AMP Protein Kinase Research Articles

CAMP regulates expression of the cyclic nucleotide transporter MRP4 (ABCC4) through the EPAC pathway.

Multidrug resistance protein 4 (MRP4/ABCC4) has been established as an independent regulator of cyclic AMP (cAMP) levels particularly in vascular smooth muscle cells and in hematopoietic cells. Here, we assessed whether cAMP in turn regulates MRP4. A significant upregulation of MRP4 mRNA and protein by long-term treatment with cAMP-enhancing agents was observed in HeLa cells, smooth muscle cells, and megakaryoblastic leukemia M07e cells. This upregulation was not affected by inhibition of protein kinase A, but could be reverted by inhibitors and siRNA of an alternative cAMP-signaling route involving exchange proteins activated by cyclic AMP (EPAC) and mitogen-activated protein kinases. A selective EPAC activator could equally induce MRP4. The transcriptional regulation was confirmed in a luciferase reporter gene assay using a vector containing a 1494-bp fragment of the promoter region of the MRP4/ABCC4 gene. Our results suggest that enhanced cAMP levels upregulate MRP4 expression, which can result in increased cAMP efflux.

A polysaccharide from Ganoderma atrum inhibits tumor growth by induction of apoptosis and activation of immune response in CT26-bearing mice.

Ganoderma atrum is one species of edible and pharmaceutical mushroom with various biological activities. Recently, a novel polysaccharide, PSG-1, was purified from G. atrum. The antitumor activity and its mechanism of action were studied. In vitro, PSG-1 has little effect on inhibiting proliferation of CT26 tumor cells. However, the tumor size was significantly decreased in PSG-1-treated mice. The results showed that PSG-1 induced apoptosis in CT26 cells. Moreover, the intracellular cyclic AMP (cAMP) level and protein kinase A (PKA) activity were markedly increased in PSG-1-treated mice. In contrast, the contents of cyclic GMP and DAG and the PKC activity were decreased. Similarly, the expression of PKA protein was upregulated, while PKC protein expression in PSG-1-treated group was lowered. Additionally, PSG-1 increased the immune organ index and serum biochemistry parameter. In general, PSG-1 enhances the antitumor immune response, induces apoptosis in CT26-bearing mice, and could be a safe and effective adjuvant for tumor therapy or functional food.

Ex vivo molecular rejuvenation improves the therapeutic activity of senescent human cardiac stem cells in a mouse model of myocardial infarction.

Cardiac stem cells (CSC) from explanted decompensated hearts (E-CSC) are, with respect to those obtained from healthy donors (D-CSC), senescent and functionally impaired. We aimed to identify alterations in signaling pathways that are associated with CSC senescence. Additionally, we investigated if pharmacological modulation of altered pathways can reduce CSC senescence in vitro and enhance their reparative ability in vivo. Measurement of secreted factors showed that E-CSC release larger amounts of proinflammatory cytokine IL1β compared with D-CSC. Using blocking antibodies, we verified that IL1β hampers the paracrine protective action of E-CSC on cardiomyocyte viability. IL1β acts intracranially inducing IKKβ signaling, a mechanism that via nuclear factor-κB upregulates the expression of IL1β itself. Moreover, E-CSC show reduced levels of AMP protein kinase (AMPK) activating phosphorylation. This latter event, together with enhanced IKKβ signaling, increases TORC1 activity, thereby impairing the autophagic flux and inhibiting the phosphorylation of Akt and cAMP response element-binding protein. The combined use of rapamycin and resveratrol enhanced AMPK, thereby restoring downstream signaling and reducing IL1β secretion. These molecular corrections reduced E-CSC senescence, re-establishing their protective activity on cardiomyocytes. Moreover ex vivo treatment with rapamycin and resveratrol improved E-CSC capacity to induce cardiac repair upon injection in the mouse infarcted heart, leading to reduced cardiomyocyte senescence and apoptosis and increased abundance of endogenous c-Kit(+) CSC in the peri-infarct area. Molecular rejuvenation of patient-derived CSC by short pharmacologic conditioning boosts their in vivo reparative abilities. This approach might prove useful for refinement of CSC-based therapies.

