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)

Upon binding to double-stranded (ds) RNA, the dsRNA-dependent protein kinase (PKR) sequentially undergoes autophosphorylation and activation. Activated PKR may exist as a dimer and phosphorylates the eukaryotic translation initiation factor 2 alpha subunit (cIF-2 alpha) to inhibit polypeptide chain initiation. Transfection of COS-1 cells with a plasmid cDNA expression vector encoding a marker gene, activates endogenous PKR, and selectively inhibits translation of the marker mRNA, dihydrofolate reductase (DHFR). This system was used to study the dsRNA binding and dimerization requirements for over-expressed PKR mutants and subdomains to affect DHFR translation. DHFR translation was rescued by expression of either an ATP hydrolysis defective mutant PKR K296P, the amino-terminal 1-243 fragment containing two dsRNA binding motifs, or the isolated first RNA binding motif (amino acids 1-123). Mutation of K64E within the dsRNA binding motif 1 destroyed dsRNA binding and the ability to rescue DHFR translation. Immunoprecipitation of T7 epitope-tagged PKR derivatives from cell lysates detected interaction between intact PKR and the amino-terminal 1-243 fragment as well as a 1-243 fragment harboring the K64E mutation. Expression of adenovirus VAI RNA, a potent inhibitor of PKR activity, did not disrupt this interaction. In contrast, intact PKR did not interact with fragments containing the first dsRNA binding motif (1-123), the second dsRNA binding motif (98-243), or the isolated PKR kinase catalytic domain (228-551). These results demonstrate that the translational stimulation mediated by the dominant negative PKR mutant does not require dimerization, but requires the ability to bind dsRNA and indicate these mutants act by competition for binding to activators.
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PMID:Double-stranded (ds) RNA binding and not dimerization correlates with the activation of the dsRNA-dependent protein kinase (PKR). 857 79

In human cells infected with herpes simplex virus 1 the double-stranded RNA-dependent protein kinase (PKR) is activated but phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2) and total shutoff of protein synthesis is observed only in cells infected with gamma(1)z34.5- mutants. The carboxyl-terminal 64 aa of gamma(1)34.5 protein are homologous to the corresponding domain of MyD116, the murine growth arrest and DNA damage gene 34 (GADD34) protein and the two domains are functionally interchangeable in infected cells. This report shows that (i) the carboxyl terminus of MyD116 interacts with protein phosphatase 1alpha in yeast, and both MyD116 and gamma(1)34.5 interact with protein phosphatase 1alpha in vitro; (ii) protein synthesis in infected cells is strongly inhibited by okadaic acid, a phosphatase 1 inhibitor; and (iii) the alpha subunit in purified eIF-2 phosphorylated in vitro is specifically dephosphorylated by S10 fractions of wild-type infected cells at a rate 3000 times that of mock-infected cells, whereas the eIF-2alpha-P phosphatase activity of gamma(1)34.5- virus infected cells is lower than that of mock-infected cells. The eIF-2alpha-P phosphatase activities are sensitive to inhibitor 2. In contrast to eIF-2alpha-P phosphatase activity, extracts of mock-infected cells exhibit a 2-fold higher phosphatase activity on [32P]phosphorylase than extracts of infected cells. These results indicate that in infected cells, gamma(1)34.5 interacts with and redirects phosphatase to dephosphorylate eIF-2alpha to enable continued protein synthesis despite the presence of activated PKR. The GADD34 protein may have a similar function in eukaryotic cells. The proposed mechanism for maintenance of protein synthesis in the face of double-stranded RNA accumulation is different from that described for viruses examined to date.
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PMID:The gamma(1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. 902 44

A novel mammalian gene, Eif4g2, with a high degree of homology to the p82 subunit of the wheat germ eukaryotic translation initiation factor eIF-(iso)4F and mammalian eIF-4G has been isolated. Zoo blot analysis indicates that Eif4g2 is a single-copy gene that is highly conserved among vertebrates. Northern blot analysis shows that Eif4g2 is ubiquitously expressed at high levels in all human and mouse tissues examined. The 3810-nucleotide Eif4g2 cDNA contains a 907-amino-acid open reading frame that codes for a polypeptide with a predicted molecular mass of 102 kDa. The Eif4g2 polypeptide exhibits an overall similarity to wheat p82 of 52%. A 248-amino-acid segment at the amino-terminal end of both peptides exhibits 63% similarity and contains conserved potential RNA binding domains and a phosphorylation site. The Eif4g2 polypeptide contains multiple potential N-linked glycosylation sites as well as protein kinase C and casein kinase II phosphorylation sites. Southern blot analysis of DNA from interspecific backcross mice shows that Eif4g2 is localized to distal mouse chromosome 7 in a region syntenic with human chromosome 11p15.
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PMID:cDNA cloning, expression analysis, and chromosomal localization of a gene with high homology to wheat eIF-(iso)4F and mammalian eIF-4G. 902 6

