MAP Kinase family regulates the nuclear localization of Nrf2

1
MAP Kinase family regulates the nuclear
localization of Nrf2

• By Ngan Nguyen
• Tory Hagen
• Linus Pauling Institute

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Outline

• Objective
• Diagram for transcription via Nrf2
• Methods
• Results
• Testing MAP Kinase family
• Electrophoretic Mobility Shift Assay
• Future Direction

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Nrf2 binds to the Antioxidant Response Element
(ARE) and promotes transcription of antioxidant
genes
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Objective

• Determine which kinase phosphorylates Nrf2
  allowing it to localize into the nucleus
• Test kinases from the MAP Kinase family, as well
  as PKA and AMPK.

Stress signaling
P
Keap1
Nrf2 released
5
Compounds for testing Nrf2 nuclear localization

• Menadione
• Yellow crystalline powder used in medicine as a
  vitamin K supplement
• Acetaminophen, APAP
• Commonly known as Tylenol
• Both compounds are known inducers of Nrf2
• Treated HepG2 with both compounds

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Nuclear Nrf2 and Gel Electrophoresis

• Subculture cells into 6-well plates
• Treat each well with a different treatment
• Harvest the cells and isolate the cytoplasm and
  nuclei
• Use SDS PAGE to separate denatured nuclear
  proteins by mass

http//www.bio-link.org/vlab/Graphics/Tools/Protei
nGel3.jpg
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Detection of nuclear Nrf2 by Western Blotting

• Transfer Proteins out of the gel onto a PVDF
  membrane,
• The membrane is then incubated with antibodies
  that will illuminate based on levels of protein
  in the cell
• The light is then detected by photographic film
  giving amount of the specific protein of interest
  in the cells

http//www.fermentas.com/techinfo/electrophoresis/
img/blotting.gif
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Is the MAP kinase family involved in Nrf2 nuclear
localization?
Stress signaling
?
P
Keap1
Nrf2 released
P
nucleus
Antioxidant enzymes
ARE
(Antioxidant Response Element)
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Testing effect of MAP Kinase Inhibitor on Nrf2
Plt0.05



By the t test, values are statistically
significant compared to vehicle (plt0.05)
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Interim Summary

• Inhibiting the MAP Kinase family appears to
  prevent or limit the amount of Nrf2 nuclear
  translocation.
• We also tried PKA and AMP kinase inhibitors, and
  they did not prevent Nrf2 nuclear translocation.

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Electrophoretic Mobility Shift Assay

• Complexes are run on a gel and transferred to a
  membrane.
• A shift in DNA position shows that there is
  protein bound to the ARE.
• Adding an antibody specific to Nrf2 would show
  more of a shift

Shift in position
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EMSA shows that transcription factors are binding
the ARE

• Biotin-ARE
• Biotin-ARE and protein
• Biotin-ARE, protein and Nrf2 antibody
• Biotin-ARE and Nrf2 antibody
• Biotin-ARE
• Biotin-ARE and protein
• Biotin-ARE, protein and Nrf2 antibody
• Biotin-ARE and Nrf2 antibody
• Biotin-ARE, protein and IgG antibody

1 2 3 4 5 6 7 8
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Vehicle
Menadione
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Conclusion

• Inhibition of the MAP Kinase family prevents Nrf2
  from entering the nucleus.
• EMSA confirms that transcription factors are
  binding to the ARE.
• The MAP Kinase family appears to be involved in
  inducing Nrf2 nuclear localization, which would
  activate the transcription of antioxidant genes

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Future Direction
Nrf2
Keap1

• Does one specific kinase in the MAP Kinase family
  phosphorylate Nrf2?

Stress signaling
P
Keap1
Nrf2 released
P
nucleus
Antioxidant enzymes
ARE
(Antioxidant Response Element)
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Acknowledgements

• Howard Hughes Medical Institute
• Dr. Tory Hagen
• Dr. Kate Shay
• Swapna Shenvi
• Alex Michels
• Jeff Monette
• Du Heath
• Judy Butler
• Dr. Regis Moreau
• Dr. Kevin Ahern