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Phosphatidylinositol 3-KinasesMAP Kinase Signaling SystemProtein KinasesProtein-Serine-Threonine KinasesProtein Kinase InhibitorsCalcium-Calmodulin-Dependent Protein Kinasessrc-Family KinasesProtein Kinase Cp38 Mitogen-Activated Protein KinasesCyclic AMP-Dependent Protein KinaseseIF-2 KinaseMitogen-Activated Protein Kinase 1p21-Activated KinasesMitogen-Activated Protein Kinase KinasesJNK Mitogen-Activated Protein KinasesMitogen-Activated Protein Kinase 3PhosphorylationEukaryotic Initiation Factor-3Protein-Tyrosine KinasesCreatine KinaseRibosomal Protein S6 KinasesCDC2 Protein KinaseCyclin-Dependent KinasesMAP Kinase Kinase KinasesCasein Kinase IIEukaryotic Initiation Factor-2Casein KinasesPyruvate KinaseMAP Kinase Kinase 1Receptor Protein-Tyrosine KinasesProtein BindingThymidine KinaseEukaryotic Initiation FactorsExtracellular Signal-Regulated MAP KinasesMAP Kinase Kinase 4Eukaryotic Initiation Factor-2BPhosphotransferases (Alcohol Group Acceptor)Protein BiosynthesisMitogen-Activated Protein KinasesSignal Transduction1-Phosphatidylinositol 4-KinaseGlycogen Synthase Kinase 3CDC2-CDC28 KinasesMolecular Sequence DataMutationI-kappa B KinaseAurora Kinasesrho-Associated KinasesIsoenzymesProtein Kinase C-alphaProtein Kinase C-deltaEukaryotic Initiation Factor-4APhosphoproteinsProto-Oncogene ProteinsKineticsAMP-Activated Protein KinasesAmino Acid SequenceProto-Oncogene Proteins c-aktDiacylglycerol KinaseRNA, MessengerTOR Serine-Threonine KinasesTransfectionBlotting, WesternEnzyme ActivationFocal Adhesion Kinase 1Protein Structure, TertiaryRibosomal Protein S6 Kinases, 90-kDaIntracellular Signaling Peptides and ProteinsBinding SitesCell LineBase SequenceMyosin-Light-Chain KinaseJanus Kinase 2Recombinant ProteinsFocal Adhesion Protein-Tyrosine KinasesSerineTyrosineSubstrate SpecificityCell Line, TumorProtein Kinase C-epsilonApoptosisSequence Homology, Amino AcidMAP Kinase Kinase Kinase 1Protein Kinase C betaCalcium-Calmodulin-Dependent Protein Kinase Type 2Eukaryotic Initiation Factor-5Recombinant Fusion ProteinsTetradecanoylphorbol AcetateAndrostadienesMAP Kinase Kinase 2Cyclin-Dependent Kinase 2Cyclic GMP-Dependent Protein KinasesChromonesEukaryotic Initiation Factor-1HeLa CellsRibosomal Protein S6 Kinases, 70-kDaCyclin-Dependent Kinase 5Cells, CulturedCell Cycle ProteinsPhosphoglycerate Kinase

Characterization of a mutant pancreatic eIF-2alpha kinase, PEK, and co-localization with somatostatin in islet delta cells. (1/1374)

Phosphorylation of eukaryotic translation initiation factor-2alpha (eIF-2alpha) is one of the key steps where protein synthesis is regulated in response to changes in environmental conditions. The phosphorylation is carried out in part by three distinct eIF-2alpha kinases including mammalian double-stranded RNA-dependent eIF-2alpha kinase (PKR) and heme-regulated inhibitor kinase (HRI), and yeast GCN2. We report the identification and characterization of a related kinase, PEK, which shares common features with other eIF-2alpha kinases including phosphorylation of eIF-2alpha in vitro. We show that human PEK is regulated by different mechanisms than PKR or HRI. In contrast to PKR or HRI, which are dependent on autophosphorylation for their kinase activity, a point mutation that replaced the conserved Lys-614 with an alanine completely abolished the eIF-2alpha kinase activity, whereas the mutant PEK was still autophosphorylated when expressed in Sf-9 cells. Northern blot analysis indicates that PEK mRNA was predominantly expressed in pancreas, though low expression was also present in several tissues. Consistent with the high levels of mRNA in pancreas, the PEK protein was only detected in human pancreatic islets, and the kinase co-localized with somatostatin, a pancreatic delta cell-specific hormone. Thus PEK is believed to play an important role in regulating protein synthesis in the pancreatic islet, especially in islet delta cells.  (+info)

Characterization of transgenic mice with targeted disruption of the catalytic domain of the double-stranded RNA-dependent protein kinase, PKR. (2/1374)

