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1.
Nat Genet ; 38(12): 1397-405, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17086182

RESUMO

Nephrotic syndrome, a malfunction of the kidney glomerular filter, leads to proteinuria, edema and, in steroid-resistant nephrotic syndrome, end-stage kidney disease. Using positional cloning, we identified mutations in the phospholipase C epsilon gene (PLCE1) as causing early-onset nephrotic syndrome with end-stage kidney disease. Kidney histology of affected individuals showed diffuse mesangial sclerosis (DMS). Using immunofluorescence, we found PLCepsilon1 expression in developing and mature glomerular podocytes and showed that DMS represents an arrest of normal glomerular development. We identified IQ motif-containing GTPase-activating protein 1 as a new interaction partner of PLCepsilon1. Two siblings with a missense mutation in an exon encoding the PLCepsilon1 catalytic domain showed histology characteristic of focal segmental glomerulosclerosis. Notably, two other affected individuals responded to therapy, making this the first report of a molecular cause of nephrotic syndrome that may resolve after therapy. These findings, together with the zebrafish model of human nephrotic syndrome generated by plce1 knockdown, open new inroads into pathophysiology and treatment mechanisms of nephrotic syndrome.


Assuntos
Mutação , Síndrome Nefrótica/enzimologia , Síndrome Nefrótica/genética , Fosfolipases Tipo C/genética , Animais , Criança , Pré-Escolar , Clonagem Molecular , Modelos Animais de Doenças , Feminino , Marcação de Genes , Genes Recessivos , Homozigoto , Humanos , Lactente , Rim/enzimologia , Rim/patologia , Masculino , Modelos Genéticos , Mutação de Sentido Incorreto , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/patologia , Fosfoinositídeo Fosfolipase C , Ratos , Deleção de Sequência , Peixe-Zebra/genética
2.
J Biol Chem ; 286(26): 23012-21, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21550986

RESUMO

To define a role for phospholipase Cε (PLCε) signaling in cardiac myocyte hypertrophic growth, PLCε protein was depleted from neonatal rat ventricular myocytes (NRVMs) using siRNA. NRVMs with PLCε depletion were stimulated with endothelin (ET-1), norepinephrine, insulin-like growth factor-1 (IGF-1), or isoproterenol and assessed for development of hypertrophy. PLCε depletion dramatically reduced hypertrophic growth and gene expression induced by all agonists tested. PLCε catalytic activity was required for hypertrophy development, yet PLCε depletion did not reduce global agonist-stimulated inositol phosphate production, suggesting a requirement for localized PLC activity. PLCε was found to be scaffolded to a muscle-specific A kinase anchoring protein (mAKAPß) in heart and NRVMs, and mAKAPß localizes to the nuclear envelope in NRVMs. PLCε-mAKAP interaction domains were defined and overexpressed to disrupt endogenous mAKAPß-PLCε complexes in NRVMs, resulting in significantly reduced ET-1-dependent NRVM hypertrophy. We propose that PLCε integrates multiple upstream signaling pathways to generate local signals at the nucleus that regulate hypertrophy.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Cardiomegalia/metabolismo , Proteínas Musculares/metabolismo , Miócitos Cardíacos/metabolismo , Membrana Nuclear/metabolismo , Fosfoinositídeo Fosfolipase C/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Animais , Cardiomegalia/genética , Células HEK293 , Humanos , Camundongos , Proteínas Musculares/genética , Especificidade de Órgãos/genética , Fosfoinositídeo Fosfolipase C/genética , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética
3.
J Physiol ; 588(Pt 24): 4871-89, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21041529

