Transcription factor regulation by mapks
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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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Transcription factor regulation by mapks

Transcription factor regulation by MAPKs

Paula MonjePostdoctoral Research AssociateThe Miami Project to Cure Paralysis

July 8th, 2004


Signaling from the cell surface to the nucleus

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


Transcription factor regulation by mapks

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


Transcription factor regulation by mapks

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


Classical mechanism of erk signaling to the nucleus

Classical mechanism of ERK signaling to the nucleus


Transcription factor regulation by mapks

Targets of phosphorylation by MAPK signaling pathways


Transcription factor regulation by mapks

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


Mapks regulate the expression of transcription factors the case of c jun and c fos

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


Transcription factor regulation by mapks

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


How do we examine signal specificity to transcription factors

How do we examine signal specificity to transcription factors?


Tools to activate and inhibit mapk signaling

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


Transcription factor regulation by mapks

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


Transcription factor regulation by mapks

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


Other approaches to activate and inhibit erk signaling

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


Transcription factor regulation by mapks

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


How do we measure transcription factor phosphorylation and activation

How do we measure transcription factor phosphorylation and activation?


R eporter s ystem s to study how signaling pathways regulate transcription

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


Luciferase r eporter s ystem s to study how signaling pathways regulate transcription

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 )


Commercially available reporter systems stratagene

Commercially available reporter systems (Stratagene)


Reporter systems using gal4 fusion proteins

Reporter systems using Gal4- fusion proteins


Transcription factor regulation by mapks

How does the Gal4 system work?


Transcription factor regulation by mapks

Gal4 reporters for specific signaling pathways

TF/TAD/unknown sequence (TF?)

JNK

ERK-JNK-p38

cAMP-PKA

p38

ERK-RSK

JNK-p38


Reporter vectors for the gal4 system

Reporter vectors for the Gal4 system


Design your own gal4 fusion protein

Design your own Gal4 fusion protein

Clone here the gene of interest


Path detect double stable reporting cell lines s trat a gene

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


Transcription factor regulation by mapks

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.


C fos is transactivated by mitogenic signals

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.


Transcription factor regulation by mapks

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.


Erk transactivates c fos and ap 1 dependent transcription

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.


Other techniques used in the study of signaling pathways and transcriptional modulation

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


Transcription factor regulation by mapks

Paula [email protected]: 305-243-7138 (lab)The Miami Project to Cure Paralysis Room 5-23


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