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Transcription factor regulation by MAPKs

Transcription factor regulation by MAPKs. Paula Monje Postdoctoral Research Associate The Miami Project to Cure Paralysis. July 8th, 2004. g. a. b. Signaling from the cell surface to the nucleus. G PCR. RTK. Ras. Raf. Intracellular Signals. MEK. Proliferation Differen t iation

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Transcription factor regulation by MAPKs

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  1. Transcription factor regulation by MAPKs Paula MonjePostdoctoral Research AssociateThe Miami Project to Cure Paralysis July 8th, 2004

  2. g a b Signaling from the cell surface to the nucleus GPCR RTK Ras Raf Intracellular Signals MEK Proliferation Differentiation Development Apoptosis CANCER ERK P P Elk-Fos

  3. MAPK ACTIVATION CASCADE SERINE/THREONINE KINASE MAPKKK Diverse multigene family S S/T DUAL THREONINE/TYROSINE KINASE MAPKK Single gene family- Share 40-50% identity- TXY SERINE/THREONINE KINASE MAPK Single gene family- Share 40-50% identity- S/T P T/S-P Target

  4. MAPK ACTIVATION MODULES Mitogens Stress/ cytokines Stimulus GPCR RTK MEKK/ Ask1/? A-Raf, B-Raf, c-Raf, c-MOS PAK/MLKs/MEKKs/Tpl-2/Ask1/ MAPKKK MEKK/? MAPKK MEK1/2 MEK5 MEK7/4 MKK3/6 JNK1/2/3 ERK1/2 ERK5 p38a//g/d MAPK MNK2 MK RSK1/2/3/4 MK2/3 MNK1 MSK1/2 TF Transcription factors - Elk-Fos-MEF-Jun-ATF

  5. Classical mechanism of ERK signaling to the nucleus

  6. Targets of phosphorylation by MAPK signaling pathways

  7. Extracellular signals regulate the expression of transcription factors (IEGs) Mitogens Stress/ cytokines GPCR RTK PKC carbachol PDGF TPA 0 20 40 60 120 20 40 60 120 20 40 60 120 time (min) c-jun MAPKs jun-B Transcription factors (IEG) c-fos fos-B Activation Post-translational modifications-Phoshorylation Expression mRNA fra-1

  8. MAPKs regulate the expression of transcription factors:the case of c-jun and c-fos ERK JNK P P P P P TCF cJun ATF2 SRF c-fos c-jun SRE AP-1 c-fos Promoter c-jun Promoter

  9. P P MAPKs regulate the activity of c-Jun and c-Fos Jun-Jun Homodimers JNK P P DBD LZ TAD Jun LZ TAD DBD LZ P bZIP P TAD DBD LZ Fos DBD LZ TAD P P P P Jun-Fos Heterodimers ERK

  10. How do we examine signal specificity to transcription factors?

  11. Tools to activate and inhibit MAPK signaling Signal ACTIVATORS INHIBITORS GTPase GyrB-MAPKKK ER-MAPKKK MAPKKK* DN-MAPKKK MAPKKK DN-MAPKK ER-MAPKK MAPKK* MAPKK DN-MAPK MAPK Fusion MAPKK-MAPK* Protein-Peptide Inhibitors Transcription Factors

  12. I VII MAPK activating protein kinase, MAPKK, MKK or MEK I VII K S S/T ATP binding site Activation motif MEK EE or MEK DD constitutively active mutant K E/D E/D MEK KR (kinase dead) or MEK AA (dominant negative) mutant I VII R A A

  13. MAPKKs (MEKs) are good candidates for intervention Dominant Negative Activated Mutant MEK1 EE MEK1 AA Signal ERK1/2 MEK5 DD MEK5 AA MAPKK ERK5 S S/T MKK3/6 EE MKK3/6 AA/KR MAPKK p38 DD/EE and AA mutants of MKK4-MKK7 do not work as activators/inhibitors of JNK signaling MKK4/7 MAPK JNK TF Activated and DN MAPKs are not functional

  14. Other approaches to activate and inhibit ERK signaling Activators Inhibitors 1. Targeting to the plasma membrane: Raf-CAAX 2. Deletion of N-terminal regulatory domain: Truncated Raf1 3. Fusion to ER hormone binding domain (ER-Raf1) - inducible by Tamoxifen 4. Oligomerization: fusion to bacterial DNA gyrase (inducible by courmermycin) Raf 1. Pharmacological inhibitors: Bay439006-ZM 336372 (c-Raf, B-Raf) • Pharmacological inhibitors: PD98059-U0126 (reversible) • Dominante negatives: MEKAA • Inhibitory peptides (ERK binding motif) MEK1/2 1. Constitutively active: MEK EE ERK1/2 1. Fusion proteins MEK1-ERK2 2. Overexpression MEK1+ERK2 1. Cell permeable inhibitor (MEK N-terminus). Blocks ERK activity by preventing its interaction with MEK TF

