RESUMO
Myc is a transcription factor that significantly contributes to cancer progression by modulating the expression of important genes through binding to a DNA sequence, CACGTG, called E-box. We find that on Myc binding to chromatin, the lysine-demethylating enzyme, LSD1, triggers a transient demethylation of lysine 4 in the histone H3. In addition, we demonstrate that Myc binds and recruits LSD1 to the E-box chromatin and the formation of this complex is stimulated by cAMP-PKA. Demethylation by LSD1 produces H(2)O(2), which locally oxidizes guanine and induces the recruitment of 8-oxoguanine-DNA glycosylase (OGG1) and of the nuclease Ape1 on the E-box chromatin. Inhibition of oxidation or silencing of LSD1, OGG1 or Ape1 significantly reduce transcription and inhibit mRNA accumulation of Myc-target genes. Collectively, these data highlight the role of transient LSD1-mediated demethylation of H3K4 leading to local DNA oxidation as driving force in the assembly of the Myc-induced transcription initiation complex.
Assuntos
Histona Desmetilases/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Gênica , Animais , Linhagem Celular , Cromatina/genética , DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Elementos E-Box , Metilação , Mitógenos/farmacologia , Oxirredução , Ratos , Transcrição Gênica/efeitos dos fármacosRESUMO
Cyclic adenosine 3'5' monophosphate (cAMP) and protein kinase A (PKA) cooperate with phosphatidylinositol 3' kinase (PI3K) signals in the control of growth and survival. To determine the molecular mechanism(s) involved, we identified and mutagenized a specific serine (residue 83) in p85alpha(PI3K), which is phosphorylated in vivo and in vitro by PKA. Expression of p85alpha(PI3K) mutants (alanine or aspartic substitutions) significantly altered the biological responses of the cells to cAMP. cAMP protection from anoikis was reduced in cells expressing the alanine version p85alpha(PI3K). These cells did not arrest in G1 in the presence of cAMP, whereas cells expressing the aspartic mutant p85D accumulated in G1 even in the absence of cAMP. S phase was still efficiently inhibited by cAMP in cells expressing both mutants. The binding of PI3K to Ras p21 was greatly reduced in cells expressing p85A in the presence or absence of cAMP. Conversely, expression of the aspartic mutant stimulated robustly the binding of PI3K to p21 Ras in the presence of cAMP. Mutation in the Ser 83 inhibited cAMP, but not PDGF stimulation of PI3K. Conversely, the p85D aspartic mutant amplified cAMP stimulation of PI3K activity. Phosphorylation of Ser 83 by cAMP-PKA in p85alpha(PI3K) was also necessary for estrogen signaling as expression of p85A or p85D mutants inhibited or amplified, respectively, the binding of estrogen receptor to p85alpha and AKT phosphorylation induced by estrogens. The data presented indicate that: (1) phosphorylation of Ser 83 in p85alpha(PI3K) is critical for cAMP-PKA induced G1 arrest and survival in mouse 3T3 fibroblasts; (2) this site is necessary for amplification of estrogen signals by cAMP-PKA and related receptors. Finally, these data suggest a general mechanism of PI3K regulation by cAMP, operating in various cell types and under different conditions.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Estrogênios/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Citoproteção , Estrogênios/metabolismo , Fase G1/efeitos dos fármacos , Fase G1/genética , Humanos , Mutação , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Serina/genética , Serina/metabolismoRESUMO
