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Role of Mitogen-activated Protein Kinase
Phosphatase During the Cellular
Response to Gentoxic Stress
:Inhibition of c-Jun N-Terminal Kinase Activity and
AP-1 Dependent Gene Activation
Liu et al. (1995) The Journal of Biological Chemistry
Introduction
UV Light
Gentoxic agents = series of
phosphorylations
lead to modification of
transcription factors and altered
gene expression
The main question - Does
MKP-1 play a role in regulating
transcriptional activation in
response to genotoxic agents?
AP1
Background Cont.
UVC damage = response, at least two
phosphorylation cascades appear to be involved.
Membrane associated tyrosine
kinases
RAF
MEK
ERK
1/2
C-Jun N-terminal
kinases (JNK )pathway
Phosphorylation
of JNK leads to
activation of c-Jun
and transcription
of gene for AP-1
Background Cont.
• Ultimately, genotoxic stress leads to
activation of either JNK or MAP Kinases or
both.
• Activity regulated via reversible phosphorylation
threonine
tyrosine
of ___________and
___________residues.
So, what de-phosphorylates
threonine and tyrosine residues?
Background Cont.
• Protein phosphatases with a high specificity for MAP kinases
- mouse
MAP kinase phosphatase 1 (MKP-1)
- human
homologue CL100
- lymphocyte-specific PAC-1 protein
• MKP-1 and PAC-1 = dephosphorylation of phosphothreonine
and phosphotyrosine residues of MAP kinases
inactivation.
P
P
MAP kinase
• Recent studies = MKP-1 inhibits RAS induced DNA synthesis
and inhibits MAP kinase regulated reporter gene expression.
4 main questions addressed
• Question 1 – Are Map kinase and JNK activated by UVC
and MMS treatments?
• Question 2 – Is MKP-1 induced by UVC and MMS
treatments?
• Question 3 – Can JNK be deactivated by rMKP-1 in
intact cells?
• Question 4 – Does MKP-1 expression inhibit AP-1
dependent gene induction?
Question 1- Map kinase and JNK activated by UVC and MMS
treatments?
-Used western blots to determine phosphorylated forms of
ERK1 and ERK2 MAP kinase activation
Western blots commonly used to detect activated proteins. Typically use
anti-phosho… antibodies for detection of phosphorylated protein, on a
nylon membrane that are marked and a picture is taken.
Treated HeLa cells with
UVC or MMS
Separated proteins
Transferred to
nylon membrane
Used
monoclonal
antibodies
against ERK1
and ERK2
Detected slower
migrating
phosphorylated
forms of ERK1 and
ERK2 using a
PAGE
Results
Question 1- Map kinase and JNK activated by UVC and MMS treatments?
UVC-irridated or MMS treated HeLa cells
Western blots, Fig. 1a
Phosphorylated
ERK 1 and ERK 2 Dephosphorylated
No phosphorylated
forms
Question 1
• ERK2 activity assessed by immunoprecipitation, using
anit-p42ERK2 antiserum.
Immunoprecipiation used to asses protein characteristics
antibody
A-sepharose
HeLa cell
Lyse cells add
phosphate
buffer + Asepharose
Immunoprecipitate
with anit-p42ERK2
PAGE to
resolve
proteins
Assayed for
phosphorylation of
ERK 2 on myelin
basic protein
Question 1 Cont.
Phosphorylation of myelin basic protein Fig. 1b
ERK2 kinase activity
>30 fold increase
ERK2 kinase activity
only 4 fold increase
Question 1 Cont.
• JNK1 activity in response to UVC and MMS using
immunocomplex kinase assay
HeLa cell
• JNK1 has been show to
phosphorylate c-Jun and activate
AP-1 when exposed to UVC.
PAGE to
resolve
proteins
Lyse cells add
phosphate
buffer + Asepharose
Immunoprecipitate with
anti-p46JNK1
Assayed for kinase
activity using GST-cJun
Question 1 Cont.
Phosphorylation of GST-c-Jun
substrate, Fig. 2
JNK1 activated 30 min post
treatment
JNK1 activated, slower, less
magnitude
Conclude – MAP kinase and JNK activated by UVC and MMS
Question 2 – Is MKP-1 induced by UVC and MMS
treatments?
Northern blots = used to see if gene of interest is
expressed/present.
18s
mRNA of interest
seperated by gel
electrophoresis
Transferred to nylon
membrane
Hybridized with
rMKP-1 cDNA probe
MKP-1
detected
Membrane
washed and
exposed to film
Question 2 – Is MKP-1 induced by UVC and MMS treatments?
Northern blots, Fig. 2
MKP1 mRNA induced 10 fold
• Maximum MKP-1 mRNA expression coincided with a
decline in MAP kinase and JNK activity.
Conclude - MKP-1 plays a role in inactivating MAP kinase and
JNK.
Question 3 – Can JNK be deactivated by rMKP-1 in intact
cells?
• Transient cotransfection assay to deliver plasmids
expressing HA-tagged JNK1 along with either the plasmid
expressing rMKP-1 (pSG5-rMKP1) or an empty psG5 vector at
psG5
Empty
EcoRI site.
JNK1
vector
rMKP-1
or
psG5
vector
HeLa
cells
• HA-JNK protein was immunoprecipitated from cell extracts
using anit-HA antiserum and immunocomplex assayed for its
ability to phosphorylated the GST-c-Jun substrate.
Question 3 Cont.
JNK activity elevated in
transfected cells following
UVC and MMS treatments
Larger amounts of rMKP-1
infected = less activation of
HA-JNK1
Conclude – Yes, JNK can be deactivated by rMKP-1 in
intact cells.
Question 4 – Does MKP-1 expression inhibit AP-1
dependent gene induction?
• Two reporter constructs (coll-CAT and jun-LUC) were used
to examine the effect rMKP-1 expression on AP-1 mediated
gene induction.
• Both constructs rely on AP-1 site for expression after UVC
treatments.
pSG5
Coll-CAT
rMKP-1 sense
Transfected with either
1 ug
HeLa
cell(s)
Transfected with either
1 ug
Jun-LUC
rMKP-1
antisense
Cells treated with
TPA, UVC, or MMS
Assayed for CAT or LUC using
luciferase assay system kit.
Question 4 Cont.
CAT or LUC activity, Fig. 5 a and b
•CAT and LUC expression enhanced by all treatments,
except treatments containing rMKP-1sense plasmid.
Conclude – rMKP1 does inhibit induction of AP-1 gene
expression, importantly rMKP1 does not act non-specifically.
4 main questions addressed
• Question 1 – Are Map kinase and JNK activated by UVC
and MMS treatments? YES
• Question 2 – Is MKP-1 induced by UVC and MMS
treatments?
YES
• Question 3 – Can JNK be deactivated by rMKP-1 in
intact cells? YES
• Question 4 – Does MKP-1 expression inhibit AP-1
dependent gene induction? YES
Discussion/conclusions
• rMKP-1 has greater influence on MAP kinase-mediated
gene activation than that mediated via JNK in response to
UVC radiation.
• JNK1 inhibited more so than MAP-kinase in response to
MMS treatments.
• Good evidence to support a role for MKP-1 regulating MAP
kinase dependent gene activation.