The interferon-inducible, double-stranded RNA-dependent protein kinase PKR has been implicated in anti-viral, anti-tumor, and apoptotic responses. Others have attempted to examine the requirement of PKR in these roles by targeted disruption at the amino terminal-encoding region of the Pkr gene. By using a strategy that aims at disruption of the catalytic domain of PKR, we have generated mice that are genetically ablated for functional PKR. Similar to the other mouse model of Pkr disruption, we have observed no consequences of loss of PKR on tumor suppression. Anti-viral response to influenza and vaccinia also appeared to be normal in mice and in cells lacking PKR. Cytokine signaling in the type I interferon pathway is normal but may be compromised in the erythropoietin pathway in erythroid bone marrow precursors. Contrary to the amino-terminal targeted Pkr mouse, tumor necrosis factor alpha-induced apoptosis and the anti-viral apoptosis response to influenza is not impaired in catalytic domain-targeted Pkr-null cells. The observation of intact eukaryotic initiation factor-2alpha phosphorylation in these Pkr-null cells provides proof of rescue by another eukaryotic initiation factor-2alpha kinase(s).  (+info)

Double-stranded-RNA-activated protein kinase PKR enhances transcriptional activation by tumor suppressor p53. (3/1374)

The tumor suppressor p53 plays a key role in inducing G1 arrest and apoptosis following DNA damage. The double-stranded-RNA-activated protein PKR is a serine/threonine interferon (IFN)-inducible kinase which plays an important role in regulation of gene expression at both transcriptional and translational levels. Since a cross talk between IFN-inducible proteins and p53 had already been established, we investigated whether and how p53 function was modulated by PKR. We analyzed p53 function in several cell lines derived from PKR+/+ and PKR-/- mouse embryonic fibroblasts (MEFs) after transfection with the temperature-sensitive (ts) mutant of mouse p53 [p53(Val135)]. Here we report that transactivation of transcription by p53 and G0/G1 arrest were impaired in PKR-/- cells upon conditions that ts p53 acquired a wild-type conformation. Phosphorylation of mouse p53 on Ser18 was defective in PKR-/- cells, consistent with an impaired transcriptional induction of the p53-inducible genes encoding p21(WAF/Cip1) and Mdm2. In addition, Ser18 phosphorylation and transcriptional activation by mouse p53 were diminished in PKR-/- cells after DNA damage induced by the anticancer drug adriamycin or gamma radiation but not by UV radiation. Furthermore, the specific phosphatidylinositol-3 (PI-3) kinase inhibitor LY294002 inhibited the induction of phosphorylation of Ser18 of p53 by adriamycin to a higher degree in PKR+/+ cells than in PKR-/- cells. These novel findings suggest that PKR enhances p53 transcriptional function and implicate PKR in cell signaling elicited by a specific type of DNA damage that leads to p53 phosphorylation, possibly through a PI-3 kinase pathway.  (+info)

Involvement of PKR in the regulation of myogenesis. (4/1374)

The involvement of the double-stranded RNA-activated protein kinase PKR in the regulation of the myogenic process was investigated. For this purpose, the murine myogenic cell line C2C12 was used. The cells were first cultivated in either growth medium or differentiation medium (DM), and the activation of PKR during differentiation was determined by monitoring its enzymatic activity and by immunoblot analysis. A significant increase in both parameters was detected already at 24 h in DM, whereas in cells grown in growth medium, the increase was evident only after 96 h, when spontaneous differentiation was observed in highly crowded cultures. Consequently, we established the direct effect of PKR activation on the myogenic process. C2C12 cells were transfected with an expression vector harboring a cDNA molecule encoding human PKR fused to the inducible metallothionein promoter. One of the clones (clone 8) expressing high levels of PKR was selected and further analyzed. In the presence of ZnCl2, which activates the promoter, the rate of cell growth of the transfected cells was clearly reduced compared to that of wild-type C2C12 cells transfected with only the neomycin-resistant gene (C2-NEO). In addition, altered morphology with partial fusion was observed. Biochemically, an increase in creatine kinase activity accompanied by an increased rate of expression of the myogenic protein troponin T and the myogenic transcription factors myoD and myogenin was detected in clone 8 cells exposed to ZnCl2. Most importantly, an induction in the level of cyclin-dependent kinase inhibitor p21WAF1 and an increase in the level of the underphosphorylated active form of the tumor suppressor protein pRb concomitant with the down-regulation of cyclin D1 and c-myc were also evident in the transfected clones. These changes were similar to those observed in normal C2C12 cells cultivated in DM. We conclude that PKR is an important regulatory protein participating in the myogenic process.  (+info)

Regulatable expression of the interferon-induced double-stranded RNA dependent protein kinase PKR induces apoptosis and fas receptor expression. (5/1374)