Phosphatidylinositol 3-kinase (PI3K) is necessary for thyroid stimulating hormone (TSH)-induced cell cycle progression. To determine the molecular mechanism linking PI3K to TSH, we have identified a serine residue in p85alpha(PI3K) phosphorylated by protein kinase A (PKA) in vitro and in vivo. Expression of an alanine mutant (p85A) abolished cyclic AMP/TSH-induced cell cycle progression and was lethal in thyroid cells (FRTL-5). The aspartic version of the p85alpha(PI3K) (p85D) inhibited apoptosis following TSH withdrawal. The p85alpha(PI3K) wild type not the p85A bound PKA regulatory subunit RIIbeta in cells stimulated with cAMP or TSH. The binding of the aspartic version of p85alpha(PI3K) to RIIbeta was independent of cAMP or TSH stimulation. Similarly, binding of PI3K to p21Ras and activation of AKT, a downstream PI3K target, were severely impaired in cells expressing the p85A mutant. Finally, we found that the catalytic activity of PI3K was stimulated by TSH in cells expressing the wild-type p85alpha(PI3K) but not in cells expressing p85A. This latter mutant did not affect the epidermal growth factor-stimulated PI3K activity. We suggest that (1) TSH-cAMP-induced PKA phosphorylates p85alpha(PI3K) at serine 83, (2) phosphorylated p85alpha(PI3K) binds RIIbeta-PKA and targets PKAII to the membrane, and (3) PI3K activity and p21Ras binding to PI3K increase and activate PI3K downstream targets. This pathway is essential for the transmission of TSH-cAMP growth signals.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Tireotropina/metabolismo , Animais , Catálise , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico , Camundongos , Mutação , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ratos , Serina/genética , Serina/metabolismo , Tireotropina/farmacologiaRESUMO
The cytotoxicity of dental monomers has been widely investigated, but the underlying mechanisms have not been elucidated. We studied the molecular mechanisms involved in cell death induced by HEMA. In human primary fibroblasts, HEMA induced a dose-dependent apoptosis that was confirmed by the activation of caspases-8, -9, and -3. We found an increase of reactive oxygen species (ROS) and NF-kappaB activation after HEMA exposure. Blocking of ROS production by anti-oxidants had no direct influence on apoptosis caused by HEMA, but inhibition of NF-kappaB increased the fraction of apoptotic cells. Accordingly, mouse embryonic fibroblasts (MEF) from p65-/- mice were more susceptible to HEMA-induced apoptosis than were wild-type controls. Our results indicate that exposure to HEMA triggers apoptosis and that this mechanism is not directly dependent upon redox signaling. Nevertheless, ROS induction by HEMA activates NF-kappaB, which exerts a protective role in counteracting apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Metacrilatos/toxicidade , NF-kappa B/fisiologia , Análise de Variância , Animais , Western Blotting , Caspases/metabolismo , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Fibroblastos/citologia , Humanos , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Pele/citologiaRESUMO
We report on a 17-year-old male with severe pre- and postnatal growth retardation, craniosynostosis, distinctive facial features, acanthosis nigricans, deafness, mental retardation and progressive multi-organ involvement, particularly of the endocrine system, including hypothyroidism, hypogonadism, transitory hypoparathyroidism, and insulin resistance. In order to find a common mechanism explaining these multiple abnormalities, we searched for a possible defect in the signal transduction pathways from membrane to nucleus involving G-protein coupled receptors (GPCR). Adenylyl cyclase activity was evaluated by assaying c-AMP in the patient's cultured fibroblasts stimulated with several drugs and toxins acting on different effectors upstream of adenylyl cyclase. The preliminary results indicate a reduced cAMP accumulation in the patient, neither caused by constitutive activation of Gi nor inhibition of Gs signaling, and probably resulting from an alteration in the adenylyl cyclase system. The differential diagnosis with syndromes showing common clinical features with our patient is discussed.
Assuntos
Adenilil Ciclases/deficiência , Deficiências do Desenvolvimento/fisiopatologia , Doenças do Sistema Endócrino/fisiopatologia , Fácies , Retardo do Crescimento Fetal/fisiopatologia , Adenilil Ciclases/genética , Adolescente , Criança , Pré-Escolar , Deficiências do Desenvolvimento/genética , Doenças do Sistema Endócrino/genética , Retardo do Crescimento Fetal/genética , Humanos , Recém-Nascido , MasculinoRESUMO
Mitosis requires cyclin-dependent kinase (cdk) 1-cyclin B activity [1]. Exit from mitosis depends on the inactivation of the complex by the degradation of cyclin B [2]. Cdk2 is also active during mitosis [3, 4]. In Xenopus egg extracts, cdk2 is primarily in complex with cyclin E, which is stable [5]. At the end of mitosis, downregulation of cdk2-cyclin E activity is accompanied by inhibitory phosphorylation of cdk2 [6]. Here, we show that cdk2-cyclin E activity maintains cdk1-cyclin B during mitosis. At mitosis exit, cdk2 is inactivated prior to cdk1. The loss of cdk2 activity follows and depends upon an increase in protein kinase A (PKA) activity. Prematurely inactivating cdk2 advances the time of cyclin B degradation and cdk1 inactivation. Blocking PKA, instead, stabilizes cdk2 activity and inhibits cyclin B degradation and cdk1 inactivation. The stabilization of cdk1-cyclin B is also induced by a mutant cdk2-cyclin E complex that is resistant to inhibitory phosphorylation. P21-Cip1, which inhibits both wild-type and mutant cdk2-cyclin E, reverses mitotic arrest under either condition. Our findings indicate that the proteolysis-independent downregulation of cdk2 activity at the end of mitosis depends on PKA and is required to activate the proteolysis cascade that leads to mitosis exit.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/fisiologia , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Quinase 2 Dependente de Ciclina , Xenopus , Proteínas de XenopusRESUMO
OBJECTIVES: The aim of the present study was to test the hypothesis that retrovirus-mediated in vivo tissue factor pathway inhibitor (TFPI) gene transfer to the arterial wall would efficiently inhibit thrombosis without causing significant changes in systemic hemostatic variables. BACKGROUND: Acute coronary syndromes (unstable angina and acute myocardial infarction) are usually caused by atherosclerotic plaque rupture, with consequent activation of the coagulation cascade and circulating platelets. Tissue factor (TF) exposure represents an early event in this pathophysiologic sequence, leading to activation of the extrinsic coagulation pathway and thrombin formation. Tissue factor pathway inhibitor is a naturally occurring inhibitor of the extrinsic pathway. METHODS: In the present study, the gene coding for rabbit TFPI was inserted in a retroviral vector under control of a tetracycline-inducible promoter. Replication-defective, infectious, recombinant retroviruses were used to transfect rabbit carotid arteries with either TFPI or a reporter gene--green fluorescent protein (GFP). RESULTS: Retroviral-mediated arterial gene transfer of TFPI resulted in potent inhibition of intravascular thrombus formation in stenotic and injured rabbit carotid arteries, whereas transfection of the contralateral carotid artery with GFP had no effect on thrombosis. No significant changes in systemic hemostatic variables (prothrombin time and partial thromboplastin time) were observed when thrombosis was inhibited. CONCLUSIONS: These data suggest that retroviral-mediated transfection of the arterial wall with TFPI might represent an attractive approach for the treatment of thrombotic disorders.
Assuntos
Lesões das Artérias Carótidas/complicações , Trombose das Artérias Carótidas/terapia , Terapia Genética , Lipoproteínas/genética , Animais , Anticoagulantes/metabolismo , Artérias Carótidas/metabolismo , Trombose das Artérias Carótidas/etiologia , Trombose das Artérias Carótidas/metabolismo , Células Cultivadas , Vetores Genéticos , Imuno-Histoquímica , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Músculo Liso Vascular/metabolismo , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/genética , TransfecçãoRESUMO
cAMP-dependent protein kinase is targeted to discrete subcellular locations by a family of specific anchor proteins (A-kinase anchor proteins, AKAPs). Localization recruits protein kinase A (PKA) holoenzyme close to its substrate/effector proteins, directing and amplifying the biological effects of cAMP signaling.AKAPs include two conserved structural modules: (i) a targeting domain that serves as a scaffold and membrane anchor; and (ii) a tethering domain that interacts with PKA regulatory subunits. Alternative splicing can shuffle targeting and tethering domains to generate a variety of AKAPs with different targeting specificity. Although AKAPs have been identified on the basis of their interaction with PKA, they also bind other signaling molecules, mainly phosphatases and kinases, that regulate AKAP targeting and activate other signal transduction pathways. We suggest that AKAP forms a "transduceosome" by acting as an autonomous multivalent scaffold that assembles and integrates signals derived from multiple pathways. The transduceosome amplifies cAMP and other signals locally and, by stabilizing and reducing the basal activity of PKA, it also exerts long-distance effects. The AKAP transduceosome thus optimizes the amplitude and the signal/noise ratio of cAMP-PKA stimuli travelling from the membrane to the nucleus and other subcellular compartments.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ancoragem à Quinase A , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Citoesqueleto/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Peroxissomos/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de SinaisRESUMO
Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.
