The NF- B/Rel family

The NF-B/Rel family

MBV4230Odd S. Gabrielsen

The NF-B/Rel familyA family of signal-responsive transcription factorsrapid response som ikke requires proteinsynteseInvolved in proinflammatory response: a first line of defense against infectious diseases and cellular stressSignal Activated NF-B immune defence activatedImmune response, inflammatory response, accute phase responseNFkB 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, AlzheimerPersistent activated in many cancers - help keeping cancer cells aliveNFkB also promoting growthActivated NF-B cyclin D expression enhanced growthDrug against NFkB = putative anti-cancer drug


The NF-B/Rel familyCharacteristic feature: homo- and heterodimeric TFs, which in non-stimulated cells are found inactive in the cytoplasm [in a complex with IB-repressors]. Active DNA-binding form: Dimers with different members of the NF-B/Rel familyInactive 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 - minutesSignal Activated NF-B immune defence activated


Signal transduction pathway

NF-B/Rel proteins


Common DBD: Rel-homology domain (RHD)RHD: 300aa conserved domain with several functionsDNA-binding (N-terminal half)dimerization (C-terminal half)IB-interaction (C-terminal half)NLS (C-terminal half)kalles ogs NRD (=NF-kB, Rel, Dorsal)Spec.DNA-bindingdimerizationIkB-interactionNLS


Homo- and heterodimersNF-B/Rel proteins = Homo- and hetero-dimeric TFs that in resting cells are retained in the cytoplasm in complex with IB.

Mature B-cells: constitutively nuclear activatorBound to kappa immunoglobuline light-chain enhancer its name


Two main classes of RHDsRel with TAD (dimeric with 1 Rel-monomers which are potent transactivators) synthesized in their mature formRel or c-Rel (as well as v-Rel)RelA (p65)RelBDrosophilas dorsal and Difp50/52 without TAD (homodimers with no transactivation properties) synthesized as precursors that are processedPrecursor forms have internal IB inhibitor functionRHD linked to inhibitory domain through Gly-rich linker (protease sensitive)Blocks DNA-binding and translocation to nucleusp105 undergoes proteolytic maturation to p50 [NF-B1]Proteolytic degradation to p50 is signal dependent, requires ATP and occurs through a ubiquitin-dependent proteasome pathwayAlso transcription from an intronic promoter expressionof IkB-p100 undergoes proteolytic maturation to p52 [NF-B2]p50/52 are distinct gene products with very similar properties


Two main classes of RHDs+TAD- TAD


RHD proteinsAnkyrinrepeatsRHD


Dimer-formationDimer-formation necessary for DNA-bindingeach subunit interacts with one half site B-sites symmetric: 5-GGGRNNYYCC-3Most combinations allowedDifferent heterodimers vary with respect topreference for different kB-seterKinetics of nuclear translocationp50/p65 rapid, p50/Rel slowabundance in different cellsException: RelB which forms dimer only with p50/p52Common form: p50/p65 (NF-kB1/RelA)most abundant, found in most cells--5-GGGRNNYYCC-3--- 3-CCCYNNRRGG-5--


3D structure - DNA interactionCrystal structures: p50-p50-DNA and p50-p65-DNATwo distinct domains1. N-terminal - specific DNA contactCompact core in the form of an antiparalell -barrel from which loops protrudeThe loop between AB = recognition loop with base contacts in major grooveCritical for specificity = R57-R59-E63C62 responsible for redox-sensitivity2. C-terminal domain responsible for dimerisation + nonspecific DNA-phosphate contact Conserved interphase explains why most heterodimers are possibleN-terminaldomainC-terminaldomain


Structure: NFkB (p50-p65) + DNASide view -barrel core with protrding loops The AB loop = recognition loop Specificity R57-R59-E63 C62 redox-sensitivity


3D structure - DNA interactionCharacteristic features of DNA-interactionEach monomer contacts a separate half siteClosing jaws mechanism for DNA-bindingThe protein encloses DNAUnusual strong binding (Kd = 10-12 M)Dissociation requires opening of the jaws through a flexible linker


3D structure - protein interactionInteraction with HMGI(Y)IFN- promoter: HMGI(Y) binds AT-rich centre of B-sites in minor grooveThe structure contains a corresponding open space

