Homologous regions of the alpha subunit of eukaryotic translational initiation factor 2 (eIF2 alpha) and the vaccinia virus K3L gene product interact with the same domain within the dsRNA-activated protein kinase (PKR)

The vaccinia virus K3L gene product, pK3, binds to the dsRNA-activated protein kinase, PKR, reducing its ability to interact with and phosphorylate eIF2 alpha. On the basis of this characteristic and the homology of pK3 to the N-terminus of eIF2 alpha, several laboratories have utilized pK3 to investigate the molecular determinants that specify substrate recognition by PKR. The data presented here demonstrate that the natural substrate, eIF2 alpha, also binds to PKR in vitro and interacts with the same or an overlapping domain within PKR. A truncated form of eIF2 alpha, representing the N-terminal 123 amino acids and containing the regions of homology to pK3, retains the ability to bind PKR. pK3, eIF2 alpha, and the truncated form of eIF2 alpha all bind to the C-terminus of PKR containing the catalytic domain, but not to the regulatory N-terminus. Variants of pK3 and eIF2 alpha, des-(75-78)-K3L (pK3 Delta GYID), and des-(80-83)-eIF2 alpha (eIF2 alpha Delta-GYID), from which the conserved amino acids GYID have been deleted, exhibit a decreased ability to interact with PKR. Similarly, the in vitro binding of pK3, eIF2 alpha, and the truncated form of eIF2 alpha to PKR can be competed with purified pK3 but not with pK3 Delta GYID. In addition, the deletion of GYID from eIF2 alpha significantly reduces its ability to be phosphorylated by PKR, demonstrating that PKR recognizes its substrate, at least in part through interaction with sequences remote from the phosphorylation site. In summary, we have shown that the region within PKR that interacts with the pseudosubstrate, pK3, is the same region that interacts with the authentic substrate, eIF2 alpha. In addition, we have shown that the N-terminal 123 amino acids of eIF2 alpha contains structural elements necessary for recognition by PKR. The results pinpoint the GYID motif, shared between pK3 and eIF2 alpha and distant from the phosphorylation site, as being important for the interaction of eIF2 alpha with PKR, as well as its phosphorylation.