Assuntos
Genes ras/fisiologia , Transdução de Sinais/fisiologia , Células 3T3 , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxirredução , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Thyrotropin (TSH) stimulates survival and growth of thyroid cells via a seven transmembrane G protein-coupled receptor. TSH elevates the intracellular cyclic AMP (cAMP) levels activating protein kinase A (PKA). Recent evidence indicates that p21 Ras is required for TSH-induced mitogenesis, but the molecular mechanism(s) is not known. Here we report that Ras p21 activity is necessary for the Go- G1 transition in TSH induced cycle and that the downstream effector of Ras upon TSH signaling is p85-p110 PI3K. We show that PI3K inhibitors block TSH-induced DNA synthesis, cAMP-PKA stimulate the formation of the complex PI3K-p21 Ras and reduce the complex Ras-Raf1 in thyroid and other cells types. Moreover, PKA phosphorylates immunoprecipitated p85 and PKA phosphorylation of cell extracts significantly stimulates the formation of the complex PI3K-Ras. We suggest that PKA phosphorylates p85 and stabilizes the complex p110-p85, enhancing the interaction PI3K and p21 Ras. Simultaneously, cAMP inhibits Raf-1-ERK signaling by decreasing Raf1 availability to Ras. Under these circumstances PI3K signaling is favored. These results indicate that PI3K is an important mediator of Ras effects in cAMP-induced proliferation and illustrates how cAMP can selectively influence Ras effector pathways.
Assuntos
Ciclo Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Transdução de Sinais , Tireoglobulina/metabolismo , Proteínas ras/metabolismo , Diferenciação Celular , Divisão Celular/fisiologia , Linhagem Celular , DNA/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Immunoblotting , Proteína Oncogênica p21(ras)/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Tireoglobulina/genética , Proteínas ras/genéticaRESUMO
cAMP-dependent protein kinase is anchored to discrete cellular compartments by a family of proteins, the A-kinase anchor proteins (AKAPs). We have investigated in vivo and in vitro the biological effects of the expression of a prototypic member of the family, AKAP75, on smooth muscle cells. In vitro expression of AKAP75 in smooth muscle cells stimulated cAMP-induced transcription, increased the levels of the cyclin-dependent kinase-2 inhibitor p27(kip1), and reduced cell proliferation. In vivo expression of exogenous AKAP75 in common carotid arteries, subjected to balloon injury, significantly increased the levels of p27(kip1) and inhibited neointimal hyperplasia. Both the effects in smooth muscle cells in vitro and in carotid arteries in vivo were specifically dependent on the amplification of cAMP-dependent protein kinase (PKA) signals by membrane-bound PKA, as indicated by selective loss of the AKAP75 biological effects in mutants defective in the PKA anchor domain or by suppression of AKAP effects by the PKA-specific protein kinase inhibitor. These data indicate that AKAP proteins selectively amplify cAMP-PKA signaling in vitro and in vivo and suggest a possible target for the inhibition of the neointimal hyperplasia after vascular injury.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Divisão Celular/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas Supressoras de Tumor , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Proteínas de Ancoragem à Quinase A , Animais , Artérias Carótidas/química , Artérias Carótidas/patologia , Proteínas de Transporte/genética , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Cloranfenicol O-Acetiltransferase/efeitos dos fármacos , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27 , DNA/biossíntese , DNA/efeitos dos fármacos , DNA Recombinante , Técnicas de Transferência de Genes , Imuno-Histoquímica , Proteínas Associadas aos Microtúbulos/análise , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Plasmídeos/genética , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Túnica Íntima/química , Túnica Íntima/patologia , Túnica Média/química , Túnica Média/patologiaRESUMO
Activation of the cAMP-dependent protein kinase A (PKA) pathway may induce cAMP-response element-binding protein (CREB) phosphorylation either directly or via cross-talk mechanisms with other signal transduction pathways. In this study, we have investigated in striatal primary cultures the mechanism by which activation of the cAMP/PKA-dependent pathway leads to CREB phosphorylation via the extracellular signal-regulated kinase (ERK)-dependent pathway. We have found that PKA-induced CREB phosphorylation and CREB-dependent transcription are mediated by calcium (Ca(2+)) release from intracellular stores and are blocked by inhibitors of the protein kinase C and ERK pathways. This mechanism appears to be mediated by the small G-protein Rap1, whose activation appears to be primed by PKA-induced Ca(2+) release but not further induced by direct or indirect PKA- or protein kinase C-dependent phosphorylation. These results suggest that, in striatal neurons, intracellular Ca(2+) release, Rap1, and ERK pathway play a crucial role in the PKA-induced CREB phosphorylation and CREB-dependent transcription.
