TRANSLATIONAL REGULATION BY THE INTERFERON-INDUCED DOUBLE-STRANDED-RNA-ACTIVATED 68-KDA PROTEIN-KINASE

Activation of the interferon-inducible 68-kDa protein kinase (referred to as P68) by double-stranded RNA catalyzes phosphorylation of the al pha subunit of eukaryotic protein synthesis initiation factor 2. We ha ve analyzed the transient expression of mutant and wild-type kinase mo lecules in transfected COS cells to examine the effects of the kinase on gene expression in the absence of other interferon-induced gene pro ducts. The wild-type P68 kinase was expressed inefficiently whereas a catalytically inactive P68 was expressed at 30- to 40 -fold higher lev els. Protein stability measurements and primer-extension analysis of h uman kinase-specific mRNA levels provided evidence that kinase express ion was regulated at the level of mRNA translation. Further, cotransfe ction experiments revealed that the domain II catalytically inactive m utant could stimulate reporter gene protein synthesis in a transdomina nt manner. We also examined the expression of mutants with deletions i n the N-terminal double-stranded RNA binding domains and found that a kinase construct lacking aa 156-243 was expressed at levels comparable to the wild type whereas a P68 construct lacking aa 91-243 was expres sed at levels 70-fold higher. Both the inactive domain II P68 mutant a nd the deletion mutant lacking aa 91-243 were less inhibitory to growt h in yeast due to the reduced ability to phosphorylate initiation fact or 2alpha in vivo. In conclusion we have demonstrated that the P68 kin ase can regulate mRNA translation primarily of its own mRNA and to a l esser extent of a heterologous mRNA and that this regulation is notabl y affected by mutations in either the catalytic or N-terminal regulato ry domains.