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Toxins 2014, 6(12), 3406-3425; doi:10.3390/toxins6123406

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

1,†
,
2,4,†
,
3
,
4,5
and
1,2,4,*
1
Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824, USA
2
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA
3
Research Technology Support Facility, Michigan State University, East Lansing, MI 48824, USA
4
Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824, USA
5
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Received: 5 November 2014 / Revised: 29 November 2014 / Accepted: 8 December 2014 / Published: 16 December 2014
(This article belongs to the Section Mycotoxins)
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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. View Full-Text
Keywords: PKR; ribotoxic stress; rRNA; deoxynivalenol; anisomycin; ricin; translational inhibitor; quantitiative Western analysis; RNA immunoprecipitation PKR; ribotoxic stress; rRNA; deoxynivalenol; anisomycin; ricin; translational inhibitor; quantitiative Western analysis; RNA immunoprecipitation
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Zhou, H.-R.; He, K.; Landgraf, J.; Pan, X.; Pestka, J.J. 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. Toxins 2014, 6, 3406-3425.

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