The kinase insert domain of interferon-induced protein kinase PKR is required for activity but not for interaction with the pseudosubstrate K3L.
Interferon-induced protein kinase (PKR) is a member of a family of kinases that regulate translation initiation through phosphorylation of eukaryotic initiation factor 2alpha. In addition to the conserved catalytic subdomains that are present in all serine/threonine kinases, the eukaryotic initiation factor 2alpha kinases possess an insert region between catalytic subdomains IV and V that has been termed the kinase insert domain. To investigate the importance of the kinase insert domain of PKR, several deletions and point mutations were introduced within this domain and analyzed for kinase activity both in vitro and in vivo. Here we show that deletion of the kinase insert sequence or mutation of serine 355, which lies within this region, abrogates kinase activity. In addition, the kinase insert domain of PKR and adjacent amino acids (LFIQME) in catalytic subdomain V are not required for binding of the pseudosubstrate inhibitor K3L from vaccinia virus. A portion of the catalytic domain of PKR between amino acids 366 and 415 confers K3L binding in vivo, suggesting a possible role for this region of PKR in substrate interaction.
Craig
A W
AW
Department of Biochemistry and McGill Cancer Centre, McGill University, Montreal, Québec H3G 1Y6, Canada.
Cosentino
G P
GP
Donzé
O
O
Sonenberg
N
N
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D004790:Enzyme Induction; D006367:HeLa Cells; D006801:Humans; D007372:Interferons / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D019892:eIF-2 Kinase
D007372:Interferons; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase