1. MAP Kinase family regulates the nuclear localization of Nrf2 By: Ngan Nguyen Tory Hagen Linus Pauling Institute

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

3. ARE Stress signaling Nrf2 P P Nrf2 released Kinase (Antioxidant Response Element) Nrf2 Antioxidant enzymes Nrf2 binds to the Antioxidant Response Element (ARE) and promotes transcription of antioxidant genes

4. 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. Nrf2 P Nrf2 released Kinase Stress signaling

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

6. 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/ProteinGel3.jpg VehicleTreatment 1 Treatment 5 Treatment 2 Treatment 3 Treatment 4

7. 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

8. Is the MAP kinase family involved in Nrf2 nuclear localization? ARE Stress signaling Nrf2 P P Nrf2 released MAPK (Antioxidant Response Element) Antioxidant enzymes ?

9. Testing effect of MAP Kinase Inhibitor on Nrf2 Nrf2 P<0.05 * * * * By the t test, values are statistically significant compared to vehicle (p<0.05)

10. 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.

11. 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 ARE ARE Nrf2 Shift in position

12. EMSA shows that transcription factors are binding the ARE 1 2 3 4 5 6 7 8 9 1.Biotin-ARE 2.Biotin-ARE and protein 3.Biotin-ARE, protein and Nrf2 antibody 4.Biotin-ARE and Nrf2 antibody 5.Biotin-ARE 6.Biotin-ARE and protein 7.Biotin-ARE, protein and Nrf2 antibody 8.Biotin-ARE and Nrf2 antibody 9.Biotin-ARE, protein and IgG antibody VehicleMenadione

13. 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

14. Future Direction Does one specific kinase in the MAP Kinase family phosphorylate Nrf2? ARE Stress signaling Nrf2 P P Nrf2 released MAPK (Antioxidant Response Element) Nrf2 Antioxidant enzymes

15. 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