Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

Cosentino, Gregory P.; Venkatesan, Sundararajan; Serluca, Farbrizio C.; Green, Simon R.; Mathews, Michael B.; Sonenberg, Nahum

doi:10.1073/pnas.92.21.9445

Cited by 164 publications

(199 citation statements)

References 53 publications

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“…As wild-type PKR is toxic to bacteria (3), we used PKR containing the K296R mutation, which is catalytically inactive yet retains the dimerization and protein-protein interaction characteristics of wt PKR (11,21,33,34,51,54,64). In vitro-translated, radiolabeled pTRS1 was incubated with GST alone or GST-PKRK296R, and protein binding was analyzed by SDS-PAGE and autoradiography.…”

Section: Fig 4 Pkr Accumulates In the Nucleus During Hcmv Infectionmentioning

confidence: 99%

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

Hakki

Marshall²,

Niro³

et al. 2006

J Virol

103

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: Fig 4 Pkr Accumulates In the Nucleus During Hcmv Infectionmentioning

confidence: 99%

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

Hakki

Marshall²,

Niro³

et al. 2006

J Virol

103

100

View full text Add to dashboard Cite

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“…HIV-1 TAR sequence appears to bind to a cellular TAR binding protein and PKR (34)(35)(36). The exceptions may be hepatitis delta virus that does not activate PKR even though in vitro it consists largely of double-stranded RNA (37), and simian viurs 40 whose large T antigen acts at a step postactivation of PKR (38).…”

Section: Discussionmentioning

confidence: 99%

The γ ₁ 34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1α to dephosphorylate the α subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase

Gross

Roizman

1997

Proc. Natl. Acad. Sci. U.S.A.

708

622

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In human cells infected with herpes simplex virus 1 the double-stranded RNA-dependent protein kinase (PKR) is activated but phosphorylation of the ␣ subunit of eukaryotic translation initiation factor 2 (eIF-2) and total shutoff of protein synthesis is observed only in cells infected with ␥ 1 z34.5 ؊ mutants. The carboxyl-terminal 64 aa of ␥ 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 1␣ in yeast, and both MyD116 and ␥ 1 34.5 interact with protein phosphatase 1␣ in vitro; (ii) protein synthesis in infected cells is strongly inhibited by okadaic acid, a phosphatase 1 inhibitor; and (iii) the ␣ 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-2␣-P phosphatase activity of ␥ 1 34.5 ؊ virus infected cells is lower than that of mock-infected cells. The eIF-2␣-P phosphatase activities are sensitive to inhibitor 2. In contrast to eIF-2␣-P phosphatase activity, extracts of mock-infected cells exhibit a 2-fold higher phosphatase activity on [ 32 P]phosphorylase than extracts of infected cells. These results indicate that in infected cells, ␥ 1 34.5 interacts with and redirects phosphatase to dephosphorylate eIF-2␣ 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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“…Moreover, the inactive mutant PKR (K296R, Figure 1a) can dimerize in vitro (Carpick et al, 1997). PKR dimerization may be mediated in part through the dsRBD, either through direct protein-protein interactions in this region (Patel et al, 1995;, or through dsRNA bridging the protein subunits (Cosentino et al, 1995; but domains within the C-terminal half of PKR are also required and dimerization (Romano et al, 1995;Tan et al, 1998) can be blocked by a peptide corresponding to amino acids 244±296 (Tan et al, 1998). While multiple deletions within this region interfere with dimerization (Romano et al, 1995), the was incubated with poly rI:rC (pIC, 60 ng) or with the indicated amounts of¯Alu RNA and kinase activity assayed as described (Chu et al, 1998).…”

Section: Activation By Dsrnamentioning

confidence: 99%

PKR; a sentinel kinase for cellular stress

1999

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Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

Abstract: The

Cited by 164 publications

References 53 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

The γ ₁ 34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1α to dephosphorylate the α subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase

PKR; a sentinel kinase for cellular stress

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Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

Abstract: The

Cited by 164 publications

References 53 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

The γ 1 34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1α to dephosphorylate the α subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase

PKR; a sentinel kinase for cellular stress

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The γ ₁ 34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1α to dephosphorylate the α subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase