Die Transkriptionsfaktoren NF-!

B und NFAT
Klaus Schulze-Osthoff
Institut fr Molekulare Medizin
Literatur:
Karin M, Greten FR. NF-kappaB: linking inflammation and immunity to cancer development and progression.
Nat Rev Immunol. 2005 Oct;5(10):749-59.
Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes Dev. 2004 Sep 15;18(18):2195-224.
Karin M, Cao Y, Greten FR, Li ZW. NF-kappaB in cancer: from innocent bystander to major culprit.
Nat Rev Cancer. 2002 Apr;2(4):301-10.
Li Q, Verma IM. NF-kappaB regulation in the immune system. Nat Rev Immunol. 2002 Oct;2(10):725-34.
Macian F. NFAT proteins: key regulators of T-cell development and function.
Nat Rev Immunol. 2005 Jun;5(6):472-84.
Crabtree GR, Olson EN. NFAT signaling: choreographing the social lives of cells. Cell. 2002 Apr;109 Suppl:S67-79.
Kiani A, Rao A, Aramburu J. Manipulating immune responses with immunosuppressive agents that target NFAT.
Immunity. 2000 Apr;12(4):359-72.
Ligand
Receptor
Signal
Processing
Amplification
Gene Expression
(Cytokines, Receptors)
Proliferation
Mitosis
Differentiation
Cell Death
Apoptosis
C
o
s
t
i
m
u
l
u
s
C
o
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t
i
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u
s
Model of Receptor-mediated Signal Transduction Model of Receptor-mediated Signal Transduction
Ligand
Receptor
Signal
Processing
Activation of
transcription factors
Cytoplasm
Nucleus
Transcription
Gene Expression
Cytokines, Receptors
Model of Receptor-mediated Gene Expression Model of Receptor-mediated Gene Expression
Transactivation
domain
NLS
(nuclear localization sequence)
DNA-binding
Domain
Transcription factors
Startpoint TATA
Oct-1 AP-1 NF-!B Oct-1 Basal Apparatus
A transcriptional Complex
IL-2 Promoter
The NF-!B family of transcription factors
NF-!B (Nuclear Factor kappa B)
First identied in B-cells, immunoglobulin !-light chain enhancer
(Sen and Baltimore, 1986)
Activated by >150 different stimuli
Regulates > 200 Genes
DNA Consensus Binding motif 5-GGGRNNYYCC-3
(R=Purine, Y=Pyrimidine)
The NF-!B signaling module
Innate
Immunity
Inflammation Apoptosis
Regulation
Cell Cycle Control
Senescence
NF-!B Activation
by multiple forms
of cellular stress
Hyperactivation in:
Inflammation: rheumatoid
Arthritis, Crohns disease.
Tumors: e.g.
Hodgkin lymphoma
N TAD
551
Rel A/p65
RHD
N TAD
619
c-Rel
N TAD LZ
Rel B
579
N GRR
ANK
900
N GRR 969
p105/p50
p100/p52
NF-!B/Rel Proteins
-

T
A
D
+

T
A
D
Ankyrin
repeats
Rel Homology
Domain
RHD: 300aa conserved domain with several functions:
DNA-binding (N-terminal half)
dimerization (C-terminal half)
IkB-interaction (C-terminal half)
NLS (C-terminal half)
NF-!B Target Genes in Endothelial Cells
Adhesion Molecules
E-selectin
P-selectin
ICAM-1
VCAM-1
MadCAM-1
Cytokines
IL-1
IL-6
IL-8
GM-CSF
MCP-1
TNF-"
Others
Cyclooxygenase-2
12-Lipoxygenase
NO synthase (iNOS)
Tissue Factor
resting
activated Endothelium
NF-!B target genes
Angeborene, unspezifische Immunabwehr
(innate immunity)
TLR2
TLR4
NF-!B
LPS
Gen
M
TNF"
IL-8
IL-12
Chemotaxis
NK-Zellakt.
Aktivierung v.
