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1.
FEBS J ; 288(7): 2418-2435, 2021 04.
Article in English | MEDLINE | ID: mdl-33051988

ABSTRACT

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.


Subject(s)
Cyclic AMP-Dependent Protein Kinase Type II/genetics , NAV1.2 Voltage-Gated Sodium Channel/genetics , Pain/genetics , Voltage-Gated Sodium Channels/genetics , Animals , Animals, Newborn , Humans , Hyperalgesia/genetics , Hyperalgesia/pathology , MAP Kinase Signaling System/drug effects , Mice , Oocytes/drug effects , Oocytes/growth & development , Rats , Scorpion Venoms/chemistry , Scorpion Venoms/pharmacology , Scorpions/chemistry , Sensory Receptor Cells , Xenopus laevis/growth & development
2.
Int J Mol Sci ; 21(9)2020 Apr 26.
Article in English | MEDLINE | ID: mdl-32357495

ABSTRACT

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.


Subject(s)
Brain/enzymology , Cyclic AMP-Dependent Protein Kinase Type II/metabolism , Cyclic AMP-Dependent Protein Kinase Type I/metabolism , Animals , Cerebral Cortex/enzymology , Corpus Striatum/enzymology , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinase Type I/genetics , Cyclic AMP-Dependent Protein Kinase Type II/genetics , Female , Male , Mice , Organ Specificity , Septal Nuclei/enzymology
3.
J Mol Cell Cardiol ; 91: 215-27, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26773602

ABSTRACT

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.


Subject(s)
Cyclic AMP/metabolism , Cyclic GMP/metabolism , Gene Regulatory Networks , Models, Cardiovascular , Myocardium/enzymology , Myocytes, Cardiac/enzymology , Phosphoric Diester Hydrolases/genetics , Animals , Computer Simulation , Cyclic AMP-Dependent Protein Kinase Type I/genetics , Cyclic AMP-Dependent Protein Kinase Type I/metabolism , Cyclic AMP-Dependent Protein Kinase Type II/genetics , Cyclic AMP-Dependent Protein Kinase Type II/metabolism , Cyclic GMP-Dependent Protein Kinases/genetics , Cyclic GMP-Dependent Protein Kinases/metabolism , Gene Expression Regulation , Isoenzymes/genetics , Isoenzymes/metabolism , Myocardial Contraction , Myocardium/cytology , Myocytes, Cardiac/cytology , Nitric Oxide/metabolism , Phosphoric Diester Hydrolases/metabolism , Signal Transduction
4.
Cell Death Dis ; 4: e516, 2013 Feb 28.
Article in English | MEDLINE | ID: mdl-23449452

ABSTRACT

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.


Subject(s)
Antibiotics, Antineoplastic/pharmacology , Apoptosis/drug effects , Cyclic AMP/metabolism , Daunorubicin/pharmacology , 1-Methyl-3-isobutylxanthine/pharmacology , Animals , Antibiotics, Antineoplastic/therapeutic use , Cell Line, Tumor , Cyclic AMP/agonists , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinase Type I/metabolism , Cyclic AMP-Dependent Protein Kinase Type II/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinase Type II/genetics , Cyclic AMP-Dependent Protein Kinase Type II/metabolism , Daunorubicin/therapeutic use , Dinoprostone/analogs & derivatives , Dinoprostone/pharmacology , Dinoprostone/therapeutic use , Disease Progression , HL-60 Cells , Humans , Leukemia, Promyelocytic, Acute/drug therapy , Leukemia, Promyelocytic, Acute/metabolism , Leukemia, Promyelocytic, Acute/pathology , Male , Mice , Mice, Inbred NOD , Mice, SCID , RNA Interference , RNA, Small Interfering/metabolism , Theophylline/pharmacology , Theophylline/therapeutic use , Transplantation, Heterologous , Tretinoin/pharmacology , Tretinoin/therapeutic use , bcl-Associated Death Protein/metabolism
5.
Nat Commun ; 2: 598, 2011 Dec 20.
Article in English | MEDLINE | ID: mdl-22186894

ABSTRACT

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.


