Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In a murine interleukin 3 (IL-3)-dependent cell line, IL-3 deprivation resulted in increased autophosphorylation of double-stranded RNA-dependent protein kinase (PKR) that has been reported to inhibit protein synthesis by phosphorylating the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha). Autophosphorylation was characterized by a shift up in mobility of PKR on SDS/PAGE gels from a 60- to a 64-kDa form. In vitro kinase studies comparing the autophosphorylated 64-kDa PKR with the nonphosphorylated 60-kDa PKR confirmed that only the 64-kDa form was active for eIF-2 alpha phosphorylation. PKR activation in vivo was associated with phosphorylation of eIF-2 alpha and inhibition of protein synthesis. Addition of IL-3 to deprived cells elicited a reciprocal response characterized by the rapid dephosphorylation of PKR and eIF-2 alpha, indicating inactivation of PKR. This was rapidly followed by the full recovery of protein synthesis. Furthermore, upon IL-3 addition, a 97-kDa phosphotyrosine-containing protein becomes rapidly and transiently associated with PKR prior to dephosphorylation of PKR and eIF-2 alpha. Genistein, a tyrosine kinase inhibitor, blocks both phosphorylation of the 97-kDa phosphoprotein and protein synthesis after IL-3 addition, suggesting a role for the 97-kDa phosphoprotein in the mechanism of inactivation of PKR and stimulation of protein synthesis. Thus, IL-3 appears to positively regulate protein synthesis by inducing the inactivation of PKR in a growth factor signaling pathway.
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PMID:Interleukin 3 stimulates protein synthesis by regulating double-stranded RNA-dependent protein kinase. 751 79

The TAR sequence at the 5'-termini of all HIV-1 mRNA species forms a stable structure that is responsible for both transcriptional and translational regulation of HIV-1. Previously we and others reported that purified TAR RNA synthesized by in vitro transcription could activate two interferon-induced enzymes, the protein kinase (PKR) and 2-5A-synthetase. Because the PKR- and 2-5A-systems block protein synthesis initiation and induce RNA decay, respectively, these findings suggested mechanisms for the control of HIV-1 replication by the interferon system. To determine if contaminating dsRNA from in vitro transcription reactions was responsible for this effect, as suggested by Gunnery et al. 1990, (Proc., Natl. Acad. Sci. USA 87, 8687), we have reexamined these findings using chemically synthesized TAR (nucleotides +1 to +57). TAR RNA is shown here to have an intrinsic ability to activate PKR and 2-5A-synthetase. In contrast, a mutant form of TAR designed to have a disrupted secondary structure did not stimulate either enzyme. Chemically synthesized TAR mimicked other dsRNA species in its ability to activate and inhibit PKR at low and high RNA concentrations, respectively. HIV-1 TAT protein inhibited activation of PKR by HIV-1 TAR RNA suggesting an escape mechanism for the virus.
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PMID:HIV-1 TAR RNA has an intrinsic ability to activate interferon-inducible enzymes. 752 41

The interferon-inducible, RNA-dependent protein kinase (PKR) is an important regulator of viral protein synthesis. Activated PKR inhibits protein synthesis by phosphorylating initiation factor eIF-2 alpha. The reovirus S4 gene, whose 1196 nucleotide mRNA transcript does not activate the PKR kinase, is efficiently expressed in vector-transfected monkey COS cells. By contrast, the 1463 nucleotide S1 gene of reovirus, which is a potent activator of PKR, is poorly expressed in COS cells. Virus genetic engineering was therefore used to examine the effect of the PKR activator sequence from the reovirus S1 gene on the expression of chimeric genes of reovirus in transfected COS cells. Chimeric S1/S4 and S4/S1/S4 reovirus constructions that included the PKR activator sequence from S1 in the sigma 3 ORF of S4 were expressed much less efficiently than wild-type S4. However, expression of sigma 3 from S4 (3'UTR/S1), which included the PKR activator sequence from S1 within the 3'-UTR of S4, was comparable to that from wild-type S4. Treatment of COS cells with 2-aminopurine, an inhibitor of PKR, increased the expression of the reovirus S1, S1/S4, and S4/S1/S4 chimeric genes but not the S4 gene or S4 (3'UTR/S1) chimera in transfected COS cells. Likewise, coexpression of the phosphotransfer-negative mutant PKR (K296R) increased the expression of reovirus S1, S1/S4 and S4/S1/S4 chimeric genes but not the S4 gene or S4 (3'UTR/S1) chimera in cotransfected COS cells. Truncated PKR(1-243) which includes the dsRNA binding domain but not the kinase catalytic subdomains was able to enhance the expression of reovirus S1, but did not affect S4 expression. The dsRNA binding protein E3L encoded by vaccinia virus also increased S1 expression similar to PKR (1-243) and PKR(K296R). These results suggest that the translational repression in vivo mediated by PKR is selective for mRNAs that possess the kinase activator region, and that the dominant negative effect of PKR on gene expression is likely mediated by the RNA binding activity of the PKR protein.
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PMID:Mechanism of interferon action. Translational control and the RNA-dependent protein kinase (PKR): antagonists of PKR enhance the translational activity of mRNAs that include a 161 nucleotide region from reovirus S1 mRNA. 752 9

