Regulation of protein synthesis and induction of oncogenesis by a cellular protein kinase inhibitor

Abstract

Thesis (Ph. D.)--University of Washington, 1998P58IPK is a cellular inhibitor of the interferon-induced, dsRNA-activated, serine/threonine kinase, PKR. Earlier, P58IPK was shown to block both PKR autophosphorylation and the phosphorylation of eIF-2alpha substrate in vitro, in a dose-dependent manner. Furthermore, murine cell lines overexpressing P58IPK were tumorigenic in nude mice, suggesting that P58IPK was an oncoprotein. The mechanism by which P58IPK inhibits PKR is unknown. In order to elucidate this mechanism as well as define the physiological role of the P58IPK protein, a structure/function study of the P58 IPK molecule was undertaken. Using site directed mutagenesis, eleven different P58IPK mutants were generated. Using both in vitro and in vivo assays, each of the mutants was characterized for the ability to block PKR phosphorylation activity in vitro, stimulate cellular protein synthesis in vivo, and induce cellular transformation and tumorigenesis in nude mice. The findings presented in this dissertation demonstrate that the sixth TPR motif (TPR6) was required for P58IPKactivity in vitro. Although the DnaJ homology region at the carboxyl terminus of P58IPK was dispensable for function in vitro, the in vivo experiments indicated that this region, along with TPR6, was required for the stimulation of cellular protein synthesis. Thus, the carboxyl terminus of P58IPK may serve a regulatory function to modulate the activity of P58IPK in the cell. The functional activity of P58IPK correlated with its ability to interact and form a complex with PKR. In addition, P58IPK prevented PKR dimerization. This ability required both TPR6 and the DnaJ homology region. Taken together, these data suggest that, through the binding of P58 IPK to PKR, P58IPK may inhibit PKR activity by interfering with the formation of active PKR dimers.To define the physiological role of the P58IPK protein, stable NIH 3T3 cell lines expressing either wild type, bovine P58IPK or DeltaTPR6, a P58IPK variant lacking TPR6, were generated and characterized. Both of the cell lines displayed in vitro hallmarks of cellular transformation and induced tumors in nude mice. By two different in vivo assays, PKR was found to be active in the DeltaTPR6-expressing cells. These results suggested that P58 IPK-induced oncogenesis may occur via a PKR-independent mechanism. Because cells overexpressing P58IPK showed constitutive activation of NF-kappaB and resisted TNF-alpha-induced apoptosis, one plausible mechanism of neoplastic transformation by P58IPK is through the activation of NF-kappaB and the subsequent suppression of apoptotic cell death.This study has identified two cellular functions for P58IPK: (i) the regulation of protein synthesis through an inhibition of PKR-mediated eIF-2alpha phosphorylation, and (ii) the regulation of transcription through the constitutive activation of NIF-kappaB. Both of these activities may regulate cellular growth and contribute to P58IPK-induced neoplastic transformation