Assuntos
Cálcio/metabolismo , Corpo Estriado/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Animais , Proteína de Ligação a CREB , Corpo Estriado/citologia , AMP Cíclico/metabolismo , Ativação Enzimática , Fosforilação , Ratos , Ratos Sprague-Dawley , Transcrição GênicaRESUMO
Cell cycle checkpoints lead to the inhibition of cell cycle progression following DNA damage. A cell-free system derived from Xenopus eggs has been established that reconstitutes the checkpoint pathway inhibiting DNA replication initiation. DNA containing double-strand breaks inhibits replication initiation in a dose-dependent manner. Upon checkpoint activation, a prereplicative complex is assembled that contains ORC, Cdc6, Cdc7, and MCM proteins but lacks Cdc45. The checkpoint is ATM dependent. Cdk2/CyclinE acts downstream of ATM and is downregulated by Cdk2 phosphorylation on tyrosine 15. Cdk2AF/CyclinE is refractory to checkpoint signaling, and Cdc25A overrides the checkpoint and restores DNA replication. This report provides the description of a DNA damage checkpoint pathway that prevents the onset of S phase independently of the transcriptional function of p53 in a vertebrate organism.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Dano ao DNA/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae , Transdução de Sinais/fisiologia , Androstadienos/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Cafeína/farmacologia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Sistema Livre de Células , Cromossomos/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , DNA/genética , Replicação do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Masculino , Mutagênese/fisiologia , Proteínas Nucleares/metabolismo , Oócitos , Inibidores de Fosfodiesterase/farmacologia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Origem de Replicação/fisiologia , Fase S/genética , Espermatozoides , Transcrição Gênica/genética , Proteínas Supressoras de Tumor , Tirosina/metabolismo , Wortmanina , Xenopus , Proteínas de XenopusRESUMO
The expansion of CAG repeats is the genetic defect underlying eight neurodegenerative diseases. A common feature of these disorders is the presence of intracellular aggregates in neuronal cells. It is still unclear the significance of these cellular inclusions in the neurodegenerative process, since cell death without aggregate formation has been reported. We have constructed a synthetic fusion protein containing 17 or 43 CAG repeats and the green fluorescent protein that recapitulates the features of CAG-expanded alleles. Expression of 43, but not 17 CAG repeats results in formation of nuclear aggregates in human neuroblastoma cells. Moreover, the normal allele (17 CAG) is sequestered in the inclusion bodies. The presence of nuclear inclusions tightly correlates with apoptosis in cells expressing the protein encoding 43 CAG repeats. Cells harboring nuclear aggregates stop proliferation and undergo apoptosis. Moreover, the inhibition of protein degradation pathway increases intracellular aggregates and cell death. These data indicate that intranuclear aggregates induce apoptosis and suggest that the degradation of unfolded proteins improves cell survival.
Assuntos
Apoptose , Corpos de Inclusão/patologia , Neuroblastoma/patologia , Peptídeos/metabolismo , Sequências Repetitivas de Aminoácidos/fisiologia , Alelos , Divisão Celular , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Sobrevivência Celular , Cisteína Endopeptidases/metabolismo , Citoplasma/metabolismo , Citoplasma/patologia , Humanos , Corpos de Inclusão/metabolismo , Microscopia de Fluorescência , Complexos Multienzimáticos/metabolismo , Neuroblastoma/metabolismo , Peptídeos/química , Peptídeos/genética , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Aminoácidos/genética , Fatores de Tempo , Transfecção , Expansão das Repetições de Trinucleotídeos/genética , Células Tumorais CultivadasRESUMO
cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases (PKAs), typically determined by their specific regulatory subunits. We describe changes in the cAMP signal transduction pathway during cell cycle progression in synchronized rat thyroid cells. Both PKA type II (PKAII) localization and nuclear cAMP signaling are significantly modified during G(0) and G(1)-S transitions. G(1) is characterized by PKA activation and amplified cAMP signal transduction. This is associated with a decrease in the concentration of RI and RII regulatory subunits and enhanced anchoring of PKAII to the Golgi-centrosome region. Just prior to S, the cAMP pathway is depressed. Up-regulation of the pathway by exogenous cAMP in G(1) inhibited the subsequent decay of the Cdk inhibitor p27 and delayed the onset of S phase. Forced translocation of endogenous PKAII to the cytosol down-regulated cAMP signaling, advancing the timing of p27 decay and inducing premature exit from G(1). These data indicate that membrane-bound PKA amplifies the transduction of cAMP signals in G(1) and that the length of G(1) is influenced by cAMP-PKA.
Assuntos
Proteínas de Ciclo Celular , Ciclo Celular/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Glândula Tireoide/citologia , Proteínas Supressoras de Tumor , Animais , Transporte Biológico , Compartimento Celular , Núcleo Celular/enzimologia , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/isolamento & purificação , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/metabolismo , Citosol/enzimologia , Regulação para Baixo , Fase G1/fisiologia , Membranas/enzimologia , Proteínas Associadas aos Microtúbulos/metabolismo , Ratos , Transdução de SinaisRESUMO
The A126 cell line, a derivative of PC12, is defective in cAMP-induced transcription and does not differentiate in the presence of cAMP. In these cells overexpression of a cAMP-dependent protein kinase (PKA) anchor protein, AKAP75, and of the PKA catalytic subunit substantially increased the fraction of PKAII bound to the membrane, stimulated the transcription of cAMP-induced genes, and induced terminal differentiation. Conversely, wild type PC12 cells expressing a derivative of the AKAP75 protein, AKAP45, which binds the PKA regulatory subunits RII, but fails to locate them to the membranes, induced translocation of PKAII to the cytosol. These cells did not efficiently accumulate PKA catalytic subunit in the nuclei when stimulated with cAMP, did not transcribe cAMP-induced genes, and failed to differentiate when exposed to cAMP. These data indicate that membrane-bound PKA positively controls the transcription of cAMP-induced genes and differentiation in PC12 cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Proteínas de Ancoragem à Quinase A , Animais , Proteínas de Transporte/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/genética , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Proteínas de Membrana/metabolismo , Fator de Crescimento Neural/farmacologia , Células PC12 , Ratos , Transcrição Gênica/genética , TransfecçãoRESUMO
The neurotransmitter dopamine (DA) at a 10 microM concentration elicited a stimulation of intracellular cyclic AMP (cAMP) accumulation in cultured astrocytes derived from embryonic rat striatum. This accumulation was partially blocked by the beta-adrenergic receptors antagonist propranolol, mimicked by the D(1) agonist SKF 38393 and by the mixed D(1)/D(2) agonist apomorphine. A regional heterogeneity in the magnitude of dopamine-induced cAMP accumulation was observed in cultured astrocytes obtained from different brain areas. The maximum effect was observed in striatal astrocytes, a lower effect in cortical astrocytes, and no increase was detected in cerebellar astrocytes. Reverse transcription-polymerase chain reaction (RT-PCR) coupled to Southern blot hybridization demonstrated that striatal astrocytes express only D(1) receptor mRNA and Western blot analysis confirmed the expression of the D(1) receptor protein in striatal astrocytes. In contrast to what found in neurons, the D(1)-dependent cAMP formation in striatal astrocytes is partially reduced by pertussis toxin (PTX) treatment. The stimulation of D(1) receptors or the activation of adenylyl cyclase by forskolin led to an increase of cytosolic and nuclear protein kinase A (PKA) catalytic activity. The presence of dopamine D(1) receptors in cultured striatal astrocytes suggests a role of dopamine in the regulation of cellular processes in striatal astrocytes.