Interaction with IBIB binding in an opening over the dimer-interphaseIB binding blocks DNA-binding

HMGI(Y)IkB--5-GGGRNNYYCC-3--- 3-CCCYNNRRGG-5--

The I-B family


The I-B proteinsN-terminal Regulatory domainAnkyrinrepeats


The IkB-familyInhibitory functionimpedes DNA-bindingblocks NLS and abolish translocation to nucleusSeveral members (at least 7 mammalian)IB- and IB-IB-and IB-Bcl-3p105 and p110IkBRCommon features: ankyrin-repeats which are necessary for RHD-interaction30-33 aa motif repeated 3 - 7xC-terminal acidic-region necessary for inhibition of DNA-bindingC-terminal PEST-sequence involved in protein-degradationSpecificityEx. IkB- inhibits DNA-binding of p65/p50 but not of p50/p50


NFkB-IkB complexIkBHMGI(Y)


SignalingIB - a key element in the canonical NFkB signaling pathway


Cytoplasmic retention due to interaction with IB-family proteinsTwo types of inactive complexes in cytoplasmTrimers = RHD-Homo-or heterodimers bound to an IBHeterodimers = Rel-protein + unprocessed RHD-precursor (p105, p110)Signal[dissociation] degradationInduction signal phosphorylation of both IB and p105 IB degradation or p105 processering active dimers that are translocated to the nucleus.One type of signal two N-terminal serines (S32 and S36) become phosphorylatedAnother type of signal two C-terminal serines become phosphorylated in p105phosphorylation probably more a signal for degradation than for dissociationUbiquitin-pathway involvedStimulation rapid degradation of IBcomplete after 10 minNo traces of IBphosphorylation of IB multiubiquitylation in K21, K22 degradation through a ubiquitin-proteasome pathway+ proteasome-inhibitors phospho-IkB remains associated with NFkBSignalSignal


Several IB-factors with different propertiesIB-: Rapid transient responseIB- best characterizedall stimuli degradation of IB-ex: TNF-rapid and transient activation of NF-kBIB-: Sustained responseOnly certain stimuli degradation of IB-ex: LPS or IL-1degradation of both IB-and IB- activation of NF-kB lasting for hoursBcl-3: repressor and activatorinhibits certain complexes like a normal IBBut may also associate with DNA-bound p50 and p52 dimers (lacking TAD) and provide transactivation properties

Signaling pathways


Upstream and downstreamUpstreamDownstream


Multiple signalling pathways activate NF-BSeveral signalling pathways converge by activation of NF-BNF-B respond to a broad range of different stimuliVirus infection (HIV, hepatite B), virus proteins (tax, E1A) and dsRNACytokines (TNF, IL-1 and IL-2)Bacterial LPSstimulation of antigen reseptor on B- and T-cellscalcium ionophoresprotein synthesis inhibitorsUV and X-raysphingomylenase/ceramidephorbol estersnitrogen oxide


Three signal transduction pathways


Signaling hits I-B through phosphorylationTwo N-terminal serines becomes phosphorylatedTNF-signalling pathways: TNF-receptor TRADD/TRAF IKK IB

IB-kinase complex central in the signaling pathwayA large 500-900 kDa IKK (IB-kinase) complex that is induced by cytokinesTwo key subunits: IKK and IKK


The IB-kinase complex central in the pathwayIB-kinase complex


The IKKb-kinase becomes activated through phosphorylationActivation loop in IKKbTwo 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 cytokinesAttenuationphosphorylated activation loop altered HLH-kinase domain interaction reduced kinase-aktivitetSer-OHSer-OHSer-PSer-PSignalUpstream kinaseinactiveactiveIKKIkBinactivePPPPAutophosphorylation


The first pathway - the classical pathwayReceptor 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) complexwhich includes the scaffold protein NEMO (NF-kB essential modulator; also named IKK), IKK and IKK kinases. The IKK complex phosphorylates IB on Ser32 and Ser36Leading to ubiquitylation and degradation via the proteasome pathwayThe free p50-p65 migrates to the nucleus where it activates target genes involved in immune response


The first pathway - the classical pathway dep on IKKTriggered by microbial and viral infections and exposure to proinflammatory cytokines


Why two kinases?In vitro: IKKa IKKb 52% identitySimilar kinase activityIn vivo: IKKa IKKbAla-mutants of IKK NFkB response dead Glu-mutants of IKK NFkB response independent of signalsAla-mutants of IKKa NFkB response unaffected Glu-mutants of IKKa NFkB response unaffectedIs IKKa totally unlinked to NFkB?


