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RNA-dependent protein kinase is aMr 68,000protein in human cells (p68 klnase) or a Mr 65,000 protein in murine cells (p65 kinase). p65/p68 is a serine/threonine kinase induced by interferon treatment and generally activated by double-stranded RNAs. Once activated, the known f i of this kinase is inhibition of protein synthesis through phosphorylation of the eukaryotic iation factor 2. Our previous work has suggested that the lack of endogenous expression (with or without interferon treatment) and interferon induction of the p65/p68 kinase in different types of cells is somehow associated with tumor growth and antitumoral action (25-27). For example, malignant embryonal carcinoma stem cells do not express this kinase even after treatment with interferon (25). This is not due to the absence of interferon receptors on embryonal carcinoma cells since another interferon-induced enzyme, (2'-5')oligoadenylate synthetase, is induced (25,28,29). In nude mice bearing human HeLa cell xenografts, treatment with speciesspecific human interferon ( results in the induction ofthe p68 kinase in the tumor and a significant inhibition of its growth (26,27).To characterize the role of the p68 kinase in tumor growth or suppression, we investigated the tumorigenicity of murine NIH 3T3 cell clones expressing either the wild-type (active) or the mutant (inactive) form of the p68 kinase. Murine cells were used in this study since the activity of the transfected human p68 kinase could be assayed independently of the endogenous p65 kinase after immunoprecipitation with monoclonal antibodies (8,30). We show that NIH 3T3 clones expressing inactive forms of the p68 kinase, produced by a point mutation of lysine-2% to arginine or to proline (18), initiate the development of rapidly growing tumors in nude Abbreviations: dsRNA, double-stranded RNA; eIF2, eukaryotic protein-synthesis initiation factor 2; EMC, encephalomyocarditis.iTo whom reprint requests should be addressed at: Virologie et Immunologie Cellulaire, Department of AIDS and Retroviruses, Institut Pasteur, 28, rue du Dr. Roux, 75015 Paris, France. §This serine/threonine protein kinase can be activated generally by dsRNAs and also by single-stranded RNAs and by heparin. In the literature, it has been referred to as dsRNA-activated protein kinase, P1/eIF2 kinase, DAI or dsI for dsRNA-activated inhibitor, and p68 (human) or p65 (murine) kinase. 232The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The human p68 kinase is an interferon‐regulated enzyme that inhibits protein synthesis when activated by double‐stranded RNA. We show here that when expressed in Saccharomyces cerevisiae, the p68 kinase produced a growth suppressing phenotype resulting from an inhibition of polypeptide chain initiation consistent with functional protein kinase activity. This slow growth phenotype was reverted in yeast by two different mechanisms: expression of the p68 kinase N‐terminus, shown to bind double‐stranded RNA in vitro and expression of a mutant form of the alpha‐subunit of yeast initiation factor 2, altered at a single phosphorylatable site. These results provide the first direct in vivo evidence that the p68 kinase interacts with the alpha‐subunit of eukaryotic initiation factor 2. Sequence similarity with a yeast translational regulator, GCN2, further suggests that this enzyme may be a functional homolog in higher eukaryotes, where its normal function is to regulate protein synthesis through initiation factor 2 phosphorylation.

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