transcription factor regulation by mapks
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
Transcription factor regulation by MAPKs
Paula MonjePostdoctoral Research Associate
The Miami Project to Cure Paralysis
July 8th, 2004
Signaling from the cell surface to the nucleus
Intracellular Signals
Raf
MEK
ERK
Elk-Fos
Ras
ProliferationDifferentiationDevelopment
ApoptosisCANCERP
P
GPCR RTK
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-
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
Classical mechanism of ERK signaling to the nucleus
Targets of phosphorylation by MAPK signaling pathways
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
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
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
How do we examine signal specificity to transcription
factors?
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
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
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
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
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
How do we measure transcription factor phosphorylation and
activation?
Reporter systems to study how signaling pathways regulate transcription
Response Element TATA Reporter Gene
LuciferaseGFPB-GalactosidaseCATSEAP
AP-1CRESRESRFP53NFkB
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
Commercially available reporter systems (Stratagene)
Reporter systems using Gal4- fusion proteins
How does the Gal4 system work?
JNKERK-JNK-p38cAMP-PKAp38ERK-RSKJNK-p38
Gal4 reporters for specific signaling pathways
TF/TAD/unknown sequence (TF?)
Reporter vectors for the Gal4 system
Design your own Gal4 fusion protein
Clone here the gene of
interest
Path-Detect double-stable reporting cell lines (Stratagene)
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.
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.
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.
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.
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