The NF-  B/Rel family

1. The NF-B/Rel family

2. 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-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

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-κB/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 IkB HMG I(Y) • --5´-GGGRNNYYCC-3´-- • - 3´-CCCYNNRRGG-5´--

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 • IκB - a key element in the canonical NFkB signaling pathway

21. 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 Signal

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. . . . . Signal transduction pathways + + NF-kB + 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

26. Signals Cytoplasm inactive Nucleus active Three signal transduction pathways

27. 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

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. 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

31. The first pathway - the classical pathway dep on IKKβ Triggered by microbial and viral infections and exposure to proinflammatory cytokines

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. A separate signalling pathwayinvolving 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 phosphorylation-dependent proteolytic removal of the IkB-like C-terminal domain of NF-κB2 B-cell maturation A role in adaptive immunity A role in innate 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. 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)

39. Connectivity map of the TNF-α/NF-κB signal transduction pathway

40. Target genes

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

42. 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

43. 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

44. 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

45. Disease links

46. 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

47. Disease links

48. 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

49. 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,

50. 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.