The Kinase Insert Domain of Interferon-induced Protein Kinase PKR Is Required for Activity but Not for Interaction with the Pseudosubstrate K3L

Abstract

Interferon-induced protein kinase (PKR) is a member of a family of kinases that regulate translation initiation through phosphorylation of eukaryotic initiation factor 2␣. In addition to the conserved catalytic subdomains that are present in all serine/threonine kinases, the eukaryotic initiation factor 2␣ 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.The double-stranded RNA (dsRNA) 1 -activated protein kinase, PKR (also called p68, DAI, and P1 kinase) is an interferon-inducible protein that plays a key role in the antiviral and antiproliferative interferon response (for a review see Ref.

A PKR-like eukaryotic initiation factor 2α kinase from zebrafish contains Z-DNA binding domains instead of dsRNA binding domains

“…Like PKZ, enzymatically inactive PKR is more highly expressed than WT (25,29). In contrast to PKZ, K296R mutants in PKR stimulate reporter protein expression 2-to 6-fold, perhaps by interacting with endogenous PKR leading to dominant negative inhibition or by sequestration of dsRNA preventing PKR activation (26,29,35,41). This finding is in agreement with the observation that the dominant negative form, PKR⌬E7, induces reporter enzyme activity in HeLa cells but not in PKR-deficient cells (33).…”

“…Like PKZ, enzymatically inactive PKR is more highly expressed than WT (25,29). In contrast to PKZ, K296R mutants in PKR stimulate reporter protein expression 2-to 6-fold, perhaps by interacting with endogenous PKR leading to dominant negative inhibition or by sequestration of dsRNA preventing PKR activation (26,29,35,41).…”

“…Splice variant B, the second most abundant PKZ variant, lacks most of the kinase insert, leaving only 15 aa, the same length as found in porcine and bovine PKR. The kinase insert domain is important for eIF2␣ phosphorylation (29). Therefore, we speculate that different PKZ isoforms may phosphorylate different targets.…”

“…The insert domain is essential for phosphorylation of eIF2␣ (29); however, it varies greatly in length even between orthologues. The length of the insert ranges from 15-30 aa in PKR to Ͼ200 in PEK ( Table 1).…”

Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein

“…1 A), which includes the Ser-51 phosphorylation site in eIF2␣. The K3L protein directly interacts with the kinase domain of PKR, as revealed by yeast 2-hybrid and in vitro interaction assays (4,(8)(9)(10)(11). Because the K3L protein and eIF2␣ were found to compete for binding to PKR (11), it is thought that they bind to the kinase by a common mechanism.…”