Heterozygous mutations in cyclic AMP phosphodiesterase-4D (PDE4D) and protein kinase A (PKA) provide new insights into the molecular pathology of acrodysostosis

Acrodysostosis without hormone resistance is a rare skeletal disorder characterized by brachydactyly, nasal hypoplasia, mental retardation and occasionally developmental delay. Recently, loss-of-function mutations in the gene encoding cAMP-hydrolyzing phosphodiesterase-4D (PDE4D) have been reported to cause this rare condition but the pathomechanism has not been fully elucidated. To understand the pathogenetic mechanism of PDE4D mutations, we conducted 3D modeling studies to predict changes in the binding efficacy of cAMP to the catalytic pocket in PDE4D mutants. Our results indicated diminished enzyme activity in the two mutants we analyzed (Gly673Asp and Ile678Thr; based on PDE4D4 residue numbering). Ectopic expression of PDE4D mutants in HEK293 cells demonstrated this reduction in activity, which was identified by increased cAMP levels. However, the cells from an acrodysostosis patient showed low cAMP accumulation, which resulted in a decrease in the phosphorylated cAMP Response Element-Binding Protein (pCREB)/CREB ratio. The reason for this discrepancy was due to a compensatory increase in expression levels of PDE4A and PDE4B isoforms, which accounted for the paradoxical decrease in cAMP levels in the patient cells expressing mutant isoforms with a lowered PDE4D activity. Skeletal radiographs of 10-week-old knockout (KO) rats showed that the distal part of the forelimb was shorter than in wild-type (WT) rats and that all the metacarpals and phalanges were also shorter in KO, as the name acrodysostosis implies. Like the G-protein α-stimulatory subunit and PRKAR1A, PDE4D critically regulates the cAMP signal transduction pathway and influences bone formation in a way that activity-compromising PDE4D mutations can result in skeletal dysplasia. We propose that specific inhibitory PDE4D mutations can lead to the molecular pathology of acrodysostosis without hormone resistance but that the pathological phenotype may well be dependent on an over-compensatory induction of other PDE4 isoforms that can be expected to be targeted to different signaling complexes and exert distinct effects on compartmentalized cAMP signaling.

Identification of 5′ AMP-activated Kinase as a Target of Reactive Aldehydes during Chronic Ingestion of High Concentrations of Ethanol

The production of reactive aldehydes including 4-hydroxy-2-nonenal (4-HNE) is a key component of the pathogenesis in a spectrum of chronic inflammatory hepatic diseases including alcoholic liver disease (ALD). One consequence of ALD is increased oxidative stress and altered β-oxidation in hepatocytes. A major regulator of β-oxidation is 5' AMP protein kinase (AMPK). In an in vitro cellular model, we identified AMPK as a direct target of 4-HNE adduction resulting in inhibition of both H2O2 and 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR)-induced downstream signaling. By employing biotin hydrazide capture, it was confirmed that 4-HNE treatment of cells resulted in carbonylation of AMPKα/β, which was not observed in untreated cells. Using a murine model of alcoholic liver disease, treatment with high concentrations of ethanol resulted in an increase in phosphorylated as well as carbonylated AMPKα. Despite increased AMPK phosphorylation, there was no significant change in phosphorylation of acetyl CoA carboxylase. Mass spectrometry identified Michael addition adducts of 4-HNE on Cys(130), Cys(174), Cys(227), and Cys(304) on recombinant AMPKα and Cys(225) on recombinant AMPKβ. Molecular modeling analysis of identified 4-HNE adducts on AMPKα suggest that inhibition of AMPK occurs by steric hindrance of the active site pocket and by inhibition of hydrogen peroxide induced oxidation. The observed inhibition of AMPK by 4-HNE provides a novel mechanism for altered β-oxidation in ALD, and these data demonstrate for the first time that AMPK is subject to regulation by reactive aldehydes in vivo.

