Mechanism of activation of the double-stranded-RNA-dependent protein kinase PKR - Role of dimerization and cellular localization in the stimulation of PKR phosphorylation of eukaryotic initiation factor-2 (elF2)

An important defense against viral infection involves inhibition of transla tion by PKR phosphorylation of the cr subunit of eIF2. Binding of viral dsR NAs to two dsRNA-binding domains (dsRBDs) in PKR leads to relief of an inhi bitory region and activation of eIF2 kinase activity. Interestingly, while deletion of the regulatory region of PKR significantly induces activity in vitro, the truncated kinase does not inhibit translation in vivo, suggestin g that these sequences carry out additional functions required for PKR cont rol. To delineate these functions and determine the order of events leading to activation of PKR, we fused truncated PKR to domains of known function and. assayed the chimeras for in vivo activity We found that fusion of a he terologous dimerization domain with the PKR catalytic domain enhanced autop hosphorylation and eIF2 kinase function in vivo. The dsRBDs also mediate ri bosome association and we proposed that such targeting increases the locali zed concentration of PKR, enhancing interaction between PKR molecules. We a ddressed this premise by linking the truncated PKR to RAS sequences mediati ng farnesylation and membrane localization and found that the fusion protei n was functional in vivo. These results indicate that cellular localization along with oligomerization enhances interaction between PKR molecules. Ala nine substitution for the phosphorylation site, threonine 446, impeded in v ivo and in vitro activity of the PKR fusion proteins, while aspartate or gl utamate substitutions partially restored the function of the truncated kina se. These results indicate that both dimerization and cellular localization play a role in transient protein-protein interactions and that trans-autop hosphorylation is the final step in the mechanism of activation of PKR.