scholarly journals TRBP Control of PACT-Induced Phosphorylation of Protein Kinase R Is Reversed by Stress

2008 ◽  
Vol 29 (1) ◽  
pp. 254-265 ◽  
Author(s):  
Aïcha Daher ◽  
Ghislaine Laraki ◽  
Madhurima Singh ◽  
Carlos E. Melendez-Peña ◽  
Sylvie Bannwarth ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Hela Cells ◽  
Rna Binding ◽  
Luciferase Assay ◽  
Serum Starvation ◽  
Protein Kinase R ◽  
Translation Inhibition ◽  
Binding Domains ◽  
Immunodeficiency Virus ◽  
Terminal Amino

ABSTRACT The TAR RNA binding Protein, TRBP, inhibits the activity of the interferon-induced protein kinase R (PKR), whereas the PKR activator, PACT, activates its function. TRBP and PACT also bind to each other through their double-stranded RNA binding domains (dsRBDs) and their Medipal domains, which may influence their activity on PKR. In a human immunodeficiency virus (HIV) long terminal repeat-luciferase assay, PACT unexpectedly reversed PKR-mediated inhibition of gene expression. In a translation inhibition assay in HeLa cells, PACT lacking the 13 C-terminal amino acids (PACTΔ13), but not full-length PACT, activated PKR and enhanced interferon-mediated repression. In contrast, in the astrocytic U251MG cells that express low TRBP levels, both proteins activate PKR, but PACTΔ13 is stronger. Immunoprecipitation assays and yeast two-hybrid assays show that TRBP and PACTΔ13 interact very weakly due to a loss of binding in the Medipal domain. PACT-induced PKR phosphorylation was restored in Tarbp2 −/− murine tail fibroblasts and in HEK293T or HeLa cells when TRBP expression was reduced by RNA interference. In HEK293T and HeLa cells, arsenite, peroxide, and serum starvation-mediated stresses dissociated the TRBP-PACT interaction and increased PACT-induced PKR activation, demonstrating the relevance of this control in a physiological context. Our results demonstrate that in cells, TRBP controls PACT activation of PKR, an activity that is reversed by stress.

scholarly journals Tilapia dsRNA-activated protein kinase R (PKR): An interferon-induced antiviral effector with translation inhibition activity

2021 ◽  
Vol 112 ◽  
pp. 74-80
Author(s):  
Zhen Gan ◽  
Jun Cheng ◽  
Jing Hou ◽  
Shannan Chen ◽  
Hongli Xia ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Inhibition Activity ◽  
Protein Kinase R ◽  
Translation Inhibition

scholarly journals Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1

2006 ◽  
Vol 80 (23) ◽  
pp. 11817-11826 ◽  
Author(s):  
Morgan Hakki ◽  
Emily E. Marshall ◽  
Katherine L. De Niro ◽  
Adam P. Geballe
Keyword(s):  
Protein Kinase ◽  
Human Cytomegalovirus ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Underlying Mechanism ◽  
Cellular Protein ◽  
Initiation Factor ◽  
Protein Kinase R ◽  
Double Stranded Rna

ABSTRACT The human cytomegalovirus (HCMV) TRS1 and IRS1 genes block the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) and the consequent shutoff of cellular protein synthesis that occur during infection with vaccinia virus (VV) deleted of the double-stranded RNA binding protein gene E3L (VVΔE3L). To further define the underlying mechanism, we first evaluated the effect of pTRS1 on protein kinase R (PKR), the double-stranded RNA (dsRNA)-dependent eIF2α kinase. Immunoblot analyses revealed that pTRS1 expression in the context of a VVΔE3L recombinant decreased levels of PKR in the cytoplasm and increased its levels in the nucleus of infected cells, an effect not seen with wild-type VV or a VVΔE3L recombinant virus expressing E3L. This effect of pTRS1 was confirmed by visualizing the nuclear relocalization of PKR-EGFP expressed by transient transfection. PKR present in both the nuclear and cytoplasmic fractions was nonphosphorylated, indicating that it was unactivated when TRS1 was present. PKR also accumulated in the nucleus during HCMV infection as determined by indirect immunofluorescence and immunoblot analysis. Binding assays revealed that pTRS1 interacted with PKR in mammalian cells and in vitro. This interaction required the same carboxy-terminal region of pTRS1 that is necessary to rescue VVΔE3L replication in HeLa cells. The carboxy terminus of pIRS1 was also required for rescue of VVΔE3L and for mediating an interaction of pIRS1 with PKR. These results suggest that these HCMV genes directly interact with PKR and inhibit its activation by sequestering it in the nucleus, away from both its activator, cytoplasmic dsRNA, and its substrate, eIF2α.