Monosodium glutamate neurotoxicity increases beta amyloid in the rat hippocampus: A potential role for cyclic AMP protein kinase

BackgroundGlutamate excitotoxicity and cyclic AMP-activated protein kinase (AMPK) are both recognized as important mediators in neurodegenerative disorders including Alzheimer's disease (AD). ObjectivesTo investigate whether oral or subcutaneous monosodium glutamate (MSG) neurotoxicity mimics some features of AD and whether these can be reversed by the AMPK activator Pioglitazone. MethodsMale Wistar rats aged 5 weeks were administered oral or subcutaneous MSG for 10 days with or without daily oral Pioglitazone. Two additional groups given only saline orally or subcutaneously acted as controls. At age 10 weeks the rats were subjected to neurobehavioral testing, then sacrificed for measurement of AMPK, β-amyloid and Fas ligand in the hippocampus. ResultsOral and subcutaneous MSG both induced a lowering of hippocampal AMPK by 43% and 31% respectively (P<0.05 for both) and >2-fold increase in hippocampal Fas ligand, a mediator of apoptosis (P<0.001 for both). MSG treatment also induced a significant increase in β-amyloid in the hippocampus by >4-fold and >5-fold in the oral and subcutaneous groups. This was associated with increased latency before crossing to the white half in the black–white alley and before the first rear in the holeboard test, suggesting increased anxiety. Pioglitazone decreased hippocampal β-amyloid accumulation and Fas ligand, but did not ameliorate the neurobehavioural deficits induced by MSG. ConclusionsMSG treatment enhances β-amyloid accumulation in the rat hippocampus. Our results suggest a role for AMPK reduction in mediating the neurotoxic effects of glutamate, including β-amyloid accumulation.

14-Deoxyandrographolide alleviates ethanol-induced hepatosteatosis through stimulation of AMP-activated protein kinase activity in rats

Andrographis paniculata (AP) is a traditional medicinal plant of Ayurveda. It grows widely in Asia and is prescribed in the treatment of liver diseases. Here we have investigated the beneficial role of 14-deoxyandrographolide (14-DAG), a bioactive diterpenoid from AP, against alcoholic steatosis in rats. 14-DAG was extracted from aerial parts (leaves and stems) of AP. Rats were fed with ethanol for 8 weeks. Animals were treated with 14-DAG during the last 4 weeks of ethanol treatment. In vitro studies were undertaken in a human hepatocellular liver carcinoma cell line culture. Hepatosteatosis was assessed from histopathological studies of liver sections. Acetyl-CoA, malonyl-CoA, and triglyceride contents were determined using commercially available kits. Fatty acid synthesis was evaluated from incorporation of 1-14C acetate. Regulation of fatty acid oxidation and lipogenesis were monitored with immunoblotting and immunoprecipitation studies. Ethanol exposure led to hepatotoxicity, as evident from the marked enhancement in the levels of AST and ALT. The values decreased almost to control levels in response to 14-DAG treatment. Results showed that ethanol feeding induced deactivation of AMP-activated protein kinase (AMPK) that led to enhanced lipid synthesis and decreased fatty acid oxidation, culminating in hepatic fat accumulation. Treatment with 14-DAG activated AMPK through induction of cyclic AMP-protein kinase A pathway. Activation of AMPK was followed by down-regulation of sterol regulatory element binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase, leading to suppression of lipogenesis. This was associated with up-regulation of sirtuin 1 and depletion of malonyl-CoA, in favor of increased fatty acid oxidation. 14-DAG controlled ethanol-induced hepatosteatosis by interfering with dysregulation of lipid metabolism. In conclusion, our results indicated that 14-DAG was capable of preventing the development of fatty liver through AMPK-mediated regulation of lipid metabolism. This finding supported the hepatoprotective role of 14-DAG, which might serve as a therapeutic option to alleviate hepatosteatosis in chronic alcoholism.

AMP-protein kinase may protect against sepsis-induced acute kidney injury through modulation of immune response and endothelial activation

Organ injury is a hallmark of sepsis, in particular acute kidney injury (AKI). Yet the mechanisms involved in sepsis-induced AKI are not well understood. Energy prioritization is an important cell defense mechanism, and thus we hypothesized that exogenous activation of AMP-protein kinase (AMPK), a master regulator of cellular energy metabolism, protects against sepsis-induced AKI.