Subject(s)
Adaptation, Physiological/genetics , Cyclic AMP-Dependent Protein Kinase Type II/metabolism , Gene Expression Regulation, Fungal/physiology , Receptors, G-Protein-Coupled/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Signal Transduction/physiology , Cloning, Molecular , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinase Type II/genetics , Escherichia coli , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein alpha Subunits/metabolism , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mutation , Phosphorylation , Plasmids , Protein Binding , Protein Subunits/genetics , Protein Subunits/metabolism , Receptors, G-Protein-Coupled/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Transformation, Bacterial
6.
BMC Biochem ; 12: 7, 2011 Feb 08.
Article in English | MEDLINE | ID: mdl-21303506

ABSTRACT

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.


Subject(s)
Cyclic AMP-Dependent Protein Kinase Type II/metabolism , Cyclic AMP-Dependent Protein Kinase Type I/metabolism , Gene Expression Regulation , Response Elements , Cyclic AMP/metabolism , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinase Type I/genetics , Cyclic AMP-Dependent Protein Kinase Type II/genetics , Genes, Reporter , HEK293 Cells , Humans , Luciferases/genetics , Luciferases/metabolism , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism
7.
Protein Pept Lett ; 17(5): 646-59, 2010 May.
Article in English | MEDLINE | ID: mdl-20441558

ABSTRACT

Two binary complexes (KAP2-C subunit and cAMP-bound KAP2) were built, to investigate molecular interaction. The binding sites of KAP2 include the acidic sequence motif (Asp73-Glu87), the inhibitor peptide/linker region (Arg93-Val118), and beta barrel of cAMP-binding domains (CBD-A/B). The binding surface on the C subunit anchoring KAP2 extends to the inhibitor binding site at the active site cleft (Glu127-Glu230), Pro243-Ser252 helix and the phosphorylated activation loop (Arg194-Thr201) of the large lobe besides some sites in the small lobe. KAP2 undergoes major conformational changes in comparison of the two complexes above, especially the linker region and Met251 at Arg234-Phe252 helix as an inflexion point of the turnaround. Additionally, the interaction between KAP2 and cAMP concentrates on two catalytic motifs (FGELAL and PRAA) of phosphate binding cassette regions and the cyclic-monophosphate and ribose of cAMP. On the other hand, WAVE1 of BAD complex maybe interacts with the D/D domain of KAP2 by each of three helical motifs (Asn24-Lys46, Pro492-Val514, and Glu525-Glu547). This is helpful for our research of molecular mechanism of PKA and further analysis of BAD complex how to modulate glycolysis and apoptosis.


Subject(s)
Cyclic AMP-Dependent Protein Kinase Type II/chemistry , Hepatocytes/enzymology , Molecular Dynamics Simulation , Amino Acid Sequence , Catalytic Domain , Cyclic AMP , Cyclic AMP-Dependent Protein Kinase Type II/genetics , Cyclic AMP-Dependent Protein Kinase Type II/metabolism , Hepatocytes/chemistry , Humans , Molecular Sequence Data , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Secondary , Sequence Alignment
8.
FEBS Lett ; 584(5): 873-7, 2010 Mar 05.
Article in English | MEDLINE | ID: mdl-20138877

ABSTRACT

The myeloid translocation gene (MTG) homologue Nervy associates with PlexinA on the plasma membrane, where it functions as an A-kinase anchoring protein (AKAP) to modulate plexin-mediated semaphorin signaling in Drosophila. Mammalian MTG16b is an AKAP found in immune cells where plexin-mediated semaphorin signaling regulates immune responses. This study provides the first evidence that MTG16b is a dual AKAP capable of binding plexins. These interactions are selective (PlexinA1 and A3 bind MTG, while PlexinB1 does not) and can be regulated by PKA-phosphorylation. Collectively, these data suggest a possible mechanism for the targeting and integration of adenosine 3',5'-cyclic monophosphate (cAMP) and semaphorin signaling in immune cells.


