role of mitogen-activated protein kinase phosphatase during the cellular response to gentoxic stress...
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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
Gentoxic agents = series of phosphorylations
lead to modification of transcription factors and altered gene expression
UV Light
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 of ___________and ___________residues.tyrosinethreonine
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.
• Recent studies = MKP-1 inhibits RAS induced DNA synthesis and inhibits MAP kinase regulated reporter gene expression.
MAP kinase
P P
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
Separated proteins
Detected slower migrating phosphorylated forms of ERK1 and ERK2 using a PAGE
Transferred to nylon membrane
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.
Used monoclonal antibodies against ERK1 and ERK2
Treated HeLa cells with UVC or MMS
ResultsQuestion 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
No phosphorylated formsDephosphorylated
Question 1
• ERK2 activity assessed by immunoprecipitation, using anit-p42ERK2 antiserum.
Immunoprecipiation used to asses protein characteristics
HeLa cell
Lyse cells add phosphate buffer + A-sepharose
Immunoprecipitate
with anit-p42ERK2
Assayed for phosphorylation of ERK 2 on myelin basic protein
PAGE to resolve proteins
antibody A-sepharose
Question 1 Cont.
ERK2 kinase activity >30 fold increase
Phosphorylation of myelin basic protein Fig. 1b
ERK2 kinase activity only 4 fold increase
• JNK1 activity in response to UVC and MMS using immunocomplex kinase assay
Question 1 Cont.
HeLa cell
Lyse cells add phosphate buffer + A-sepharose
Immunoprecipitate with anti-p46JNK1
• JNK1 has been show to phosphorylate c-Jun and activate AP-1 when exposed to UVC.
Assayed for kinase activity using GST-c-Jun
PAGE to resolve proteins
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.
mRNA of interest seperated by gel electrophoresis
Transferred to nylon membrane
Hybridized with rMKP-1 cDNA probe
Membrane washed and exposed to film
18s
MKP-1 detected
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 EcoRI site.
• 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.
psG5 vector
rMKP-1
Empty psG5 vector
JNK1
or HeLa cells
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.
HeLa cell(s)
Transfected with either
rMKP-1 sense
rMKP-1 antisense
Transfected with either
pSG5
Cells treated with TPA, UVC, or MMS
Assayed for CAT or LUC using luciferase assay system kit.
Coll-CAT
1 ug
Jun-LUC
1 ug
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?
• 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?
YES
YES
YES
YES
Discussion/conclusions
• Good evidence to support a role for MKP-1 regulating MAP kinase dependent gene activation.
• 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.
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