RESUMO

Calcium can be mobilized in pancreatic ß-cells via a mechanism of Ca(2+)-induced Ca(2+) release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR in ß-cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide-specific phospholipase C- (PLC-) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC- gene expression in mice disrupts the action of exendin-4 to facilitate CICR in the ß-cells of these mice. Thus, in the present study, in which wild-type (WT) C57BL/6 mouse ß-cells were loaded with the photolabile Ca(2+) chelator NP-EGTA, the UV flash photolysis-catalysed uncaging of Ca(2+) generated CICR in only 9% of the ß-cells tested, whereas CICR was generated in 82% of the ß-cells pretreated with exendin-4. This action of exendin-4 to facilitate CICR was reproduced by cAMP analogues that activate protein kinase A (6-Bnz-cAMP-AM) or Epac2 (8-pCPT-2'-O-Me-cAMP-AM) selectively. However, in ß-cells of PLC- KO mice, and also Epac2 KO mice, these test substances exhibited differential efficacies in the CICR assay such that exendin-4 was partly effective, 6-Bnz-cAMP-AM was fully effective, and 8-pCPT-2'-O-Me-cAMP-AM was without significant effect. Importantly, transduction of PLC- KO ß-cells with recombinant PLC- rescued the action of 8-pCPT-2'-O-Me-cAMP-AM to facilitate CICR, whereas a K2150E PLC- with a mutated Ras association (RA) domain, or a H1640L PLC- that is catalytically dead, were both ineffective. Since 8-pCPT-2'-O-Me-cAMP-AM failed to facilitate CICR in WT ß-cells transduced with a GTPase activating protein (RapGAP) that downregulates Rap activity, the available evidence indicates that a signal transduction 'module' comprised of Epac2, Rap and PLC- exists in ß-cells, and that the activities of Epac2 and PLC- are key determinants of CICR in this cell type.


Assuntos
Cálcio/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Peptídeos/farmacologia , Fosfoinositídeo Fosfolipase C/metabolismo , Receptores de Glucagon/agonistas , Peçonhas/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Exenatida , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1 , Fatores de Troca do Nucleotídeo Guanina/genética , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Knockout , Fosfoinositídeo Fosfolipase C/genética , Tapsigargina/farmacologia
4.
Am J Physiol Endocrinol Metab ; 298(3): E622-33, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20009023

RESUMO

Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2'-O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. RT-QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was detectable. Under conditions of islet perifusion, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) potentiated first- and second-phase 10 mM glucose-stimulated insulin secretion (GSIS) while failing to influence insulin secretion measured in the presence of 3 mM glucose. The insulin secretagogue action of 8-pCPT-2'-O-Me-cAMP-AM was associated with depolarization and an increase of [Ca(2+)](i) that reflected both Ca(2+) influx and intracellular Ca(2+) mobilization in islet beta-cells. As expected for an Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Unexpectedly, treatment of human islets with an inhibitor of PKA activity (H-89) or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS) nearly abolished the action of 8-pCPT-2'-O-Me-cAMP-AM to potentiate GSIS. It is concluded that there exists a permissive role for PKA activity in support of human islet insulin secretion that is both glucose dependent and Epac regulated. This permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or postpriming steps of Ca(2+)-dependent exocytosis.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/análogos & derivados , Glucose/administração & dosagem , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Células Cultivadas , AMP Cíclico/administração & dosagem , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
5.
Biochem J ; 415(3): 439-48, 2008 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-18588506

RESUMO

In the present study, we examined the role of PLC delta 1 (phospholipase C delta 1) in the regulation of cellular proliferation. We demonstrate that RNAi (RNA interference)-mediated knockdown of endogenous PLC delta 1, but not PLC beta 3 or PLC epsilon, induces a proliferation defect in Rat-1 and NIH 3T3 fibroblasts. The decreased proliferation was not due to an induction of apoptosis or senescence, but was associated with an approx. 60% inhibition of [(3)H]thymidine incorporation. Analysis of the cell cycle with BrdU (bromodeoxyuridine)/propidium iodide-labelled FACS (fluorescence-activated cell sorting) demonstrated an accumulation of cells in G(0)/G(1)-phase and a corresponding decrease in cells in S-phase. Further examination of the cell cycle after synchronization by serum-starvation demonstrated normal movement through G(1)-phase but delayed entry into S-phase. Consistent with these findings, G(1) cyclin (D2 and D3) and CDK4 (cyclin-dependent kinase 4) levels and associated kinase activity were not affected. However, cyclin E-associated CDK2 activity, responsible for G(1)-to-S-phase progression, was inhibited. This decreased activity was accompanied by unchanged CDK2 protein levels and paradoxically elevated cyclin E and cyclin E-associated CDK2 levels, suggesting inhibition of the cyclin E-CDK2 complex. This inhibition was not due to altered stimulatory or inhibitory phosphorylation of CDK2. However, p27, a Cip/Kip family CKI (CDK inhibitor)-binding partner, was elevated and showed increased association with CDK2 in PLC delta 1-knockdown cells. The result of the present study demonstrate a novel and critical role for PLC delta 1 in cell-cycle progression from G(1)-to-S-phase through regulation of cyclin E-CDK2 activity and p27 levels.


