Date of Award

1-1-2011

Document Type

Campus Access Dissertation

Department

Biological Sciences

First Advisor

Rekha C Patel

Abstract

PKR (protein kinase, RNA activated) is an interferon (IFN)-induced serine-threonine protein kinase, which plays a crucial role in IFN's antiviral and antiproliferative actions. In addition, PKR plays a regulatory role in signal transduction leading to apoptosis in response to a variety of stresses. PACT (PKR activator) activates PKR by direct protein-protein interaction in response to stress signals, which leads to phosphorylation of the translation initiation factor eIF2α and cellular apoptosis. In addition to positive regulation by PACT, PKR activity in cells is also negatively regulated by human immunodeficiency virus trans-activating region (TAR) RNA-binding protein (TRBP). The purpose of this study is to understand the mechanism of PACT-mediated PKR activation and to define its functional importance in stress-induced apoptosis. In particular, we have investigated the role of stress-induced phosphorylation of serine 287 in PACT. Using yeast two-hybrid analysis and co-immunoprecpitation assays we demonstrate that stress-induced phosphorylation of PACT at serine 287 significantly increases its interaction with PKR by weakening its interaction with TRBP. These results demonstrate that stress-induced PACT phosphorylation functions to free PACT from the inhibitory interaction with TRBP and also to enhance its interaction with PKR. Any functional significance of PACT-PACT interaction in the efficient activation of PKR was also investigated. Our results establish that there is a stronger interaction between two PACT molecules when PACT is phosphorylated on serines 246 and 287, and this is essential for efficient PKR activation. Using a point mutant of PACT that is deficient in PACT-PACT interaction, we demonstrate that PACT-PACT interaction promoted by stress-induced phosphorylation is essential for efficient PKR activation.

We further investigated the involvement of PACT in PKR activation in response to endoplasmic reticulum (ER) stressor tunicamycin. Our results establish that PACT is phosphorylated in response to tunicamycin, causing its increased association with PKR leading to PKR activation. Thus, our results uncover a novel functional role of PACT-mediated PKR activation in tunicamycin-induced ER stress response pathway and regulation of apoptosis.

Rights

© 2011, Madhurima Singh

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