PKR activity modulation by phosphomimetic mutations of serine residues located three aminoacids upstream of double-stranded RNA binding motifs

Abstract

Eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), better known as PKR, plays a key role in the response to viral infections and cellular homeostasis by regulating mRNA translation. Upon binding dsRNA, PKR is activated through homodimerization and subsequent autophosphorylation on residues Thr446 and Thr451. In this study, we identified a novel PKR phosphorylation site, Ser6, located 3 amino acids upstream of the first double-stranded RNA binding motif (DRBM1). Another Ser residue occurs in PKR at position 97, the very same position relative to the DRBM2. Ser or Thr residues also occur 3 amino acids upstream DRBMs of other proteins such as ADAR1 or DICER. Phosphoinhibiting mutations (Ser-to-Ala) introduced at Ser6 and Ser97 spontaneously activated PKR. In contrast, phosphomimetic mutations (Ser-to-Asp) inhibited PKR activation following either poly (I:C) transfection or virus infection. These mutations moderately affected dsRNA binding or dimerization, suggesting a model where negative charges occurring at position 6 and 97 tighten the interaction of DRBMs with the kinase domain, thus keeping PKR in an inactive closed conformation even in the presence of dsRNA. This study provides new insights on PKR regulation mechanisms and identifies Ser6 and Ser97 as potential targets to modulate PKR activity for therapeutic purposes.

Move and countermove: the integrated stress response in picorna- and coronavirus-infected cells

“…Instead, it interferes with dsRNA association to PKR by binding and hijacking host kinases, RSKs 41• , 42•• . How this suppresses PKR activation is unknown, but this observation adds to the notion that regulation of PKR activation is more complex than generally assumed, as also recently proposed by others 43 , 44 . Additionally, it has been suggested that L interferes with the interaction between eIF4G and G3BP to suppress SG formation [27•] .…”

Cardiovirus leader proteins retarget RSK kinases toward alternative substrates to perturb nucleocytoplasmic traffic

“…Other lentiviral vectors used for protein expression were derived from pCCLsin.PPT.hPGK.GFP.pre. 47 - TM945 is a Prom CMV -MCS-IRES-mCherry construct 48 - TM952 is a Prom CMV -MCS-IRES-neo construct 49 - BLP10 is a TM952 derivative coding for BioID2-HA-linker(3xGGGS)-MuRSK2. This construct expresses murine RSK2, which differs from human RSK2 by a single amino acid. - BLP20 is a TM952 derivative coding for As1-RSK2 (hu-RSK2 with Analog-sensitive kinase 1 (As1) mutation (Leu 147 → Gly). - BLP21 is a TM952 derivative coding for As2-RSK2 (hu-RSK2 with Analog-sensitive kinase 2 (As2) mutation (Leu 147 → Ala). - CL13 is a TM945 derivative expressing the 10 C-terminal aminoacids of L followed by the entire capsid precursor of virus DA1. - TM1116, TM1117, TM1118 and TM1119 are TM952 derivatives carrying the sequences coding for 3xHA-Human RSK1, RSK2, RSK3 and RSK4 respectively 1 .…”

“…-pLVX-EF1alpha-2xGFP:NES-IRES-2xRFP:NLS was a gift from Fred Gage (Addgene plasmid # 71396) (Mertens et al, 2015) Other lentiviral vectors used for protein expression were derived from pCCLsin.PPT.hPGK.GFP.pre. (Follenzi et al, 2000) -TM945 is a PromCMV-MCS-IRES-mCherry construct (Hermant et al, 2013) -TM952 is a PromCMV-MCS-IRES-neo construct (Cesaro et al, 2021) -BLP10 is a TM952 derivative coding for BioID2-HA-linker(3xGGGS)-MuRSK2. Note that this construct expresses murine RSK2, which differs from human RSK2 by a single amino acid.…”

Mechanisms of circular RNA degradation

“…Moreover, PKR plays a key role in the viral infection response and intracellular homeostasis by regulating the translation of mRNAs. After dsRNA binds to PKR, PKR is activated through homozygous dimerization and autophosphorylation of Thr446 and Thr451 residues 32 . Upon inflammation or virus infection, circRNAs are globally degraded by activated RNase L, releasing PKR for its aberrant activation, as seen in SLE patients with reduced degradation of circRNAs leading to increased aberrant activation of PKR (Fig.…”