RAX, a Cellular Activator for Double-stranded RNA-dependent Protein Kinase during Stress Signaling

Takahiko Ito,Mingli Yang,W Stratford May

doi:10.1074/jbc.274.22.15427

Abstract

The double-stranded (ds) RNA-dependent protein kinase (PKR) regulates protein synthesis by phosphorylating the alpha subunit of eukaryotic initiation factor-2. PKR is activated by viral induced dsRNA and thought to be involved in the host antiviral defense mechanism. PKR is also activated by various nonviral stresses such as growth factor deprivation, although the mechanism is unknown. By screening a mouse cDNA expression library, we have identified an ubiquitously expressed PKR-associated protein, RAX. RAX has a high sequence homology to human PACT, which activates PKR in the absence of dsRNA. Although RAX also can directly activate PKR in vitro, overexpression of RAX does not induce PKR activation or inhibit growth of interleukin-3 (IL-3)-dependent cells in the presence of IL-3. However, IL-3 deprivation as well as diverse cell stress treatments including arsenite, thapsigargin, and H2O2, which are known to inhibit protein synthesis, induce the rapid phosphorylation of RAX followed by RAX-PKR association and activation of PKR. Therefore, cellular RAX may be a stress-activated, physiologic activator of PKR that couples transmembrane stress signals and protein synthesis.

Highlights

Eukaryotic cells rapidly and reversibly halt protein synthesis in response to a variety of stresses including virus infection and cytotoxic chemical injury [1, 2]
The paradigm for PKR activation following viral infection indicates that dsRNA activates this important regulator of protein synthesis, but the mechanism of activation in the absence of viral infection is not clear
The in vitro studies with purified RAX indicate that RAX, like PACT, can directly activate PKR, confirming that PKR can be activated by cellular proteins in the absence of dsRNA

Summary

Introduction

Eukaryotic cells rapidly and reversibly halt protein synthesis in response to a variety of stresses including virus infection and cytotoxic chemical injury [1, 2]. This fundamental homeostatic mechanism is thought to involve the phosphorylation of the ␣ subunit of eukaryotic initiation factor-2 (eIF2␣), which regulates protein synthesis rate at translational initiation. In interferon-treated cells, virus infection leads to activation of PKR by autophosphorylation, followed by eIF2␣ phosphorylation and inhibition of protein synthesis [5, 6]. We previously reported that in interleukin-3 (IL-3)-dependent cells, IL-3 deprivation induced activation of PKR in close association with a decreased rate of total protein synthesis [7]. Results here indicate that the RAX-PKR association and any resulting cellular activation of PKR may be regulated by an unique, stress-induced signaling mechanism featuring RAX phosphorylation

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Results

Conclusion