scholarly journals Protein Kinase R Mediates the Inflammatory Response Induced by Hyperosmotic Stress

2016 ◽  
Vol 37 (4) ◽  
Author(s):  
Kenneth T. Farabaugh ◽  
Mithu Majumder ◽  
Bo-Jhih Guan ◽  
Raul Jobava ◽  
Jing Wu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Rna Binding ◽  
Nuclear Factor Κb ◽  
Hyperosmotic Stress ◽  
Nuclear Accumulation ◽  
Intestinal Epithelial ◽  
Protein Kinase R ◽  
Oxide Synthase

ABSTRACT High extracellular osmolarity results in a switch from an adaptive to an inflammatory gene expression program. We show that hyperosmotic stress activates the protein kinase R (PKR) independently of its RNA-binding domain. In turn, PKR stimulates nuclear accumulation of nuclear factor κB (NF-κB) p65 species phosphorylated at serine-536, which is paralleled by the induction of a subset of inflammatory NF-κB p65-responsive genes, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and IL-1β. The PKR-mediated hyperinduction of iNOS decreases cell survival in mouse embryonic fibroblasts via mechanisms involving nitric oxide (NO) synthesis and posttranslational modification of proteins. Moreover, we demonstrate that the PKR inhibitor C16 ameliorates both iNOS amplification and disease-induced phenotypic breakdown of the intestinal epithelial barrier caused by an increase in extracellular osmolarity induced by dextran sodium sulfate (DSS) in vivo. Collectively, these findings indicate that PKR activation is an essential part of the molecular switch from adaptation to inflammation in response to hyperosmotic stress.

scholarly journals Regulation of PACT-Mediated Protein Kinase Activation by the OV20.0 Protein of Orf Virus

2015 ◽  
Vol 89 (22) ◽  
pp. 11619-11629 ◽  
Author(s):  
Yeu-Yang Tseng ◽  
Guan-Ru Liao ◽  
Ganes C. Sen ◽  
Fong-Yuan Lin ◽  
Wei-Li Hsu
Keyword(s):  
Protein Kinase ◽  
Rna Binding ◽  
Small Ruminants ◽  
Kinase Domain ◽  
Orf Virus ◽  
Infection Model ◽  
Content Type ◽  
Binding Domains ◽  
Mouse Infection Model

ABSTRACTDouble-stranded RNA (dsRNA)-activated protein kinase (PKR), a major component of the cellular antiviral system, is activated by the binding of either dsRNA or the cellular PKR activator, the PACT protein. The suppression of PKR activation is one of the main strategies that viruses employ to circumvent interferon signaling. Orf virus (ORFV), a parapoxvirus from thePoxviridaefamily, causes contagious pustular dermatitis in small ruminants. Previous studies have demonstrated that various OV20.0 isoforms, encoded by the OV20.0L gene, are able to inhibit PKR activation both by sequestering dsRNA and by physically interacting with PKRin vitro. Thus, this gene acts as a virulence factor of ORFV when tested using a mouse infection model. In the present study, the regions within OV20.0 that interact with dsRNA and with PKR have been mapped. Furthermore, this study demonstrates for the first time that OV20.0 is also able to interact with the dsRNA binding domain of PACT and that the presence of dsRNA strengthened the interaction of these two molecules. The presence of OV20.0 diminishes PKR phosphorylation when this is stimulated by PACT. Nevertheless, the association of OV20.0 with PKR, rather than with PACT, was found to be essential for reducing PACT-mediated PKR phosphorylation. These observations elucidate a new strategy whereby innate immunity can be evaded by ORFV.IMPORTANCEOur previous study indicated that ORFV's two OV20.0 isoforms act as a PKR antagonist via sequestering the PKR activator, dsRNA, and by interacting with PKR, leading to an inhibition of PKR activation (Y. Y. Tseng, F. Y. Lin, S. F. Cheng, D. Tscharke, S. Chulakasian, C. C. Chou, Y. F. Liu, W. S. Chang, M. L. Wong, and W. L. Hsu, J Virol89:4966–4979, 2015, doi:10.1128/JVI.03714-14). In the current study, the possible mechanisms by which OV20.0 protein counteracts PKR activation were studied in depth. OV20.0 is able to bind PKR and its two activators, dsRNA and PACT. In addition, OV20.0 binds directly to the RNA binding domains (RBDs) of PKR, and this interaction does not require dsRNA. Moreover, OV20.0 interacts with or occupies the RBD2 and the kinase domain of PKR, which then prevents PACT binding to PKR. Finally, OV20.0 associates with PACT via the RBDs, which may reduce the ability of PACT to induce PKR activation. The findings in this study provide new concepts in relation to how ORFV modulates PKR activation.