The vaccinia virus (VV) E3L gene, which encodes a potent inhibitor of the interferon (IFN)-induced, double-stranded RNA (dsRNA)-dependent protein kinase, PKR, is thought to be involved in the IFN-resistant phenotype of VV. The E3L gene products, p25 and p20, act as inhibitors of PKR, presumably by binding and sequestering activator dsRNA from the kinase. In this study we demonstrate that VV with the E3L gene specifically deleted (vP1080) was sensitive to the antiviral effects of IFN and debilitated in its ability to rescue vesicular stomatitis virus from the antiviral effects of IFN. Infection of L929 cells with E3L-minus virus led to rRNA degradation typical of activation of the 2'-5'-oligoadenylate synthetase/RNase L system, and extracts of infected cells lacked the PKR-inhibitory activity characteristic of wild-type VV. The reovirus S4 gene, which encodes a dsRNA-binding protein (sigma 3) that can also inhibit PKR activation by binding and sequestering activator dsRNA, was inserted into vP1080. The resultant virus (vP1112) was partially resistant to the antiviral effects of IFN in comparison with vP1080. Further studies demonstrated that transient expression of the reovirus sigma 3 protein rescued E3L-minus VV replication in HeLa cells. In these studies, rescue by sigma 3 mutants correlated with their ability to bind dsRNA. Finally, vP112 was also able to rescue the replication of the IFN-sensitive virus vesicular stomatitis virus in a manner similar to that of wild-type VV. Together, these results suggest that the reovirus S4 gene can replace the VV E3L gene with respect to interference with the IFN-induced antiviral activity.
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PMID:Reversal of the interferon-sensitive phenotype of a vaccinia virus lacking E3L by expression of the reovirus S4 gene. 752 85

This report describes the production and characterization of a mouse monoclonal antibody (mAb) TJ4C4, which is directed against the interferon-induced double-stranded RNA-activated protein kinase p68 (PKR). The mAb TJ4C4 was produced against p68 which was isolated using a partially purified p68 preparation from human cells. The specificity of this mAb is demonstrated in competitive inhibition assays using recombinantly produced p68 protein and by immunoprecipitation. This mAb is of particular value in that it detects an epitope on p68 which is not destroyed in routinely prepared, formalin-fixed paraffin-embedded tissues, or in a variety of other commonly used clinical fixatives. The inhibition of mAb TJ4C4 by recombinantly produced p68, on human tissues sections, validates the specificity of this mAb in histological studies. Therefore, mAb TJ4C4 serves as a specific probe to study p68 expression in clinically derived tissue specimens.
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PMID:Characterization of the mouse monoclonal antibody TJ4C4 directed at human p68 kinase. 752 82

