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

Paula MonjeMPaula@miamiproject.med.miami.edu

Phone: 305-243-7138 (lab)The Miami Project to Cure Paralysis

Room 5-23