Transcript Signaling pathways

The NF-B/Rel family
MBV4230
The NF-B/Rel family

A family of signal-responsive transcription factors


rapid response som ikke requires proteinsyntese
Involved in proinflammatory response: a first line of
defense against infectious diseases and cellular
stress

Signal  Activated NF-B  immune defence activated


Immune response, inflammatory response, accute phase response
NFkB also a major anti-apoptopic factor
aberrant activation of NF-B = one of the primary causes of a wide range of
human diseases like in Inflammatory diseases, Rheumatoid arthritis, Asthma,
Atherosclerosis, Alzheimer
 Persistent activated in many cancers - help keeping them alive


NFkB also promoting growth


Activated NF-B  cyclin D expression enhanced  growth
Drug against NFkB = putative anti-cancer drug
MBV4230
The NF-B/Rel family

Characteristic feature: homo- and heterodimeric TFs,
which in non-stimulated cells are found inactive in the
cytoplasm [in a complex with IB-repressors].
Active DNA-binding form: Dimers with different members of the NF-B/Rel family
 Inactive cytoplasmic form: inhibitory factor/domain in addition


Upon stimulation, active NF-B rapidly translocates to
the nucleus where it binds B-sites and activates target
genes.

Rapid response - minutes

Signal Activated NF-B  immune defence activated
MBV4230
Signal transduction pathway
Signals
Cytoplasm
inactive
Nucleus
active
NF-B/Rel proteins
MBV4230
Common DBD:
Rel-homology domain (RHD)

RHD: 300aa conserved domain with several
functions

DNA-binding (N-terminal half)
dimerization (C-terminal half)
IB-interaction (C-terminal half)
NLS (C-terminal half)

kalles også NRD (=NF-kB, Rel, Dorsal)



Spec.DNA-binding
dimerization
IkB-interaction
NLS
MBV4230
Homo- and
heterodimers

NF-B/Rel proteins
= Homo- and
hetero-dimeric TFs
that in resting cells
are retained in the
cytoplasm in
complex with IB.

Mature B-cells:
constitutively
nuclear activator

Bound to kappa
immunoglobuline lightchain enhancer  its
name
MBV4230
Two main classes of RHDs

Rel with TAD (dimeric with ≥ 1 Rel-monomers which are potent
transactivators) synthesized in their mature form
Rel or c-Rel (as well as v-Rel)
 RelA (p65)
 RelB
 Drosophilas dorsal and Dif


p50/52 without TAD (homodimers with no transactivation properties)
synthesized as precursors that are processed
Precursor forms have internal IB inhibitor function
 RHD linked to inhibitory domain through Gly-rich linker (protease sensitive)
 Blocks DNA-binding and translocation to nucleus
 p105 undergoes proteolytic maturation to p50 [NF-B1]
 Proteolytic degradation to p50 is signal dependent, requires ATP and occurs through a
ubiquitin-dependent proteasome pathway
 Also transcription from an intronic promoter expression of IkB-
 p100 undergoes proteolytic maturation to p52 [NF- B2]
 p50/52 are distinct gene products with very similar properties

MBV4230
Two main classes of RHDs
- TAD
Rel homology domain
p105
p50
C-terminal IB-like domains
p100
+TAD
p52
RelA(p65)
cRel
RelB
Acitvation domains
MBV4230
RHD proteins
Ankyrin
repeats
RHD
MBV4230
Dimer-formation

Dimer-formation necessary for DNAbinding
each subunit interacts with one half site
 B-sites symmetric: 5´-GGGRNNYYCC-3´


Most combinations allowed

Different heterodimers vary with respect to


preference for different kB-seter
Kinetics of nuclear translocation




p50/p65 rapid, p50/Rel slow
abundance in different cells
Exception: RelB which forms dimer only with p50/p52
Common form: p50/p65 (NF-kB1/RelA)

most abundant, found in most cells
–--5´-GGGRNNYYCC-3´-–- 3´-CCCYNNRRGG-5´--
MBV4230
3D structure - DNA interaction

Crystal structures:



p50-p50-DNA and p50-p65-DNA
Two distinct domains
1. N-terminal - specific DNA contact





