The objective of this proposal is to further elucidate the biological role of the dsRNA-dependent protein kinase, PKR, and substrates, in the cell. Evidence suggests that this kinase plays a crucial role in interferon's (IFN's) response to viral infection and tumorigenesis and functions as a signal transducer in the regulation of dsRNA-induced genes and apoptosis. We have attempted to further understand the functions of PKR, in vitro and in vivo, using inducible-cell lines, yeast-two hybrid screens and genetically engineered animal models that lack this kinase. We anticipate that our proposal will not only contribute towards elucidating the biological properties of PKR but will also shed light into the mechanisms of cell signaling, tumorigenesis and IFN-mediated actions in the cell. More specifically: 1.) Using proteomic and DNA array approaches, genes/proteins that are regulated through the PKRelF2alpha translation pathway have been identified and will be studied as described. The role of the elF2 pathway in tumorigenesis will also be further analyzed as part of this aim. 2.) Using phospho-labelled embryonic fibroblasts (EFs) containing or lacking PKR, we have used a similar proteomics approach (MS/Mass spectrometry) to identify novel candidate substrates of this kinase. We will identify the sites of phosphorylation on these candidate substrates and evaluate the biological importance of these modifications. 3.) We have recently identified the NFAR proteins as substrates for PKR. We will confirm the identity of the site(s) of phosphorylation and determine the significance of such post-translational effects in NFAR and PKR function.
This aim will also encompass further determining the function of the NFAR proteins. We propose that these aims will lend insight into mechanisms of cell signaling, tumorigenesis and interferon-mediated actions in the cell.
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