Monozyten,
Makrophagen
Endothelien
IFN#
Lipoproteine
NF-!B as a regulator of apoptotic gene expression
Anti-apoptotic target genes:
Bcl-2 Proteins: Bcl-2, Bcl-x
L
, A1
Inhibitior of apoptosis proteins: XIAP, cIAPs
c-FLIP
death substrates death substrates
CASP-3 CASP-3
Bcl-2
Bcl-x
L
Bcl-2
Bcl-x
L
CASP-9 CASP-9 CASP-8 CASP-8
CASP-8
CD95
DISC
apoptosis apoptosis
Cyt Cyt c c Apaf Apaf-1 -1
c-FLIP
Bid
IAPs
Pro-apoptotic target genes:
Bcl-2 Proteins: Bak, Bax
Death receptors: Fas, TNF-R1, TRAIL-R2
Death receptor ligands: FasL, TRAIL
Adaptor proteins: TRAF-1, -2, -6
p53, Gadd45, c-Myc
NF-!B and Tumorigenesis
c-Rel amplications in tumors.
Bcl-3 and p65 translocation in leukemias.
I!B" mutations in Hodgkins lymphoma.
Activation by viral oncogenes
(HTLV-1 Tax, EBV-LMP1).
Target genes: cyclin D, c-Myc.
N-terminal
Regulatory domain
Ankyrin
repeats
The Inhibitor of kappaB proteins
Inhibitory function
impedes DNA-binding
blocks NLS and abolish translocation to nucleus
Several members (at least 7 mammalian)
I!B-" and I!B-$
I!B-# and I!B-%
Bcl-3
p105 and p110
Common features:
ankyrin-repeats which are necessary for RHD-interaction
30-33 aa motif repeated 3 - 7x
C-terminal acidic-region necessary for inhibition of DNA-binding
C-terminal PEST-sequence involved in protein-degradation
Specicity
Ex. IkB-" inhibits DNA-binding of
p65/p50 but not of p50/p50
The I!B family
I!B proteins have different properties
I!B-": Rapid transient response
I!B-" best characterized
all stimuli & degradation of I!B-"
ex: TNF-" & rapid and transient activation of NF-!B
I!B-$: Sustained response
Only certain stimuli & degradation of I!B-$
ex: LPS or IL-1 & degradation of both I!B-" and I!B-$ & activation of
NF-kB lasting for hours
Bcl-3: repressor and activator
inhibits certain complexes like a normal I!B
But may also associate with DNA-bound p50 and p52 dimers (lacking
TAD) and provide transactivation properties
Pathways of receptor-mediated NF-!B activation
There are many other
levels of control!
Signaltransduktion der
Signaltransduktion der
Ser/Thr-Phosphatase Calcineurin
Ser/Thr-Phosphatase Calcineurin
und
und
des Transkriptionsfaktors NFAT
des Transkriptionsfaktors NFAT
Borel JF, Feurer C, Gubler HU, Stahelin H.
Biological effects of cyclosporin A: a new
antilymphocytic agent.
Agents Actions. 1976 Jul;6(4):468-75.
