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The NF- B/Rel family Slide 2 MBV4230 Odd S. Gabrielsen 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 cancer cells alive NFkB also promoting growth Activated NF- B cyclin D expression enhanced growth Drug against NFkB = putative anti-cancer drug Slide 3 MBV4230 Odd S. Gabrielsen 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 Slide 4 MBV4230 Odd S. Gabrielsen Signal transduction pathway Cytoplasm inactive Nucleus active Signals Slide 5 NF-B/Rel proteins Slide 6 MBV4230 Odd S. Gabrielsen 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 ogs NRD (=NF-kB, Rel, Dorsal) Spec.DNA-binding dimerization IkB-interaction NLS Slide 7 MBV4230 Odd S. Gabrielsen 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 Slide 8 MBV4230 Odd S. Gabrielsen 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 Slide 9 MBV4230 Odd S. Gabrielsen Two main classes of RHDs p105 p50 p100 p52 RelA(p65) cRel RelB Rel homology domain C-terminal I B-like domains Acitvation domains +TAD - TAD Slide 10 MBV4230 Odd S. Gabrielsen RHD proteins Ankyrin repeats RHD Slide 11 MBV4230 Odd S. Gabrielsen 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-- Slide 12 MBV4230 Odd S. Gabrielsen 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 = R 57 -R 59 -E 63 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-terminal domain C-terminal domain Slide 13 MBV4230 Odd S. Gabrielsen 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 Slide 14 MBV4230 Odd S. Gabrielsen 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 Slide 15 MBV4230 Odd S. Gabrielsen 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 HMG I(Y) IkB --5-GGGRNNYYCC-3-- - 3-CCCYNNRRGG-5-- Slide 16 The I- B family Slide 17 MBV4230 Odd S. Gabrielsen The I- B proteins N-terminal Regulatory domain Ankyrin repeats Slide 18 MBV4230 Odd S. Gabrielsen 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 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 Slide 19 MBV4230 Odd S. Gabrielsen NF B-I B complex IkB HMG I(Y) Slide 20 MBV4230 Odd S. Gabrielsen Signaling IB - a key element in the canonical NF B signaling pathway Slide 21 MBV4230 Odd S. Gabrielsen Cytoplasmic retention due to interaction with I B-family proteins Signal Two types of inactive complexes in cytoplasm Trimers = RHD-Homo-or heterodimers bound to an I B Heterodimers = Rel-protein + unprocessed RHD-precursor (p105, p110) Signal [dissociation] 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-proteasome pathway + proteasome-inhibitors phospho-IkB remains associated with NFkB Slide 22 MBV4230 Odd S. Gabrielsen 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 Slide 23 Signaling pathways Slide 24 MBV4230 Odd S. Gabrielsen Upstream and downstream NF- k B Signal transduction pathways + +.. +.. Upstream Downstream Slide 25 MBV4230 Odd S. Gabrielsen NF- k B Signal transduction pathways + +.. +.. 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 Slide 26 MBV4230 Odd S. Gabrielsen Three signal transduction pathways Cytoplasm inactive Nucleus active Signals Slide 27 MBV4230 Odd S. Gabrielsen Signaling hits I- B through phosphorylation Two N-terminal serines becomes phosphorylated TNF-signalling pathways: TNF-receptor TRADD/TRAF 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 Slide 28 MBV4230 Odd S. Gabrielsen The I B-kinase complex central in the pathway I B-kinase complex Slide 29 MBV4230 Odd S. Gabrielsen 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 NF B-activation of cytokines Attenuation phosphorylated activation loop altered HLH-kinase domain interaction reduced kinase-aktivitet Ser-OH Ser-P Signal Upstream kinase inactive active IKK IBIB inactive PP P P Autophosphorylation Slide 30 MBV4230 Odd S. Gabrielsen The first pathway - the classical pathway Receptor triggered by pro-inflammatory cytokines such as tumour necrosis factor (TNF)- 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 IB-kinase (IKK) complex which includes the scaffold protein NEMO (NF-kB essential modulator; also named IKK ), IKK and IKK kinases. The IKK complex phosphorylates IB 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 Slide 31 MBV4230 Odd S. Gabrielsen The first pathway - the classical pathway dep on IKK Triggered by microbial and viral infections and exposure to proinflammatory cytokines Slide 32 MBV4230 Odd S. Gabrielsen Why two kinases? Ser-OH Ser-P Signal upstream kinase inactive active IKK IBIB In vitro: IKK IKK 52% identity Similar kinase activity In vivo: IKK IKK Ala-mutants of IKK NF B response dead Glu-mutants of IKK NF B response independent of signals Ala-mutants of IKK NF B response unaffected Glu-mutants of IKK NF B response unaffected Is IKK totally unlinked to NF B? Slide 33 MBV4230 Odd S. Gabrielsen 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 , epidermis 5-10x thicker than normal, highly undifferentiated s l Normal number of B- and T-cells, but B-cells not fully differentiated Slide 34 MBV4230 Odd S. Gabrielsen 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 Slide 35 MBV4230 Odd S. Gabrielsen The solution Processing depends on IKK Target of IKK Slide 36 MBV4230 Odd S. Gabrielsen A separate signalling pathway involving only IKK Affects NF-B2 (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, IKK- dependent + another kinase NIK. Induce the phosphorylation- dependent proteolytic removal of the IkB-like C- terminal domain of NF-B2 A role in innate immunity B-cell maturation A role in adaptive immunity Slide 37 MBV4230 Odd S. Gabrielsen 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 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 I B-like C- terminal domain of NF- B2. Slide 38 MBV4230 Odd S. Gabrielsen 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) Slide 39 MBV4230 Odd S. Gabrielsen Connectivity map of the TNF-/NF-B signal transduction pathway Slide 40 MBV4230 Odd S. Gabrielsen Seminar Slide 41 Target genes Slide 42 MBV4230 Odd S. Gabrielsen Upstream and downstream NF- k B Signal transduction pathways + +.. +.. Upstream Downstream Slide 43 MBV4230 Odd S. Gabrielsen 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 Slide 44 MBV4230 Odd S. Gabrielsen 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 Slide 45 MBV4230 Odd S. Gabrielsen 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. Angiogenesis Metastasis Slide 46 Disease links Slide 47 MBV4230 Odd S. Gabrielsen 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 Slide 48 MBV4230 Odd S. Gabrielsen Disease links Slide 49 MBV4230 Odd S. Gabrielsen 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 Slide 50 MBV4230 Odd S. Gabrielsen 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, Slide 51 MBV4230 Odd S. Gabrielsen 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-X L, 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. Slide 52 MBV4230 Odd S. Gabrielsen 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. Slide 53 MBV4230 Odd S. Gabrielsen Blocking the response Redox-dependency Antioxidants and alkylating agens inhibit response to many stimuli and inhibit phosphorylation and degradation of I B H 2 O 2 activates NF- B Induction of ROI (reactive oxygen intermediates) a possible common element? Proteasome inhibitors Slide 54 MBV4230 Odd S. Gabrielsen 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.