The Regulatory and Kinase Domains but Not the Interdomain Linker Determine Human Double-stranded RNA-activated Kinase (PKR) Sensitivity to Inhibition by Viral Non-coding RNAs

Sunita, S.; Schwartz, Samantha L.; Conn, Graeme L.

doi:10.1074/jbc.m115.679738

Cited by 7 publications

(15 citation statements)

References 51 publications

(54 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similarly, Conn and coworkers that reported that removal of the linker did not block activation of PKR by dsRNA or inhibition by VAI RNA. 16 They found that the functional properties of hPKR and a linker-deleted construct are identical, whereas we detected some quantitative differences in RNA-independent and dependent activation and RNA binding affinities. Interestingly, rat PKR was reported to bind viral RNA inhibitor with the same affinity as the human enzyme but the inhibition potency was greatly reduced.…”

Section: Discussionmentioning

confidence: 52%

See 1 more Smart Citation

Role of the Interdomain Linker in RNA-Activated Protein Kinase Activation

2015

View full text Add to dashboard Cite

PKR is a key component of the interferon-induced antiviral pathway in higher eukaryotes. Upon recognition of viral dsRNA, PKR is activated via dimerization and autophosphorylation. PKR contains two N-terminal dsRNA binding domains (dsRBD) and a C-terminal kinase domain. The dsRBDs and the kinase are separated by a long, unstructured ~80 amino acid linker in the human enzyme. The length of the N-terminal portion of the linker varies among PKR sequences and it is completely absent in one ortholog. Here, we characterize the effects of deleting the variable region from the human enzyme to produce PKRΔV. The linker deletion results in quantitative but not qualitative changes in catalytic activity, RNA binding and conformation. PKRΔV is somewhat more active and exhibits more cooperative RNA binding. As we previously observed for the full-length enzyme, PKRΔV is flexible in solution and adopts a range of compact and extended conformations. The conformational ensemble is biased towards compact states which may be related to weak interactions between the dsRBD and kinase domains. PKR retains RNA-induced autophosphorylation upon complete removal of the linker, indicating that the C-terminal, basic region is also not required for activity. PKR is a key component of the interferon-induced antiviral pathway in higher eukaryotes.1,2 Upon recognition of intracellular dsRNA of 30 bp or longer it is activated via dimerization and autophophorylation.3 Activated PKR phosphorylates eIF2α, which locks the eIF2A heterotrimer in a GDP bound complex incapable of binding Met-tRNA and initiating translation. Consequently, protein synthesis is halted, leading to apoptosis and containment of the viral infection. In addition, PKR is involved in a plethora of signaling pathways involving stress response and inflammation and regulates cellular growth and proliferation, nutrient signaling and metabolism.4–6

show abstract

Section: Discussionmentioning

confidence: 52%

“…Indeed, it was recently reported that deletions of the linker region from hPKR do not affect dsRNA binding, activation or inhibition by noncoding viral RNA inhibitors. 16 …”

mentioning

confidence: 99%

Role of the Interdomain Linker in RNA-Activated Protein Kinase Activation

2015

View full text Add to dashboard Cite

show abstract

“…We next tested the effect that these differences in PKR binding had on the ability of each nc886 conformer to inhibit PKR autophosphorylation in the presence of a dsRNA activator using an established slot-blot radiometric kinase assay (Sunita et al 2015). PKR autophosphorylation was measured in the presence of a fixed concentration of poly(rI:rC) dsRNA and increasing amounts of each nc886 RNA conformer.…”

Section: Nc886 Rna Conformers Have Opposing Activities Against Pkrmentioning

confidence: 99%

“…PKR activation assays PKR (0.1 μg) was incubated with 0-10 μM of full-length or variant nc886 RNA for 10 min at 25°C in 50 mM Tris buffer (pH 7.8) containing 50 mM KCl, 2.5 mM DTT, 10% glycerol, 20 μM ATP, 1 μCi [γ 32 P]-ATP, and 2 mM MgCl 2 . After incubation, reactions were quenched with excess ice-cold phosphate buffered saline containing 200 μM ATP and applied to a Bio-Dot SF (Bio-Rad) microfiltration system as described previously (Sunita et al 2015). Membranes were exposed to a phosphor storage screen and the extent of phosphorylation was determined using analysis by a Typhoon FLA 7000 PhosphorImager and ImageQuant software (GE Healthcare).…”