Assuntos
Proliferação de Células , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Fase G1 , Fosfolipase C delta/metabolismo , Fase S , Animais , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Humanos , Camundongos , Células NIH 3T3 , Fosfolipase C delta/genética , Fosforilação , Interferência de RNA , Ratos
6.
Circ Res ; 97(12): 1305-13, 2005 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-16293787

RESUMO

Phospholipase C (PLC) epsilon is a recently identified enzyme regulated by a wide range of molecules including Ras family small GTPases, Rho A, Galpha(12/13), and Gbetagamma with primary sites of expression in the heart and lung. In a screen for human signal transduction genes altered during heart failure, we found that PLCepsilon mRNA is upregulated. Two murine models of cardiac hypertrophy confirmed upregulation of PLCepsilon protein expression or PLCepsilon RNA. To identify a role for PLCepsilon in cardiac function and pathology, a PLCepsilon-deficient mouse strain was created. Echocardiography indicated PLCepsilon(-/-) mice had decreased cardiac function, and direct measurements of left ventricular contraction demonstrated that PLCepsilon(-/-) mice had a decreased contractile response to acute isoproterenol administration. Cardiac myocytes isolated from PLCepsilon(-/-) mice had decreased beta-adrenergic receptor (betaAR)-dependent increases in Ca2+ transient amplitudes, likely accounting for the contractile deficiency in vivo. This defect appears to be independent from the ability of the betaAR system to produce cAMP and regulation of sarcoplasmic reticulum Ca2+ pool size. To address the significance of these functional deficits to cardiac pathology, PLCepsilon(-/-) mice were subjected to a chronic isoproterenol model of hypertrophic stress. PLCepsilon(-/-) mice were more susceptible than wild-type littermates to development of hypertrophy than wild-type littermates. Together, these data suggest a novel PLC-dependent component of betaAR signaling in cardiac myocytes responsible for maintenance of maximal contractile reserve and loss of PLCepsilon signaling sensitizes the heart to development of hypertrophy in response to chronic cardiac stress.


Assuntos
Cardiomegalia/prevenção & controle , Contração Miocárdica , Receptores Adrenérgicos beta/fisiologia , Fosfolipases Tipo C/fisiologia , Animais , Cálcio/metabolismo , Cardiomegalia/enzimologia , Insuficiência Cardíaca/enzimologia , Humanos , Isoproterenol/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Fosfoinositídeo Fosfolipase C , RNA Mensageiro/análise , Retículo Sarcoplasmático/metabolismo , Fosfolipases Tipo C/genética
7.
Methods Enzymol ; 407: 195-217, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16757325

RESUMO

Ras proteins function as signaling nodes that are activated by diverse extracellular stimuli. Equally complex for this family of molecular switches is the multitude of downstream effectors and the pathways that they traverse to translate extracellular signals into a spectrum of cellular consequences. To better understand the individual and collective roles of these effector signaling networks, both genetic and pharmacological tools have been developed. By either stimulating or ablating specific components in a cascade downstream of Ras activation, one can gain insight into the specific signaling underlying a particular Ras phenotype, for example, malignant transformation. In this chapter, we describe the use of activating and dominant-negative mutations, both artificial and naturally occurring, of Ras and its effectors, as well as pharmacological inhibitors used to probe the effector pathways (Raf kinase, phosphoinositol 3-kinase, Tiam1, phospholipase C epsilon, and RalGEF) implicated in Ras-mediated oncogenesis.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas ras/metabolismo , Butadienos/farmacologia , Células Epiteliais/fisiologia , Feminino , Flavonoides/farmacologia , Genes ras/genética , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Mutação , Nitrilas/farmacologia , Ovário , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoinositídeo Fosfolipase C/metabolismo , Estrutura Terciária de Proteína , Interferência de RNA , Transdução de Sinais , Quinases raf/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Proteínas ras/genética
8.
Biochem J ; 378(Pt 1): 129-39, 2004 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-14567755

