DRBP76, a Double-stranded RNA-binding Nuclear Protein, Is Phosphorylated by the Interferon-induced Protein Kinase, PKR

Patel, Rekha C.; Vestal, Deborah J.; Xu, Zeyuan; Bandyopadhyay, Smarajit; Guo, Weidong; Erme, Scott M.; Williams, Bryan R.G.; Sen, Ganes C.

doi:10.1074/jbc.274.29.20432

Cited by 121 publications

(125 citation statements)

References 31 publications

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“…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). While the functional significance of these interactions is unclear, PKR relocalization to the nucleus during HCMV infection may serve a role distinct from its role in the eIF2␣ pathway.…”

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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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%

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

Hakki

Marshall²,

Niro³

et al. 2006

J Virol

104

100

View full text Add to dashboard Cite

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“…L18 inhibits both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2a in vitro and in vivo. Although overexpression of L18 in tumors may promote protein synthesis and cell growth through inhibition of PKR activity, there are a large number of dsRNA-binding proteins that also bind PKR (Patel et al, 1999) and identifying any one of these as the crucial PKR regulator in tumorigenesis without more direct evidence is speculative.…”

Section: Pkr Viral Mediated Apoptosis and Heat Shockmentioning

confidence: 99%

PKR; a sentinel kinase for cellular stress

1999

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“…Some of these mechanisms mediated by PKR in response to dsRNA might be attributed either to protein-protein interaction or to the phosphorylation of substrates other than eIF2␣. Accordingly, PKR has been shown to interact constitutively or in a stimulus-dependent manner with a number of proteins, which may serve as PKR substrates, thus regulating protein translation and transcriptional activity (3,4,14,15,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). A role for PKR in transcriptional activity in the inflammatory response was suggested by the observation that PKR-null primary fibroblasts and mice had defects in the activation of p38 mitogen-activated protein kinase (MAPK) and downstream proinflammatory gene expression in response to different stimuli (29).…”

Section: Expression Of Kinase-inactive Pkr (K296r) In Cells Inhibitedmentioning

confidence: 99%

Protein Kinase R (PKR) Interacts with and Activates Mitogen-activated Protein Kinase Kinase 6 (MKK6) in Response to Double-stranded RNA Stimulation

Silva¹,

Whitmore²,

Xu³

et al. 2004

Journal of Biological Chemistry

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105

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The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) has been invoked in different signaling pathways. In cells pre-exposed to the PKR inhibitor 2-aminopurine or in PKR-null cells, the activation of p38 mitogen-activated protein kinase (MAPK) following dsRNA stimulation is attenuated. We found that the p38 MAPK activator MKK6, but not its close relatives MKK3 or MKK4, exhibited an increased affinity for PKR following the exposure of cells to poly(rI:rC), a dsRNA analog. In vitro kinase assays revealed that MKK6 was efficiently phosphorylated by PKR, and this could be inhibited by 2-aminopurine. Expression of kinase-inactive PKR (K296R) in cells inhibited the poly(IC)-induced phosphorylation of MKK3/6 detected by phosphospecific antiserum but did not affect the poly(IC)-induced gel migration retardation of MKK3.This suggests that poly(IC)-mediated in vivo activation of MKK6, but not MKK3, is through PKR. Consistent with this observation, PKR was capable of activating MKK6 as assessed in a coupled kinase assay containing the components of the p38 MAPK pathway. Our results indicate that the interaction of MKK6 and PKR provides a mechanism for regulating p38 MAPK activation in response to dsRNA stimulation. Double-stranded RNAs (dsRNA) 1 formed during virus infection can be potent stimulating agents triggering cellular responses through distinct targets and pathways. DsRNA-dependent interferon synthesis as well as NF-B activation may require the binding of dsRNA with cellular membrane component(s) that includes Toll-like receptor 3 (TLR-3) (1). However, other dsRNA sensor cellular components have also been implicated in the activation of cellular pathways triggered by dsRNA (2-4).The dsRNA-dependent protein PKR is a serine/threonine kinase, the expression of which is induced by interferons. This enzyme is activated in response to dsRNA of viral or synthetic origin, most notably the synthetic polyribonucleotide duplex, poly(rI):poly(rC) (pIC), and also by cytokines and cellular stress signals (5). Once activated, PKR functions as one of the mediators of antiviral and antiproliferative activities of interferons (6 -12). PKR has two double-stranded RNA-binding motifs located in the NH 2 -terminal domain (5, 13), which bind pIC or viral dsRNA intermediates generated during a viral infection and also permit the recruitment of other dsRNA-binding domain-containing proteins (14, 15). The carboxyl-terminal half of PKR harbors the kinase catalytic domain (9). On binding dsRNA, PKR dimerizes and undergoes autophosphorylation at multiple sites (5, 16). The direct antiviral activity of PKR is a consequence of the phosphorylation and activation of its major substrate eukaryotic translation initiation factor eIF2␣, thereby inhibiting protein synthesis and impeding virus multiplication (6,10,12).In addition to playing a role in protein synthesis inhibition, PKR also functions in other biological processes including apoptosis, transcriptional regulation, and cell growth, independent of the phosphorylation of eIF2␣ (5). ...

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DRBP76, a Double-stranded RNA-binding Nuclear Protein, Is Phosphorylated by the Interferon-induced Protein Kinase, PKR

Cited by 121 publications

References 31 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

PKR; a sentinel kinase for cellular stress

Protein Kinase R (PKR) Interacts with and Activates Mitogen-activated Protein Kinase Kinase 6 (MKK6) in Response to Double-stranded RNA Stimulation

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