Assuntos
Astrócitos/metabolismo , Neostriado/metabolismo , Receptores de Dopamina D1/biossíntese , Animais , Astrócitos/efeitos dos fármacos , Southern Blotting , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dopamina/fisiologia , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Immunoblotting , Imuno-Histoquímica , Neostriado/citologia , Neostriado/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Passage through mitosis resets cells for a new round of chromosomal DNA replication [1]. In late mitosis, the pre-replication complex - which includes the origin recognition complex (ORC), Cdc6 and the minichromosome maintenance (MCM) proteins - binds chromatin as a pre-requisite for DNA replication. S-phase-promoting cyclin-dependent kinases (Cdks) and the kinase Dbf4-Cdc7 then act to initiate replication. Before the onset of replication Cdc6 dissociates from chromatin. S-phase and M-phase Cdks block the formation of a new pre-replication complex, preventing DNA over-replication during the S, G2 and M phases of the cell cycle [1]. The nuclear membrane also contributes to limit genome replication to once per cell cycle [2]. Thus, at the end of M phase, nuclear membrane breakdown and the collapse of Cdk activity reset cells for a new round of chromosomal replication. We showed previously that protein kinase A (PKA) activity oscillates during the cell cycle in Xenopus egg extracts, peaking in late mitosis. The oscillations are induced by the M-phase-promoting Cdk [3] [4]. Here, we found that PKA oscillation was required for the following phase of DNA replication. PKA activity was needed from mitosis exit to the formation of the nuclear envelope. PKA was not required for the assembly of ORC2, Cdc6 and MCM3 onto chromatin. Inhibition of PKA activity, however, blocked the release of Cdc6 from chromatin and subsequent DNA replication. These data suggest that PKA activation in late M phase is required for the following S phase.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Replicação do DNA/fisiologia , Proteínas de Saccharomyces cerevisiae , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Cromatina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Densitometria , Ativação Enzimática , Glutationa Transferase/farmacologia , Immunoblotting , Masculino , Mitose/efeitos dos fármacos , Complexo de Reconhecimento de Origem , Proteínas Recombinantes de Fusão/farmacologia , Espermatozoides/fisiologia , Xenopus/genética , Xenopus/crescimento & desenvolvimentoRESUMO
A common feature of CAG-expansion neurodegenerative diseases is the presence of intranuclear aggregates in neuronal cells. We have used a synthetic fusion protein containing at the NH2 terminus the influenza hemoagglutinin epitope (HA), a polyglutamine stretch (polyQ) of various size (17, 36, 43 CAG) and a COOH tail encoding the green fluorescent protein (GFP). The fusion proteins were expressed in COS-7 and neuroblastoma SK-N-BE cells. We found that the formation of aggregates largely depends on the length of polyglutamine tracts and on the levels of expression of the fusion protein. Moreover, transglutaminase overexpression caused an increase of insoluble aggregates only in cells expressing the mutant expanded protein. Conversely, treatment of cells with cystamine, a transglutaminase inhibitor, reduced the percentage of aggregates. We found also that the inhibition of the proteasome ubiquitin-dependent degradation increased the formation of intranuclear aggregates. These data suggest that length of polyglutamine tract, its expression, unbalance between cellular transglutaminase activity, and the ubiquitin-degradation pathway are key factors in the formation of intranuclear aggregates.
Assuntos
Peptídeos/fisiologia , Transglutaminases/fisiologia , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Animais , Células COS , Cistamina/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Cobaias , Modelos Biológicos , Proteínas Recombinantes de Fusão , Fatores de Tempo , Transglutaminases/farmacologia , Expansão das Repetições de Trinucleotídeos , Células Tumorais Cultivadas , Ubiquitinas/fisiologiaRESUMO
cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases, typically determined by their specific regulatory subunits. In the brain the major regulatory isoform RIIbeta and the RII-anchor protein, AKAP150 (rat) or 75 (bovine), are differentially expressed. Cortical neurons express RIIbeta and AKAP75; conversely, granule cerebellar cells express predominantly RIalpha and RIIalpha. Cortical neurons accumulate PKA catalytic subunit and phosphorylated cAMP responsive element binding protein very efficiently into nuclei upon cAMP induction, whereas granule cerebellar cells fail to do so. Down-regulation of RIIbeta synthesis by antisense oligonucleotides inhibited cAMP-induced nuclear signaling in cortical neurons. Expression in cerebellar granule cells of RIIbeta and AKAP75 genes by microinjection of specific expression vectors, markedly stimulated cAMP-induced transcription of the lacZ gene driven by a cAMP-responsive element promoter. These data indicate that the composition of PKA in cortical and granule cells underlies the differential ability of these cells to transmit cAMP signals to the nucleus.