The NF-κB/Rel family
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 Immune response, inflammatory response, accute phase response 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 Activated NF-κB cyclin D expression enhanced growth Drug against NFkB = putative anti-cancer drug
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
Signal transduction pathway Signal Cytoplasm inactive Nucleus active
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) Spec.DNA-binding dimerization IkB-interaction NLS kalles også NRD (=NF-kB, Rel, Dorsal)
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
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
Two main classes of RHDs Rel homology domain +TAD - TAD p105 p50 p100 p52 RelA(p65) crel RelB C-terminal IκB-like domains Acitvation domains
RHD proteins Ankyrin repeats RHD
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 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 --
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 Conserved interphase explains why most heterodimers are possible C-terminal domain N-terminal domain
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
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 d = 10-12 M) Dissociation requires opening of the jaws through a flexible linker
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 --
The I-κB family
The I-κB proteins Ankyrin repeats N-terminal Regulatory domain
The IκB-family Inhibitory function impedes DNA-binding blocks NLS and abolish translocation to nucleus Several members (at least 7 mammalian) IκB-α and IκB-β IκB-γ and IκB-ε Bcl-3 p105 and p110 IkBR Common features: ankyrin-repeats which are necessary for RHD-interaction 30-33 aa motif repeated 3-7x Specificity Ex. IkB-α inhibits DNA-binding of p65/ p50 but not of p50/p50 C-terminal acidic-region necessary for inhibition of DNA-binding C-terminal PEST-sequence involved in protein-degradation
NFκB-IκB complex IkB HMG I(Y)
Signaling IκB - a key element in the canonical NFκB signaling pathway
Cytoplasmic retention due to interaction with IκB-family proteins Two types of inactive complexes in cytoplasm Trimers = RHD-Homo-or heterodimers bound to an IκB Heterodimers = Rel-protein + unprocessed RHD-precursor (p105, p110) Signal [dissociation] degradation Signal Signal 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 multiubiquitylation in K21, K22 degradation through a ubiquitin-proteasome pathway + proteasome-inhibitors phospho-ikb remains associated with NFkB
MBV4230 Several IκB-factors with different properties IκB-α: Rapid transient response IκB-α best characterized all stimuli degradation of IκB-α ex: TNF-α rapid and transient activation of NF-kB IκB-β: Sustained response 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
Signaling pathways
Upstream and downstream Upstream Signal transduction pathways. + + NF-kB Downstream +..
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 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 +..
Three signal transduction pathways Signal Cytoplasm inactive Nucleus active
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
The IκB-kinase complex central in the pathway IκB-kinase complex
The IKKβ-kinase becomes activated through phosphorylation Activation loop in IKKβ 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 Signal Upstream kinase Ser-P Ser-OH Ser-P inactive Signal phosphorylation phosphorylation of loop necessary for NFκB-activation of cytokines Attenuation phosphorylated activation loop altered HLH-kinase domain interaction reduced kinase-aktivitet IκB active P P PP inactive Autophosphorylation
The first pathway - the classical pathway Receptor triggered by pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α 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 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
The first pathway - the classical pathway dep on IKKβ Triggered by microbial and viral infections and exposure to proinflammatory cytokines
Why two kinases? In vitro: IKKα IKKβ 52% identity Similar kinase activity Signal upstream kinase In vivo: 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 inactive Ser-OH Ser-OH IκB active Ser-P Ser-P
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
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
The solution Processing depends on IKKα Target of IKKβ
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 C- terminal domain of NF-κB2 A role in innate immunity B-cell maturation A role in adaptive immunity
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.
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 C- terminal cluster, which is recognized by the p38- activated casein kinase 2 (CK2)
Connectivity map of the TNF-α/NF-κB signal transduction pathway
Target genes
Upstream and downstream Upstream Signal transduction pathways. + + NF-kB Downstream +..
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
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
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
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
Disease links
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
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,
MBV4230 Mechanisms of NF-κB activation promoting leukemia Mechanisms by which NF-κB activation can contribute to leukaemia and lymphogenesis 1. 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. 2. Gene errors: Persistent NF-κB activation can also be brought about by chromosomal rearrangements that affect genes that encode NF-κB or I-κB. 3. 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. 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-X L, ciaps and A1/BFL1. 1. 2. 3. 4. Tumour cells in which NF-κB is constitutively active are highly resistant to anticancer drugs or ionizing radiation.
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.
Blocking the response Redox-dependency Antioxidants and alkylating agens inhibit response to many stimuli and inhibit phosphorylation and degradation of IκB H 2 O 2 activates NF-κB Induction of ROI (reactive oxygen intermediates) a possible common element? Proteasome inhibitors
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.
Example of previously given question for the exam Signalling pathway Describe briefly the key elements of signalling pathways involving IKK and NF-κB. 54