Compact core in the form of an antiparalell -barrel
from which loops protrude
The loop between AB = recognition loop with base
contacts in major groove
Critical for specificity = R57-R59-E63
C62 responsible for redox-sensitivity
2. C-terminal domain responsible
for dimerisation + nonspecific DNAphosphate contact

C-terminal
domain
Conserved interphase explains why most heterodimers
are possible
N-terminal
domain
MBV4230
Structure: NFB (p50-p65) + DNA
Side view
• -barrel core with protrding loops
• The AB loop = recognition loop
• Specificity R57-R59-E63
• C62 redox-sensitivity
MBV4230
3D structure - DNA interaction

Characteristic features of DNA-interaction


Each monomer contacts a separate half site
“Closing jaws” mechanism for DNA-binding
 The protein encloses DNA
 Unusual strong binding (Kd = 10-12 M)
 Dissociation requires opening of the jaws through a flexible
linker
MBV4230
3D structure - protein interaction

Interaction with HMGI(Y)
IFN- promoter: HMGI(Y) binds AT-rich centre of B-sites in minor groove
 The structure contains a corresponding open space


Interaction with IB
IB binding in an opening over the dimer-interphase
 IB binding blocks DNA-binding




due to steric effect ?
due to hinge-effect ?
due to induced change of geometry in C-terminal domain  reduced non-specific
DNA-binding?
The I-B family
MBV4230
The I-B proteins
Ankyrin
repeats
N-terminal
Regulatory domain
MBV4230
The IB-family

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
IkBR
Specificity
Ex. IkB- inhibits DNA-binding of
p65/p50 but not of p50/p50
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
MBV4230
NFB-IB complex
IkB
HMG
I(Y)
MBV4230
Signaling

The chain of
events in
the
canonical
NFB
signaling
pathway
MBV4230
Cytoplasmic retention due to
interaction with IB-family proteins

Two types of inactive complexes in the cytoplasm
1. Trimers = RHD-Homo-or heterodimers bound to an IB-repressor
 2. Heterodimers = Rel-protein + unprocessed RHD-precursor (p105, p110)


Model: Signal  dissociation (?) and degradation
Induction signal  phosphorylation of both IB and p105  IB degradation or p105
processering  active dimers that are translocated to the nucleus.
 One type of signal  two N-terminal serines (S32 and S36) become phosphorylated
 Another type of signal  two C-terminal serines become phosphorylated in p105
 phosphorylation probably more a signal for degradation than for dissociation


Ubiquitin-pathway involved
Stimulation  rapid degradation of IB
 complete after 10 min
 No traces of IB
 phosphorylation of IB  multiubiquitylation in K21, K22  degradation through a
ubiquitin-dependent proteasome pathway
 I presence of proteasome-inhibitors phosphorylated IkB remains associated with NFkB

MBV4230
Several IB-factors
with 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-kB


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 NFkB 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

Signaling pathways
MBV4230
Upstream and downstream
Upstream
Signal transduction
pathways
+
..
+
NF-kB
Downstream
+
..
MBV4230
Signaling

The chain of
events in the
NFB signaling
pathway

The system = a
total of 50 geneproducts, but only
1 component is
regulated: the IKK
complex
MBV4230
Multiple signalling pathways
activate NF-B

Several signalling pathways converge
by activation of NF-B

NF-B respond to a broad range of different stimuli










Virus infection (HIV, hepatite B), virus proteins (tax, E1A)
and dsRNA
Cytokines (TNF, IL-1 and IL-2)
Bacterial LPS
stimulation of antigen reseptor on B- and T-cells
calcium ionophores
protein synthesis inhibitors
UV and X-ray
sphingomylenase/ceramide
phorbol esters
nitrogen oxide
MBV4230
One type of signaling hits I-B
through phosphorylation

Two N-terminal serines
becomes phosphorylated


TNF-signalling pathways: TNF-receptor 
TRADD/TRAF  NIK  IKK IB 
IB-kinase complex central in
the signaling pathway


A large 500-900 kDa IKK (IB-kinase)
complex that is induced by cytokines
Two key subunits: IKK and IKK

Each with three domains: KD (kinase domain)
+ LZ (leucine zipper) + HLH (helix-loop-helix)
?
Kinase?
MBV4230
The IB-kinase complex
central in the pathway
IB-kinase complex
MBV4230
The IKK-kinase becomes
activated through phosphorylation




