female apoE(-/-) mice were fed an atherogenic diet for 16 weeks from 10 weeks of age. Second, female apoE(-/-) mice were ovariectomized (ovx) or sham operated at 8 weeks of age, and 2-weeks afterwards, one-third of each ovx-group received conjugated equine estrogens (CEE) (0, 2.5 or 5.0 µg/day) for 16 weeks. Atherosclerotic lesions were examined after experimental periods. To clarify anti-atherogenic effect of 17ß-estradiol (E2) on artery, neutral cholesteryl ester hydrolase (N-CEase) activity in aorta and peritoneal macrophages, and E2-treated J774A.1 cells were measured. RESULTS: First, atherosclerotic lesion in female mice was significantly less than male mice without any changes in serum lipids and lipoprotein profile. N-CEase activity in aorta and peritoneal macrophages in female mice was significantly higher than male mice. Second, atherosclerotic lesion in ovx-group was significantly greater than sham-group. CEE-replacement to ovx-group decreased atherosclerotic lesion in a dose-dependent manner. N-CEase activity in aorta and peritoneal macrophages was decreased in ovx-group compared to sham-group, and restored by CEE-replacement in macrophages. To study detailed mechanisms, J774A.1 cells were treated with E2. E2 significantly increased N-CEase activity, and cAMP-dependent protein kinase (A-kinase) type II activity and the protein in cytosol fraction without any changes of total protein of A-kinase type II. CONCLUSION: These results suggest that gender difference of atherogenesis is partly accounted for activation of N-CEase through estrogen-dependent translocation of A-kinase type II in macrophages.

Posttranscriptional activation of gene expression in Xenopus laevis oocytes by microRNA-protein complexes (microRNPs).

MicroRNA-protein complexes (microRNPs) can activate translation of target reporters and specific mRNAs in quiescent (i.e., G0) mammalian cell lines. Induced quiescent cells, like folliculated immature oocytes, have high levels of cAMP that activate protein kinase AII (PKAII) to maintain G0 and immature states. We report microRNA-mediated up-regulated expression of reporters in immature Xenopus laevis oocytes, dependent on Xenopus AGO or human AGO2 and on FXR1, as in mammalian cells. Importantly, we find that maintenance of cAMP levels and downstream PKAII signaling are required for microRNA-mediated up-regulated expression in oocytes. We identify an important, endogenous cell state regulator, Myt1 kinase, as a natural target of microRNA-mediated up-regulation in response to xlmiR16, ensuring maintenance of oocyte immaturity. Our data reveal the physiological relevance of cAMP/PKAII-controlled posttranscriptional gene expression activation by microRNAs in maintenance of the immature oocyte state.

Protein kinase A type I activates a CRE-element more efficiently than protein kinase A type II regardless of C subunit isoform.

BACKGROUND: Protein kinase A type I (PKAI) and PKAII are expressed in most of the eukaryotic cells examined. PKA is a major receptor for cAMP and specificity is achieved partly through tissue-dependent expression and subcellular localization of subunits with different biochemical properties. In addition posttranslational modifications help fine tune PKA activity, distribution and interaction in the cell. In spite of this the functional significance of two forms of PKA in one cell has not been fully determined. Here we have tested the ability of PKAI and PKAII formed by expression of the regulatory (R) subunits RIα or RIIα in conjunction with Cα1 or Cß2 to activate a co-transfected luciferace reporter gene, controlled by the cyclic AMP responsive element-binding protein (CREB) in vivo. RESULTS: We show that PKAI when expressed at equal levels as PKAII was significantly (p < 0.01) more efficient in inducing Cre-luciferace activity at saturating concentrations of cAMP. This result was obtained regardless of catalytic subunit identity. CONCLUSION: We suggest that differential effects of PKAI and PKAII in inducing Cre-luciferace activity depend on R and not C subunit identity.