Direct activation of ribosome-associated double-stranded RNA-dependent protein kinase (PKR) by deoxynivalenol, anisomycin and ricin: a new model for ribotoxic stress response induction.

Hui-Ren Zhou,James Pestka,Jeff Landgraf,Xiao Pan,Kaiyu He

doi:10.3390/toxins6123406

Abstract

Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is a critical upstream mediator of the ribotoxic stress response (RSR) to the trichothecene deoxynivalenol (DON) and other translational inhibitors. Here, we employed HeLa cell lysates to: (1) characterize PKR’s interactions with the ribosome and ribosomal RNA (rRNA); (2) demonstrate cell-free activation of ribosomal-associated PKR and (3) integrate these findings in a unified model for RSR. Robust PKR-dependent RSR was initially confirmed in intact cells. PKR basally associated with 40S, 60S, 80S and polysome fractions at molar ratios of 7, 2, 23 and 3, respectively. Treatment of ATP-containing HeLa lysates with DON or the ribotoxins anisomycin and ricin concentration-dependently elicited phosphorylation of PKR and its substrate eIF2α. These phosphorylations could be blocked by PKR inhibitors. rRNA immunoprecipitation (RNA-IP) of HeLa lysates with PKR-specific antibody and sequencing revealed that in the presence of DON or not, the kinase associated with numerous discrete sites on both the 18S and 28S rRNA molecules, a number of which contained double-stranded hairpins. These findings are consistent with a sentinel model whereby multiple PKR molecules basally associate with the ribosome positioning them to respond to ribotoxin-induced alterations in rRNA structure by dimerizing, autoactivating and, ultimately, evoking RSR.

Highlights

Many toxins produced by microbes and plants inhibit translation by binding to the ribosome and interfering with initiation, elongation and/or termination [1]
Western blotting revealed that DON (500 ng/mL) induced robust p38 phosphorylation in 5 min in HeLa cells, which was maximal at 15 min and lasted up to at least 2 h (Figure 1A)
Concentration-dependently suppressed the p38 and Jun N-terminal kinase (JNK) (Figure 1C). These findings recapitulated prior findings in mononuclear phagocytes that DON induces ribotoxic stress response (RSR) in a PKR-dependent manner, indicating that HeLa cells are appropriate for the investigation of PKR-ribosome interactions

Summary

Introduction

Many toxins produced by microbes and plants inhibit translation by binding to the ribosome and interfering with initiation, elongation and/or termination [1]. Besides translation inhibition, these biotoxins are capable of activating mitogen-activated protein kinases (MAPKs) via a process known as the ribotoxic stress response (RSR) [2,3,4,5]. The prototypical ribotoxin deoxynivalenol (DON), a trichothecene mycotoxin, triggers activation of p38 and c-Jun N-terminal kinase (JNK) MAPKs with robust downstream sequelae including cytokine and chemokine gene expression as well as programmed cell death in leukocytes [6,7]. Cell culture studies on the effects of DON and other ribotoxins strongly indicate that double-stranded RNA-activated protein kinase (PKR) is a critical upstream sensor and transducer of RSR [7,8,9,10]

Results

Discussion

Conclusion