The interferon-inducible double-stranded RNA protein kinase PKR controls protein synthesis through the phosphorylation of eukaryotic translation initiation factor (eIF)-2. In addition to its demonstrated role in translational control, several reports have suggested a transcriptional role for PKR. Here we report that PKR is involved in IFN- and dsRNA-signaling pathways by modulating the function of the signal transducer and activator of transcription STAT1. We also show that PKR associates with STAT1 in mouse and human cells. The association is not a kinase-substrate interaction since STAT1 phosphorylation is not modified by PKR in vitro or in vivo. In addition, the formation of the PKR-STAT1 complex is not dependent upon the enzymatic activity of PKR but does require the dsRNA-binding domain of PKR. Moreover, there is a concomitant decrease in PKR-STAT1 interaction and increase in STAT1 DNA binding in response to IFNs or dsRNA. These findings suggest that PKR plays an important role in IFN and dsRNA-signaling pathways by modulating the transcriptional function of STAT1.
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PMID:Physical association between STAT1 and the interferon-inducible protein kinase PKR and implications for interferon and double-stranded RNA signaling pathways. 913 45

The interferon-inducible, double-stranded (ds) RNA-dependent serine/threonine protein kinase (PKR) plays a role in viral pathogenesis, cell growth, and differentiation and is implicated as a tumor suppressor gene. Expression of a trans-dominant negative, catalytically inactive mutant PKR protected NIH3T3 cells from apoptosis in response to either treatment with tumor necrosis factor alpha (TNF alpha), serum deprivation. In cells expressing mutant PKR, TNF alpha, but not dsRNA induced transcription from a nuclear factor kappa B-dependent promoter, demonstrating specificity for dsRNA in signaling through the PKR pathway. Serum or platelet-derived growth factor addition to serum-deprived mutant PKR-expressing cells induced transcription of the early response genes c-fos and c-jun, indicating that the immediate early response signaling was intact. Overexpression of wild-type PKR in a transient DNA transfection system was sufficient to induce apoptosis. TNF alpha-induced apoptosis correlated with increased phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha), the primary physiological substrate of the PKR. Furthermore, forced expression of a nonphosphorylatable S51A mutant eIF-2 alpha partially protected cells from TNF alpha-induced apoptosis, and expression of a S51D mutant eIF-2 alpha, a mutant that mimics phosphorylated eIF-2 alpha, was sufficient to induce apoptosis. Taken together, these studies identify a novel requirement for PKR in stress-induced apoptosis that is mediated through eIF-2 alpha phosphorylation.
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PMID:Phosphorylation of eukaryotic translation initiation factor 2 mediates apoptosis in response to activation of the double-stranded RNA-dependent protein kinase. 944 91

Phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) is a common cellular mechanism to limit protein synthesis in stress conditions. Baculovirus PK2, which resembles the C-terminal half of a protein kinase domain, was found to inhibit both human and yeast eIF2alpha kinases. Insect cells infected with wild-type, but not pk2-deleted, baculovirus exhibited reduced eIF2alpha phosphorylation and increased translational activity. The negative regulatory effect of human protein kinase RNA-regulated (PKR), an eIF2alpha kinase, on virus production was counteracted by PK2, indicating that baculoviruses have evolved a unique strategy for disrupting a host stress response. PK2 was found in complex with PKR and blocked kinase autophosphorylation in vivo, suggesting a mechanism of kinase inhibition mediated by interaction between truncated and intact kinase domains.
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PMID:Disruption of cellular translational control by a viral truncated eukaryotic translation initiation factor 2alpha kinase homolog. 953 7

The interferon-inducible, double-stranded (ds) RNA-dependent protein kinase (PKR) regulates protein synthesis initiation by phosphorylating the alpha-subunit of eukaryotic translation initiation factor 2 (eIF-2). The amino-terminal half of PKR contains two dsRNA binding domains, and the kinase domain resides in the carboxy-terminal half of the protein. PKR is a ribosomal-associated protein. In this report, we provide evidence that PKR contains three ribosome interaction sites, two that are localized in each of the dsRNA binding domains and one that is localized in the kinase domain. All three domains can associate with polysomes independently. The ribosome association of the dsRNA binding domains requires dsRNA binding activity. Ribosome interaction of either the individual or the combined dsRNA binding domains was disrupted by 0.1 M KCl. In contrast, the ribosome interaction of intact PKR and the isolated kinase domain was largely resistant to 0.5 M KCl. These results indicate that all three domains of PKR contribute to the high-affinity ribosomal association. After dissociation of polysomes with EDTA, both intact PKR and the isolated kinase domain were primarily associated with the 60S ribosomal subunit. Coexpression of the adenovirus VAI RNA, an RNA polymerase III gene product that binds and inactivates PKR, disrupted ribosomal association of intact PKR, but not of the isolated PKR kinase domain. The results support a model where VAI RNA induces a major conformational change in PKR to prohibit ribosome association of all interaction sites. In contrast, other inhibitors of PKR including vaccinia virus E3L and K3L gene products, and the HIV trans-activating response (TAR) element binding protein TRBP, did not disrupt ribosome association of PKR. The results suggest a novel mechanism by which viral RNAs may inactivate PKR through disrupting ribosome association.
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PMID:Identification and requirement of three ribosome binding domains in dsRNA-dependent protein kinase (PKR). 975 71

