Transcription factor regulation by MAPKs
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Transcript of Transcription factor regulation by MAPKs
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Transcription factor regulation by MAPKs
Paula MonjePostdoctoral Research Associate
The Miami Project to Cure Paralysis
July 8th, 2004
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Signaling from the cell surface to the nucleus
Intracellular Signals
Raf
MEK
ERK
Elk-Fos
Ras
ProliferationDifferentiationDevelopment
ApoptosisCANCERP
P
GPCR RTK
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MAPKKK
MAPKK
S S/T
MAPK
TXY
SERINE/THREONINE KINASE
SERINE/THREONINE KINASE
DUAL THREONINE/TYROSINE KINASE
MAPK ACTIVATION CASCADE
S/T P
Target
T/S-P
Single gene family- Share 40-50% identity-
Diverse multigene family
Single gene family- Share 40-50% identity-
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MAPKKK
MAPKK
MAPK
MAPK ACTIVATION MODULES
MEK1/2
A-Raf, B-Raf, c-Raf, c-MOS
ERK1/2
GPCR
MEK7/4
PAK/MLKs/MEKKs/Tpl-2/Ask1/
JNK1/2/3
MEKK/Ask1/?
MKK3/6
p38
MEKK/?
ERK5
MEK5
RTK StimulusStress/cytokines
Mitogens
MKRSK1/2/3/4MSK1/2 MNK1
MNK2MK2/3
Transcription factors - Elk-Fos-MEF-Jun-ATF TF
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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)
carbachol PDGF TPA0 20 40 60 20120 40 60 120 20 40 60 120time (min)
c-jun
jun-B
c-fos
fos-B
fra-1
PKC GPCRRTKStress/
cytokines
Mitogens
Transcription factors (IEG)
ExpressionmRNA
ActivationPost-translational
modifications-Phoshorylation
MAPKs
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MAPKs regulate the expression of transcription factors:
the case of c-jun and c-fos
SRE
TCF
ERK
SRF
c-fos Promoter
c-fos
P P
AP-1
cJun
JNK
ATF2
c-jun Promoter
c-jun
P P P
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Jun-Jun Homodimers
Jun-Fos Heterodimers
JNK
LZPP
PP
P
DBD LZPP
ERK
MAPKs regulate the activity of c-Jun and c-Fos
DBD LZ
DBD LZ
DBD LZ
Fos
JunbZIP
TAD
TAD
TAD
TAD
P P P
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How do we examine signal specificity to transcription
factors?
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Tools to activate and inhibit MAPK signaling
MAPKKK
MAPKK
MAPK
Signal
Transcription Factors
GTPase
INHIBITORSACTIVATORS
DN-MAPKKK
DN-MAPKK
DN-MAPK
GyrB-MAPKKKER-MAPKKK
MAPKKK*
ER-MAPKKMAPKK*
Fusion MAPKK-MAPK*Protein-Peptide Inhibitors
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K S S/T
I VII
ATP binding site Activation motif
MAPK activating protein kinase, MAPKK, MKK or MEK
R A A
I VII
MEK KR (kinase dead) or MEK AA (dominant negative) mutant
MEK EE or MEK DD constitutively active mutant
K
I VII
E/D E/D
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MAPKK
MAPK
Signal
TF
MAPKK
S S/T
MEK1 EE
ERK1/2
MKK4/7
JNK
MKK3/6 EE
p38
ERK5
MEK1 AA
MEK5 DD MEK5 AA
MKK3/6 AA/KR
Activated Mutant Dominant Negative
DD/EE and AA mutants of MKK4-MKK7 do not work as activators/inhibitors of
JNK signaling
Activated and DN MAPKs are not functional
MAPKKs (MEKs) are good candidates for
intervention
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Other approaches to activate and inhibit ERK signaling
Raf
MEK1/2
ERK1/2
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)
1. Pharmacological inhibitors: PD98059-U0126 (reversible)
2. Dominante negatives: MEKAA3. Inhibitory peptides (ERK
binding motif)
