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. 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

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. tyrosinethreonine So, what de-phosphorylates threonine and tyrosine residues?

6. 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 PP

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 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

9. 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 No phosphorylated forms Dephosphorylated

10. Question 1 ERK2 activity assessed by immunoprecipitation, using anit-p42 ERK2 antiserum. Immunoprecipiation used to asses protein characteristics HeLa cell Lyse cells add phosphate buffer + A- sepharose Immunoprecipitate with anit-p42 ERK2 Assayed for phosphorylation of ERK 2 on myelin basic protein PAGE to resolve proteins antibody A-sepharose

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

12. 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-p46 JNK1 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

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

15. 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.

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. 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

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. 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

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? 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

21. 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.