Abstract P2-09-15: Effect of [4-valine 5-glutamine] desmopressin, a novel vasopressin analog, on human breast cancer cell growth in two-dimensional (2D) and three-dimensional (3D) cultures

Abstract Background: Desmopressin (dDAVP), a synthetic nonapeptide derivative of arginine vasopressin, is a safe hemostatic compound that acts as a selective agonist for the vasopressin V2 membrane receptor (V2R). This receptor is expressed in endothelium and also in some tumor cells. V2R stimulation is associated with increased levels of cyclic AMP and cyclic AMP-dependent protein kinase activation. We previously reported that dDAVP can inhibit progression of residual metastatic cells in preclinical models. Among other mechanisms, the compound induces an agonist effect on V2R present in tumor cells. This nonapeptide was substituted in positions 4 and 5 searching for compounds with improved biological activity. Such positions belong to the conformational peptide loop which has a key role in ligand-receptor interaction. After an extensive in vitro and in vivo characterization, novel peptide compound [4-valine 5-glutamine] dDAVP ([V4Q5]dDAVP) exerted a remarkable and improved antitumor effect in comparison to parental peptide dDAVP. [V4Q5]dDAVP inhibited tumor growth in both syngeneic F3II and xenogeneic MDA-MB-231 breast cancer models, modulating tumor aggressiveness and vasculature. In this study, to further understand direct cytostatic action of this novel vasopressin analog on tumor cells, we assessed the effect of [V4Q5]dDAVP on clonogenic and three dimensional growth of V2R-expressing human breast carcinoma cell lines. Methods and results: We used MCF7 and triple negative MDA-MB-231 human breast cancer cell lines to conduct the experiments. Vasopressin V2 membrane receptor expression in tumor cell lines was assessed by RT-PCR and immunofluorescence. We first examined peptide ability to modify in vitro low density cell growth by the clonogenic growth assay. Incubation with [V4Q5]dDAVP (100-1500 nM) during one week significantly reduced the number of tumor cell colonies showing a concentration dependant manner and an IC50 value of 1135 nM. To assess the effect of novel analog on three dimensional growth of cancer cells we used two different assays. The first one evaluates the ability of basement membrane extract (BME)-embedded single cells to proliferate and form spheroids. The second assay evaluates growth of single fully established spheroids (Approx. 300 mm diameter) transplanted individually on a non-adherent surface. [V4Q5]dDAVP, at a concentration of 1000 nM, was able to significantly reduce the number of BME-embedded spheroids formed from single cells after 14 days of treatment. In a similar manner, incubation of established and isolated spheroids with [V4Q5]dDAVP using the same concentration for 9 days, caused a decrease in multicellular spheroids final diameter. Cellular metabolic activity analysis by the MTS assay showed no significant differences between treated and control groups. Conclusions: We report for the first time the inhibitory effect of [V4Q5]dDAVP on colony and spheroid formation and growth of human breast carcinoma cells. Our results using three dimensional models suggest that V2R stimulation in cancer cells may affect early stages of microtumor establishment. Further analysis of involved signaling pathways in treated 3D cultures is needed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-15.

Map4k4 suppresses Srebp-1 and adipocyte lipogenesis independent of JNK signaling

Adipose tissue lipogenesis is paradoxically impaired in human obesity, promoting ectopic triglyceride (TG) deposition, lipotoxicity, and insulin resistance. We previously identified mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a sterile 20 protein kinase reported to be upstream of c-Jun NH2-terminal kinase (JNK) signaling, as a novel negative regulator of insulin-stimulated glucose transport in adipocytes. Using full-genome microarray analysis we uncovered a novel role for Map4k4 as a suppressor of lipid synthesis. We further report here the surprising finding that Map4k4 suppresses adipocyte lipogenesis independently of JNK. Thus, while Map4k4 silencing in adipocytes enhances the expression of lipogenic enzymes, concomitant with increased conversion of (14)C-glucose and (14)C-acetate into TGs and fatty acids, JNK1 and JNK2 depletion causes the opposite effects. Furthermore, high expression of Map4k4 fails to activate endogenous JNK, while Map4k4 depletion does not attenuate JNK activation by tumor necrosis factor α. Map4k4 silencing in cultured adipocytes elevates both the total protein expression and cleavage of sterol-regulated element binding protein-1 (Srebp-1) in a rapamycin-sensitive manner, consistent with Map4k4 signaling via mechanistic target of rapamycin complex 1 (mTORC1). We show Map4k4 depletion requires Srebp-1 upregulation to increase lipogenesis and further show that Map4k4 promotes AMP-protein kinase (AMPK) signaling and the phosphorylation of mTORC1 binding partner raptor (Ser792) to inhibit mTORC1. Our results indicate that Map4k4 inhibits adipose lipogenesis by suppression of Srebp-1 in an AMPK- and mTOR-dependent but JNK-independent mechanism.

