The double-stranded RNA (dsRNA)-binding domain of the human p68 kinase has been loaliz to the N-terminal half of the enzyme by using progressive deletion analysis and in vitro binding assays. To further define the domains responsible for binding to dsRNA, we cloned the mouse dsRNA-activated p65 kinase and used sequence alignment to identify conserved domains in the N-terminal region.Deletions in either of two 12-amino-acid-long and arginine-or lysine-rich regions abrogated binding to dsRNA. Moreover, in an in vivo growth inhibition assay in the yeast Saccharomyces cerevisiae, these mutants failed to exhibit a slow-growth phenotYpe.The double-stranded RNA (dsRNA)-activated protein kinase is an interferon (IFN)-induced enzyme, existing as a 68-kDa protein in human cells (p68) and a 65-kDa protein in mouse cells (p65). It was originally discovered based on the inhibitory effect of dsRNA on protein synthesis in a cell-free system (see ref. 1 for a review). Once activated by dsRNA, the protein becomes autophosphorylated and in turn phosphorylates its specific substrate, the a subunit of eukaryotic initiation factor 2, eIF2a (1, 2), leading to a decline in the rate of protein synthesis in cells (3)(4)(5)(6). dsRNA, in addition to being an activator of the p68 kinase, is also an effective inducer of IFN genes and IFN-inducible genes; this induction could be blocked by 2-aminopurine, an inhibitor of the p68 kinase (7-11). Furthermore, dsRNA has been shown to activate the nuclear transcriptional factor NFKB via the phosphorylation and deattachment of its inhibitor, IKB (12), and NFKB is involved in regulating IFN-P gene expression.Consequently, it has been suggested that the dsRNAactivated kinase may also phosphorylate other substrates that are involved in signaling or transcriptional activation of IFN and IFN-inducible genes.We have reported (13) the isolation and characterization of the cDNA for human p68 kinase. This cDNA contains all of the conserved domains represented in protein kinases (14), and the sequence confirmed previous observations (15, 16) that p68 is a member of the serine/threonine kinases. The in vitro translated product of the cDNA is able to bind to dsRNA, be recognized by a monoclonal antibody to native p68 kinase, autophosphorylate, and phosphorylate eIF2a in vitro (13,17,18). When expressed in the yeast Saccharomyces cerevisiae, the human p68 kinase exhibits a growthsuppressing phenotype, which is correlated with phosphorylation of yeast eIF2a (18). Therefore, these studies provide a basis for further analysis of the relationship between structure and function of the p68 kinase.To reveal the mechanism for the regulation of p68 kinase activity by dsRNA, we have performed a deletion analysis of the p68 kinase to reveal domains important for binding to dsRNA. We have cloned and sequenced § the murine p65 kinase and used sequence alignment between p68 and p65 to identify conserved regions in the N-terminal domain. Sitedirected mutagenesis of these segments indicates that positively charged dom...