Liu, Farmer, Lane, Friedman, Weissman, Schreiber
Calcineurin is a common target of cyclophilin-cyclosporin
A and FKBP-FK506 complexes. (1991) Cell
Tolypocladium inflatum, a white mold from Norway
The Billion Dollar The Billion Dollar Molecule Molecule: :
The Quest For The Perfect Drug The Quest For The Perfect Drug
by Barry Werth (1995)
HO
H
OCH
3
H
3
C
H
3
C
HO
O
H
3
C
H
3
C
OCH
3
OCH
3
O O
O
CH
3
O
N
O
HO
FK506
(Tacrolimus; Prograf

)
Rapamycin
(Sirolimus)
HO
H
OCH
3
H
3
C
OH
O
CH
3
O
H
3
C
O O
O
O CH
3
N
O
HO
O
CH
3
O
H
3
C
CH
3
H
3
C
CH
3
O
N
H
N
CH
3
CH
3
CH
3
CH
3
OH
CH
3
CH
3
CH
3
O
O
N
H
CH
3
O
N
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3 CH
3 CH
3
CH
3
CH
3
CH
3
CH
3
O O
O
O
O
O
O
N
N
N
N
N
N
N
H H
H
H
H
H
H
Cyclosporin A
(CsA; SandImmun

)
T cell receptor-mediated activation (Ca
2+
dependent)
- Production of IL2, IL3, IL4
- Dephosphorylation and nuclear translocation of NFAT
- Activation-induced cell death (via TCR or CD2)
- Expression of CD95-ligand (Apo1L, FasL)
- Degranulation of cytotoxic T cells
B cell Receptor-mediated activation
Degranulation of Mast cells
Intermediate target: Cyclophilins, FK506-binding proteins
Target: Calcineurin
MW: 822 Da MW: 914 Da MW: 1203 Da
IL2,3,4,6- receptor-mediated activation (Ca
2+
independent)
Cell cycle (progression from G1 to S phase)
p70S6 kinase
Intermediate target: FK506-binding proteins
Target: TOR, (FRAP, RAFT1); PI3-kinase like protein
Immunosuppressive Immunosuppressive Drugs Drugs
Tolypocladium inflatum Streptomyces hygroscopicus Streptomyces tsukubaensis
Immunophilin Size (kDa) Drug affinity (nM) Subcellular location
The The family family of of immunophilins immunophilins
CyPA 17.7 2.6 (Ki) Cytosol, nucleus
CyPB 21.0 6.9 (Ki) Secretory pathway
CyPC 22.8 Secretory pathway
CyPD 18.0 10 (Ki) Mitochondria
FKBP FK506 Rapamycin
Cyclophilin CsA
FKBP12 11.8 0.4 (Kd) 0.2 (Kd) Cytosol
FKBP13 13.2 ER
FKBP25 25.4 160 (Ki) 0.9 (Ki) Nucleus
FKBP52 51.8 10 (Ki) 8.0 (Ki) Cytosol
Rotamase activity (cis-trans peptidyl-prolyl isomerase activity)
"Gain "Gain of of Function" Function" Model Model
Single components: inactive
Cyclophilin Cyclophilin FKBP12 FKBP12 FKBP12 FKBP12
CsA CsA FK506 FK506 Rapa Rapa
Calcineurin
Cyclophilin
CsA
Calcineurin
Cyclophilin
CsA CsA
Calcineurin
FKBP12
FK506
Calcineurin
FKBP12
FK506
TOR
FKBP12
Rapa
TOR
FKBP12
Rapa
Target X Target X Target Y
Complexes: active inhibitors
CsA CsA FK506 FK506 Rapa Rapa
Cyclophilin Cyclophilin FKBP12 FKBP12 FKBP12 FKBP12
CsA CsA FK506 FK506 Rapa Rapa
Calcineurin Calcineurin TOR
Calcineurin (Phosphatase 2B)
Ser/Thr-Phosphatase 1, 2A, 2B, 2C
Regulatory B-chain (18 kDa; 30% sequence homology with CaM)
B-chain has 4 Ca
2+
binding EF-fingers
Catalytic A-Chain (59 kDa)
Catalytic domain, B-chain binding domain, CaM-binding domain,
autoinhibitory domain
Substrates: NFAT, myosin light chain kinase etc.
Inhibitors: AKAP79, CAIN/CABIN, CHP, MCIP1,2,3
Crystal Crystal structure structure of of calcineurin calcineurin
Kissinger et al. Nature (1995)
Agouron Pharmaceuticals
Cn-B Cn-B CaM
Catalytic
domain
Cn-B binding
domain
CaM binding
domain
Autoinhibitory
domain
Catalytic
center
Substrate
binding site
CaM
Ca
2+
Calcineurin inactive Calcineurin active
Plasma membrane
IP
3
-Receptor
CRAC
Ca
2+
Ca
2+
Calcineurin
TCR/CD3
complex
IP
3
P P
P
P
P P
NFAT NFAT
NFAT
TATA
Transcription of
IL-2,-3,-4,-5,-8,-13, IFN#, GM-CSF, TNF", CD95L
PLC#
Ca
2+
Ca
2+
Ca
2+
Ca
2+
Ca
2+
Nucleus
ER
Signal Signal Transduction Transduction of of Calcineurin Calcineurin and NFAT and NFAT
Cn-B CaM
FK506
CsA
PIP
2
DAG
PKC
Ca2+ release leads to NF-AT activation.