Subject(s)
A Kinase Anchor Proteins/metabolism , Cell Adhesion Molecules/metabolism , Nerve Tissue Proteins/metabolism , Phosphoproteins/metabolism , Repressor Proteins/metabolism , Tumor Suppressor Proteins/metabolism , A Kinase Anchor Proteins/genetics , Animals , COS Cells , Cell Adhesion Molecules/genetics , Chlorocebus aethiops , Cyclic AMP-Dependent Protein Kinase Type I/genetics , Cyclic AMP-Dependent Protein Kinase Type I/metabolism , Cyclic AMP-Dependent Protein Kinase Type II/genetics , Cyclic AMP-Dependent Protein Kinase Type II/metabolism , Humans , Immunoblotting , Immunoprecipitation , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Nerve Tissue Proteins/genetics , Phosphoproteins/genetics , Phosphorylation , Protein Binding , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Repressor Proteins/genetics , Tumor Suppressor Proteins/genetics
9.
J Biol Chem ; 284(3): 1583-92, 2009 Jan 16.
Article in English | MEDLINE | ID: mdl-18945669

ABSTRACT

Protein kinase A (PKA)-dependent phosphorylation is regulated by targeting of PKA to its substrate as a result of binding of regulatory subunit, R, to A-kinase-anchoring proteins (AKAPs). We investigated the effects of disrupting PKA targeting to AKAPs in the heart by expressing the 24-amino acid regulatory subunit RII-binding peptide, Ht31, its inactive analog, Ht31P, or enhanced green fluorescent protein by adenoviral gene transfer into rat hearts in vivo. Ht31 expression resulted in loss of the striated staining pattern of type II PKA (RII), indicating loss of PKA from binding sites on endogenous AKAPs. In the absence of isoproterenol stimulation, Ht31-expressing hearts had decreased +dP/dtmax and -dP/dtmin but no change in left ventricular ejection fraction or stroke volume and decreased end diastolic pressure versus controls. This suggests that cardiac output is unchanged despite decreased +dP/dt and -dP/dt. There was also no difference in PKA phosphorylation of cardiac troponin I (cTnI), phospholamban, or ryanodine receptor (RyR2). Upon isoproterenol infusion, +dP/dtmax and -dP/dtmin did not differ between Ht31 hearts and controls. At higher doses of isoproterenol, left ventricular ejection fraction and stroke volume increased versus isoproterenol-stimulated controls. This occurred in the context of decreased PKA phosphorylation of cTnI, RyR2, and phospholamban versus controls. We previously showed that expression of N-terminal-cleaved cTnI (cTnI-ND) in transgenic mice improves cardiac function. Increased cTnI N-terminal truncation was also observed in Ht31-expressing hearts versus controls. Increased cTnI-ND may help compensate for reduced PKA phosphorylation as occurs in heart failure.


Subject(s)
A Kinase Anchor Proteins/metabolism , Cyclic AMP-Dependent Protein Kinase Type II/metabolism , Myocardial Contraction/physiology , Myocardium/metabolism , Peptides/metabolism , Troponin I/metabolism , A Kinase Anchor Proteins/genetics , Adenoviridae , Animals , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cardiotonic Agents/pharmacology , Cyclic AMP-Dependent Protein Kinase Type II/genetics , Gene Expression , Isoproterenol/pharmacology , Male , Mice , Myocardial Contraction/drug effects , Peptides/genetics , Phosphorylation/drug effects , Phosphorylation/physiology , Rats , Ryanodine Receptor Calcium Release Channel/genetics , Ryanodine Receptor Calcium Release Channel/metabolism , Stroke Volume/drug effects , Stroke Volume/physiology , Transduction, Genetic , Troponin I/genetics
10.
Mol Cell Endocrinol ; 300(1-2): 94-103, 2009 Mar 05.
Article in English | MEDLINE | ID: mdl-19111595

