title: 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.
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.
authors.affiliation: Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824, USA. zhouh@msu.edu.
authors.forename: Hui-Ren
authors.initials: HR
authors.lastname: Zhou
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA. daxiaopenny@gmail.com.
authors.forename: Kaiyu
authors.initials: K
authors.lastname: He
authors.affiliation: Research Technology Support Facility, Michigan State University, East Lansing, MI 48824, USA. landgra1@msu.edu.
authors.forename: Jeff
authors.initials: J
authors.lastname: Landgraf
authors.affiliation: Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824, USA. daxiaopenny@gmail.com.
authors.forename: Xiao
authors.initials: X
authors.lastname: Pan
authors.affiliation: Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824, USA. Pestka@msu.edu.
authors.forename: James J
authors.initials: JJ
authors.lastname: Pestka
mesh_terms: D000841:Anisomycin / Q000633:toxicity*; D003001:Cloning, Molecular; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D011500:Protein Synthesis Inhibitors; D012337:RNA, Ribosomal, 18S / Q000737:chemistry; D012339:RNA, Ribosomal, 28S / Q000737:chemistry; D012270:Ribosomes / Q000378:metabolism; D012276:Ricin / Q000633:toxicity*; D017423:Sequence Analysis, RNA; D014255:Trichothecenes / Q000633:toxicity*; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D011500:Protein Synthesis Inhibitors; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S; D014255:Trichothecenes; D000841:Anisomycin; D012276:Ricin; D019892:eIF-2 Kinase; D048051:p38 Mitogen-Activated Protein Kinases; C007262:deoxynivalenol