Nuclear Localization of the Interferon-Inducible Protein Kinase PKR in Human Cells and Transfected Mouse Cells

Jeffrey, Ian W.; Kadereit, Suzanne; Meurs, Éliane; Metzger, Thomas; Bachmann, Michael; Schwemmle, Martin; Hovanessian, Ara G.; Clemens, Michael J.

doi:10.1006/excr.1995.1126

Cited by 113 publications

(103 citation statements)

References 41 publications

(52 reference statements)

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“…There are limited data concerning the movement of PKR within the cell in response to viral infection. While adenovirus infection and interferon treatment do not alter PKR localization (40,41), infection of interferon-treated HeLa cells with encephalomyocarditis virus (EMCV) results in the displacement of PKR from ribosomal subunits and the aggregation of PKR in the perinuclear area (30). However, the PKR that aggregates in the perinuclear area during EMCV infection is activated and is thought to contribute to the inhibition of EMCV replication (30).…”

Section: Discussionmentioning

confidence: 99%

“…While PKR is normally found predominantly in the cytoplasm, it is also present in lower quantities in the nucleolus, where it is thought, although not proven, to play a role in ribosome biogenesis (40,41). Additionally, there is indirect evidence implicating PKR in the transcriptional up-regulation of select genes in response to viral infection or dsRNA (75), and PKR has been demonstrated to directly interact with several proteins present in the nucleus that may affect regulation of the cell cycle (22,23,55).…”

Section: Discussionmentioning

confidence: 99%

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

Hakki

Marshall²,

Niro³

et al. 2006

J Virol

104

100

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The human cytomegalovirus (HCMV) TRS1 and IRS1 genes block the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2␣) 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 (VV⌬E3L). To further define the underlying mechanism, we first evaluated the effect of pTRS1 on protein kinase R (PKR), the double-stranded RNA (dsRNA)-dependent eIF2␣ kinase. Immunoblot analyses revealed that pTRS1 expression in the context of a VV⌬E3L 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 VV⌬E3L 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 VV⌬E3L replication in HeLa cells. The carboxy terminus of pIRS1 was also required for rescue of VV⌬E3L 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, eIF2␣.Double-stranded RNA (dsRNA) activates the host cell response to viral infection in numerous ways, one of which involves the interferon-induced, dsRNA-dependent protein kinase R (PKR) (reviewed in reference 43). After binding to dsRNA, PKR dimerizes, autophosphorylates, and then phosphorylates the eukaryotic initiation factor 2 (eIF2) on its ␣ subunit. Phosphorylated eIF2␣ sequesters the guanine nucleotide exchange factor eIF2B, resulting in the inhibition of protein synthesis at the level of translation initiation. Since viruses depend on the cellular translational machinery, the shutoff of host cell protein synthesis inhibits viral replication and spread.Many viruses have mechanisms for inhibiting the PKR-mediated phosphorylation of eIF2␣ (56). The vaccinia virus (VV) E3L protein prevents the activation of PKR by binding to and sequestering dsRNA via a carboxy-terminal double-stranded RNA binding domain (dsRBD) (39). VV from which the E3L gene has been deleted (VV⌬E3L) exhibits a dsRNA-dependent restriction of host cell range, and this phenotype can be reversed by expression of the dsRBD from pE3L or dsRNAbinding proteins from other viruses (4, 47, 62). We previously found that the products of the human cytomegalovirus (HCMV) genes TRS1 and IRS1 restore the host cell range of VV⌬E3L, inhibit the phosphorylation of eIF2␣, and prevent the shutoff...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Hakki

Marshall²,

Niro³

et al. 2006

J Virol

104

100

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“…However, electron micrograph analysis and immunogold labeling has also indicated that a fraction of PKR resides in the nucleus (48,49). Since the NFARs have a consensus bipartite nuclear localization signal (Fig.…”

Section: Isolation and Identification Of Nfar-1 And -2-mentioning

confidence: 99%

Characterization of Two Evolutionarily Conserved, Alternatively Spliced Nuclear Phosphoproteins, NFAR-1 and -2, That Function in mRNA Processing and Interact with the Double-stranded RNA-dependent Protein Kinase, PKR

