AUTOPHOSPHORYLATION IN THE ACTIVATION LOOP IS REQUIRED FOR FULL KINASE-ACTIVITY IN-VIVO OF HUMAN AND YEAST EUKARYOTIC INITIATION-FACTOR 2-ALPHA KINASES PKR AND GCN2

The human double-stranded RNA-dependent protein kinase (PKR) is an imp ortant component of the interferon response to virus infection, The ac tivation of PKR is accompanied by autophosphorylation at multiple site s, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity, Several protein kinases are activa ted by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop, We show that Thr-446 and Th r-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity, Mutation of either residue to Ala impaire d translational control by PKR in yeast cells and COS1 cells and led t o tumor formation in mice, These mutations also impaired autophosphory lation and eukaryotic initiation factor 2 subunit alpha (eIF2 alpha) p hosphorylation by PKR in vitro. Whereas the Ala-446 substitution subst antially reduced PKR function, the mutant kinase containing Ala-451 wa s completely inactive, PKR specifically phosphorylated Thr-446 and Thr -451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain Ii of PKR partially restored kinase activity when combined with the Ala-451 mutation. Thi s finding suggests that the interaction between subdomain X and the ac tivation loop, described previously for MAP kinase, is a regulatory fe ature conserved in PKR. We found that the yeast eIF2 alpha kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR, Thr- 887 was more critically required than was Thr-882 for GCN2 kinase acti vity, paralleling the relative importance of Thr-446 and Thr-451 in PK R. These results indicate striking similarities between GCN2 and PKR i n the importance of autophosphorylation and the conserved Thr residues in the activation loop.