RESUMO

PLCepsilon (phospholipase Cepsilon) is a novel PLC that has a CDC25 guanine nucleotide exchange factor domain and two RA (Ras-association) domains of which the second (RA2) is critical for Ras activation of the enzyme. In the present studies, we examined hormonal stimulation to elucidate receptor-mediated pathways that functionally regulate PLCepsilon. We demonstrate that EGF (epidermal growth factor), a receptor tyrosine kinase agonist, and LPA (lysophosphatidic acid), S1P (sphingosine 1-phosphate) and thrombin, GPCR (G-protein-coupled receptor) agonists, stimulate PLCepsilon overexpressed in COS-7 cells. EGF stimulated PLCepsilon in an RA2-dependent manner through Ras and Rap. In contrast, LPA, S1P and thrombin stimulated PLCepsilon by both RA2-independent and -dependent mechanisms. To determine the G-proteins that mediate the effects of these GPCR agonists, we co-expressed constitutively active G-proteins with PLCepsilon and found that G(alpha12), G(alpha13), Rho, Rac and Ral stimulate PLCepsilon in an RA2-independent manner; whereas TC21, Rap1A, Rap2A and Rap2B stimulate PLCepsilon in an RA2-dependent manner similar to H-Ras. Of these G-proteins, we show that G(alpha12)/G(alpha13) and Rap partly mediate the effects of LPA, S1P and thrombin to stimulate PLCepsilon. In addition, the stimulation by LPA and S1P is also partly sensitive to pertussis toxin. These studies demonstrate diverse hormonal regulation of PLCepsilon by distinct and overlapping pathways.


Assuntos
Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Hormônios/farmacologia , Fosfolipases Tipo C/metabolismo , Proteínas ras/metabolismo , Animais , Células COS , Chlorocebus aethiops , Isoenzimas/metabolismo , Lisofosfolipídeos/antagonistas & inibidores , Toxina Pertussis/farmacologia , Fosfoinositídeo Fosfolipase C , Estrutura Terciária de Proteína , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/antagonistas & inibidores , Fosfolipases Tipo C/química , Proteínas rho de Ligação ao GTP
9.
Methods Mol Biol ; 237: 55-64, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-14501038

RESUMO

Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP(3)). The PLC beta isoforms of PLCs are activated by G proteins after hormone or neurotransmitter stimulation of G protein-coupled receptors (GPCR). PLC epsilon is a recently identified PLC isoform that is activated by Ras and G beta gamma subunit although the physiological role of this enzyme is not well understood. Methods for purification of PLC beta and PLC epsilon from Sf9 cells are described. In the case of hexahistidine (6-His)-tagged PLC beta the purification involves two steps, affinity chromatography with Ni-NTA agarose followed by heparin Sepharose chromatography. 6-His-tagged PLC epsilon can be purified in a single step with nickel nitrilotriacetic acid-agarose (Ni-NTA) affinity chromatography.


Assuntos
Cromatografia de Afinidade/métodos , Isoenzimas/isolamento & purificação , Fosfolipases Tipo C/isolamento & purificação , Animais , Linhagem Celular , Insetos , Fosfoinositídeo Fosfolipase C , Fosfolipase C beta , Proteínas Recombinantes/isolamento & purificação , Transdução de Sinais
10.
J Biol Chem ; 284(3): 1514-22, 2009 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-18957419

RESUMO

Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)epsilon that plays a critical role in maximal beta-adrenergic receptor (betaAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we demonstrate that PLCepsilon phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLCepsilon-stimulated Rap1 GEF activity are both required for PLCepsilon-mediated enhancement of sarcoplasmic reticulum Ca2+ release and that PLCepsilon significantly enhances Rap activation in response to betaAR stimulation in the heart. Downstream of PLCepsilon hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both betaAR- and Epac-stimulated increases in CICR in PLCepsilon+/+ myocytes but had no effect in PLCepsilon-/- myocytes. betaAR and Epac activation caused membrane translocation of PKCepsilon in PLCepsilon+/+ but not PLCepsilon-/- myocytes and small interfering RNA-mediated PKCepsilon knockdown significantly inhibited both betaAR and Epac-mediated CICR enhancement. Further downstream, the Ca2+/calmodulin-dependent protein kinase II (CamKII) inhibitor, KN93, inhibited betaAR- and Epac-mediated CICR in PLCepsilon+/+ but not PLCepsilon-/- myocytes. Epac activation increased CamKII Thr286 phosphorylation and enhanced phosphorylation at CamKII phosphorylation sites on the ryanodine receptor (RyR2) (Ser2815) and phospholamban (Thr17) in a PKC-dependent manner. Perforated patch clamp experiments revealed that basal and betaAR-stimulated peak L-type current density are similar in PLCepsilon+/+ and PLCepsilon-/- myocytes suggesting that control of sarcoplasmic reticulum Ca2+ release, rather than Ca2+ influx through L-type Ca2+ channels, is the target of regulation of a novel signal transduction pathway involving sequential activation of Epac, PLCepsilon, PKCepsilon, and CamKII downstream of betaAR activation.


