No abstract

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.

Communicated by A.M.MichelsonRecently, the existence of 40-kd and 46-kd 2-5A synthetases in interferon-treated cells has been confirmed by cloning and characterization of cDNA corresponding to these small size enzymes. By the use of specific monoclonal antibodies, we describe here two forms of high mol. wt 2-5A synthetases of 69 and 100 kd in human cells. The monoclonal antibodies immunoprecipitate either a 69-or a 100-kd 2-5A synthetase. These purified 2-5A synthetases in immune complex preparations are active, i.e. addition of poly(l).poly(C) and ATP results in the synthesis of 2-5A. Both 2-5A synthetases are composed of several subspecies with similar isoelectric points in the range of 7-8 but have different subcellular localizations: 100-kd synthetase is recovered from the microsomal pellet whereas 69-kd synthetase is found to be associated with cell membranes as well as with the microsomal pellet. Different types of interferon-treated human cells express both or either forms of these enzymes. The 69and 100-kd 2-5A synthetases were also identified by electrophoretic transfer inmmunoblot analysis using rabbit polyclonal antibodies against a synthetic peptide common on both 46and 40-kd 2-5A synthetases. These results indicate that small and large size isozymes share a common peptide sequence.

We present evidence that the HIV-1 Tat protein and the RNA-dependent cellular protein kinase, PKR, interact with each other both in vitro and in vivo. Using GST fusion chromatography, we demonstrate that PKR, interacts directly with the HIV-1 Tat protein. The region in Tat sufficient for binding PKR maps within amino acids 20 to 72. In in vitro assays, the two-exon form of Tat (Tat 86) was phosphorylated by PKR, while the one exon form of Tat (Tat 72) inhibited PKR autophosphorylation and substrate phosphorylation. The ability of Tat to interact with PKR was demonstrated in both yeast and mammalian cells. Expression of PKR in yeast results in a growth suppressor phenotype which was reversed by coexpression of a one exon form of Tat. Expression of Tat 72 in HeLa cells resulted in direct interaction with PKR as detected by coimmunprecipitation with a Tat antibody. Tat and PKR also form a coimmunoprecipitable complex in cell-free extracts prepared from productively infected T lymphocytes. The interaction of Tat with PKR provides a potential mechanism by which HIV could suppress the interferon system.