Structure/Function of PKR and eIF-2a using vaccinia virus K3L Gene Product

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Jagus 9305314 The objective of this proposal is to elucidate the mechanism of action the dsRNA-dependent elF-2cx specific protein kinase, PKR, at the molecular structural level, utilizing a vaccinia virus gene product, pK3, that functions as a pseudosubstrate. PKR, a potent inhibitor of protein synthesis, is an enzyme that serves a regulatory role in host defense against viral infection, cell proliferation, cell differentiation, and the inflammatory response. The specific aims are 1) to identify the substrate binding domain(s) of PKR using pK3, the vaccinia virus homologue of elF-2cx that binds stably to PKR; 2) to confirm the results of the invitro studies in vivo, using wild-type and variant forms of PKR transiently expressed in COS-1 cells, and 3) to extrapolate the conclusions of elF-2cx itself. The methods to be employed are recombinant DNA techniques, polymerase chain reaction, site-specific mutagenesis, deoxynucleotide sequencing, in vitro transcription and translation, protein kinase assays, gel electrophoresis, immunoblotting, immunoprecipitation, and expression of recombinant proteins in bacteria and mammalian cells. PKR plays a key role in the inhibition of viral growth by interferon, in the maintenance of low proliferation rates, the initiation of differentiation, and the production of cytokines and cellular adhesion molecules. Despite the importance of this enzymes and wealth of information recently uncovered on its regulatory domains, little is currently known about how it interacts with its only known cellular substrate, elF02cx. This proposal represents a strategy to identify substrate binding domains in PKR using a vaccinia virus homologue to elF-cx that binds stably to PKR. The ability of pK3 to form tight associations with PKR, as well as its homology to elF-2cx, forms the basis of a simple binding assay to investigate functional domains of PKR and pK3, and by extrapolation of elF-2cx. %%% The synthesis of proteins is accomplished by a c omplex machinery involving hundreds of proteins and is regulated by the protein synthesis factor, elF-2. elF-2 exists in two forms, an active and an inactive form. The change from an active to an inactive form of elF-2 is accomplished by a modifying protein called PKR. To increase our understanding of the mechanism of action of PKR at a molecular level, we will identify the site within the molecule that interacts with elF-2. The gene for PKR will be mutated and the variant PKR's will be expressed in bacteria and purified. The activities of the variant PKR's will be compared for their ability to bind to a competitor for elF-2, pK3, a protein produced in vaccinia virus infected cells. The results from these studies using isolated components will be confirmed by expressing the mutant PKR's in a model cell system and monitoring their ability to modify elF-2. PKR plays a key role in many cellular processes such as the inhibition of viral growth by interferon, the initiation of cell differentiation, and the production of molecules that promote cell- cell interaction. In addition, PKR seems to function as a tumor suppressor gene. ***