Download Signalling pathway

The NF-κB/Rel family
MBV4230
The NF-κB/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-κB  immune defence activated


NFkB also a major anti-apoptopic factor



aberrant activation of NF-κB = 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 cancer cells alive
NFkB also promoting growth


Immune response, inflammatory response, accute phase response
Activated NF-κB  cyclin D expression enhanced  growth
Drug against NFkB = putative anti-cancer drug
Odd S. Gabrielsen
MBV4230
The NF-κB/Rel family

Characteristic feature: homo- and heterodimeric TFs,
which in non-stimulated cells are found inactive in the
cytoplasm [in a complex with IκB-repressors].


Active DNA-binding form: Dimers with different members of the NF-κB/Rel family
Inactive cytoplasmic form: inhibitory factor/domain in addition

Upon stimulation, active NF-κB rapidly translocates to the
nucleus where it binds κB-sites and activates target
genes.

Rapid response - minutes

Signal → Activated NF-κB → immune defence activated
Odd S. Gabrielsen
MBV4230
Signal transduction pathway
Signal
Cytoplasm
inactive
Nucleus
active
Odd S. Gabrielsen
NF-κB/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)
 IκB-interaction (C-terminal half)

NLS (C-terminal half)

kalles også NRD (=NF-kB, Rel, Dorsal)
Spec.DNA-binding
dimerization
IkB-interaction
NLS
Odd S. Gabrielsen
MBV4230
Homo- and
heterodimers

NF-κB/Rel proteins
= Homo- and
hetero-dimeric TFs
that in resting cells
are retained in the
cytoplasm in
complex with IκB.

Mature B-cells:
constitutively
nuclear activator

Bound to kappa
immunoglobuline lightchain enhancer → its
name
Odd S. Gabrielsen
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 IκB 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-κB1]
 Proteolytic degradation to p50 is signal dependent, requires ATP and occurs through a ubiquitindependent proteasome pathway
 Also transcription from an intronic promoter → expression of IkB-γ
 p100 undergoes proteolytic maturation to p52 [NF-kB2]
 p50/52 are distinct gene products with very similar properties

Odd S. Gabrielsen
MBV4230
Two main classes of RHDs
- TAD
Rel homology domain
p105
p50
C-terminal IκB-like domains
p100
+TAD
p52
RelA(p65)
cRel
Acitvation domains
RelB
Odd S. Gabrielsen
MBV4230
RHD proteins
Ankyrin
repeats
RHD
Odd S. Gabrielsen
MBV4230
Dimer-formation

Dimer-formation necessary for DNAbinding



each subunit interacts with one half site
κB-sites symmetric: 5´-GGGRNNYYCC-3´
Most combinations allowed

Different heterodimers vary with respect to


preference for different kB-seter
Kinetics of nuclear translocation




–--5´-GGGRNNYYCC-3´-–- 3´-CCCYNNRRGG-5´--
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
Odd S. Gabrielsen
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 β-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
N-terminal
domain
Conserved interphase explains why most heterodimers are
possible
Odd S. Gabrielsen
MBV4230
Structure: NFκB (p50-p65) + DNA
Side view
• β-barrel core with protrding loops
• The AB loop = recognition loop
• Specificity R57-R59-E63
• C62 redox-sensitivity
Odd S. Gabrielsen
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 (K = 10-12 M)
d

Dissociation requires opening of the jaws through a flexible
linker
Odd S. Gabrielsen
MBV4230
3D structure - protein interaction

Interaction with HMGI(Y)

IFN-β promoter: HMGI(Y) binds AT-rich
centre of κB-sites in minor groove
 The structure contains a corresponding
open space

IkB
Interaction with IκB

IκB binding in an opening over the
dimer-interphase
 IκB binding blocks DNA-binding
HMG
I(Y)
–--5´-GGGRNNYYCC-3´-–- 3´-CCCYNNRRGG-5´--
Odd S. Gabrielsen
The I-κB family
MBV4230
The I-κB proteins
Ankyrin
repeats
N-terminal
Regulatory domain
Odd S. Gabrielsen
MBV4230
The IκB-family

Inhibitory function



Several members (at least 7 mammalian)


