Binding of double-stranded RNA to protein kinase PKR is required for dimerization and promotes critical autophosphorylation events in the activation loop

Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphor ylates translation initiation factor 2 alpha to inhibit protein synthesis i n virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DR BMs I and II) required for activation by dsRNA, There is strong evidence th at PKR activation requires dimerization, but the role of dsRNA in dimer for mation is controversial. By making alanine substitutions predicted to remov e increasing numbers of side chain contacts between the DRBMs and dsRNA we found that dimerization of full-length PKR in yeast was impaired by the min imal combinations of mutations required to impair dsRNA binding in vitro. M utation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization withou t affecting dsRNA binding, destroyed both activities in our assays. By cont rast, deletion of a second dimerization region that overlaps the kinase dom ain had no effect on PKR dimerization in yeast, Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activati on loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 pho sphorylation is stimulated by dsRNA binding, Our results provide strong evi dence that dsRNA binding is required for dimerization of full-length PKR mo lecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2 alpha kinase function of PKR.