1. Pharmacological inhibitors: Bay439006-ZM 336372 (c-Raf, B-Raf)
1. Fusion proteins MEK1-ERK22. Overexpression MEK1+ERK2
Activators Inhibitors
1. Constitutively active: MEK EE
TF
1. Cell permeable inhibitor (MEK N-terminus). Blocks ERK activity
by preventing its interaction with MEK
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MEKK
MEK4/7
JNK1. Fusion proteins MEK7-JNK1
1. Truncated MEKK1(regulatory domain deleted)
1. Inhibitory ProteinJIP-1 (JNK Inhibitory Protein).
2. Pharmacological inbihitors: SP600125 (binds to ATP binding site in JNK).
Blocks JNK1-2-3 activity3. Inhibitory peptide: cJun (delta
domain).TF
Activators Inhibitors
Approaches to activate and inhibit JNK signaling
MLKs1. Pharmacological Inhibitors:
CEP1347
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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
Response Element TATA Reporter Gene
LuciferaseGFPB-GalactosidaseCATSEAP
AP-1CRESRESRFP53NFkB
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Luciferase reporter systems to study how signaling pathways regulate transcription
pAP-1 Luc
7 x ( TGA C TAA )
Extracellular Stimuli
7 x AP-1
Kinase
P P PP
Fos Jun Luc
Luc LucLuc
LucLuc
Luc
LucLuc
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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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JNKERK-JNK-p38cAMP-PKAp38ERK-RSKJNK-p38
Gal4 reporters for specific signaling pathways
TF/TAD/unknown sequence (TF?)
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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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ProliferationTransformation
SRE AP-1
P P P PP
P
JunTCFSRF
Raf
MEK
ERK
c-Fos
Ras
ERK signaling to c-Fos and AP-1
activation
PDGF c-sis
c-Fos
Monje et.al., MCB 2003.
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c-Fos is transactivated by mitogenic signals
Gal4 RELuciferaseGal4 DBD
FL / TADP
Serum/PDGF
P
pGal4-Luc
Gal4-c-Fos
PDGFSerum + +
0
40
80
120
160
Luci
fera
se a
ctiv
ity
pGal4-Luc
+ +
Gal4 Reporter Systemc-Fos
FLc-Fos TAD
Gal4DBD:
Use of Gal4 reporters to study how extracellular signals trigger transcriptional activation
Monje et.al., MCB 2003.
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c-Fos activation by PDGF requires ERK signaling
0
40
80
120
Luc
ifera
se a
ctiv
ity(a
rbitr
ary
units
)
c-FosTAD PDGF U0126 SP600125 SB203580
pGal4-Luc
anti-c-Fos
anti-P-ERK
anti- ERK
+ + + + +
+
+
+
+
+ + +
+
++
++
++
4244
4244
35
50
MW(kDa)
PDGF
ERKJNK p38P -TAD
Gal4 RELucGal4 DBD
c-Fos TADP P
pGal4-Luc
Gal4-c-Fos
SP SBU0126
Use of Gal4 reporters to study which signaling pathways activate transcription
Monje et.al., MCB 2003.
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ERK transactivates c-Fos and AP-1-dependent transcription
c-FosFL c-FosTAD MEKEE+ERK2
Luci
fera
se a
ctiv
ity(a
rbitr
ary
units
)
0
50
100
150
200pGal4-Luc pAP1-Luc
Luci
fera
se a
ctiv
ity(a
rbitr
ary
units
)
0
100
200
300
400
c-Fos c-Jun MEKEE+ERK2
++ ++++ +
+ ++ +++
+ ++
+ +
Use of luciferase reporters to study how activated kinases stimulate transcription
7 x ( TGA C TAA )
7 x AP-1
Luc
MEKEE+ERK2
P P PP
Fos Jun
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-Immunocytochemistry (phospho-specific antibodies)
* Gel shift assays
* CHIP assays