Hematopoietic cell kinase associates with the 40S ribosomal subunit and mediates the ribotoxic stress response to deoxynivalenol in mononuclear phagocytes

Heekyong Bae, Jennifer S. Gray, Maoxiang Li, Laura Vines, Joon Kim, James J. Pestka

Research output: Contribution to journalArticle

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Abstract

The trichothecene deoxynivalenol (DON) binds to eukaryotic ribosomes and triggers p38-driven proinflammatory gene expression in the macrophage-a response that is dependent on both double-stranded RNA-activated protein kinase (PKR) and hematopoietic cell kinase (Hck). Here we elucidated critical linkages that exist among the ribosome and these kinases during the course of DON-induced ribotoxic stress in mononuclear phagocytes. Similar to PKR inhibitors, Hck inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine (PP2) suppressed p38 activation and p38-driven interleukin 8 (IL-8) expression in the U937 human monocyte cell line. U937 cells stably transfected with a PKR antisense vector (U9K-A1) displayed marked reduction of DON-induced p38 activation and IL-8 expression as compared to cells transfected with empty vector (U9K-C2), with both responses being completely ablated by PP2. Western analysis of sucrose density gradient fractions revealed that PKR and Hck interacted with the 40S ribosomal subunit in U9K-C2 but not U9K-A1 cells. Subsequent transfection and immunoprecipitation studies with HeLa cells indicated that Hck interacted with ribosomal protein S3. Consistent with U937 cells, DON induced p38 association with the ribosome and phosphorylation in peritoneal macrophages from wild-type but not PKR-deficient mice. DON-induced phosphorylation of ribosome-associated Hck in RAW 264.7 murine macrophages was also suppressed by 2-aminopurine (2-AP). Both 2-AP and PP2 inhibited DON-induced phosphorylation of p38 as well as two kinases, apoptosis signal-regulating kinase 1 and mitogen-activated protein kinase 3/6, known to be upstream of p38. Taken together, PKR and Hck were critical for DON-induced ribosomal recruitment of p38, its subsequent phosphorylation, and, ultimately, p38-driven proinflammatory cytokine expression.

Original languageEnglish
Pages (from-to)444-452
Number of pages9
JournalToxicological Sciences
Volume115
Issue number2
DOIs
Publication statusPublished - 2010 Feb 24

Fingerprint

Proto-Oncogene Proteins c-hck
Eukaryotic Small Ribosome Subunits
Phagocytes
Phosphorylation
Ribosomes
Macrophages
2-Aminopurine
eIF-2 Kinase
U937 Cells
Interleukin-8
Mitogen-Activated Protein Kinase 6
Phosphotransferases
MAP Kinase Kinase Kinase 5
Chemical activation
MAP Kinase Kinase 3
Trichothecenes
Mitogen-Activated Protein Kinase 3
Double-Stranded RNA
Peritoneal Macrophages
deoxynivalenol

Keywords

  • Deoxynivalenol (DON)
  • Protein kinase
  • Ribosome
  • Translation

ASJC Scopus subject areas

  • Toxicology

Cite this

Hematopoietic cell kinase associates with the 40S ribosomal subunit and mediates the ribotoxic stress response to deoxynivalenol in mononuclear phagocytes. / Bae, Heekyong; Gray, Jennifer S.; Li, Maoxiang; Vines, Laura; Kim, Joon; Pestka, James J.

In: Toxicological Sciences, Vol. 115, No. 2, 24.02.2010, p. 444-452.

Research output: Contribution to journalArticle

Bae, Heekyong ; Gray, Jennifer S. ; Li, Maoxiang ; Vines, Laura ; Kim, Joon ; Pestka, James J. / Hematopoietic cell kinase associates with the 40S ribosomal subunit and mediates the ribotoxic stress response to deoxynivalenol in mononuclear phagocytes. In: Toxicological Sciences. 2010 ; Vol. 115, No. 2. pp. 444-452.
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