Detection of protein kinase homologues and viral RNA-binding domains utilizing polyclonal antiserum prepared against a baculovirus-expressed ds RNA-activated 68,000-Da protein kinase

Barber, Glen N.; Tomita, Joseph T.; Garfinkel, Michele S.; Meurs, Éliane; Hovanessian, Ara G.; Katze, Michael G.

doi:10.1016/0042-6822(92)90242-h

Cited by 53 publications

(47 citation statements)

References 46 publications

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“…Immunoblot analysis. Expression of GAL4 fusion constructs in yeast transfectants was verified by immunoblot analysis using the indicated antibodies and enhanced chemiluminescence (Amersham) as described previously (2,3). Yeast protein extracts were prepared from 5-ml cultures of each yeast transfectant.…”

Section: Methodsmentioning

confidence: 99%

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Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58^IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation

Gale

Tan

Wambach

et al. 1996

Molecular and Cellular Biology

104

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Expression of the double-stranded RNA-activated protein kinase (PKR) is induced by interferons, with PKR activity playing a pivotal role in establishing the interferon-induced antiviral and antiproliferative states. PKR is directly regulated by physical association with the specific inhibitor, P58 IPK , a cellular protein of the tetratricopeptide repeat (TPR) family, and K3L, the product of the corresponding vaccinia virus gene. P58 IPK and K3L repress PKR activation and activity. To investigate the mechanism of P58 IPK -and K3L-mediated PKR inhibition, we have used a combination of in vitro and in vivo binding assays to identify the interactive regions of these proteins. The P58 IPK -interacting site of PKR was mapped to a 52-amino-acid aa segment (aa 244 to 296) spanning the ATP-binding region of the protein kinase catalytic domain. The interaction with PKR did not require the C-terminal DNA-J homology region of P58 IPK but was dependent on the presence of the eukaryotic initiation factor 2-␣ homology region, mapping to the 34 aa within the sixth P58 IPK TPR motif. Consistent with other TPR proteins, P58 IPK formed multimers in vivo: the N-terminal 166 aa were both necessary and sufficient for complex formation. A parallel in vivo analysis to map the K3L-binding region of PKR revealed that like P58 IPK , K3L interacted exclusively with the PKR protein kinase catalytic domain. In contrast, however, the K3L-binding region of PKR was localized to within aa 367 to 551, demonstrating that each inhibitor bound PKR in unique, nonoverlapping domains. These data, taken together, suggest that P58 IPK and K3L may mediate PKR inhibition by distinct mechanisms. Finally, we will propose a model of PKR inhibition in which P58 IPK or a P58 IPK complex binds PKR and interferes with nucleotide binding and autoregulation, while formation of a PKR-K3L complex interferes with active-site function and/or substrate association.

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Section: Methodsmentioning

confidence: 99%

“…3 -CYC1-lacZ; Clontech] was transfected with the indicated combination of AD and BD plasmids via the lithium acetate method (6). All colonies which contained expression constructs were tested for respective fusion protein expression by immunoblot analysis prior to the two-hybrid assay (see below).…”

Section: Methodsmentioning

confidence: 99%

Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58^IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation

Gale

Tan

Wambach

et al. 1996

Molecular and Cellular Biology

104

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“…However, it rapidly became clear that PKR exerted a powerful growth suppressive and toxic effect on the host and few clones expressing recombinant PKR could be isolated. [43][44][45] One possibility for these observations could include that viralspecific sequences commonly used in eucaryotic expression plasmids could be activating PKR and subsequently eIF2a to inhibit translation within the host cell. Certainly, attempts to express PKR using baculoviruses and retroviruses were met with failure probably for these reasons.…”

Section: Pkr and The Control Of Translationmentioning

confidence: 99%

The dsRNA-dependent protein kinase, PKR and cell death

Barber

2005

Cell Death Differ

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“…3 Overexpression of PKR has been suggested to inhibit cell proliferation in yeast, insect, and mammalian cells. [51][52][53] Several studies showed that the expression of PKR mediates apoptosis. 54,55 In contrast, the expression of catalytically inactive mutants of PKR in NIH3T3 cells results in tumorigenicity in nude mice, which is attributed to a dominant-negative effect of mutant PKR.…”

Section: Er Stress and Interferon Responses: The Eif2a Connectionmentioning

confidence: 99%

Viruses, endoplasmic reticulum stress, and interferon responses

2006

Cell Death Differ

373

316

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Viral infection induces endoplasmic reticulum (ER) stress and interferon responses. While viral double-stranded RNA intermediates trigger interferon responses, viral polypeptides synthesized during infection stimulate ER stress. Among the interferon-regulated gene products, the double-stranded RNA-dependent protein kinase (PKR) plays a key role in limiting viral replication. Thus, to establish productive infection, viruses have evolved mechanisms to overcome the deleterious effects of PKR. It has become clear that ER stress causes translational attenuation and transcriptional upregulation of genes encoding proteins that facilitate folding or degradation of proteins. Notably, prolonged ER stress triggers apoptosis. Therefore, viruses are confronted with the consequences of ER stress. Emerging evidence suggests that viruses not only interfere with the interferon system involving PKR but also manipulate the programs emanating from the ER in a complex way, which may facilitate viral replication or pathogenesis. This review highlights recent progress in these areas.

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Detection of protein kinase homologues and viral RNA-binding domains utilizing polyclonal antiserum prepared against a baculovirus-expressed ds RNA-activated 68,000-Da protein kinase

Cited by 53 publications

References 46 publications

Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58^IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation

Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58^IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation

The dsRNA-dependent protein kinase, PKR and cell death

Viruses, endoplasmic reticulum stress, and interferon responses

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Detection of protein kinase homologues and viral RNA-binding domains utilizing polyclonal antiserum prepared against a baculovirus-expressed ds RNA-activated 68,000-Da protein kinase

Cited by 53 publications

References 46 publications

Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation

Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation

The dsRNA-dependent protein kinase, PKR and cell death

Viruses, endoplasmic reticulum stress, and interferon responses

Contact Info

Product

Resources

About

Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58^IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation

Interaction of the Interferon-Induced PKR Protein Kinase with Inhibitory Proteins P58^IPKand Vaccinia Virus K3L Is Mediated by Unique Domains: Implications for Kinase Regulation