AUTOPHOSPHORYLATION SITES PARTICIPATE IN THE ACTIVATION OF THE DOUBLE-STRANDED-RNA-ACTIVATED PROTEIN-KINASE PKR

The interferon-induced RNA-dependent protein kinase PKR is found in ce lls in a latent state. In response to the binding of double-stranded R NA, the enzyme becomes activated and autophosphorylated on several ser ine and threonine residues. Consequently, it has been postulated that autophosphorylation is a prerequisite for activation of the kinase. We report the identification of PKR sites that are autophosphorylated in vitro concomitantly with activation and examine their roles in the ac tivation of PKR, Mutation of one site, threonine 258, results in a kin ase that is less efficient in autophosphorylation and in phosphorylati ng its substrate, the initiation factor eIF2, in vitro. The mutant kin ase is also impaired in vivo, displaying reduced ability to inhibit pr otein synthesis in yeast and mammalian cells and to induce a slow-grow th phenotype in Saccharomyces cerevisiae. Mutations at two neighboring sites, serine 242 and threonine 255, exacerbated the effect. Taken to gether with earlier results (S. B. Lee, S. R. Green, M. B. Mathews, an d M. Esteban, Proc. Natl. Acad. Sci. USA 91:10551-10555, 1994), these data suggest that the central part of the PKR molecule, lying between its RNA-binding and catalytic domains, regulates kinase activity via a utophosphorylation.