The NF-B/Rel family

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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

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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 IB-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

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Signal transduction pathway

Cytoplasminactive

Nucleusactive

Signals

NF-B/Rel proteins

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Common DBD: Rel-homology domain (RHD) RHD: 300aa conserved domain with several

functions DNA-binding (N-terminal half) dimerization (C-terminal half) IB-interaction (C-terminal half) NLS (C-terminal half) kalles også NRD (=NF-kB, Rel, Dorsal)

Spec.DNA-binding dimerizationIkB-interaction

NLS

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Homo- and heterodimers

NF-B/Rel proteins = Homo- and hetero-dimeric TFs that in resting cells are retained in the cytoplasm in complex with IB.

Mature B-cells: constitutively nuclear activator Bound to kappa

immunoglobuline light-chain enhancer its name

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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 IB 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 expressionof IkB-

p100 undergoes proteolytic maturation to p52 [NF-B2] p50/52 are distinct gene products with very similar properties

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Two main classes of RHDs

p105

p50

p100

p52

RelA(p65)

cRel

RelB

Rel homology domain

C-terminal IB-like domains

Acitvation domains

+TA

D-

TA

D

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RHD proteins

Ankyrinrepeats

RHD

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Dimer-formation

Dimer-formation necessary for DNA-binding 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´--

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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 DNA-phosphate contact

Conserved interphase explains why most heterodimers are possible

N-terminaldomain

C-terminaldomain

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Structure: NFB (p50-p65) + DNA

Side view

• -barrel core with protrding loops • The AB loop = recognition loop• Specificity R57-R59-E63• C62 redox-sensitivity

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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

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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 IB IB binding in an opening over the dimer-interphase IB 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

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The I-B proteins

N-terminal Regulatory domain

Ankyrinrepeats

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The IB-family

Inhibitory function impedes DNA-binding blocks NLS and abolish translocation to nucleus

Several members (at least 7 mammalian) IB- and IB- IB-and IB- Bcl-3 p105 and p110 IkBR

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

SpecificityEx. IkB- inhibits DNA-binding of p65/p50 but not of p50/p50

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NFB-IB complex

IkB

HMGI(Y)

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Signaling

The chain of events in the canonical NFB signaling pathway

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Cytoplasmic retention due to interaction with IB-family proteins Two types of inactive complexes in the cytoplasm

1. Trimers = RHD-Homo-or heterodimers bound to an IB-repressor 2. Heterodimers = Rel-protein + unprocessed RHD-precursor (p105, p110)

Model: Signal dissociation (?) and degradation Induction signal phosphorylation of both IB and p105 IB 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 IB

complete after 10 min No traces of IB

phosphorylation of IB multiubiquitylation in K21, K22 degradation through a ubiquitin-dependent proteasome pathway

I presence of proteasome-inhibitors phosphorylated IkB remains associated with NFkB

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Several IB-factors with different properties IB-: Rapid transient response

IB- best characterized all stimuli degradation of IB- ex: TNF-rapid and transient activation of NF-kB

IB-: Sustained response Only certain stimuli degradation of IB- ex: LPS or IL-1degradation of both IB-and IB- activation of NF-

kB lasting for hours

Bcl-3: repressor and activator inhibits certain complexes like a normal IB But may also associate with DNA-bound p50 and p52 dimers (lacking TAD)

and provide transactivation properties

Signaling pathways

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Upstream and downstream

NF-kB

Signal transductionpathways

+ +..

+ ..

Upstream

Downstream

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Signaling

The chain of events in the NFB signaling pathway

The system = a total of 50 gene-products, but only 1 component is regulated: the IKK complex

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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

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One type of signaling hits I-B through phosphorylation

Two N-terminal serines becomes phosphorylated TNF-signalling pathways: TNF-receptor

TRADD/TRAF NIK IKK IB

IB-kinase complex central in the signaling pathway A large 500-900 kDa IKK (IB-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?