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


impedes DNA-binding
blocks NLS and abolish translocation to nucleus
IκB-α and IκB-β
IκB-γ and IκB-ε
Bcl-3
p105 and p110
IkBR
Specificity
Ex. IkB-α 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
Odd S. Gabrielsen
MBV4230
NFκB-IκB complex
IkB
HMG
I(Y)
Odd S. Gabrielsen
MBV4230
Signaling

IκB - a key
element in
the
canonical
NFκB
signaling
pathway
Odd S. Gabrielsen
MBV4230
Cytoplasmic retention due to
interaction with IκB-family proteins

Two types of inactive complexes in cytoplasm
Signal
Trimers = RHD-Homo-or heterodimers bound to an IκB
 Heterodimers = Rel-protein + unprocessed RHD-precursor (p105, p110)


Signal→[dissociation] → degradation
Induction signal → phosphorylation of both IκB and p105 → IκB 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 IκB
 complete after 10 min
 No traces of IκB
 phosphorylation of IκB




Signal
→ multiubiquitylation in K21, K22
→ degradation through a ubiquitin-proteasome pathway
+ proteasome-inhibitors → phospho-IkB remains associated with NFkB
Odd S. Gabrielsen
MBV4230
Several IκB-factors
with different properties

IκB-α: Rapid transient response



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IκB-β: Sustained response



IκB-α best characterized
all stimuli → degradation of IκB-α
ex: TNF-α → rapid and transient activation of NF-kB
Only certain stimuli → degradation of IκB-β
ex: LPS or IL-1 → degradation of both IκB-α and IκB-β → activation of NF-kB
lasting for hours
Bcl-3: repressor and activator


inhibits certain complexes like a normal IκB
But may also associate with DNA-bound p50 and p52 dimers (lacking TAD) and
provide transactivation properties
Odd S. Gabrielsen
Signaling pathways
MBV4230
Upstream and downstream
Upstream
Signal transduction
pathways
+
.
.
+
NF-kB
Downstream
+
.
.
Odd S. Gabrielsen
MBV4230
Multiple signalling pathways
activate NF-κB

Several signalling pathways converge
by activation of NF-κB

NF-κB respond to a broad range of different stimuli

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
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Virus infection (HIV, hepatite B), virus proteins (tax, E1A) and
dsRNA
Cytokines (TNFα, 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
Signal transduction
pathways
..
+
+
NF-kB
+
..
Odd S. Gabrielsen
MBV4230
Three signal transduction pathways
Signal
Cytoplasm
inactive
Nucleus
active
Odd S. Gabrielsen
MBV4230
Signaling hits I-κB through
phosphorylation

Two N-terminal serines
becomes phosphorylated


TNF-signalling pathways: TNF-receptor →
TRADD/TRAF → IKK → IκB →→
IκB-kinase complex central in
the signaling pathway

A large 500-900 kDa IKK (IκB-kinase)
complex that is induced by cytokines
 Two key subunits: IKKα and IKKβ
Odd S. Gabrielsen
MBV4230
The IκB-kinase complex
central in the pathway
IκB-kinase complex
Odd S. Gabrielsen
MBV4230
The IKKβ-kinase becomes
activated through phosphorylation

Activation loop in IKKβ



Ser-OH
Ser-P
Ser-OH
Ser-P
inactive
Signal → phosphorylation


Two serines bocomes phosphorylated
in a signal dep manner (IL1, TNF)
Ala-mutants block the signalling
pathway, Glu-mutants lead to a
IKKß
constitutive active kinase
Signal
Upstream kinase
inactive
Autophosphorylation
phosphorylation of loop necessary for
NFκB-activation of cytokines
Attenuation

active
P
P
PP
IκB
phosphorylated activation loop →
altered HLH-kinase domain
interaction → reduced kinase-aktivitet
Odd S. Gabrielsen
MBV4230
The first pathway
- the classical pathway

Receptor triggered by pro-inflammatory cytokines


Recruitment of various adaptors




including TRADD (TNF-receptor associated death domain protein), RIP (receptor interacting
protein and TRAF2 (TNF-receptor-associated factor 2) to the cytoplasmic membrane.
Recruitment and activation of the classical IκB-kinase (IKK)
complex


such as tumour necrosis factor (TNF)-α
which includes the scaffold protein NEMO (NF-kB essential modulator; also named IKKγ), IKKα
and IKKβ kinases.
The IKK complex phosphorylates IκBα on Ser32 and Ser36
Leading to ubiquitylation and degradation via the proteasome
pathway
The free p50-p65 migrates to the nucleus where it activates
target genes involved in immune response
Odd S. Gabrielsen
MBV4230
The first pathway
- the classical pathway dep on IKKβ
Triggered by microbial and
viral infections and
exposure to
proinflammatory cytokines
Odd S. Gabrielsen
MBV4230
Why two kinases?




