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Phosphorylation within the bipartite NLS alters the localization and toxicity of the ER stress response factor

Jonathan C Bartko1, Yinghui Li2, George Sun2

  • 1Departments of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, United States.

Cellular Signalling
|July 17, 2020
PubMed

Insights

Phosphorylation of CCAAT/enhancer-binding protein homologous protein (CHOP/DDIT3) at Ser107 regulates its nuclear import and cellular response to endoplasmic reticulum stress, impacting cell survival and toxicity.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Stress Response

Background:

  • Nuclear-cytoplasmic trafficking regulates cellular responses to oxidant and endoplasmic reticulum (ER) stress.
  • CCAAT/enhancer-binding protein homologous protein (CHOP/DDIT3), a bZIP transcription factor, is crucial in mediating cellular adaptation to redox and ER stress.
  • CHOP influences cell growth, differentiation, and survival pathways.

Purpose of the Study:

  • To investigate the role of post-translational modifications, specifically phosphorylation, in regulating the subcellular localization and function of CHOP/DDIT3.
  • To identify the specific residue and its impact on CHOP's nuclear-cytoplasmic ratio and cellular toxicity under stress conditions.

Main Methods:

  • Functional analyses to characterize the nuclear localization signal (NLS) of CHOP.
  • Phylogenetic analyses and mass spectrometry to identify phosphorylation sites.
  • Site-directed mutagenesis (S107A and S107E) to study the functional consequences of phosphorylation.
  • Cellular injury assays using thapsigargin to assess the role of CHOP phosphorylation in ER stress-induced damage.

Main Results:

  • CHOP/DDIT3 possesses a conserved bipartite NLS within its DNA-binding domain.
  • Serine 107 (S107) within the bipartite NLS is phosphorylated under normal conditions.
  • Phosphorylation at S107, mimicked by the S107E mutation, alters the nuclear-to-cytoplasmic ratio of CHOP.
  • The S107A mutation abrogated ER stress-induced cellular injury observed with wild-type CHOP upon thapsigargin treatment.

Conclusions:

  • Phosphorylation of CHOP/DDIT3 at S107 within its bipartite NLS is a key regulatory mechanism controlling its subcellular localization.
  • This phosphorylation event modulates the toxic potential of CHOP/DDIT3 during acute ER stress.
  • Targeting the kinase/phosphatase networks regulating this phosphorylation site may offer therapeutic strategies to mitigate cellular damage in ER stress-related conditions.
Keywords:
ApoptosisDDIT3/CHOPGrowth arrestNuclear localization signalNuclear-cytoplasmic transportPost-translational modification

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