Two serines bocomes phosphorylated
in a signal dep manner (IL1, TNF)
Ala-mutants block the signalling
pathway, Glu-mutants lead to a
constitutive active kinase
IKKß
Ser-OH
Ser-P
Ser-OH
Ser-P
inactive
Signal  phosphorylation


Signal
Upstream kinase
Activation loop in IKK
phosphorylation of loop necessary for
NFB-activation of cytokines
Attenuation

active
phosphorylated activation loop 
altered HLH-kinase domain
interaction  reduced kinase-aktivitet
P
P
PP
inactive
Autophosphorylation
IB
MBV4230
Stimulus-specific signal
transduction pathways?
MBV4230
Stimulus-specific signalling
pathways?

Novel IKK-candidates



IKK possibly the kinase in an
independent IKK-complex
which is responsive to phorbol
esters (PMA/TPA) and T-cell
receptor, but not to TNF and
IL1.
Possibly more
Novel IKK-kinase
candidates
Signal 2
Signal 3
Signal 1
Alternative
Upstream cascade from
membrane-receptors to the IKK-complexes
IKK-complex where TRAF
and NIK are involved
 Alternative inputs probably
through MEKK1 and Akt/PKB

Alternative
IKK-kinases
MBV4230
Why two kinases?

In vitro: IKK ≈ IKK



Signal
upstream kinase
52% identity
Similar kinase activity
In vivo: IKK ≠ IKK
Ala-mutants of IKKß  NFB
response dead
 Glu-mutants of IKKß  NFB
response independent of signals
 Ala-mutants of IKK  NFB
response unaffected
 Glu-mutants of IKK  NFB
response unaffected


IKKß
Ser-OH
Ser-P
Ser-OH
Ser-P
inactive
active
Is IKK totally unlinked
to NFB?
IB
MBV4230
The next indication:
KO phenotypes of IKK ≠ IKK

Knock-out of of IKK  loss of B- and T-cell response
Normal development
 Mice dead at day 13.5, liver destroyed due to massive apoptosis
 Lack of IKK  lack of active NFkB  lack of protection against apoptosis 
massive cell death
 Lost T-cell response because Apoptosis important for T-cell development


Knock-out of of IKK 

undifferentiated
, epidermis 5-10x thicker than normal, highly
s
l
 Normal number of B- and T-cells, but B-cells not fully differentiated

MBV4230
A separate signaling pathway
through IKK




A desparate postdoc looked at all the 50
components - all behaved normal, except one
The proteolytic maturation of the p100
precursor to p52 [NF-B2] was defective in
the IKK
processing depends on NIK
Hypothesis: NIK acts through IKK
MBV4230
The solution
Target of
IKK
Processing depends
on IKK
MBV4230
Model - two divergent pathways
through the IKK complex
Signal 2
NIK
TNF-R
Altered processing
of p100
Affect B-cell
maturation
A role in adaptive immunity
A role in innate immunity
MBV4230
Two kinases
- two main signaling pathways

The canonical NF-B activation
pathway (left)
Applies to RelA-p50 and c-Rel-p50
 Retained in cytoplasm by IB
 Triggered by microbial and viral
infections and exposure to
proinflammatory cytokines
 Depends mainly on the IKK subunit of
the IKK complex.


The second pathway (right)
Affects NF-B2, which preferentially
dimerizes with RELB.
 Triggered by members of the tumournecrosis factor (TNF) cytokine family
 Depends selectively on activation of the
IKK subunit + another kinase NIK.
 Induce the phosphorylation-dependent
proteolytic removal of the IB-like Cterminal domain of NF-B2.