52% identity
Similar kinase activity
In vivo: IKKα ≠ IKKβ





Signal
upstream
kinase
In vitro: IKKα ≈ IKKβ
Ala-mutants of IKKß → NFκB
response dead
Glu-mutants of IKKß → NFκB
response independent of signals
Ala-mutants of IKKα → NFκB
response unaffected
Glu-mutants of IKKα → NFκB
response unaffected
Is IKKα totally unlinked to
NFκB?
IKKß
Ser-OH
Ser-P
Ser-OH
Ser-P
inactive
active
IκB
Odd S. Gabrielsen
MBV4230
The next indication:
KO phenotypes of IKKα ≠ IKKβ

Knock-out of of IKKβ  loss of B- and T-cell response





Normal development
Mice dead at day 13.5, liver destroyed due to massive apoptosis
Lack of IKKβ → 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 IKKα  specific B-cell problem



Skin phenotype, epidermis 5-10x thicker than normal, highly undifferentiated
NFkB response normal
Normal number of B- and T-cells, but B-cells not fully differentiated
Odd S. Gabrielsen
MBV4230
A separate signaling pathway
through IKKα




A desparate postdoc looked at all the 50
components - all behaved normal, except one
The proteolytic maturation of the p100
precursor to p52 [NF-κB2] was defective in the
IKKα(-/-)
This processing depends on NIK
Hypothesis: NIK acts through IKKα
Odd S. Gabrielsen
MBV4230
The solution
Processing depends
on IKKα
Target of
IKKβ
Odd S. Gabrielsen
MBV4230
A separate signalling pathway
involving only IKKα
Affects NF-κB2 (p100), which
preferentially dimerizes with RelB.
Triggered by by cytokines such as
lymphotoxin b, B-cell activating factor
(BAFF) or the CD40 ligand and by viruses
such as human T-cell leukaemia virus.
NEMO-independent,
IKKα- dependent + another
kinase NIK.
Induce the phosphorylationdependent proteolytic
removal of the IkB-like Cterminal domain of NF-κB2
B-cell maturation
A role in innate immunity A role in adaptive immunity
Odd S. Gabrielsen
MBV4230
Two kinases
- two main signaling pathways

The canonical NF-κB activation
pathway (left)

Applies to RelA-p50 and c-Rel-p50
 Retained in cytoplasm by IκB
 Triggered by microbial and viral infections
and exposure to proinflammatory cytokines
 Depends mainly on the IKKβ subunit of the
IKK complex.

The second pathway (right)
Affects NF-κB2, which preferentially
dimerizes with RELB.
 Triggered by members of the tumour-necrosis
factor (TNF) cytokine family
 Depends selectively on activation of the IKKα
subunit + another kinase NIK.
 Induce the phosphorylation-dependent
proteolytic removal of the IκB-like C-terminal
domain of NF-κB2.

Odd S. Gabrielsen
MBV4230
A third signalling pathway
independent on both IKKs




classified as atypical
because it is
independent of IKK
proteasome still required
triggered by DNA
damage such as UV or
doxorubicin
UV radiation induces
IkBa degradation via the
proteasome, but the
targeted serine residues
are located within a Cterminal cluster, which is
recognized by the p38activated casein kinase 2
(CK2)
Odd S. Gabrielsen
MBV4230
Connectivity map of the TNF-α/NF-κB
signal transduction pathway
Odd S. Gabrielsen
Target genes
MBV4230
Upstream and downstream
Upstream
Signal transduction
pathways
+
.
.
+
NF-kB
Downstream
+
.
.
Odd S. Gabrielsen
MBV4230
Families of target genes

