Mechanism of PKR Activation: Dimerization and Kinase Activation in the Absence of Double-stranded RNA

Lemaire, Peter A.; Lary, Jeffrey W.; Cole, James L.

doi:10.1016/j.jmb.2004.10.031

Cited by 96 publications

(206 citation statements)

References 49 publications

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“…29 The full length K296R mutant binds and is phosphorylated by the wild-type enzyme, and has been widely used to study PKR autophosphorylation and dimerization. 1,11,17,19,20,21,25,26,30 Important for the NMR studies, this mutant can not autophoshorylate during bacterial expression, and therefore does not dimerize. 21,30 Limited proteolysis experiments on the full-length protein identified a domain coinciding with the product of caspase cleavage during apoptosis, D251-C551.…”

Section: Resultsmentioning

confidence: 99%

“…1,15,21,30 In addition, multiple phosphorylation sites along the protein may serve to prevent the RBD from re-associating with the kinase, to fine-tune enzymatic activity, or to mediate various non-covalent interactions of PKR. A major challenge that remains is to elucidate the sequence and mechanism of autophosphorylation events that occur during PKR activation.…”

Section: Supplementary Materialsmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Supplementary Materialsmentioning

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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“…Although PKR exists in a monomer-dimer equilibrium (29), at the maximum enzyme concentration employed in the titration in Figure 2 only about 20% dimer is present. However, to avoid potential complications associated with variable extents of PKR dimerization during the course of a titration we have devised an alternative reverse anisotropy titration where the enzyme concentration is fixed and the ligand concentration is varied.…”

Section: Equilibrium Binding Of Atp To Pkrmentioning

confidence: 98%

“…Protein concentrations were measured by absorbance using ε 280 = 4.33 × 10 4 M −1 cm −1 . At enzyme concentrations greater than 0.5 μM, PKR undergoes dsRNA -independent autophosphorylation upon addition of ATP (29). PKR was phosphorylated by incubation in phosphorylation buffer (20 mM HEPES, 50 mM KCl, 5 mM MgCl 2 , 0.1 mM EDTA, 1 mM DTT, pH 7.5) with 5 mM ATP at a protein concentration of 10-15 μM.…”

Section: Reagents and Materialsmentioning

confidence: 99%

“…Thus, there appears to be no strong correlation between activation of PKR and enhanced nucleotide binding affinity; indeed, dsRNA binding slightly weakens nucleotide binding. Because PKR undergoes dsRNA-independent autophosphorylation at the higher protein concentrations used in the fluorescence measurements (29) it is not possible to directly characterize binding of mant-ATP to wild type, unphosphorylated enzyme without complications from the catalytic reaction. Thus, we have employed a catalytically inactive K296R mutant.…”

Section: Equilibrium Binding Of Atp To Pkrmentioning

confidence: 99%

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Unactivated PKR Exists in an Open Conformation Capable of Binding Nucleotides

et al. 2006

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The dsRNA-activated protein kinase, PKR, plays a pivotal role in the cellular antiviral response. PKR contains an N-terminal dsRNA binding domain (dsRBD) and a C-terminal kinase domain. An autoinhibition model has been proposed in which latent PKR exists in a closed conformation where the substrate binding cleft of the kinase is blocked by the dsRBD. Binding to dsRNA activates the enzyme by inducing an open conformation and enhancing dimerization. We have tested this model by characterizing the affinity and kinetics of nucleotide substrate binding to PKR. The fluorescent nucleotide mant-AMPPNP binds to unactivated PKR with K d ~ 30 μM and the affinity is not strongly affected by autophosphorylation or binding to dsRNA. Biphasic binding kinetics are observed where the fast phase depends on nucleotide concentration but the slow phase is ligand-independent. The kinetic data fit to a two-step model of ligand binding followed by a slow conformation change. The kinetics are also not strongly affected by phosphorylation state or dsRNA binding. Thus, the equilibrium and kinetic data indicate that substrate accessibility of the kinase is not modulated by PKR activation state as predicted by the autoinhibition model. In atomic force microscopy images, monomers of the latent protein are resolved with three separate regions linked by flexible, bridge-like structures. Resolution of the individual domains in the images supports a model in which unactivated PKR exists in an open conformation where the kinase domain is accessible and capable of binding substrate.PKR is a soluble protein kinase which is activated by dsRNA (1). PKR is constitutively expressed in most mammalian cell types but it is induced by type 1 interferons and plays a central role in the innate immunity defense against viral infection (2). In addition, PKR has been implicated in a variety of cellular signal transduction pathways (3). PKR is a member of the family of protein kinases which phosphorylate eIF2α, resulting in a blockage of translational initiation (4). Production of dsRNA during viral infection leads to PKR activation and inhibition of protein synthesis and apoptosis (5). PKR contains an N-terminal double-stranded RNA binding domain (dsRBD) and a C-terminal kinase domain. The dsRBD consists of two tandem copies of a widely distributed double stranded RNA binding * To whom correspondence may be addressed: (860) 486-4333 (telephone), james.cole@uconn.edu. Table S1: Relative contribution of the fast phase for association of mant-AMPNP with PKR. This material is available free of charge via the Internet at http://pubs.acs.org. Supporting Information NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 August 2. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript motif (6). In an NMR structure of a PKR dsRBD construct, each of the dsRNA binding motifs adopts the canonical αβββα fold with a ~20 amino acid intervening region that is unstructured in the absence of dsRNA (7). The PKR kinas...

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Implication of double‐stranded RNA signaling in the etiology of autoimmune myasthenia gravis

Cufi

Dragin

Weiss

et al. 2012

Annals of Neurology

116

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Mechanism of PKR Activation: Dimerization and Kinase Activation in the Absence of Double-stranded RNA

Cited by 96 publications

References 49 publications

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

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

Unactivated PKR Exists in an Open Conformation Capable of Binding Nucleotides

Implication of double‐stranded RNA signaling in the etiology of autoimmune myasthenia gravis

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