Importance of adrenergic pathways in women's cancers.

The importance of adrenergic pathways in cancer has long been suspected, but now there is mounting epidemiological, preclinical, and clinical evidence of its importance in gynecologic cancers. To date, most of these effects are mediated primarily through the beta 2 adrenergic receptor activation of the tumor cell cyclic AMP-protein kinase A signaling pathway. This review will discuss the current knowledge about the neuroendocrine stress response in gynecologic tumor biology.

Dissociation of rugose‐dependent short‐term memory component from memory consolidation in Drosophila

Extensive investigations show several molecular and neuroanatomical mechanisms underlying short-lived and long-lasting memory in Drosophila. At the molecular level, the genetic pathway of memory formation, which was obtained through mutant research, seems to occur sequentially. So far, studies of Drosophila mutants appear to support the idea that mutants defective in short-term memory (STM) are always associated with long-term memory (LTM) impairment. At the neuroanatomical level, distinct memory traces are partially independently distributed. However, whether memory phase dissociation also exists at the molecular level remains unclear. Here, we report on molecular separation of STM and consolidated memory through genetic dissection of rugose mutants. Mutants in the rugose gene, which encodes an evolutionarily conserved A-kinase anchor protein, show immediate memory defects as assayed through aversive olfactory conditioning. Intriguingly, two well-defined consolidated memory components, anesthesia-resistant memory and protein synthesis-dependent LTM, are both normal in spite of the defective immediate memory after 10-session massed and spaced training. Moreover, rugose genetically interacts with cyclic AMP-protein kinase A signaling during STM formation. Considering our previous study that AKAP Yu specifically participates in LTM formation, these results suggest that there exists a molecular level of memory phase dissociation with distinct AKAPs in Drosophila.

Modeling mutant phenotypes and oscillatory dynamics in the Saccharomyces cerevisiae cAMP-PKA pathway

Background The cyclic AMP-Protein Kinase A (cAMP-PKA) pathway is an evolutionarily conserved signal transduction mechanism that regulates cellular growth and differentiation in animals and fungi. We present a mathematical model that recapitulates the short-term and long-term dynamics of this pathway in the budding yeast, Saccharomyces cerevisiae. Our model is aimed at recapitulating the dynamics of cAMP signaling for wild-type cells as well as single (pde1Δ and pde2Δ) and double (pde1Δpde2Δ) phosphodiesterase mutants.Results Our model focuses on PKA-mediated negative feedback on the activity of phosphodiesterases and the Ras branch of the cAMP-PKA pathway. We show that both of these types of negative feedback are required to reproduce the wild-type signaling behavior that occurs on both short and long time scales, as well as the the observed responses of phosphodiesterase mutants. A novel feature of our model is that, for a wide range of parameters, it predicts that intracellular cAMP concentrations should exhibit decaying oscillatory dynamics in their approach to steady state following glucose stimulation. Experimental measurements of cAMP levels in two genetic backgrounds of S. cerevisiae confirmed the presence of decaying cAMP oscillations as predicted by the model.Conclusions Our model of the cAMP-PKA pathway provides new insights into how yeast respond to alterations in their nutrient environment. Because the model has both predictive and explanatory power it will serve as a foundation for future mathematical and experimental studies of this important signaling network.

The genetic architecture of biofilm formation in a clinical isolate of Saccharomyces cerevisiae.