Immune response

Cytokines,

Chemokines
Cytokine and immuno-receptors



Adhesion molecules
Acute-phase proteins

Stress-responsive genes
NF-κB is both being activated by and inducing the expression of inflammatory cytokines
NF-κB activation can spread from cell to cell
Odd S. Gabrielsen
MBV4230
Negative feedback:
Attenuation of respons

Negative loop: IκB-α under direct control of NF-κB





Activated NF-κB translocated to the nucleus will activate expression of IκB-α
Newly synthesized IκB-α will bind up and inactivate remaining NF-κB in the
cytoplasma
Excess IκB-α will migrate to the nucleus and inactivate DNA-bound NF-κB
(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 IκB-α
which then binds up and inactivates NF-κB in the cytoplasm, leading to reduced
immune- and inflammatory response
Odd S. Gabrielsen
MBV4230
Target genes:
Link to cancer

Tumorigenesis requires
6 types of alterations


Hanahan & Weinberg 2000
Several of these can be
caused by perturbation in
NF-κB or linked signaling
molecules

Tumour cells in which NF-κB is
constitutively active are highly resistant
to anticancer drugs or ionizing radiation.
Angiogenesis
Metastasis
Odd S. Gabrielsen
Disease links
MBV4230
Viruses exploit NF-κB

several patogenic viruses exploit the NF-κB
system for their own profit

Incorporation of κB-sites in virus DNA cause enhanced expression of
virus-genes when the immune response is activated
 Virus proteins activate NF-κB
Odd S. Gabrielsen
MBV4230
Disease links
Odd S. Gabrielsen
MBV4230
Constitutively
nuclear NF-κB

Disruption of the regulatory
mechanism → aberrant
activation of NFκB = one of the
primary causes of a wide range
of human diseases

Inflammatory diseases




Rheumatoid arthritis
Asthma
Atherosclerosis
Alzheimer
Odd S. Gabrielsen
MBV4230
Link: inflammation - cancer

A causal connection between inflammation and cancer
has been suspected for many years.

NF-κB might serve as the missing link between these
two processes.


NF-κB becomes activated in response to inflammatory stimuli
Constitutive activation of NF-κB has been associated with cancer,
Odd S. Gabrielsen
MBV4230
Mechanisms of NF-κB activation
promoting leukemia

Mechanisms by which NF-κB
activation can contribute to
leukaemia and lymphogenesis
1.
2.
3.
4.
Input: NF-κB can be constitutively activated in
myeloid and lymphoid cells in response to growth
factors and cytokines or the expression of certain
viral oncoproteins.
Gene errors: Persistent NF-κB activation can also be
brought about by chromosomal rearrangements that
affect genes that encode NF-κB or I-κB.
Autocrine loop: Once NF-κB is activated, it can lead
to the production of cytokines and growth factors,
such as CD40 ligand (CD40L), that further
propagates its activation.
Growth - apoptosis: It also activates the
transcription of cell-cycle regulators, such as cyclins
D1 and D2, which promote G1- to S-phase transition,
or inhibitors of apoptosis, such as BCL-XL, cIAPs
and A1/BFL1.
1
.
3
.
2
.
4
.
Tumour cells in which NF-κB is constitutively active
are highly resistant to anticancer drugs or ionizing radiation.
Odd S. Gabrielsen
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-κB ligand (RANKL), which binds to the receptor
activator of NF-κB (RANK). This pathway, which leads to
activation of NF-κB, depends on the IKKα subunit of the IKK
complex.

After nuclear translocation, NF-κB 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.
Odd S. Gabrielsen
MBV4230
Blocking the response


Redox-dependency

Antioxidants and alkylating agens inhibit response to many stimuli and
inhibit phosphorylation and degradation of IκB

H2O2 activates NF-κB

Induction of ROI (reactive oxygen intermediates) a possible common
element?
Proteasome inhibitors
Odd S. Gabrielsen
MBV4230
Therapeutic inhibition
of NFκB



Numerous
inhibitors of NFκB under
development.
Difficult to
develop cancer
specific
inhibitors.
Understanding
the two
pathways
should lead to
better
therapeutics.
Odd S. Gabrielsen
MBV4230
Example of previously given
question for the exam

Signalling pathway

Describe briefly the key elements of signalling pathways involving IKK
and NF-κB.
54
Odd S. Gabrielsen