PKR is an interferon-induced dsRNA-dependent protein kinase involved in the antiviral response as well as in cell growth and differentiation. Studies using a transdominant negative mutant of PKR also have implicated the kinase in tumor suppression and apoptosis. However, functional studies of PKR have been hampered by the lack of a suitable expression system. In this study, we used a tetracycline-regulated inducible system in NIH3T3 cells to investigate the involvement of PKR in programmed cell death (apoptosis). We show that expression of wild-type PKR causes apoptosis and correlates with increased mRNA levels for the Fas receptor, a member of the tumor necrosis family of proteins. Expression of an inactive form of PKR (K296R) or the vector alone did not induce apoptosis or elevate Fas mRNA levels. Our results clearly demonstrate that expression of an active form of PKR triggers apoptosis, possibly through upregulation of the Fas receptor.  (+info)

Failure of measles virus to activate nuclear factor-kappa B in neuronal cells: implications on the immune response to viral infections in the central nervous system. (6/1374)

Neurons are postmitotic cells that foster virus persistence. These cells lack the HLA class I molecules required for clearance of infected cells. Previously, we showed that HLA class I is induced by measles virus (MV) on glial cells, which is primarily mediated by IFN-beta. In contrast, MV was unable to induce HLA class I or IFN-beta in neuronal cells. This failure was associated with lack of NF-kappa B binding to the positive regulatory domain II element of the IFN-beta promoter, which is essential for virus-induced IFN-beta gene activity. In this study, we demonstrate that the failure to activate NF-kappa B in neuronal cells is due to the inability of MV to induce phosphorylation and degradation of I kappa B, the inhibitor of NF-kappa B. In contrast, TNF-alpha induced degradation of I kappa B alpha in the neuronal cells, suggesting that failure to induce I kappa B alpha degradation is likely due to a defect in virus-mediated signaling rather than to a defect involving neuronal I kappa B alpha. Like MV, mumps virus and dsRNA failed to induce I kappa B alpha degradation in the neuronal cells, suggesting that this defect may be specific to viruses. Autophosphorylation of the dsRNA-dependent protein kinase, a kinase possibly involved in virus-mediated I kappa B alpha phosphorylation, was intact in both cell types. The failure of virus to induce I kappa B alpha phosphorylation and consequently to activate NF-kappa B in neuronal cells could explain the repression of IFN-beta and class I gene expression in virus-infected cells. These findings provide a potential mechanism for the ability of virus to persist in neurons and to escape immune surveillance.  (+info)

Full peptide synthesis, purification, and characterization of six Tat variants. Differences observed between HIV-1 isolates from Africa and other continents. (7/1374)

AIDS in Africa is characterized by the equal distribution of mortality between the two genders because of highly virulent human immunodeficiency virus type 1 (HIV-1) strains. The viral protein Tat trans-activates viral gene expression and is essential for HIV-1 replication. We chemically synthesized six different Tat proteins, with sizes ranging from 86 to 101 residues, from HIV-1 isolates located in different parts of the world including highly virulent African strains. Protein purification, mass spectroscopy, and amino acid analysis showed that the synthesis was successful in each case but with different yields. We show that all have the ability to bind the HIV long terminal repeat (LTR) RNA trans-activation response element (TAR) region, involved in Tat-mediated trans-activation, but structural heterogeneities are revealed by circular dichroism. These Tat synthetic proteins cross membranes but differ in their ability to trans-activate an HIV LTR-reporter gene in stably transfected HeLa cells. Two Tat proteins from virulent African HIV-1 strains were much more active than those from Europe and the United States. The interferon-induced kinase (PKR), involved in cell antiviral defense, phosphorylates only Tat variants corresponding to less or nonvirulent HIV-1 isolates. Our results indicate that the high virulence of some African HIV-1 strains could be related to Tat activity.  (+info)

NFkappaB activation is required for interferon regulatory factor-1-mediated interferon beta induction. (8/1374)

The interferon regulatory factor 1 (IRF-1) acts as a transcriptional inducer of the interferon beta (IFN-beta) gene and interferon-stimulated genes. Here we report that IRF-1-mediated IFN-beta induction depends on NFkappaB activity. IRF-1 by itself initiates NFkappaB activation by inducing a reduction in cellular MAD3/IkappaBalpha, an inhibitor of NFkappaB. After nuclear translocation, NFkappaB synergizes with IRF-1 on the cis-elements positive regulatory domain (PRD)II and PRDI/III to induce transcription of the IFN-beta gene. In contrast with IFN-beta transcription induced by dsRNA or virus, c-Jun/ATF-2 binding to PRDIV is not involved. Recombinant MAD3/IkappaBalpha is phosphorylated in vitro by extracts from IRF-1-expressing cells. IRF-1-dependent MAD3/IkappaBalpha degradation is not detectable in cells expressing a dominant negative mutant of the protein kinase PKR, suggesting that PKR mediates MAD3/IkappaBalpha degradation.  (+info)