Biofilms are microbial communities that form on surfaces. They are the primary form of microbial growth in nature and can have detrimental impacts on human health. Some strains of the budding yeast Saccharomyces cerevisiae form colony biofilms, and there is substantial variation in colony architecture between biofilm-forming strains. To identify the genetic basis of biofilm variation, we developed a novel version of quantitative trait locus mapping, which leverages cryptic variation in a clinical isolate of S. cerevisiae. We mapped 13 loci linked to heterogeneity in biofilm architecture and identified the gene most closely associated with each locus. Of these candidate genes, six are members of the cyclic AMP-protein kinase A pathway, an evolutionarily conserved cell signaling network. Principal among these is CYR1, which encodes the enzyme that catalyzes production of cAMP. Through a combination of gene expression measurements, cell signaling assays, and gene overexpression, we determined the functional effects of allelic variation at CYR1. We found that increased pathway activity resulting from protein coding and expression variation of CYR1 enhances the formation of colony biofilms. Four other candidate genes encode kinases and transcription factors that are targets of this pathway. The protein products of several of these genes together regulate expression of the sixth candidate, FLO11, which encodes a cell adhesion protein. Our results indicate that epistatic interactions between alleles with both positive and negative effects on cyclic AMP-protein kinase A signaling underlie much of the architectural variation we observe in colony biofilms. They are also among the first to demonstrate genetic variation acting at multiple levels of an integrated signaling and regulatory network. Based on these results, we propose a mechanistic model that relates genetic variation to gene network function and phenotypic outcomes.

Age-dependent alterations in cAMP signaling contribute to synaptic plasticity deficits following traumatic brain injury

The elderly have comparatively worse cognitive impairments from traumatic brain injury (TBI) relative to younger adults, but the molecular mechanisms that underlie this exacerbation of cognitive deficits are unknown. Experimental models of TBI have demonstrated that the cyclic AMP-protein kinase A (cAMP-PKA) signaling pathway is downregulated after brain trauma. Since the cAMP-PKA signaling pathway is a key mediator of long-term memory formation, we investigated whether the TBI-induced decrease in cAMP levels is exacerbated in aged animals. Aged (19months) and young adult (3months) male Fischer 344 rats received sham surgery or mild (1.4–1.6 atmospheres, atm) or moderate (1.7–2.1atm) parasagittal fluid-percussion brain injury. At various time points after surgery, the ipsilateral parietal cortex, hippocampus, and thalamus were assayed for cAMP levels. Mild TBI lowered cAMP levels in the hippocampus of aged, but not young adult animals. Moderate TBI lowered cAMP levels in the hippocampus and parietal cortex of both age groups. In the thalamus, cAMP levels were significantly lowered after moderate, but not mild TBI. To determine if the TBI-induced decreases in cAMP had physiological consequences in aged animals, hippocampal long-term potentiation (LTP) in the Schaffer collateral pathway of the CA1 region was assessed. LTP was significantly decreased in both young adult and aged animals after mild and moderate TBI as compared to sham surgery animals. Rolipram rescued the LTP deficits after mild TBI for young adult animals and caused a partial recovery for aged animals. However, rolipram did not rescue LTP deficits after moderate TBI in either young adult or aged animals. These results indicate that the exacerbation of cognitive impairments in aged animals with TBI may be due to decreased cAMP levels and deficits in hippocampal LTP.

Functional control of the Candida albicans cell wall by catalytic protein kinase A subunit Tpk1

The cyclic AMP protein kinase A pathway governs numerous biological features of the fungal pathogen Candida albicans. The catalytic protein kinase A subunits, Tpk1 (orf19.4892) and Tpk2 (orf19.2277), have divergent roles, and most studies indicate a more pronounced role for Tpk2. Here we dissect two Tpk1-responsive properties: adherence and cell wall integrity. Homozygous tpk1/tpk1 mutants are hyperadherent, and a Tpk1 defect enables biofilm formation in the absence of Bcr1, a transcriptional regulator of biofilm adhesins. A quantitative gene expression-based assay reveals that tpk1/tpk1 and bcr1/bcr1 genotypes show mixed epistasis, as expected if Tpk1 and Bcr1 act mainly in distinct pathways. Overexpression of individual Tpk1-repressed genes indicates that cell surface proteins Als1, Als2, Als4, Csh1 and Csp37 contribute to Tpk1-regulated adherence. Tpk1 is also required for cell wall integrity, but has no role in the gene expression response to cell wall inhibition by caspofungin. Interestingly, increased expression of the adhesin gene ALS2 confers a cell wall defect, as manifested in hypersensitivity to the cell wall inhibitor caspofungin and a shallow cell wall structure. Our findings indicate that Tpk1 governs C. albicans cell wall properties through repression of select cell surface protein genes.