Ca2+ binds to calmodulin.
Ca-calmodulin complex activates calcineurin.
Calcineurin dephosphorylates NF-AT.
NF-AT activates Transcription.
FK506 and cyclosporin block NF-AT activation
by inhibiting calcineurin.
CsA and FK508 are essential drugs to prevent
transplant rejection.
The The Family Family of NFAT ( of NFAT (N Nuclear uclear F Factor of actor of A Activated ctivated T T cells) cells)
NFAT1 (c2, c)
NFATc (c1, p)
NFAT3 (c4)
NFAT (c3, x)
NFAT
Yes
Yes
No
Yes
80%90% of total NFAT in
resting immune cells
One isoform induced
upon activation
Highly expressed
in thymocytes
Implicated in cardiac
hypertrophy
Increased IL-4 production, Th2 bias
(delayed shutoff of IL-4 transcription)
Embryonic lethality (heart valve defect)
Lymphocytes produce less IL-4
Viable and fertile
Detailed phenotype not reported yet
Defect in positive selection of
thymocytes
Present in
Immune cells? Other features Phenotype of knockout mice
DNA-Binding
Domain
TAD-B
Regulatory
Domain
SP 1-3
NLS
TAD-B
Cn-B
Ca
2+
CaM
Cn-B CaM
Cn-B CaM
P P
P P
NFAT
P P
P P
Cn-B CaM
FK506
FKBP12
Cn-B CaM
P P
P P
NFAT
Cn-B CaM
P P
P P
NFAT
Mechanism Mechanism of Ca of Ca
2+ 2+
/CaM mediated activation /CaM mediated activation of of
calcineurin calcineurin and and inhbition by inhbition by FK506 FK506
Cyclosporine and
Cyclosporine and
Tacrolimus
Tacrolimus
(FK506)
(FK506)
Calcineurin Calcineurin Inhibitors- block lymphocyte transcription Inhibitors- block lymphocyte transcription
Bind to specific cytoplasmic Bind to specific cytoplasmic prolyl isomerases prolyl isomerases
( (immunophilins immunophilins) )
Cyclosporine > Cyclosporine > cyclophilin cyclophilin
Tacrolimus Tacrolimus > > FK506-binding protein (100x more potent) FK506-binding protein (100x more potent)
Lead to inhibition of Lead to inhibition of calcineurin calcineurin (cytoplasmic (cytoplasmic phosphatase phosphatase); );
required for activation of required for activation of NF-AT, NF-AT, a T cell specific a T cell specific
transcription factor that regulates IL-2 production transcription factor that regulates IL-2 production
Sirolimus
Sirolimus
(
(
Rapamycin
Rapamycin
)
)
Binds FKBP-12 intracellular binding protein
Specifically blocks T cell proliferation by inhibiting
mTOR (mammalian target of Rapamycin)
mTOR- serine-threonine kinase; controls cell cycle
progression
Synergizes with tacrolimus and cyclosporine
Cn-B CaM
P
P
P
P
Cn-B
NFAT
P
P
P
P
Cn-B CaM
Cn-B CaM
Ca
2+
CaM
CsA
FK506
NFAT-Kinase
(GSK-1, CK2)
Nucleus
Cytoplasm
Model of Model of the activation the activation of of calcineurin calcineurin and NFAT and NFAT
Cn-B CaM
Calcineurin
active
Calcineurin
inactive
Side Side Effects Effects of of Cyclosporine Cyclosporine