  15. Approaches to activate and inhibit JNK signaling Activators Inhibitors MEKK MLKs 1. Pharmacological Inhibitors: CEP1347 1. Truncated MEKK1 (regulatory domain deleted) MEK4/7 JNK 1. Inhibitory Protein JIP-1 (JNK Inhibitory Protein). 2. Pharmacological inbihitors: SP600125 (binds to ATP binding site in JNK). Blocks JNK1-2-3 activity 3. Inhibitory peptide: cJun (delta domain). 1. Fusion proteins MEK7-JNK1 TF

  16. How do we measure transcription factor phosphorylation and activation?

  17. Reporter Gene Response Element TATA AP-1 CRE SRE SRF P53 NFkB Luciferase GFP B-Galactosidase CAT SEAP Reporter systems to study how signaling pathways regulate transcription

  18. Luc Luc Luc Luc Luc Luc Luc Luc Luc Luciferase reporter systems to study how signaling pathways regulate transcription Extracellular Stimuli Kinase P P P P Jun Fos pAP-1 Luc 7 x AP-1 7 x ( TGA C TAA )

  19. Commercially available reporter systems (Stratagene)

  20. Reporter systems using Gal4- fusion proteins

  21. How does the Gal4 system work?

  22. Gal4 reporters for specific signaling pathways TF/TAD/unknown sequence (TF?) JNK ERK-JNK-p38 cAMP-PKA p38 ERK-RSK JNK-p38

  23. Reporter vectors for the Gal4 system

  24. Design your own Gal4 fusion protein Clone here the gene of interest

  25. Path-Detect double-stable reporting cell lines (Stratagene)

  26. PDGF c-sis ERK signaling to c-Fos and AP-1 activation Ras Raf MEK ERK P Proliferation Transformation P P P P c-Fos P c-Fos Jun TCF SRF AP-1 SRE Monje et.al., MCB 2003.

  27. Use of Gal4 reporters to study how extracellular signals trigger transcriptional activation c-Fos is transactivated by mitogenic signals pGal4-Luc Serum/ PDGF 160 120 80 Luciferase activity P P 40 FL/TAD Gal4-c-Fos 0 Gal4 DBD Luciferase + + PDGF Serum pGal4-Luc Gal4 RE + + Gal4 Reporter System Gal4DBD: c-Fos TAD c-Fos FL Monje et.al., MCB 2003.

  28. Use of Gal4 reporters to study which signaling pathways activate transcription 120 pGal4-Luc c-Fos activation by PDGF requires ERK signaling 80 Luciferase activity (arbitrary units) PDGF 40 MW (kDa) 0 50 JNK ERK p38 anti-c-Fos P-TAD SB SP U0126 35 44 P P anti-P-ERK 42 c-Fos TAD 44 anti- ERK Gal4-c-Fos 42 Gal4 DBD Luc + + + + + + + + + c-FosTAD PDGF U0126 SP600125 SB203580 pGal4-Luc Gal4 RE + + + + + + + + + + Monje et.al., MCB 2003.

  29. Use of luciferase reporters to study how activated kinases stimulate transcription ERK transactivates c-Fos and AP-1-dependent transcription pGal4-Luc pAP1-Luc MEKEE +ERK2 200 400 150 300 Luciferase activity (arbitrary units) Luciferase activity (arbitrary units) 100 200 P P P 50 100 P Jun Fos 0 0 Luc + + + + + + c-FosFL c-FosTAD MEKEE+ERK2 c-Fos c-Jun MEKEE+ERK2 + + + + + + 7 x AP-1 + + + + + + 7 x ( TGA C TAA ) Monje et.al., MCB 2003.

  30. Other techniques used in the study of signaling pathways and transcriptional modulation * Kinase assays * Western blotting-Immunocytochemistry (phospho-specific antibodies) * Gel shift assays * CHIP assays

  31. Paula MonjeMPaula@miamiproject.med.miami.eduPhone: 305-243-7138 (lab)The Miami Project to Cure Paralysis Room 5-23

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