ABSTRACT

Following tropic hormone challenge, steroidogenic tissues utilize PKA to phosphorylate unique subsets of proteins necessary to facilitate steroidogenesis. This includes the PKA-dependent expression and activation of the steroidogenic acute regulatory protein (STAR), which mediates the rate-limiting step of steroidogenesis by inducing the transfer of cholesterol from the outer to the inner mitochondrial membrane. Since both type I and type II PKA are present in steroidogenic tissues, we have utilized cAMP analog pairs that preferentially activate each PKA subtype in order to examine their impact on STAR synthesis and activity. In MA-10 mouse Leydig tumor cells Star gene expression is more dependent upon type I PKA, while the post-transcriptional regulation of STAR appears subject to type II PKA. These experiments delineate the discrete effects that type I and type II PKA exert on STAR-mediated steroidogenesis, and suggest complimentary roles for each subtype in coordinating steroidogenesis.


Subject(s)
Cyclic AMP-Dependent Protein Kinase Type II/metabolism , Cyclic AMP-Dependent Protein Kinase Type I/metabolism , Phosphoproteins/metabolism , Steroids/biosynthesis , Animals , Cell Line, Tumor , Cyclic AMP/analogs & derivatives , Cyclic AMP/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinase Type I/genetics , Cyclic AMP-Dependent Protein Kinase Type II/genetics , Enzyme Activation , Isoenzymes/genetics , Isoenzymes/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Leydig Cell Tumor , Mice
11.
J Biol Chem ; 283(48): 33708-18, 2008 Nov 28.
Article in English | MEDLINE | ID: mdl-18824551

ABSTRACT

A-kinase anchoring proteins (AKAPs) target protein kinase A (PKA) to a variety of subcellular locations. Conventional AKAPs contain a 14-18-amino acid sequence that forms an amphipathic helix that binds with high affinity to the regulatory (R) subunit of PKA type II. More recently, a group of dual specificity AKAPs has been classified on the basis of their ability to bind the PKA type I and the PKA type II isozymes. In this study we show that dual specificity AKAPs contain an additional PKA binding determinant called the RI Specifier Region (RISR). A variety of protein interaction assays and immunoprecipitation and immunolocalization experiments indicates that the RISR augments RI binding in vitro and inside cells. Cellular delivery of the RISR peptide uncouples RI anchoring to Ezrin leading to release of T cell inhibition by cAMP. Likewise, expression of mutant Ezrin forms where RI binding has been abrogated by substitution of the RISR sequence prevents cAMP-mediated inhibition of T cell function. Thus, we propose that the RISR acts in synergy with the amphipathic helix in dual specificity anchoring proteins to enhance anchoring of PKA type I.


Subject(s)
A Kinase Anchor Proteins/metabolism , Cyclic AMP-Dependent Protein Kinase Type I/metabolism , Cytoskeletal Proteins/metabolism , T-Lymphocytes/enzymology , A Kinase Anchor Proteins/genetics , Amino Acid Sequence/physiology , Animals , Cattle , Cyclic AMP/genetics , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinase Type I/genetics , Cyclic AMP-Dependent Protein Kinase Type II/genetics , Cyclic AMP-Dependent Protein Kinase Type II/metabolism , Cytoskeletal Proteins/genetics , Humans , Jurkat Cells , Protein Structure, Secondary/physiology , Protein Structure, Tertiary/physiology , T-Lymphocytes/cytology
12.
Circ Res ; 103(8): 836-44, 2008 Oct 10.
Article in English | MEDLINE | ID: mdl-18757829