Assuntos
Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Miocárdio/enzimologia , Fosfoinositídeo Fosfolipase C/metabolismo , Animais , Benzilaminas/farmacologia , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Membrana Celular/enzimologia , Membrana Celular/genética , Ativação Enzimática/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos , Camundongos Knockout , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/enzimologia , Fosfatidilinositol 4,5-Difosfato , Fosfoinositídeo Fosfolipase C/genética , Fosforilação/fisiologia , Proteína Quinase C-épsilon/genética , Proteína Quinase C-épsilon/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/fisiologia , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/enzimologia , Retículo Sarcoplasmático/genética , Sulfonamidas/farmacologia
11.
Islets ; 1(3): 260-5, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-21099281

RESUMO

Epac2 is a cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF) that is proposed to mediate stimulatory actions of the second messenger cAMP on mouse islet insulin secretion. Here we have used methods of islet perifusion to demonstrate that the acetoxymethyl ester (AM-ester) of an Epac-selective cAMP analog (ESCA) penetrates into mouse islets and is capable of potentiating both first and second phases of glucose-stimulated insulin secretion (GSIS). When used at low concentrations (1-10 µM), 8-pCPT-2'-O-Me-cAMP-AM activates Rap1 GTPase but exhibits little or no ability to activate protein kinase A (PKA), as validated in assays of in vitro PKA activity (phosphorylation of Kemptide), Ser (133) CREB phosphorylation status, RIP1-CRE-Luc reporter gene activity, and PKA-dependent AKAR3 biosensor activation. Since quantitative PCR demonstrates Epac2 mRNA to be expressed at levels ca. 5.3-fold greater than that of Epac1, available evidence indicates that Epac2 does in fact mediate stimulatory actions of cAMP on mouse islet GSIS.


Assuntos
AMP Cíclico/análogos & derivados , Glucose/farmacologia , Fatores de Troca do Nucleotídeo Guanina/agonistas , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Animais , Células Cultivadas , AMP Cíclico/administração & dosagem , AMP Cíclico/química , AMP Cíclico/farmacologia , Combinação de Medicamentos , Sinergismo Farmacológico , Glucose/administração & dosagem , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Camundongos
12.
J Biol Chem ; 284(16): 10728-36, 2009 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-19244230

RESUMO

To ascertain the identities of cyclic nucleotide-binding proteins that mediate the insulin secretagogue action of cAMP, the possible contributions of the exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) were evaluated in a pancreatic beta cell line (rat INS-1 cells). Assays of Rap1 activation, CREB phosphorylation, and PKA-dependent gene expression were performed in combination with live cell imaging and high throughput screening of a fluorescence resonance energy transfer-based cAMP sensor (Epac1-camps) to validate the selectivity with which acetoxymethyl esters (AM-esters) of cAMP analogs preferentially activate Epac or PKA. Selective activation of Epac or PKA was achieved following exposure of INS-1 cells to 8-pCPT-2'-O-Me-cAMP-AM or Bt(2)cAMP-AM, respectively. Both cAMP analogs exerted dose-dependent and glucose metabolism-dependent actions to stimulate insulin secretion, and when each was co-administered with the other, a supra-additive effect was observed. Because 2.4-fold more insulin was secreted in response to a saturating concentration (10 microm) of Bt(2)cAMP-AM as compared with 8-pCPT-2'-O-Me-cAMP-AM, and because the action of Bt(2)cAMP-AM but not 8-pCPT-2'-O-Me-cAMP-AM was nearly abrogated by treatment with 3 microm of the PKA inhibitor H-89, it is concluded that for INS-1 cells, it is PKA that acts as the dominant cAMP-binding protein in support of insulin secretion. Unexpectedly, 10-100 microm of the non-AM-ester of 8-pCPT-2'-O-Me-cAMP failed to stimulate insulin secretion and was a weak activator of Rap1 in INS-1 cells. Moreover, 10 microm of the AM-ester of 8-pCPT-2'-O-Me-cAMP stimulated insulin secretion from mouse islets, whereas the non-AM-ester did not. Thus, the membrane permeability of 8-pCPT-2'-O-Me-cAMP in insulin-secreting cells is so low as to limit its biological activity. It is concluded that prior reports documenting the failure of 8-pCPT-2'-O-Me-cAMP to act in beta cells, or other cell types, need to be re-evaluated through the use of the AM-ester of this cAMP analog.


