Liu et al. (1995) The Journal of Biological Chemistry

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.