ABSTRACT

Protein kinase A (PKA) is a key regulatory enzyme that, on activation by cAMP, modulates a wide variety of cellular functions. PKA isoforms type I and type II possess different structural features and biochemical characteristics, resulting in nonredundant function. However, how different PKA isoforms expressed in the same cell manage to perform distinct functions on activation by the same soluble intracellular messenger, cAMP, remains to be established. Here, we provide a mechanism for the different function of PKA isoforms subsets in cardiac myocytes and demonstrate that PKA-RI and PKA-RII, by binding to AKAPs (A kinase anchoring proteins), are tethered to different subcellular locales, thus defining distinct intracellular signaling compartments. Within such compartments, PKA-RI and PKA-RII respond to distinct, spatially restricted cAMP signals generated in response to specific G protein-coupled receptor agonists and regulated by unique subsets of the cAMP degrading phosphodiesterases. The selective activation of individual PKA isoforms thus leads to phosphorylation of unique subsets of downstream targets.


Subject(s)
Cyclic AMP-Dependent Protein Kinase Type II/metabolism , Cyclic AMP-Dependent Protein Kinase Type I/metabolism , Cyclic AMP/metabolism , Myocytes, Cardiac/enzymology , Signal Transduction , A Kinase Anchor Proteins/metabolism , Animals , Animals, Newborn , Biosensing Techniques , CHO Cells , Calcium-Binding Proteins/metabolism , Cricetinae , Cricetulus , Cyclic AMP-Dependent Protein Kinase Type I/genetics , Cyclic AMP-Dependent Protein Kinase Type II/genetics , Fluorescence Recovery After Photobleaching , Fluorescence Resonance Energy Transfer , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Myocytes, Cardiac/drug effects , Phosphoric Diester Hydrolases/metabolism , Phosphorylation , Rats , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/metabolism , Recombinant Fusion Proteins/metabolism , Signal Transduction/drug effects , Time Factors , Transfection , Troponin I/metabolism
13.
Proc Natl Acad Sci U S A ; 105(1): 276-81, 2008 Jan 08.
Article in English | MEDLINE | ID: mdl-18172198

ABSTRACT

Agouti lethal yellow (A(y)) mice express agouti ectopically because of a genetic rearrangement at the agouti locus. The agouti peptide is a potent antagonist of the melanocortin 4 receptor (MC4R) expressed in neurons, and this leads to hyperphagia, hypoactivity, and increased fat mass. The MC4R signals through Gs and is thought to stimulate the production of cAMP and activation of downstream cAMP effector molecules such as PKA. Disruption of the RIIbeta regulatory subunit gene of PKA results in release of the active catalytic subunit and an increase in basal PKA activity in cells where RIIbeta is highly expressed. Because RIIbeta is expressed in neurons including those in the hypothalamic nuclei where MC4R is prominent we tested the possibility that the RIIbeta knockout might rescue the body weight phenotypes of the A(y) mice. Disruption of the RIIbeta PKA regulatory subunit gene in mice leads to a 50% reduction in white adipose tissue and resistance to diet-induced obesity and hyperglycemia. The RIIbeta mutation rescued the elevated body weight, hyperphagia, and obesity of A(y) mice. Partial rescue of the A(y) phenotypes was even observed on an RIIbeta heterozygote background. These results suggest that the RIIbeta gene mutation alters adiposity and locomotor activity by modifying PKA signaling pathways downstream of the agouti antagonism of MC4R in the hypothalamus.


Subject(s)
Cyclic AMP-Dependent Protein Kinase Type II/genetics , Cyclic AMP-Dependent Protein Kinase Type II/physiology , Cyclic AMP-Dependent Protein Kinases/chemistry , Gene Expression Regulation , Obesity/genetics , Obesity/therapy , Adipose Tissue/metabolism , Animals , Body Weight , Disease Models, Animal , Heterozygote , Hypothalamus/metabolism , Leptin/metabolism , Mice , Mice, Knockout , Models, Biological , Motor Activity , Phenotype , Receptor, Melanocortin, Type 4/metabolism
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