The NF-  B/Rel family

1. The NF-B/Rel family

2. 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

3. 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

4. Signals Cytoplasm inactive Nucleus active Signal transduction pathway

5. NF-B/Rel proteins

6. 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

7. 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

8. 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

9. Rel homology domain p105 C-terminal IkB-like domains p50 p100 p52 RelA(p65) cRel Acitvation domains RelB Two main classes of RHDs - TAD +TAD

10. RHD proteins Ankyrin repeats RHD

11. 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´--

12. 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

13. Structure: NFkB (p50-p65) + DNA Side view • -barrel core with protrding loops • The AB loop = recognition loop • Specificity R57-R59-E63 • C62 redox-sensitivity

14. 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

15. 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?

16. The I-B family

17. The I-B proteins Ankyrin repeats N-terminal Regulatory domain

18. 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

19. NFkB-IkB complex IkB HMG I(Y)

20. Signaling • The chain of events in the canonical NFkB signaling pathway

21. 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

22. 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

23. Signaling pathways

24. . . . . Signal transduction pathways + + NF-kB + Upstream and downstream Upstream Downstream

25. 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

26. 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

27. 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?

28. The IB-kinase complex central in the pathway IB-kinase complex

29. 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

30. Stimulus-specific signal transduction pathways?

31. 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

32. Signal upstream kinase IKKß Ser-OH Ser-P Ser-OH Ser-P inactive active IkB 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?

33. 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

34. 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

35. The solution Processing depends on IKKa Target of IKKb

36. 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

37. 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.

38. Target genes

39. . . . . Signal transduction pathways + + NF-kB + Upstream and downstream Upstream Downstream

40. 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

41. 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

42. 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

43. Disease links

44. 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

45. Disease links

46. 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

47. Link: inflammation - cancer • A causal connection between inflammation and cancer has been suspected for many years. • NF-Bmight serve as the missing link between these two processes. • NF-Bbecomes activated in response to inflammatory stimuli • Constitutive activation of NF-Bhas been associated with cancer,

48. 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.

49. 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.

50. 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