Saunders

Perkins

Balachandran

et al. 2001

Journal of Biological Chemistry

114

148

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We report here the isolation and characterization of two proteins, NFAR-1 and -2, which were isolated through their ability to interact with the dsRNA-dependent protein kinase, PKR. The NFAR proteins, of 90 and 110 kDa, are derived from a single gene through alternative splicing and are evolutionarily conserved nuclear phosphoproteins that interact with doublestranded RNA. Both NFAR-1 and -2 are phosphorylated by PKR, reciprocally co-immunoprecipitate with PKR, and colocalize with the kinase in a diffuse nuclear pattern within the cell. Transfection studies indicate that the NFARs regulate gene expression at the level of transcription, probably during the processing of pre-mRNAs, an activity that was increased in fibroblasts lacking PKR. Subsequent functional analyses indicated that amino acids important for NFAR's activity were localized to the C terminus of the protein, a region that was found to specifically interact with FUS and SMN, proteins also known as regulators of RNA processing. Accordingly, both NFARs were found to associate with both pre-mRNAs and spliced mRNAs in post-transcriptional studies, similar to the known splicing factor ASF/ SF-2. Collectively, our data indicate that the NFARs may facilitate double-stranded RNA-regulated gene expression at the level of post-transcription and possibly contribute to host defense-related mechanisms in the cell.

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“…8,13,14 Although PKR has been identified in nuclear and cytoplasmic fractions, most activities attributed to PKR occur in the cytoplasm; hence, the role of nuclear PKR has remained unclear. Jeffrey et al 15 found, in NIH3T3 cells overexpressing exogenous PKR and Daudi Burkitt's lymphoma cells treated with interferon, the ratio of cytoplasmic to nuclear PKR to be 5:1. Both nuclear and cytoplasmic PKR were reported to be 68 kDa; the only difference was in post-translational modification (PTM).…”

Section: Introductionmentioning

confidence: 99%

“…Whereas, cytoplasmic PKR presented with multiple isoelectric points (pIs), nuclear PKR presented with a single, basic pI, suggesting this protein was not highly phosphorylated. 15 Recent studies have attributed clinical and pathological significance to nuclear PKR. 9,13,16 We initially reported that CD34 þ cells from myelodysplastic syndrome (MDS) patients contain elevated levels of p-T451 PKR as compared with controls.…”

Section: Introductionmentioning

confidence: 99%

Multiple forms of PKR present in the nuclei of acute leukemia cells represent an active kinase that is responsive to stress

et al. 2010

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A number of cancers possess constitutive activity of the dsRNA-dependent kinase, PKR. Inhibition of PKR in these cancers leads to tumor cell death. We recently reported the increased presence of PKR phosphorylated on Thr451 (p-T451 PKR) in clinical samples from myelodysplastic syndrome (MDS) patients and acute leukemia cell lines. Whereas p-T451 PKR in low-risk patient samples or PTEN-positive acute leukemia cell lines was mostly cytoplasmic, in high-risk patient samples and acute leukemia cell lines deficient in PTEN, p-T451 PKR was mainly nuclear. As nuclear activity of PKR has not been previously characterized, we examined the status of nuclear PKR in acute leukemia cell lines. Using antibodies to N-terminus, C-terminus and the kinase domain in conjunction with a proteomics approach, we found that PKR exists in diverse molecular weight forms in the nucleus. Analysis of PKR transcripts by reverse transcriptase-PCR, and PKR-derived peptides by MS/MS revealed that these forms were the result of post-translational modifications (PTMs). Biochemical analysis demonstrated that nuclear PKR is an active kinase that can respond to stress. Given the association of PKR with PTEN and the Fanconi complex, these results indicate that PKR likely has other previously unrecognized roles in nuclear signaling that may contribute to leukemic development.

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Nuclear Localization of the Interferon-Inducible Protein Kinase PKR in Human Cells and Transfected Mouse Cells

Cited by 113 publications

References 41 publications

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

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

Characterization of Two Evolutionarily Conserved, Alternatively Spliced Nuclear Phosphoproteins, NFAR-1 and -2, That Function in mRNA Processing and Interact with the Double-stranded RNA-dependent Protein Kinase, PKR

Multiple forms of PKR present in the nuclei of acute leukemia cells represent an active kinase that is responsive to stress

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