The RNA-binding activity of the interferon-inducible, RNA-dependent protein kinase PKR, expressed from the human PKR cDNA, was quantitated using a gel mobility-shift assay. The N-terminal R-domain truncation Wt(1-243) and the full-length catalytic mutant K296R(21-551) were analyzed for their abilities to bind adenovirus VAI RNA, human immunodeficiency virus TAR RNA, and the synthetic homopolymer pI:pC RNA. The N-terminal 243 amino acid residue form of PKR [Wt(1-243)] bound VAI RNA with similar affinity as the 551 amino acid residue full-length catalytic mutant [K296R(1-551)]. The dissociation constant for VAI RNA was approximately 2 x 10(-9) M for both the K296R(1-551) and Wt(1-243) proteins. The K64E mutation significantly impaired the VAI RNA-binding activity as measured with the full-length double-point mutant PKR protein, K64E/K296R(1-551). Using a gel-shift competition assay, the dissociation constants of K296R(1-551) and Wt(1-243) for VAI(1-160) RNA and pI:pC RNA were comparable. By contrast, the dissociation constants of K296R(1-551) and Wt(1-243) for TAR(1-82) RNA were both about 1 x 10(-7) M. These results suggest that the RNA-binding affinity of PKR is approximately 100-fold lower for TAR RNA than for either VAI RNA or pI:pC RNA and that the full-length and N-terminal R-domain forms of PKR bind RNA with similar affinity.
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PMID:Mechanism of interferon action: RNA-binding activity of full-length and R-domain forms of the RNA-dependent protein kinase PKR--determination of KD values for VAI and TAR RNAs. 753 Mar 96

Polyclonal antibodies raised against purified and urea-denatured double-stranded protein kinase (PKR) from human origin cross-reacted by immunoblotting with a 48-kD protein (p48) induced by the three types of interferon (IFN), alpha, beta, and gamma. The induction of p48 is IFN dose dependent and its accumulation occurs a few hours after the addition of IFN. The induction of p48 is blocked by actinomycin D. Analysis by two-dimensional gel isoelectric-focusing, revealed p48 as a single spot with an isoelectric point (pI) of 6.8. In the same experiment the PKR was revealed as several subspecies with pI values in the pH range of 7.4-8.0. Cell fractionation experiments indicated that PKR and p48 have different subcellular localizations: PKR was found to be associated with the microsomal pellet as shown previously whereas p48 was recovered in the microsomal supernatant fraction. In addition to these differences, PKR and p48 were found to be differentially expressed in some human cells treated with the three types of IFN. For example, in HeLa cells, IFN-alpha or IFN-beta induced similarly both PKR and p48 whereas IFN-gamma induced mainly p48. In U937 cells in which PKR was not expressed with or without IFN treatment, p48 was strongly induced by all three types of IFN. These results suggest different mechanisms for the induction of PKR and p48. In view of its presence in different types of human cells and its induction by different types of IFN, it is possible to suggest that p48 might play an important role in mediating some of the action of IFN.
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PMID:Characterization of an interferon-induced 48-kD protein immunologically related to the double-stranded RNA-activated protein kinase PKR. 753 1

The gene encoding the RNA-dependent protein kinase (PKR) was cloned from mouse genomic DNA and characterized by restriction mapping, Southern blot analysis and sequencing. The Southern analyses were consistent with the presence of a single copy of the Pkr gene in the mouse haploid genome. The genomic nucleotide (nt) sequence, when compared to that of previously determined cDNA nt sequences, revealed 16 exons encoding the 515-amino-acid PKR.
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PMID:Sequence of the murine interferon-inducible RNA-dependent protein kinase (PKR) deduced from genomic clones. 753 17

This review describes the structure and function of the double-stranded RNA-dependent protein kinase (PKR) and its interaction with RNA activators and inhibitors. The abilities of small virally-encoded RNAs such as VAI RNA of adenovirus, the Epstein-Barr virus encoded (EBER) RNAs and the Tat-responsive region RNA of HIV-1 to bind to and regulate PKR are reviewed, and the physiological implications of such regulation for the control of viral replication and cell growth are discussed. The potential effects on the activity of PKR of other proteins that bind double-stranded RNA and/or small viral and cellular RNAs are also considered.
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PMID:Regulation of the interferon-inducible eIF-2 alpha protein kinase by small RNAs. 753 82

Interferons modulate a number of biological functions including cell growth and differentiation, the immune response, and virus replication. The antiviral activity of interferons (IFNs), the property that led to their discovery, is mediated by multiple cellular proteins. Among the IFN-induced cellular proteins with antiviral activity are the protein kinase PKR, the enzymes of the 2', 5'-oligoadenylate pathway, and the Mx proteins. The antiviral activities of these proteins differ significantly against viruses of the myxoviridae, rhabdoviridae, and picornaviridae virus families.
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PMID:Interferon-induced proteins and their mechanisms of action. 753 27


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