In herpes simplex virus-infected cells, viral gamma134.5 protein blocks the shutoff of protein synthesis by activated protein kinase R (PKR) by directing the protein phosphatase 1alpha to dephosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). The amino acid sequence of the gamma134.5 protein which interacts with the phosphatase has high homology to a domain of the eukaryotic protein GADD34. A class of compensatory mutants characterized by a deletion which results in the juxtaposition of the alpha47 promoter next to US11, a gamma2 (late) gene in wild-type virus-infected cells, has been described. In cells infected with these mutants, protein synthesis continues even in the absence of the gamma134.5 gene. In these cells, PKR is activated but eIF-2alpha is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2alpha. We report the following: (i) in cells infected with these mutants, US11 protein was made early in infection; (ii) US11 protein bound PKR and was phosphorylated; (iii) in in vitro assays, US11 blocked the phosphorylation of eIF-2alpha by PKR activated by poly(I-C); and (iv) US11 was more effective if present in the reaction mixture during the activation of PKR than if added after PKR had been activated by poly(I-C). We conclude the following: (i) in cells infected with the compensatory mutants, US11 made early in infection binds to PKR and precludes the phosphorylation of eIF-2alpha, whereas US11 driven by its natural promoter and expressed late in infection is ineffective; and (ii) activation of PKR by double-stranded RNA is a common impediment countered by most viruses by different mechanisms. The gamma134.5 gene is not highly conserved among herpesviruses. A likely scenario is that acquisition by a progenitor of herpes simplex virus of a portion of the cellular GADD34 gene resulted in a more potent and reliable means of curbing the effects of activated PKR. US11 was retained as a gamma2 gene because, like many viral proteins, it has multiple functions.
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PMID:The herpes simplex virus US11 protein effectively compensates for the gamma1(34.5) gene if present before activation of protein kinase R by precluding its phosphorylation and that of the alpha subunit of eukaryotic translation initiation factor 2. 976 1

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) provides a fundamental control step in the regulation of protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha), a process that prevents polypeptide chain initiation. In such a manner, activated PKR inhibits cell growth and induces apoptosis, whereas disruption of normal PKR signaling results in unregulated cell growth. Therefore, tight control of PKR activity is essential for regulated cell growth. PKR is activated by dsRNA binding to two conserved dsRNA binding domains within its amino terminus. We isolated a ribosomal protein L18 by interaction with PKR. L18 is a 22-kDa protein that is overexpressed in colorectal cancer tissue. L18 competed with dsRNA for binding to PKR, reversed dsRNA binding to PKR, and did not directly bind dsRNA. Mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding and L18 interaction, suggesting that the two interactive sites overlap. L18 inhibited both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2alpha in vitro. Overexpression of L18 by transient DNA transfection reduced eIF-2alpha phosphorylation and stimulated translation of a reporter gene in vivo. These results demonstrate that L18 is a novel regulator of PKR activity, and we propose that L18 prevents PKR activation by dsRNA while PKR is associated with the ribosome. Overexpression of L18 may promote protein synthesis and cell growth in certain cancerous tissue through inhibition of PKR activity.
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PMID:Double-stranded RNA-activated protein kinase (PKR) is negatively regulated by 60S ribosomal subunit protein L18. 989 Oct 46

Protein kinase CK2 forms complexes with some protein substrates what may be relevant for the physiological control of this protein kinase. In previous studies in rat liver cytosol we had detected that the trimeric form of eukaryotic translation initiation factor 2 (eIF-2) co-eluted with protein kinase CK2. We have now observed that the ratio between eIF-2 and cytosolic CK2 contents in testis, liver and brain is quite similar, being eIF-2 levels about 5-fold higher than those of CK2. Furthermore eIF-2 was present in liver samples immunoprecipitated with anti-CK2alpha/alpha' antibodies, confirming the existence of complexes containing both proteins. Nonetheless, these complexes would represent only a fraction of total cytosolic CK2 and eIF-2. We had also observed that rat liver membrane glycoproteins obtained through chromatography on wheat-germ lectin-Sepharose contain CK2 activity which copurifies with grp94/endoplasmin. We have now confirmed that this activity was due to the presence of protein kinase CK2 as evidenced by immunodetection with antibodies against CK2alpha/alpha'. The fractions enriched in grp94/endoplasmin and CK2 also contained another 55-kDa polypeptide (p55) phosphorylated by CK2 which has been identified as calreticulin by N-terminal sequencing. Calreticulin and grp94/endoplasmin could be partially resolved from CK2 by chromatography on heparin-agarose and almost completely on ConA-Sepharose. However, phosphorylation of immunoprecipitated grp94/endoplasmin was enhanced by its preincubation with purified CK2 prior to immunoprecipitation, what confirms the easy reassociation between these proteins. The association of protein kinase CK2 with eIF-2 and with grp94/endoplasmin may serve to locate the enzyme in the cellular machinery involved in protein synthesis and folding, and reinforces the possible involvement of CK2 in these processes.
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PMID:Association of protein kinase CK2 with eukaryotic translation initiation factor eIF-2 and with grp94/endoplasmin. 1009 97


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