Identification of the heparin-binding domains of the interferon-induced protein kinase, PKR.
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 alpha-subunit of the protein synthesis initiation factor 2 (eIF2alpha) 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.
Fasciano
Stephen
S
Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA.
Hutchins
Brian
B
Handy
Indhira
I
Patel
Rekha C
RC
D000595:Amino Acid Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D017931:DNA Primers; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007372:Interferons / Q000494:pharmacology; D007700:Kinetics; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014158:Transcription, Genetic / Q000187:drug effects; D019892:eIF-2 Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D006493:Heparin; D007372:Interferons; D019892:eIF-2 Kinase