Assuntos
AMP Cíclico/análogos & derivados , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Insulina/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , AMP Cíclico/química , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina/genética , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Isoquinolinas/metabolismo , Camundongos , Inibidores de Proteínas Quinases/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sulfonamidas/metabolismo , Proteínas rap1 de Ligação ao GTP/genética
13.
J Biol Chem ; 282(28): 20104-15, 2007 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-17502376

RESUMO

Inositol 1,4,5-trisphosphate (IP(3)) receptors are endoplasmic reticulum (ER) membrane calcium channels that, upon activation, become substrates for the ER-associated degradation (ERAD) pathway. Although it is clear that IP(3) receptors are polyubiquitinated upon activation and are transferred to the proteasome by a p97-based complex, currently nothing is known about the proteins that initially select activated IP(3) receptors for ERAD. Here, we sought to identify novel proteins that associate with and mediate the ERAD of endogenous activated IP(3) receptors. SPFH2, an uncharacterized SPFH domain-containing protein, rapidly associated with IP(3) receptors in a manner that preceded significant polyubiquitination and the association of p97 and related proteins. SPFH2 was found to be an ER membrane protein largely residing within the ER lumen and in resting and stimulated cells was linked to ERAD pathway components, apparently via endogenous substrates undergoing degradation. Suppression of SPFH2 expression by RNA interference markedly inhibited IP(3) receptor polyubiquitination and degradation and the processing of other ERAD substrates. Overall, these studies identify SPFH2 as a key ERAD pathway component and suggest that it may act as a substrate recognition factor.


Assuntos
Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Animais , Retículo Endoplasmático/genética , Células HeLa , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/fisiologia , RNA Interferente Pequeno/genética , Ratos , Fator 2 Associado a Receptor de TNF , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo
14.
Proc Natl Acad Sci U S A ; 104(39): 15543-8, 2007 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-17878312

RESUMO

Phospholipase Cepsilon (PLCepsilon) has been suggested to transduce signals from small GTPases, but its biological function has not yet been clarified. Using astrocytes from PLCepsilon-deficient mice, we demonstrate that endogenous G protein-coupled receptors (GPCRs) for lysophosphatidic acid, sphingosine 1-phosphate, and thrombin regulate phosphoinositide hydrolysis primarily through PLCepsilon. Stimulation by lysophospholipids occurs through G(i), whereas thrombin activates PLC through Rho. Further studies reveal that PLCepsilon is required for thrombin- but not LPA-induced sustained ERK activation and DNA synthesis, providing a novel mechanism for GPCR and Rho signaling to cell proliferation. The requirement for PLCepsilon in this pathway can be explained by its role as a guanine nucleotide exchange factor for Rap1. Thus, PLCepsilon serves to transduce mitogenic signals through a mechanism distinct from its role in generation of PLC-derived second messengers.


Assuntos
Astrócitos/citologia , Receptores Acoplados a Proteínas G/metabolismo , Fosfolipases Tipo C/fisiologia , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Astrócitos/metabolismo , Carbacol/farmacologia , Proliferação de Células , GTP Fosfo-Hidrolases/metabolismo , Genótipo , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fosfoinositídeo Fosfolipase C , Transdução de Sinais
15.
J Biol Chem ; 282(8): 5488-95, 2007 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-17178726

