MAP Kinase family regulates the
nuclear localization of Nrf2
By: Ngan Nguyen
Tory Hagen
Linus Pauling Institute
Outline
 Objective

Diagram for transcription via Nrf2
 Methods
 Results


Testing MAP Kinase family
Electrophoretic Mobility Shift Assay
 Future Direction
Nrf2
Nrf2 binds to the Antioxidant
Response Element (ARE) and
promotes transcription of
antioxidant genes
Stress
signaling
Kinase
Nrf2
P
Nrf2 released
Nrf2
ARE
(Antioxidant Response Element)
P
Antioxidant
enzymes
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
Kinase
Nrf2
P
Nrf2 released
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

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
Vehicle
Treatment
3
Treatment
1
Treatment
4
Treatment
2
Treatment
5
http://www.bio-link.org/vlab/Graphics/Tools/ProteinGel3.jpg
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
Is the MAP kinase family
involved in Nrf2 nuclear
localization?
Stress
signaling
MAPK
?
Nrf2
P
Nrf2 released
Nrf2
ARE
(Antioxidant Response Element)
P
Antioxidant
enzymes
Testing effect of MAP Kinase Inhibitor
on Nrf2
Nrf2
250
P<0.05
Relative Nuclear Nrf2
200
*
150
*
*
100
50
6
UO
12
4h
rs
e
ad
ad
M
en
* By the t test, values are statistically significant compared to vehicle (p<0.05)
io
n
M
en
1
hr
io
n
e
4
UO
hr
s
12
6
hr
AP
AP
1
AP
AP
Ve
hi
c
le
1h
Ve
rU
O
hi
c
12
6
le
0
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.
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
Nrf2
ARE
Shift in
position
ARE
EMSA shows that transcription factors
are binding the ARE
1
2
3
Vehicle
4
5
6
7
Menadione
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
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
Nrf2
Future Direction
 Does one specific kinase in
the MAP Kinase family
phosphorylate Nrf2?
Stress
signaling
MAPK
Nrf2
P
Nrf2 released
Nrf2
ARE
(Antioxidant Response Element)
P
Antioxidant
enzymes
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