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Eif-2 KinasePhosphatidylinositol 3-KinasesProtein KinasesSystème Signalisation Map KinaseProtein-Serine-Threonine KinasesInhibiteurs Des Protéines-KinasesCalcium-Calmodulin-Dependent Protein KinasesFacteur D'Initiation Eucaryote 2Src-Family KinasesProtéine Kinase CP38 Mitogen-Activated Protein KinasesCyclic Amp-Dependent Protein KinasesMitogen-Activated Protein Kinase 1Phosphorylationp21-Activated KinasesRéticulocytesMitogen-Activated Protein Kinase KinasesJnk Mitogen-Activated Protein KinasesMitogen-Activated Protein Kinase 3Facteur D'Initiation Eucaryote 4FProtein-Tyrosine KinasesCréatine KinaseCdc2 Protein KinaseCyclin-Dependent KinasesMap Kinase Kinase KinasesRibosomal Protein S6 KinasesCasein KinasesCasein Kinase IiPyruvate KinaseMap Kinase Kinase 1CinétiqueRécepteur Thymidine KinaseExtracellular Signal-Regulated Map KinasesMap Kinase Kinase 4Données Séquence MoléculairePhosphotransferases (Alcohol Group Acceptor)1-Phosphatidylinositol 4-KinaseBiosynthèse Des ProtéinesMitogen-Activated Protein KinasesCdc2-Cdc28 KinasesI-Kappa B KinaseInitiation Traductionnelle De La Chaine PeptidiqueTransduction SignalGlycogen Synthase Kinase 3Aurora Kinasesrho-Associated KinasesFacteur D'Initiation Eucaryote 4AIsoenzymesLiaison Protein Kinase C-DeltaProtein Kinase C-AlphaSéquence NucléotidiqueOryctolagus CuniculusPhosphoproteinesArn MessagerProtéinesSéquence Des Acides AminésMutationProtéines Des Proto-OncogènesTransfectionDiacylglycérol KinaseAMP-Activated Protein KinasesActivation EnzymatiqueFar-Western BlotProtéines Du Proto-Oncogène C-AktFocal Adhesion Kinase 1Spécificité Selon SubstratSérineMyosin Light Chain KinaseLignée CellulaireJanus Kinase 2Facteurs D'Initiation EucaryoteAcétate TétradécanoylphorbolFocal Adhesion Protein-Tyrosine Kinases90-Kda Ribosomal Protein S6 KinasesTyrosineProtéines Et Peptides De Signalisation IntracellulaireProtéines RecombinaisonProtein Kinase C-EpsilonArn Transfert MéthionineHomologie Séquentielle Acides AminésMap Kinase Kinase Kinase 1Site FixationCalcium-Calmodulin-Dependent Protein Kinase Type 2TOR Serine-Threonine KinasesAndrostadiènesProtein Kinase C betaMap Kinase Kinase 2ApoptoseCyclin-Dependent Kinase 2Cyclic Gmp-Dependent Protein KinasesClonage MoléculaireLignée Cellulaire TumoraleProtéines De Fusion RecombinantesGtpCellules 3T3Cyclin-Dependent Kinase 5Phosphoglycérate KinaseStructure Tertiaire Protéine

Influenza virus NS1 protein counteracts PKR-mediated inhibition of replication. (49/1374)

The availability of an influenza virus NS1 gene knockout virus (delNS1 virus) allowed us to establish the significance of the biological relationship between the influenza virus NS1 protein and double-stranded-RNA-activated protein kinase (PKR) in the life cycle and pathogenicity of influenza virus. Our results show that the lack of functional PKR permits the delNS1 virus to replicate in otherwise nonpermissive hosts, suggesting that the major function of the influenza virus NS1 protein is to counteract or prevent the PKR-mediated antiviral response.  (+info)

PKR stimulates NF-kappaB irrespective of its kinase function by interacting with the IkappaB kinase complex. (50/1374)

