Molecular basis for PKR activation by PACT or dsRNA

Li, Shoudong; Peters, Gregory A.; Ding, Keyang; Zhang, Xiaolun; Qin, Jun; Sen, Ganes C.

doi:10.1073/pnas.0602317103

Cited by 110 publications

(116 citation statements)

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“…Two models currently describe PKR activation: the autoinhibition and dimerization models (50). It was suggested that binding of either dsRNA or PACT to PKR may interrupt intramolecular interaction between the kinase and dsRNA-binding domains, leading to an open conformation of the PKR molecule and allowing autophosphorylation to occur (49). NF90 is phosphorylated by PKR in its RBD (6).…”

Section: /2mentioning

confidence: 99%

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NF90 Exerts Antiviral Activity through Regulation of PKR Phosphorylation and Stress Granules in Infected Cells

Wen

Huang

Mok

et al. 2014

The Journal of Immunology

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NF90 was shown to exhibit broad antiviral activity against several viruses, but detailed mechanisms remain unclear. In this study, we examined the molecular basis for the inhibitory effect of NF90 on virus replication mediated through protein kinase (PKR)-associated translational regulation. We first verified the interaction between NF90 and PKR in mammalian cells and showed that NF90 interacts with PKR through its C-terminal and that the interaction is independent of NF90 RNA-binding properties. We further showed that knockdown of NF90 resulted in significantly lower levels of PKR phosphorylation in response to dsRNA induction and influenza virus infection. We also showed that high concentrations of NF90 exhibit negative regulatory effects on PKR phosphorylation, presumably through competition for dsRNA via the C-terminal RNA-binding domain. PKR activation is essential for the formation of stress granules in response to dsRNA induction. Our results showed that NF90 is a component of stress granules. In NF90-knockdown cells, dsRNA treatment induced significantly lower levels of stress granules than in control cells. Further evidence for an NF90–PKR antiviral pathway was obtained using an NS1 mutated influenza A virus specifically attenuated in its ability to inhibit PKR activation. This mutant virus replicated indistinguishably from wild-type virus in NF90-knockdown cells, but not in scrambled control cells or Vero cells, indicating that NF90’s antiviral function occurs through interaction with PKR. Taken together, these results reveal a yet-to-be defined host antiviral mechanism in which NF90 upregulation of PKR phosphorylation restricts virus infection.

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Section: /2mentioning

confidence: 99%

“…Phosphorylation of PKR is essential for activation of its function, and it can be regulated by host RNA-binding proteins other than dsRNA (47)(48)(49). Two models currently describe PKR activation: the autoinhibition and dimerization models (50).…”

Section: /2mentioning

confidence: 99%

NF90 Exerts Antiviral Activity through Regulation of PKR Phosphorylation and Stress Granules in Infected Cells

Wen

Huang

Mok

et al. 2014

The Journal of Immunology

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“…48 Convincing NMR titration, immunoprecipitation and mutagenesis data was presented to show that this region in the N-lobe of the kinase is responsible for the autoinhibitory kinase-RBD2 interaction. In contrast, we observe only minor changes in the implicated region and attribute these to RBD1 binding.…”

Section: Note Added In Proofmentioning

confidence: 99%

Mapping of the Auto-inhibitory Interactions of Protein Kinase R by Nuclear Magnetic Resonance

Gelev

Aktaş

Marintchev

et al. 2006

Journal of Molecular Biology

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SummaryThe dsRNA-dependent protein kinase (PKR) is a key mediator of the anti-viral and antiproliferative effects of interferon. Unphosphorylated PKR is characterized by inhibitory interactions between the kinase and RNA binding domains (RBDs), but the structural details of the latent state and its unraveling during activation are not well understood. To study PKR regulation by NMR we assigned a large portion of the backbone resonances of the catalytically inactive K296R kinase domain, and performed 15 N-HSQC titrations of this kinase domain with the RBDs. Chemical shift perturbations in the kinase indicate that RBD2 binds to the substrate eIF2α docking site in the kinase C-lobe. Consistent with these results, a mutation in the eIF2α docking site, F495A displays weaker interactions with the RBD. The full-length RBD1+2 binds more strongly to the kinase domain than RBD2 alone. The observed chemical shift changes extend from the eIF2α binding site into the kinase N-lobe and inside the active site, consistent with weak interactions between the N-terminal part of the RBD and the kinase.

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“…On the other hand, domain 3, which, in vitro, can activate PKR by itself, binds, with low affinity, to a specific region in the kinase domain of PKR (20). We have recently identified the PACT binding motif (PBM) in this region of PKR (21). Moreover, we demonstrated intramolecular interaction between PBM and dsRBM2 of PKR.…”

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confidence: 99%

Phosphorylation of Specific Serine Residues in the PKR Activation Domain of PACT Is Essential for Its Ability to Mediate Apoptosis

Peters

Sen

2006

Journal of Biological Chemistry

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Activation of the latent protein kinase, PKR, by extracellular stresses and triggering of resultant cellular apoptosis are mediated by the protein, PACT, which itself gets phosphorylated in stressed cells. We have analyzed the underlying biochemical mechanism by carrying out alanine-scanning mutagenesis of the PKR activation domain of PACT. Among the indispensable residues identified were two serine residues, whose phosphorylation was essential for the cellular actions of PACT. Two-dimensional gel analysis, Western analysis using phosphoamino acid-specific antiserum, and in vivo 32 P labeling of PACT demonstrated that constitutive phosphorylation of one of the two residues, Ser 246 , was required for stress-induced phosphorylation of the other, Ser 287 . Substitution of either of them by threonine or aspartic acid, but not alanine, was tolerated. Substitution of both residues with the phosphoserine mimetic, aspartic acid, produced a mutant PACT that, unlike the wild-type protein, caused PKR activation and apoptosis, even in unstressed cells. These results indicate that phosphorylation of specific serine residues in the activation domain of PACT is the major mode of transmission of cellular stress response to PKR.

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Molecular basis for PKR activation by PACT or dsRNA

Abstract: antiviral mechanism ͉ autoinhibition ͉ NMR ͉ peptide activator ͉ protein kinase

Cited by 110 publications

References 50 publications

NF90 Exerts Antiviral Activity through Regulation of PKR Phosphorylation and Stress Granules in Infected Cells

NF90 Exerts Antiviral Activity through Regulation of PKR Phosphorylation and Stress Granules in Infected Cells

Mapping of the Auto-inhibitory Interactions of Protein Kinase R by Nuclear Magnetic Resonance

Phosphorylation of Specific Serine Residues in the PKR Activation Domain of PACT Is Essential for Its Ability to Mediate Apoptosis

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