RESUMO

Recently we demonstrated that PLC(epsilon) plays an important role in beta-adrenergic receptor (betaAR) stimulation of Ca(2+)-induced Ca(2+) release (CICR) in cardiac myocytes. Here we have reported for the first time that a pathway downstream of betaAR involving the cAMP-dependent Rap GTP exchange factor, Epac, and PLC(epsilon) regulates CICR in cardiac myocytes. To demonstrate a role for Epac in the stimulation of CICR, cardiac myocytes were treated with an Epac-selective cAMP analog, 8-4-(chlorophenylthio)-2'-O-methyladenosine-3',5'-monophosphate (cpTOME). cpTOME treatment increased the amplitude of electrically evoked Ca(2+) transients, implicating Epac for the first time in cardiac CICR. This response is abolished in PLC(epsilon)(-/-) cardiac myocytes but rescued by transduction with PLC(epsilon), indicating that Epac is upstream of PLC(epsilon). Furthermore, transduction of PLC(epsilon)(+/+) cardiac myocytes with a Rap inhibitor, RapGAP1, significantly inhibited isoproterenol-dependent CICR. Using a combination of cpTOME and PKA-selective activators and inhibitors, we have shown that betaAR-dependent increases in CICR consist of two independent components mediated by PKA and the novel Epac/(epsilon) pathway. We also show that Epac/PLC(epsilon)-dependent effects on CICR are independent of sarcoplasmic reticulum loading and Ca(2+) clearance mechanisms. These data define a novel endogenous PKA-independent betaAR-signaling pathway through cAMP-dependent Epac activation, Rap, and PLC(epsilon) that enhances intracellular Ca(2+) release in cardiac myocytes.


Assuntos
Sinalização do Cálcio/fisiologia , Miócitos Cardíacos/enzimologia , Receptores Adrenérgicos beta/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Camundongos , Camundongos Knockout , Miócitos Cardíacos/citologia , Fosfoinositídeo Fosfolipase C , Retículo Sarcoplasmático/enzimologia , Retículo Sarcoplasmático/genética , Transdução Genética , Fosfolipases Tipo C/deficiência , Proteínas rap de Ligação ao GTP/metabolismo
16.
J Biol Chem ; 281(5): 2639-48, 2006 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-16314422

RESUMO

Phospholipase Cepsilon (PLCepsilon) is one of the newest members of the phosphatidylinositol-specific phospholipase C (PLC) family. Previous studies have suggested that G-protein-coupled receptors (GPCRs) stimulate phosphoinositide (PI) hydrolysis by activating PLCbeta isoforms through G(q) family G proteins and Gbetagamma subunits. Using RNA interference to knock down PLC isoforms, we demonstrate that the GPCR agonists endothelin (ET-1), lysophosphatidic acid (LPA), and thrombin, acting through endogenous receptors, couple to both endogenous PLCepsilon and the PLCbeta isoform, PLCbeta3, in Rat-1 fibroblasts. Examination of the temporal activation of these PLC isoforms, however, reveals agonist- and isoform-specific profiles. PLCbeta3 is activated acutely within the first minute of ET-1, LPA, or thrombin stimulation but does not contribute to sustained PI hydrolysis induced by LPA or thrombin and accounts for only part of ET-1 sustained stimulation. PLCepsilon, on the other hand, predominantly accounts for sustained PI hydrolysis. Consistent with this observation, reconstitution of PLCepsilon in knockdown cells dose-dependently increases sustained, but not acute, agonist-stimulated PI hydrolysis. Furthermore, combined knockdown of both PLCepsilon and PLCbeta3 additively inhibits PI hydrolysis, suggesting independent regulation of each isoform. Importantly, ubiquitination of inositol 1,4,5-trisphosphate receptors correlates with sustained, but not acute, activation of PLCepsilon or PLCbeta3. In conclusion, GPCR agonists ET-1, LPA, and thrombin activate endogenous PLCepsilon and PLCbeta3 in Rat-1 fibroblasts. Activation of these PLC isoforms displays agonist-specific temporal profiles; however, PLCbeta3 is predominantly involved in acute and PLCepsilon in sustained PI hydrolysis.