The interferon (IFN)-induced double-stranded RNA-activated protein kinase PKR mediates inhibition of protein synthesis through phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) and is also involved in the induction of the IFN gene through the activation of the transcription factor NF-kappaB. NF-kappaB is retained in the cytoplasm through binding to its inhibitor IkappaBalpha. The critical step in NF-kappaB activation is the phosphorylation of IkappaBalpha by the IkappaB kinase (IKK) complex. This activity releases NF-kappaB from IkappaBalpha and allows its translocation to the nucleus. Here, we have studied the ability of PKR to activate NF-kappaB in a reporter assay and have shown for the first time that two catalytically inactive PKR mutants, PKR/KR296 and a deletion mutant (PKR/Del42) which lacks the potential eIF2alpha-binding domain, can also activate NF-kappaB. This result indicated that NF-kappaB activation by PKR does not require its kinase activity and that it is independent of the PKR-eIF2alpha relationship. Transfection of either wild-type PKR or catalytically inactive PKR in PKR(0/0) mouse embryo fibroblasts resulted in the activation of the IKK complex. By using a glutathione S-transferase pull-down assay, we showed that PKR interacts with the IKKbeta subunit of the IKK complex. This interaction apparently does not require the integrity of the IKK complex, as it was found to occur with extracts from cells deficient in the NF-kappaB essential modulator, one of the components of the IKK complex. Therefore, our results reveal a novel pathway by which PKR can modulate the NF-kappaB signaling pathway without using its kinase activity.  (+info)

The B56alpha regulatory subunit of protein phosphatase 2A is a target for regulation by double-stranded RNA-dependent protein kinase PKR. (51/1374)

PKR is a cellular serine/threonine kinase that phosphorylates eukaryotic translation initiation factor 2alpha (eIF2alpha) to regulate protein synthesis. PKR also plays a role in the regulation of transcription, programmed cell death and the cell cycle, processes which likely involve other substrates. In a yeast two-hybrid screen, we isolated human protein phosphatase 2A (PP2A) regulatory subunit B56alpha as a PKR-interacting protein. The interaction between B56alpha and PKR was confirmed by in vitro binding assays as well as by in vivo coimmunoprecipitation, and this interaction is dependent on the catalytic activity of PKR. Moreover, recombinant B56alpha was efficiently phosphorylated by PKR in vitro and an isoelectric point shift in B56alpha was detected in extracts from cells induced with the PKR activator pIC. An in vitro dephosphorylation assay showed that when B56alpha was phosphorylated by PKR, the activity of PP2A trimeric holoenzyme was increased. A functional interaction between B56alpha and PKR was observed in cotransfection assays, where a B56alpha-mediated increase in luciferase expression was inhibited by cotransfection with wild-type PKR. This is likely due to a decreased level of eIF4E phosphorylation caused by an increase in PP2A activity following PKR phosphorylation of B56alpha. Taken together, our data indicate that PKR can modulate PP2A activity by phosphorylating B56alpha to regulate cellular activities.  (+info)

Regulation of the RNA-dependent protein kinase by triple helix formation. (52/1374)

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phos-phorylates the alpha-subunit of the translation initiation factor eIF-2, inhibiting its function. PKR is activated in vitro by binding to double-stranded RNA (dsRNA) molecules of approximately 30 bp or longer. Here we show that triple helix forming oligonucleotides (TFOs) inhibit dsRNA binding to the isolated RNA binding domain of PKR. The inhibition is specific to the targeted RNA and dependent on TFO length. Binding to a 30 bp duplex is inhibited by a 28 nt TFO and a 20 nt TFO with an IC(50) of 35 +/- 2 and 210 +/- 22 nM, respectively. An 18 nt TFO partially inhibits binding. The activation of the kinase domain of PKR by a 30 bp RNA duplex is also inhibited by a 28 nt TFO. Inhibition of binding is most effective when the triple helix is formed prior to addition of the protein. These results indicate that triplex formation can be used to prevent the binding of an RNA binding protein with dsRNA-binding motifs.  (+info)

Human breast cancer cells contain elevated levels and activity of the protein kinase, PKR. (53/1374)