Interactions Between the Kinetochore Complex and the Protein Kinase A Pathway inSaccharomyces cerevisiae

The kinetochore is a large structure composed of multiple protein subcomplexes that connect chromosomes to spindle microtubules to enable accurate chromosome segregation. Significant advances have been made in the identification of kinetochore proteins and elucidation of kinetochore structure; however, comparatively little is known about how cellular signals integrate with kinetochore function. In the budding yeast Saccharomyces cerevisiae, the cyclic AMP protein kinase A signaling pathway promotes cellular growth in response to glucose. In this study, we find that decreasing protein kinase A activity, either by overexpressing negative regulators of the pathway or deleting the upstream effector Ras2, improves the viability of ipl1 and spc24 kinetochore mutants. Ipl1/Aurora B is a highly conserved kinase that corrects attachment of sister kinetochores that have attached to the same spindle pole, whereas Spc24 is a component of the conserved Ndc80 kinetochore complex that attaches directly to microtubules. Unexpectedly, we find that kinetochore mutants have increased phosphorylation levels of protein kinase A substrates, suggesting that the cyclic AMP protein kinase A signaling pathway is stimulated. The increase in protein kinase A activity in kinetochore mutants is not induced by activation of the spindle checkpoint or a metaphase delay because protein kinase A activity remains constant during an unperturbed cell cycle. Finally, we show that lowering protein kinase A activity can rescue the chromosome loss defect of the inner kinetochore ndc10 mutant. Overall, our data suggest that the increased protein kinase A activity in kinetochore mutants is detrimental to cellular growth and chromosome transmission fidelity.

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

McCune Albright syndrome

Inheritance MAS is not inherited. It is a sporadic genetic disorder, caused by a mutation in the GNAS gene encoding the alpha subunit of the stimulatory G protein (GSalpha), cyclic AMP protein kinase dependent cellular signaling pathway involving G protein coupled receptors. An arginine or occasionally serine, leucine or glycine to histidine transposition at residue 201 attenuates GTpase activity. This results in constitutive activation of adenylyl cyclase. The somatic mutation occurs in early embryogenesis, resulting in widespread tissue distribution of abnormalities. The post zygotic mutation is responsible for the mosaic pattern of tissue distribution and the extreme variability of clinical changes, varying from multi system disease to almost unrecognized disorders with single organ involvement. Increased adenylate cyclase activity in bone from clinical fibrous dysplastic lesions is caused by expression of Cfos proto-oncogene in bone. Recent identification of increased fibroblast growth factor (FGF23) activity in fibrous dysplastic lesions has linked the bony abnormalities to a recognized marker of bone metabolism.

AMPK Potentiates Hypertonicity-induced Apoptosis by Suppressing NFκB/COX-2 in Medullary Interstitial Cells

Cells residing in the hypertonic, hypoxic renal medulla depend on dynamic adaptation mechanisms to respond to changes in energy supply and demand. The serine/threonine kinase 5'-AMP protein kinase (AMPK) is a sensor of cellular energy status, but whether it contributes to the survival of cells in the renal medulla is unknown. Here, hypertonic conditions induced a decrease in AMPK phosphorylation within 12 hours in renal medullary interstitial cells (RMIC), followed by a gradual return to baseline levels. Activation of AMPK markedly increased hypertonicity-induced apoptosis of RMICs and suppressed both hypertonicity-induced NFκB nuclear translocation and cyclooxygenase-2 (COX-2) activation; overexpression of COX-2 significantly attenuated these effects. AMPK activation also markedly reduced generation of reactive oxygen species and nuclear expression of tonicity-responsive enhancer-binding protein, which prevented upregulation of osmoprotective genes. In vivo, pharmacologic activation of AMPK led to massive apoptosis of RMICs and renal dysfunction in the setting of water deprivation in mice. Taken together, these results identify a critical role for AMPK in the maintenance of RMIC viability and suggest that AMPK modulates the NFκB-COX-2 survival pathway in the renal medulla. Furthermore, this study raises safety concerns for the development of AMPK activators as anti-diabetic drugs, especially for patients prone to dehydration.