The NFBRel family

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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
  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

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Signal transduction pathway
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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)
• I?B-interaction (C-terminal half)
• NLS (C-terminal half)
• kalles også NRD (NF-kB, Rel, Dorsal)

dimerization IkB-interaction NLS
Spec.DNA-binding
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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 I?B.
• 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 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

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Two main classes of RHDs
- TAD
TAD
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RHD proteins
Ankyrin repeats
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

C-terminal domain
N-terminal domain
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Structure NFkB (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 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?

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The I-?B family
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The I-?B proteins
Ankyrin repeats
N-terminal Regulatory domain
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The IkB-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
• 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

Specificity Ex. IkB-? inhibits DNA-binding of
p65/p50 but not of p50/p50
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NFkB-IkB complex
IkB
HMG I(Y)
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Signaling

• The chain of events in the canonical NFkB
  signaling pathway

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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

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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 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

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Signaling pathways
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Upstream and downstream
Upstream
Downstream
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Signaling

• The chain of events in the NFkB 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 ??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?
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The I?B-kinase complex central in the pathway
I?B-kinase complex
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The IKKb-kinase becomes activated through
phosphorylation
Signal Upstream kinase

• Activation loop in IKKb
• 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
  NFkB-activation of cytokines
• Attenuation
• phosphorylated activation loop ? altered
  HLH-kinase domain interaction ? reduced
  kinase-aktivitet

IKKß
Ser-OH
Ser-P
Ser-OH
Ser-P
P
P
P
P
inactive
active
inactive
Autophosphorylation
IkB
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Stimulus-specific signal transduction pathways?
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Stimulus-specific signalling pathways?

• Novel IKK-candidates
• IKKe 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

Signal 2
Signal 3
Signal 1
Alternative IKK-kinases
Alternative IKK-complexes
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Why two kinases?

• In vitro IKKa IKKb
• 52 identity
• Similar kinase activity
• In vivo IKKa ? IKKb
• Ala-mutants of IKKß ?? NFkB response dead
• Glu-mutants of IKKß ?? NFkB response independent
  of signals
• Ala-mutants of IKKa ?? NFkB response unaffected
• Glu-mutants of IKKa ?? NFkB response unaffected
• Is IKKa totally unlinked to NFkB?

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The next indication KO phenotypes of IKKa ? IKKb

• 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????? ????????????????????????
• ??????????????, epidermis 5-10x thicker than
  normal, highly undifferentiated
• ???????????s???????l
• Normal number of B- and T-cells, but B-cells not
  fully differentiated

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A separate signaling pathway through IKKa

• 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 depends on IKKa
Target of IKKb
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Model - two divergent pathways through the IKK
complex
Signal 2
NIK
TNF-R
Altered processing of p100
A role in innate immunity
Affect B-cell maturation
A role in adaptive immunity
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Two kinases- two main signaling pathways

• The canonical NF-kB activation pathway (left)
• Applies to RelA-p50 and c-Rel-p50
• Retained in cytoplasm by IkB
• Triggered by microbial and viral infections and
  exposure to proinflammatory cytokines
• Depends mainly on the IKKb subunit of the IKK
  complex.
• The second pathway (right)
• Affects NF-kB2, which preferentially dimerizes
  with RELB.
• Triggered by members of the tumour-necrosis
  factor (TNF) cytokine family
• Depends selectively on activation of the IKKa
  subunit another kinase NIK.
• Induce the phosphorylation-dependent proteolytic
  removal of the IkB-like C-terminal domain of
  NF-kB2.

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Target genes
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Upstream and downstream
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-kB is both being activated by and inducing the
expression of inflammatory cytokines NF-kB
activation can spread from cell to cell
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Negative feedbackAttenuation 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

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Target genesLink 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.

Angiogenesis Metastasis
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Disease links
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Viruses exploit NF-kB

• several patogenic viruses exploit the NF-kB
  system for their own profit
• Incorporation of kB-sites in virus DNA cause
  enhanced expression of virus-genes when the
  immune response is activated
• Virus proteins activate NF-kB

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Disease links
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Constitutivelynuclear NF-kB

• Disruption of the regulatory mechanism ? aberrant
  activation of NFkB 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-kB activation promoting leukemia

• Mechanisms by which NF-kB activation can
  contribute to leukaemia and lymphogenesis
• Input NF-kB 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-kB activation can also
  be brought about by chromosomal rearrangements
  that affect genes that encode NF-kB or I-kB.
• Autocrine loop Once NF-kB 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.
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-kB ligand (RANKL), which
  binds to the receptor activator of NF-kB (RANK).
  This pathway, which leads to activation of NF-kB,
  depends on the IKKa subunit of the IKK complex.
• After nuclear translocation, NF-kB 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 I?B
• H2O2 activates NF-?B
• Induction of ROI (reactive oxygen intermediates)
  a possible common element?
• Proteasome inhibitors

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Therapeutic inhibition of NFkB

• Numerous inhibitors of NF-kB under development.
• Difficult to develop cancer specific inhibitors.
• Understanding the two pathways should lead to
  better therapeutics.