Assuntos
Isoenzimas/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Fosfolipases Tipo C/metabolismo , Animais , Células Cultivadas , Endotelina-1/metabolismo , Fibroblastos/citologia , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Lisofosfolipídeos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoinositídeo Fosfolipase C , Fosfolipase C beta , Isoformas de Proteínas/genética , Ratos , Trombina/metabolismo , Fatores de Tempo , Fosfolipases Tipo C/genética
17.
J Biol Chem ; 280(41): 34530-7, 2005 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-16103111

RESUMO

Inositol 1,4,5-trisphosphate (IP(3)) receptors form tetrameric, IP(3)-gated channels in endoplasmic reticulum membranes that govern the release of Ca(2+) from this organelle. In response to activation of certain G protein-coupled receptors that persistently elevate IP(3) concentration, IP(3) receptors are ubiquitinated and degraded by the ubiquitin-proteasome pathway. IP(3) receptor ubiquitination is mediated by the ubiquitin-conjugating enzyme, (mam)Ubc7, a component of the endoplasmic reticulum-associated degradation pathway. However, the mechanism by which ubiquitinated IP(3) receptors are transferred to the proteasome is not known. Here, we examine this process and show in several mammalian cell types that the ATPase p97 associates with IP(3) receptors in response to hormonal stimuli that induce IP(3) receptor ubiquitination. To examine the functional relevance of the p97 interaction with IP(3) receptors, we stably and specifically reduced p97 protein levels by 62 +/- 3% in Rat-1 fibroblasts using RNA interference. In these cells, endothelin-1-induced IP(3) receptor degradation was markedly retarded and the accumulation of ubiquitinated IP(3) receptors was markedly enhanced. These effects were reversed by expression of exogenous p97. In addition, Ufd1 and Npl4, which complex with p97, also associated with IP(3) receptors upon hormonal stimulation. We conclude that the p97-Ufd1-Npl4 complex couples ubiquitinated IP(3) receptors to proteasomal degradation and, thus, plays a key role in IP(3) receptor processing. These data also establish that the p97-Ufd1-Npl4 complex mediates endoplasmic reticulum-associated degradation in mammalian cells.


Assuntos
Adenosina Trifosfatases/fisiologia , Canais de Cálcio/metabolismo , Proteínas de Transporte/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas Nucleares/fisiologia , Proteínas/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Citosol/metabolismo , DNA Complementar/metabolismo , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Fibroblastos/metabolismo , Imunoprecipitação , Receptores de Inositol 1,4,5-Trifosfato , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Interferência de RNA , Ratos , Frações Subcelulares , Fatores de Tempo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo
18.
J Biol Chem ; 277(12): 9966-75, 2002 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-11788587

RESUMO

Oncogenic Ras and activated forms of the Ras-related protein TC21/R-Ras2 share similar abilities to alter cell proliferation. However, in contrast to Ras, we found previously that TC21 fails to activate the Raf-1 serine/threonine kinase. Thus, TC21 must utilize non-Raf effectors to regulate cell function. In this study, we determined that TC21 interacts strongly with some (RalGDS, RGL, RGL2/Rlf, AF6, and the phosphatidylinositol 3-kinase (PI3K) catalytic subunit p110delta), and weakly with other Ras small middle dotGTP-binding proteins. In addition, library screening identified novel TC21-interacting proteins. We also determined that TC21, similar to Ras, mediates activation of phospholipase Cepsilon. We then examined if RalGDS, a RalA guanine nucleotide exchange factor, or PI3K are effectors for TC21-mediated signaling and cell proliferation in murine fibroblasts. We found that overexpression of full-length RalGDS reduced the focus forming activity of activated TC21. Furthermore, expression of activated Ras, but not TC21, enhanced GTP loading on RalA. In fact, TC21 attenuated insulin-stimulated RalA small middle dotGTP formation. In contrast, like Ras, expression of activated TC21 resulted in membrane translocation and an increase in the PI3K-dependent phosphorylation of Akt, and inhibition of PI3K activity interfered with TC21 focus formation. Finally, unlike Ras, TC21 did not activate the Rac small GTPase, indicating that Ras may not activate Rac by PI3K. Taken together, these results suggest that PI3K, but not RalGDS, is an important mediator of cell proliferation by TC21.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Ligação ao GTP , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas ral de Ligação ao GTP , Fator ral de Troca do Nucleotídeo Guanina/metabolismo , Células 3T3 , Animais , Células COS , Divisão Celular , Membrana Celular/metabolismo , Células Cultivadas , Cromonas/farmacologia , DNA/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , GTP Fosfo-Hidrolases/metabolismo , Biblioteca Gênica , Camundongos , Morfolinas/farmacologia , Fosfoinositídeo Fosfolipase C , Fosforilação , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Fosfolipases Tipo C/metabolismo
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