scholarly journals Activation of dsRNA-Dependent Protein Kinase R by MicroRNA-378 Sustains Metabolic Inflammation in Hepatic Insulin Resistance

2021 ◽  
Author(s):  
Hao Wang ◽  
Yongyan Song ◽  
Yuxin Wu ◽  
Virender Kumar ◽  
Ram I Mahato ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Protein Kinase ◽  
Er Stress ◽  
Rna Binding ◽  
Hepatic Insulin Resistance ◽  
Protein Kinase R ◽  
Dependent Protein Kinase ◽  
Metabolic Inflammation ◽  
Mitochondrial Fatty Acid ◽  
Dependent Protein

<a>MicroRNAs (miRNAs) are noncoding small RNAs that regulate various pathophysiological cellular processes. Here we reported that expression of the miR-378 family was significantly induced by metabolic inflammatory inducers, a high-fructose diet, and inflammatory cytokine TNF</a>a. Hepatic miRNA profiling revealed that expression of miR-378a was highly upregulated which, in turn, targeted the 3’-UTR of PPARa mRNA, impaired mitochondrial fatty acid b-oxidation and induced mitochondrial and ER stress. More importantly, the upregulated miR-378a can directly bind to and activate the dsRNA-dependent protein kinase R (PKR) to sustain the metabolic stress. <i>In vivo</i>, genetic depletion of miR-378a prevented PKR activation, ameliorated inflammatory stress and insulin resistance. Counterbalancing the upregulated miR-378a using nanoparticles encapsulated with an anti-miR-378a oligonucleotide restored PPARa activity, inhibited PKR activation and ER stress, and improved insulin sensitivity in the fructose-fed mice. <i>Conclusion: </i>Our study delineated a novel mechanism of miRNA-378a in the pathogenesis of metabolic inflammation and insulin resistance through targeting metabolic signaling at both mRNA (e.g., PPARa) and protein (e.g., PKR) molecules. This novel finding of functional interaction between miRNAs (e.g., miR-378a) and cellular RNA binding protein(s) (e.g., PKR) is biologically significant as it greatly broadens the potential targets of miRNAs in cellular pathophysiological processes.

scholarly journals C114 Is a Novel IL-11-inducible Nuclear Double-stranded RNA-binding Protein That Inhibits Protein Kinase R

2003 ◽  
Vol 278 (25) ◽  
pp. 22838-22845 ◽  
Author(s):  
Zhan Yin ◽  
Jennifer Haynie ◽  
Bryan R. G. Williams ◽  
Yu-Chung Yang
Keyword(s):  
Protein Kinase ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Protein Kinase R ◽  
Double Stranded Rna

scholarly journals Suppression of NYVAC Infection in HeLa Cells Requires RNase L but Is Independent of Protein Kinase R Activity

2015 ◽  
Vol 90 (4) ◽  
pp. 2135-2141 ◽  
Author(s):  
Mercedes Fernández-Escobar ◽  
José Luis Nájera ◽  
Sara Baldanta ◽  
Dolores Rodriguez ◽  
Michael Way ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Hela Cells ◽  
Rnase L ◽  
Abortive Infection ◽  
Protein Kinase R ◽  
Viral Spread ◽  
Viral Genes ◽  
Cell Components

Protein kinase R (PKR) and RNase L are host cell components that function to contain viral spread after infections. In this study, we analyzed the role of both proteins in the abortive infection of human HeLa cells with the poxvirus strain NYVAC, for which an inhibition of viralA27LandB5Rgene expression is described. Specifically, the translation of these viral genes is independent of PKR activation, but their expression is dependent on the RNase L activity.

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