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Published on: May 18, 2017

Tyrosine phosphorylation controls nuclear export of Fyn, allowing Nrf2 activation of cytoprotective gene expression

James W Kaspar1, Anil K Jaiswal

  • 1Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201, USA.

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
|November 25, 2010
PubMed

Insights

Fyn kinase negatively regulates the antioxidant response by exporting Nrf2 from the nucleus. Inhibiting Fyn

Area of Science:

  • Cellular signaling pathways
  • Molecular biology
  • Biochemistry

Background:

  • Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor regulating cellular defense against oxidative stress.
  • Fyn, an Src family kinase, acts as a negative regulator of Nrf2 activity.
  • Understanding the precise mechanisms of Fyn-mediated Nrf2 regulation is crucial for developing therapeutic strategies against oxidative stress-related diseases.

Purpose of the Study:

  • To elucidate the role of Fyn in the regulation of Nrf2 nuclear export and subsequent antioxidant gene expression.
  • To identify the specific site and mechanism of Fyn phosphorylation controlling its nuclear export.
  • To investigate the functional consequences of Fyn nuclear export inhibition on Nrf2 target gene activation and cellular survival.

Main Methods:

  • Cell culture (HepG2, Hepa-1 cells)
  • Site-directed mutagenesis of Fyn (Y213F, NES mutant)
  • Western blotting and immunofluorescence for protein localization
  • Chromatin immunoprecipitation (ChIP) assays
  • Quantitative real-time PCR (qRT-PCR)
  • Mass spectrometry
  • Cell viability assays (etoposide treatment)

Main Results:

  • Antioxidant treatment induced rapid nuclear export of Fyn in HepG2 cells.
  • Mutation of Fyn at Tyrosine 213 (Y213F) or a putative nuclear export signal (NES) inhibited Fyn nuclear export.
  • Mass spectrometry confirmed Tyrosine 213 as the site of Fyn phosphorylation.
  • FynY213F mutant significantly reduced Nrf2 binding to the NQO1 promoter and decreased NQO1 expression.
  • FynY213F mutation increased susceptibility to etoposide-induced cell death in Hepa-1 cells.

Conclusions:

  • Fyn nuclear export, regulated by Tyrosine 213 phosphorylation, is a critical step in the pre-induction regulation of Nrf2.
  • Inhibition of Fyn nuclear export prevents Nrf2 activation of antioxidant genes, leading to increased cellular vulnerability.
  • Targeting Fyn nuclear export represents a potential therapeutic strategy to enhance cellular antioxidant defenses.

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