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The IB-kinase complex central in the pathway

IB-kinase complex

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The IKK-kinase becomes activated through phosphorylation

Activation loop in IKK 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

Signal phosphorylation phosphorylation of loop necessary for

NFB-activation of cytokines

Attenuation phosphorylated activation loop

altered HLH-kinase domain interaction reduced kinase-aktivitet

Ser-OH

Ser-OH

Ser-P

Ser-P

SignalUpstream kinase

inactive active

IKKß

IB

inactiveP P

PP

Autophosphorylation

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Stimulus-specific signal transduction pathways?

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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 Upstream cascade from

membrane-receptors to the IKK-complex where TRAF and NIK are involved

Alternative inputs probably through MEKK1 and Akt/PKB

AlternativeIKK-kinases

AlternativeIKK-complexes

Signal 1

Signal 2

Signal 3

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Why two kinases?

Ser-OH

Ser-OH

Ser-P

Ser-P

Signalupstream kinase

inactive active

IKKß

IB

In vitro: IKK ≈ IKK 52% identity Similar kinase activity

In vivo: IKK ≠ IKK Ala-mutants of IKKß NFB

response dead Glu-mutants of IKKß NFB

response independent of signals Ala-mutants of IKKNFB

response unaffected Glu-mutants of IKKNFB

response unaffected

Is IKK totally unlinked to NFB?

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The next indication: KO phenotypes of IKK ≠ IKK

Knock-out of of IKKloss 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 , epidermis 5-10x thicker than normal, highly

undifferentiated sl Normal number of B- and T-cells, but B-cells not fully differentiated

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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

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The solution Processing dependson IKK

Target ofIKK

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Model - two divergent pathways through the IKK complex

TNF-R

Altered processingof p100

NIK

Signal 2

Affect B-cellmaturation

A role in adaptive immunity

A role in innate immunity

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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 IB 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 tumour-

necrosis factor (TNF) cytokine family Depends selectively on activation of the

IKK subunit + another kinase NIK. Induce the phosphorylation-dependent

proteolytic removal of the IB-like C-terminal domain of NF-B2.

Target genes

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Upstream and downstream

NF-kB

Signal transductionpathways

+ +..

+ ..

Upstream

Downstream

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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 cytokinesNF-B activation can spread from cell to cell

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Negative feedback:Attenuation of respons Negative loop: IB- under direct control of NF-B

Activated NF-B translocated to the nucleus will activate expression of IB- Newly synthesized IB-will bind up and inactivate remaining NF-B in the

cytoplasma Excess IB-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 IB-

which then binds up and inactivates NF-B in the cytoplasm, leading to reduced immune- and inflammatory response

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Target genes:Link to cancer Tumorigenesis requires

6 types of alterations 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.

AngiogenesisMetastasis

Disease links

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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

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Disease links

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Constitutivelynuclear NF-B

Disruption of the regulatory mechanism aberrant activation of NFB = one of the primary causes of a wide range of human diseases Inflammatory diseases

Rheumatoid arthritis Asthma

Atherosclerosis Alzheimer

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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,

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Mechanisms of NF-B activation promoting leukemia

Mechanisms by which NF-B activation can contribute to leukaemia and lymphogenesis 1. 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. Autocrine loop: Once NF-B is activated, it 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 S-phase transition, or inhibitors of apoptosis, such as BCL-XL, cIAPs and A1/BFL1.

1. 2.

3.4.

Tumour cells in which NF-B is constitutively active are highly resistant to anticancer drugs or ionizing radiation.

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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.

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Blocking the response

Redox-dependency Antioxidants and alkylating agens inhibit response to many stimuli and

inhibit phosphorylation and degradation of IB H2O2 activates NF-B Induction of ROI (reactive oxygen intermediates) a possible common

element?

Proteasome inhibitors

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Therapeutic inhibition of NFB Numerous

inhibitors of NF-B under development.

Difficult to develop cancer specific inhibitors.

Understanding the two pathways should lead to better therapeutics.