PKR is a double-stranded (ds) RNA activated protein kinase whose expression is induced by interferon. Activated PKR phosphorylates its cellular substrate, eIF2, an essential initiation factor of translation. Prior evidence from a murine model system suggested that PKR may act as a tumor suppressor, but the evidence from human tumors is equivocal. To study PKR function in human breast cancer, PKR activity was measured in mammary carcinoma cell lines and nontransformed mammary epithelial cell lines. If PKR functioned as a tumor suppressor in this system, its activity would be higher in nontransformed cells than in carcinoma cells. On the contrary, PKR autophosphorylation and the phosphorylation of its substrate, the alpha-subunit of eIF2, is 7 - 40-fold higher in lysates prepared from breast carcinoma cell lines than in those from nontransformed epithelial cell lines. Correspondingly, a larger proportion of eIF2alpha is present in a phosphorylated state in carcinoma cell lines than in nontransformed cell lines. Protein synthesis is not inhibited by the high eIF2alpha phosphorylation in carcinoma cells, probably because they contain higher levels of eIF2B, the initiation factor that is inhibited by eIF2alpha phosphorylation. The dramatically lower PKR activity in nontransformed cell lines is partially due to lower PKR protein levels (2 - 4-fold) as well as to the presence of a PKR inhibitor. The nontransformed cells contain P58, a known cellular inhibitor of PKR that physically interacts with PKR and may be responsible for the low PKR activity in these cells. Taken together, these observations call into question the role of PKR as a tumor suppressor and suggest a positive regulatory role of PKR in growth control of breast cancer cells.  (+info)

Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase. (54/1374)

NO synthase 2 (NOS2) is induced in airway epithelium by influenza virus infection. NOS2 induction late in the course of viral infection may occur in response to IFN-gamma, but early in infection gene expression may be induced by the viral replicative intermediate dsRNA through the dsRNA-activated protein kinase (PKR). Since PKR activates signaling pathways important in NOS2 gene induction, we determined whether PKR is a component in the signal transduction pathway leading to NOS2 gene expression after viral infection of airway epithelium. We show that NOS2 gene expression in human airway epithelial cells occurs in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including NF-kappaB and IFN regulatory factor 1. NOS2 expression is markedly diminished and IFN regulatory factor 1 and NF-kappaB activation are substantially impaired in PKR null cells. Strikingly, NOS2 induction in response to LPS is abolished in PKR null cells, confirming a central role for PKR in the general signaling pathway to NOS2.  (+info)

Perk is essential for translational regulation and cell survival during the unfolded protein response. (55/1374)

Malfolded proteins in the endoplasmic reticulum (ER) inhibit translation initiation. This response is believed to be mediated by increased phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) and is hypothesized to reduce the work load imposed on the folding machinery during stress. Here we report that mutating the gene encoding the ER stress-activated eIF2alpha kinase PERK abolishes the phosphorylation of eIF2alpha in response to accumulation of malfolded proteins in the ER resulting in abnormally elevated protein synthesis and higher levels of ER stress. Mutant cells are markedly impaired in their ability to survive ER stress and inhibition of protein synthesis by cycloheximide treatment during ER stress ameliorates this impairment. PERK thus plays a major role in the ability of cells to adapt to ER stress.  (+info)

Potential role of PKR in double-stranded RNA-induced macrophage activation. (56/1374)

In this study, the role of the double-stranded (ds) RNA-dependent protein kinase (PKR) in macrophage activation was examined. dsRNA [polyinosinic:polycytidylic acid (poly IC)]-stimulated inducible nitric oxide synthase, interleukin (IL)-1alpha and IL-1beta mRNA expression, nitrite formation and IL-1 release are attenuated in RAW264.7 cells stably expressing dominant negative (dn) mutants of PKR. The transcriptional regulator nuclear factor (NF)-kappaB is activated by dsRNA, and appears to be required for dsRNA-induced macrophage activation. While dnPKR mutants prevent macrophage activation, they fail to attenuate dsRNA-induced IkappaB degradation or NF-kappaB nuclear localization. The inhibitory actions of dnPKR on dsRNA-induced macrophage activation can be overcome by treatment with interferon (IFN)-gamma, an event associated with PKR degradation. Furthermore, dsRNA + IFN-gamma stimulate inducible nitric oxide synthase expression, IkappaB degradation and NF-kappaB nuclear localization to similar levels in macrophages isolated from PKR(-/-) and PKR(+/+) mice. These findings indicate that both NF-kappaB and PKR are required for dsRNA-induced macrophage activation; however, dsRNA-induced NF-kappaB activation occurs by PKR-independent mechanisms in macrophages. In addition, the PKR dependence of dsRNA-induced macrophage activation can be overcome by IFN-gamma.  (+info)