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

1. 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

2. UV Light Introduction 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

3. Background Cont. UVC damage = response, at least two phosphorylation cascades appear to be involved. Membrane associated tyrosine kinases RAF MEK ERK 1/2

4. C-Jun N-terminal kinases (JNK )pathway Phosphorylation of JNK leads to activation of c-Jun and transcription of gene for AP-1

5. Background Cont. • Ultimately, genotoxic stress leads to activation of either JNK or MAP Kinases or both. • Activity regulated via reversible phosphorylation of ___________and ___________residues. threonine tyrosine So, what de-phosphorylates threonine and tyrosine residues?

6. P P MAP kinase 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.

7. 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?

8. 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 Detected slower migrating phosphorylated forms of ERK1 and ERK2 using a PAGE Separated proteins Used monoclonal antibodies against ERK1 and ERK2 Transferred to nylon membrane

9. Phosphorylated ERK 1 and ERK 2 Dephosphorylated Results Question 1- Map kinase and JNK activated by UVC and MMS treatments? UVC-irridated or MMS treated HeLa cells Western blots, Fig. 1a No phosphorylated forms

10. HeLa cell PAGE to resolve proteins Lyse cells add phosphate buffer + A-sepharose Assayed for phosphorylation of ERK 2 on myelin basic protein Immunoprecipitate with anit-p42ERK2 Question 1 • ERK2 activity assessed by immunoprecipitation, using anit-p42ERK2 antiserum. Immunoprecipiation used to asses protein characteristics antibody A-sepharose

11. Question 1 Cont. Phosphorylation of myelin basic protein Fig. 1b ERK2 kinase activity >30 fold increase ERK2 kinase activity only 4 fold increase

12. HeLa cell PAGE to resolve proteins Lyse cells add phosphate buffer + A-sepharose Assayed for kinase activity using GST-c-Jun Immunoprecipitate with anti-p46JNK1 Question 1 Cont. • JNK1 activity in response to UVC and MMS using immunocomplex kinase assay • JNK1 has been show to phosphorylate c-Jun and activate AP-1 when exposed to UVC.

13. 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

14. 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 18s Transferred to nylon membrane MKP-1 detected Membrane washed and exposed to film Hybridized with rMKP-1 cDNA probe

15. MKP1 mRNA induced 10 fold Question 2 – Is MKP-1 induced by UVC and MMS treatments? Northern blots, Fig. 2 • 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.

16. 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. psG5 vector Empty psG5 vector JNK1 rMKP-1 HeLa cells or • 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.

17. 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.

18. pSG5 Coll-CAT rMKP-1 sense 1 ug 1 ug Jun-LUC rMKP-1 antisense Assayed for CAT or LUC using luciferase assay system kit. Cells treated with TPA, UVC, or MMS 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 Transfected with either

19. 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.

20. 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

21. 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.