Section: Pkr Inhibition Assaysmentioning

confidence: 99%

Human noncoding RNA 886 (nc886) adopts two structurally distinct conformers that are functionally opposing regulators of PKR

Calderon

Conn

2017

RNA

Self Cite

View full text Add to dashboard Cite

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) senses dsRNA produced during viral infection and halts cellular protein synthesis to block viral replication. How basal PKR activity is controlled in the absence of infection was unclear until the recent identification of a potential endogenous regulator, the cellular noncoding RNA 886 (nc886). However, nc886 adopts two distinct conformations for which the structural details and potential functional differences remain unclear. Here, we isolated and separately dissected the function of each form of nc886 to more clearly define the molecular mechanism of nc886-mediated PKR inhibition. We show that nc886 adopts two stable, noninterconverting RNA conformers that are functionally nonequivalent using complementary RNA structure probing and mutational analyses combined with PKR binding and activity assays. One conformer acts as a potent inhibitor, while the other is a pseudoinhibitor capable of weakly activating the kinase. We mapped the nc886 region necessary for high affinity binding and potent inhibition of PKR to an apical stem-loop structure present in only one conformer of the RNA. This structural feature is not only critical for inhibiting PKR autophosphorylation, but also the phosphorylation of its cellular substrate, the eukaryotic translation initiation factor 2α subunit. The identification of different activities of the nc886 conformers suggests a potential mechanism for producing a gradient of PKR regulation within the cell and reveals a way by which a cellular noncoding RNA can mask or present a structural feature to PKR for inhibition.

show abstract

“…PKR exists in an ensemble of extended and compact conformations (Anderson and Cole 2008;VanOudenhove et al 2009). Interestingly, removal of this interdomain linker did not abolish dsRNA-dependent autophosphorylation nor inhibition by viral RNAs in vitro (Sunita et al 2015;Husain et al 2016). However, the linker's carboxy-terminal portion, a 23-a.a. "basic patch," is required for full activation and enhances PKR's basal activity (Husain et al 2016).…”

Section: Bridging the Regulatory And Effector Domainsmentioning

confidence: 99%

The search for a PKR code—differential regulation of protein kinase R activity by diverse RNA and protein regulators

Bou‐Nader

Gordon

Henderson

et al. 2019

RNA

View full text Add to dashboard Cite

The interferon-inducible protein kinase R (PKR) is a key component of host innate immunity that restricts viral replication and propagation. As one of the four eIF2α kinases that sense diverse stresses and direct the integrated stress response (ISR) crucial for cell survival and proliferation, PKR's versatile roles extend well beyond antiviral defense. Targeted by numerous host and viral regulators made of RNA and proteins, PKR is subject to multiple layers of endogenous control and external manipulation, driving its rapid evolution. These versatile regulators include not only the canonical doublestranded RNA (dsRNA) that activates the kinase activity of PKR, but also highly structured viral, host, and artificial RNAs that exert a full spectrum of effects. In this review, we discuss our deepening understanding of the allosteric mechanism that connects the regulatory and effector domains of PKR, with an emphasis on diverse structured RNA regulators in comparison to their protein counterparts. Through this analysis, we conclude that much of the mechanistic details that underlie this RNA-regulated kinase await structural and functional elucidation, upon which we can then describe a "PKR code," a set of structural and chemical features of RNA that are both descriptive and predictive for their effects on PKR.

show abstract

The Regulatory and Kinase Domains but Not the Interdomain Linker Determine Human Double-stranded RNA-activated Kinase (PKR) Sensitivity to Inhibition by Viral Non-coding RNAs

Cited by 7 publications

References 51 publications

Role of the Interdomain Linker in RNA-Activated Protein Kinase Activation

Role of the Interdomain Linker in RNA-Activated Protein Kinase Activation

Human noncoding RNA 886 (nc886) adopts two structurally distinct conformers that are functionally opposing regulators of PKR

The search for a PKR code—differential regulation of protein kinase R activity by diverse RNA and protein regulators

Contact Info

Product

Resources

About