Target genes
MBV4230
Upstream and downstream
Upstream
Signal transduction
pathways
+
..
+
NF-kB
Downstream
+
..
MBV4230
Families of target genes

Immune response






Cytokines,
Chemokines
Cytokine and immuno-receptors
Adhesion molecules
Acute-phase proteins
Stress-responsive genes
NF-B is both being activated by and inducing the expression of inflammatory cytokines
NF-B activation can spread from cell to cell
MBV4230
Negative feedback:
Attenuation of respons

Negative loop: IB- under direct control of NF-B
Activated NF-B translocated to the nucleus will activate expression of IB-
 Newly synthesized IB-will bind up and inactivate remaining NF-B in the
cytoplasma
 Excess IB-will migrate to the nucleus and inactivate DNA-bound NF-B
(contains both NLS and nuclear eksport signal)
 A20 protein another strongly induced negative feedback protein


Immunosupressive effect of glucocorticoids

Probably a direct effect of glucocorticoids enhancing the expression of IBwhich then binds up and inactivates NF-B in the cytoplasm, leading to
reduced immune- and inflammatory response
MBV4230
Target genes:
Link to cancer

Tumorigenesis requires
types of alterations


6
Hanahan & Weinberg 2000
Several of these can be
caused by perturbation in
NF-B or linked signaling
molecules

Tumour cells in which NF-B is
constitutively active are highly resistant
to anticancer drugs or ionizing radiation.
Angiogenesis
Metastasis
Disease links
MBV4230
Viruses exploit NF-B

several patogenic viruses exploit the NF-B
system for their own profit


Incorporation of B-sites in virus DNA cause enhanced expression of
virus-genes when the immune response is activated
Virus proteins activate NF-B
MBV4230
Disease links
MBV4230
Constitutively
nuclear NF-B

Disruption of the regulatory
mechanism  aberrant
activation of NFB = one of the
primary causes of a wide range
of human diseases

Inflammatory diseases


Rheumatoid arthritis
Asthma
Atherosclerosis
 Alzheimer

MBV4230
Link: inflammation - cancer

A causal connection between inflammation and
cancer has been suspected for many years.

NF-B might serve as the missing link between these
two processes.
NF-B becomes activated in response to inflammatory stimuli
 Constitutive activation of NF-B has been associated with cancer,

MBV4230
Mechanisms of NF-B activation
promoting leukemia

Mechanisms by which NF-B
activation can contribute to
leukaemia and lymphogenesis
1.
2.
Input: NF-B can be constitutively activated in
myeloid and lymphoid cells in response to
growth factors and cytokines or the expression
of certain viral oncoproteins.
2. Gene errors: Persistent NF-B activation can
also be brought about by chromosomal
rearrangements that affect genes that encode
NF-B or I-B.
3.
3. Autocrine loop: Once NF-B is activated, it
4.
can lead to the production of cytokines and
growth factors, such as CD40 ligand (CD40L),
that further propagates its activation.
4. Growth - apoptosis: It also activates the
transcription of cell-cycle regulators, such as
cyclins D1 and D2, which promote G1- to Sphase transition, or inhibitors of apoptosis, such
as BCL-XL, cIAPs and A1/BFL1.
Tumour cells in which NF-B is constitutively active
1.
are highly resistant to anticancer drugs or ionizing radiation.
MBV4230
Breast cancer: Signalling pathways
that stimulate proliferation

Signaling induction of cyclin D1.
Two signalling pathways contribute to the induction of
cyclin D1 transcription in mammary epithelial cells.
 One pathway, which leads to activation of transcription
factor AP1, is activated by growth factors (GF), which bind
to receptor tyrosine kinases (RTK). This pathway relies on
activation of RAS and MAPK cascades.
 The second pathway is activated by the TNF-family
receptor activator of NF-B ligand (RANKL), which binds
to the receptor activator of NF-B (RANK). This pathway,
which leads to activation of NF-B, depends on the IKK
subunit of the IKK complex.


After nuclear translocation, NF-B
activates cyclin D1 expression, leading to
cell-cycle progression.

The expression of GFs and RANKL is regulated by various
hormonal stimuli during mammary-gland development.
Aberrant and persistent activation of either pathway can
lead to deregulated proliferation of mammary epithelial
cells.
MBV4230
Blocking the response

Redox-dependency




Antioxidants and alkylating agens inhibit response to many stimuli and
inhibit phosphorylation and degradation of IB
H2O2 activates NF-B
Induction of ROI (reactive oxygen intermediates) a possible common
element?
Proteasome inhibitors
MBV4230
Therapeutic inhibition
of NFB



Numerous
inhibitors of
NF-B under
development.
Difficult to
develop cancer
specific
inhibitors.
Understanding
the two
pathways
should lead to
better
therapeutics.