Characterization of purified double-stranded RNA-activated eIF-2 alpha kinase from rabbit reticulocytes.

The initiation of protein synthesis in reticulocyte lysates is inhibited by low levels (1-50 ng/ml) of doublestranded RNA (dsRNA) due to the activation of a CAMPindependent protein kinase (dsI) which phosphorylates the a-subunit (38,000 daltons) of eukaryotic initiation factor 2 (eIF-2a). In lysates, dsI is present in an inactive precursor form (latent dsI) associated with the ribosomal complement. Latent dsI is solubilized by extraction with high salt and can be purified in its latent state. Activation of latent dsI at any stage of purification requires dsRNA and ATP and is accompanied by the phosphorylation of a polypeptide doublet of approximately 67,000 and 68,500 daltons (67K/68.5K). Several criteria indicate that the 67K and 68.5K components represent different phosphorylated states of the same polypeptide. (a) During activation, the 67K component is phosphorylated first, followed by increasing phosphorylation of the 68.5K component; a pulsechase analysis suggests that the 67K polypeptide is a precursor of the 68.5K component. (b) Treatment of the phosphorylated 68.5K polypeptide with crude lysate protein phosphatases produces a shift to the 67K polypeptide. (c) Protease digestion of the two polypeptides yields similar phosphopeptide patterns. Latent dsI has been purified to near homogeneity by affinity chromatography on an agarose-poly(1) poly(C) matrix, from which latent dsI can be eluted with 2 M KC1 and 100 p~ ATP. Activation of this dsI still gives rise to the phosphorylated 67K/68.5K doublet. Labeling with the ”’1labeled Bolton-Hunter reagent reveals a single major component of about 67,000 daltons before activation, and a polypeptide doublet of 67,000 and 68,500 daltons after activation. From our findings, we conclude that (i) the phosphorylated 67K/68.5K doublet is active dsI; (ii) activation of the isolated dsI is rapid and proceeds through an autokinase mechanism; (iii) eIF-2a kinase activity increases with increasing autophosphorylation of dsI; and (iv) maximal activity is achieved when relative phosphate levels are significantly higher in the 68.513 polypeptide than in the 67K polypeptide.