Transcription factor regulation by MAPKs

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Transcription factor regulation by MAPKs
Paula MonjePostdoctoral Research AssociateThe
Miami Project to Cure Paralysis
July 8th, 2004
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Signaling from the cell surface to the nucleus
GPCR
RTK
Ras
Raf
Intracellular Signals
MEK
Proliferation Differentiation Development Apoptosi
s CANCER

ERK
P
P
Elk-Fos
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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
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MAPK ACTIVATION MODULES
Mitogens
Stress/ cytokines
Stimulus
GPCR
RTK
MAPKKK
A-Raf, B-Raf, c-Raf, c-MOS
PAK/MLKs/MEKKs/Tpl-2/Ask1/
MEKK/ Ask1/?
MEKK/?
MAPKK
MEK1/2
MEK7/4
MKK3/6
MEK5
ERK1/2
JNK1/2/3
p38a/?/g/d
ERK5
MAPK
MNK2
MK
RSK1/2/3/4
MK2/3
MNK1
MSK1/2
TF
Transcription factors - Elk-Fos-MEF-Jun-ATF
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Classical mechanism of ERK signaling to the
nucleus
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Targets of phosphorylation by MAPK signaling
pathways
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Extracellular signals regulate the expression of
transcription factors (IEGs)
Mitogens
Stress/ cytokines
GPCR
RTK
PKC
carbachol
PDGF
TPA
0
20
40
60
20
120
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-Phosho
rylation
Expression mRNA
fra-1
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MAPKs regulate the expression of transcription
factorsthe 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
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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
DBD LZ
TAD
Fos
DBD LZ
TAD
P
P
P
P
Jun-Fos Heterodimers
ERK
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How do we examine signal specificity to
transcription factors?
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Tools to activate and inhibit MAPK signaling
Signal
INHIBITORS
ACTIVATORS
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
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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
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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
MAPK
MKK4/7
JNK
TF
Activated and DN MAPKs are not functional
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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)

1. Pharmacological inhibitors PD98059-U0126
   (reversible)
2. Dominante negatives MEKAA
3. Inhibitory peptides (ERK binding motif)

MEK1/2
1. Constitutively active MEK EE
ERK1/2
1. Fusion proteins MEK1-ERK2 2. Overexpression
MEK1ERK2
1. Cell permeable inhibitor (MEK N-terminus).
Blocks ERK activity by preventing its interaction
with MEK
TF
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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
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How do we measure transcription factor
phosphorylation and activation?
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Reporter systems to study how signaling pathways
regulate transcription
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Luciferase reporter systems to study how
signaling pathways regulate transcription
Extracellular Stimuli
Kinase
P
P
P
P
Fos
Jun
pAP-1 Luc
7 x AP-1
7 x ( TGA C TAA )
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Commercially available reporter systems
(Stratagene)
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Reporter systems using Gal4- fusion proteins
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How does the Gal4 system work?
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Gal4 reporters for specific signaling pathways
TF/TAD/unknown sequence (TF?)
JNK ERK-JNK-p38 cAMP-PKA p38 ERK-RSK JNK-p38
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Reporter vectors for the Gal4 system
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Design your own Gal4 fusion protein
Clone here the gene of interest
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Path-Detect double-stable reporting cell lines
(Stratagene)
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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
SRE
AP-1
Monje et.al., MCB 2003.
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c-Fos is transactivated by mitogenic signals
Use of Gal4 reporters to study how extracellular
signals trigger transcriptional activation
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.
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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
ERK
JNK
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
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Gal4 DBD
Luc









c-FosTAD PDGF
U0126
SP600125 SB203580
Gal4 RE
pGal4-Luc










Monje et.al., MCB 2003.
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ERK transactivates c-Fos and AP-1-dependent
transcription
Use of luciferase reporters to study how
activated kinases stimulate 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
Fos
Jun
0
0
Luc






c-FosFL c-FosTAD
MEKEEERK2
c-Fos c-Jun
MEKEEERK2






7 x AP-1






7 x ( TGA C TAA )
Monje et.al., MCB 2003.
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Other techniques used in the study of signaling
pathways and transcriptional modulation
Kinase assays Western blotting-Immunocytochemi
stry (phospho-specific antibodies) Gel shift
assays CHIP assays
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Paula MonjeMPaula_at_miamiproject.med.miami.eduPhon
e 305-243-7138 (lab)The Miami Project to Cure
Paralysis Room 5-23