Identification of the heparin‐binding domains of the interferon‐induced protein kinase, PKR

Abstract

PKR is an interferon‐induced serine‐threonine protein kinase that plays an important role in the mediation of the antiviral and antiproliferative actions of interferons. PKR is present at low basal levels in cells and its expression is induced at the transcriptional level by interferons. PKR's kinase activity stays latent until it binds to its activator. In the case of virally infected cells, double‐stranded (ds) RNA serves as PKR's activator. The dsRNA binds to PKR via two copies of an evolutionarily conserved motif, thus inducing a conformational change, unmasking the ATP‐binding site and leading to autophosphorylation of PKR. Activated PKR then phosphorylates the α‐subunit of the protein synthesis initiation factor 2 (eIF2α) thereby inducing a general block in the initiation of protein synthesis. In addition to dsRNA, polyanionic agents such as heparin can also activate PKR. In contrast to dsRNA‐induced activation of PKR, heparin‐dependent PKR activation has so far remained uncharacterized. In order to understand the mechanism of heparin‐induced PKR activation, we have mapped the heparin‐binding domains of PKR. Our results indicate that PKR has two heparin‐binding domains that are nonoverlapping with its dsRNA‐binding domains. Although both these domains can function independently of each other, they function cooperatively when present together. Point mutations created within these domains rendered PKR defective in heparin‐binding, thereby confirming their essential role. In addition, these mutants were defective in kinase activity as determined by both in vitro and in vivo assays.

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

“…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.…”

Mechanistic characterization of the 5′-triphosphate-dependent activation of PKR: Lack of 5′-end nucleobase specificity, evidence for a distinct triphosphate binding site, and a critical role for the dsRBD

“…Despite these similarities, distinct differences in the 59-end requirements of ppp-ssRNA and dsRNA exist-requiring and not requiring a 59-triphosphate, respectively-suggesting potential mechanistic disparities between these two classes of activators. Indeed, ssRNA displays coarse structural similarity to a non-RNA activator of PKR, the polyanion heparin, which has been shown to bind basic residues near the active site in the kinase domain (Fasciano et al 2005a). To elucidate the mechanism of activation of PKR by ppp-ssRNA, we sought to elucidate the role, if any, of the dsRBD in such activation.…”

“…Because ssRNA and heparin are both non-dsRNA polyanion activators, we thought that they might have similar mechanisms of activation. Heparin has been shown to bind clusters of basic amino acids at the entrance to the kinase active site that are nonoverlapping with the dsRBD, suggesting a primarily electrostatic as opposed to structure-based mechanism of activation (Fasciano et al 2005a). Furthermore, it has been shown that heparin can activate a truncated version of PKR that does not contain the dsRBD, although the extent of activation was strongly diminished relative to fulllength PKR (Patel et al 1994;George et al 1996;Fasciano et al 2005a).…”

“…Heparin has been shown to bind clusters of basic amino acids at the entrance to the kinase active site that are nonoverlapping with the dsRBD, suggesting a primarily electrostatic as opposed to structure-based mechanism of activation (Fasciano et al 2005a). Furthermore, it has been shown that heparin can activate a truncated version of PKR that does not contain the dsRBD, although the extent of activation was strongly diminished relative to fulllength PKR (Patel et al 1994;George et al 1996;Fasciano et al 2005a). We thus anticipated that ppp-ssRNA activators might compete for activation by heparin while short dsRNA would not compete; indeed, such a result would suggest that the 59-triphosphate-binding site overlaps with the cluster of basic residues that serve as the binding site for heparin.…”

Nucleoside modifications modulate activation of the protein kinase PKR in an RNA structure-specific manner

“…We recently demonstrated that PKR can also be activated by relatively unstructured ssRNA transcripts if a 59-triphosphate is present, wherein the 59-triphosphate likely serves as a pathogen-associated molecular pattern (PAMP) (Nallagatla et al 2007). PKR can also be activated by non-RNA molecules including heparin, dextran sulfate, and poly-L-glutamate (Hovanessian and Galabru 1987;Fasciano et al 2005). Despite identification of these diverse PKR activators, a clear understanding of the RNA molecular patterns that affect PKR activation has not been achieved.…”