The next indication: KO phenotypes of IKKa IKKbKnock-out of of IKKloss of B- and T-cell responseNormal developmentMice dead at day 13.5, liver destroyed due to massive apoptosisLack 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 undifferentiatedslNormal number of B- and T-cells, but B-cells not fully differentiated


A separate signaling pathway through IKKaA desparate postdoc looked at all the 50 components - all behaved normal, except oneThe proteolytic maturation of the p100 precursor to p52 [NF-B2] was defective in the IKK

processing depends on NIKHypothesis: NIK acts through IKK


The solutionProcessing dependson IKKaTarget ofIKKb


A separate signalling pathwayinvolving only IKKAffects 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 immunityB-cell maturationA role in adaptive immunity


Two kinases- two main signaling pathwaysThe canonical NF-kB activation pathway (left)Applies to RelA-p50 and c-Rel-p50Retained in cytoplasm by IkBTriggered by microbial and viral infections and exposure to proinflammatory cytokinesDepends 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 familyDepends 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.


A third signalling pathway independent on both IKKsclassified as atypical because it is independent of IKKproteasome still requiredtriggered by DNA damage such as UV or doxorubicinUV 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-/NF-B signal transduction pathway


Seminar

Target genes


Upstream and downstreamUpstreamDownstream


Families of target genesImmune responseCytokines, ChemokinesCytokine and immuno-receptorsAdhesion moleculesAcute-phase proteinsStress-responsive genes

NF-kB is both being activated by and inducing the expression of inflammatory cytokinesNF-kB activation can spread from cell to cell


Negative feedback:Attenuation of responsNegative loop: IB- under direct control of NF-BActivated NF-B translocated to the nucleus will activate expression of IB-Newly synthesized IB-will bind up and inactivate remaining NF-B in the cytoplasmaExcess IB-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 proteinImmunosupressive effect of glucocorticoidsProbably a direct effect of glucocorticoids enhancing the expression of IB-which then binds up and inactivates NF-B in the cytoplasm, leading to reduced immune- and inflammatory response


Target genes:Link to cancerTumorigenesis requires 6 types of alterationsHanahan & Weinberg 2000Several of these can be caused by perturbation in NF-B or linked signaling moleculesTumour cells in which NF-B is constitutively active are highly resistant to anticancer drugs or ionizing radiation.AngiogenesisMetastasis

Disease links


Viruses exploit NF-kBseveral patogenic viruses exploit the NF-kB system for their own profitIncorporation of kB-sites in virus DNA cause enhanced expression of virus-genes when the immune response is activatedVirus proteins activate NF-kB


Disease links


Constitutivelynuclear NF-kBDisruption of the regulatory mechanism aberrant activation of NFkB = one of the primary causes of a wide range of human diseasesInflammatory diseasesRheumatoid arthritisAsthmaAtherosclerosisAlzheimer


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 stimuliConstitutive activation of NF-B has been associated with cancer,


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.


Breast cancer: Signalling pathways that stimulate proliferationSignaling 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.


Blocking the responseRedox-dependencyAntioxidants and alkylating agens inhibit response to many stimuli and inhibit phosphorylation and degradation of IBH2O2 activates NF-BInduction of ROI (reactive oxygen intermediates) a possible common element?Proteasome inhibitors


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

The NF- B/Rel family

Documents

Transcript of The NF- B/Rel family

NF- κ B Signaling Adam Johnson. What is NF- κ B? Transcription Factor Dimer of Rel and NF- κ B proteins RelA RelB c-Rel p50 p52.

Kinetis K24F Sub-Family Data Sheet - NXP Semiconductors · Kinetis K24F Sub-Family Data Sheet 120 MHz ARM® Cortex®-M4-based Microcontroller with FPU The K24 product family members

Kostas Tsingos - Ifigenia Tsoka family tree

The NF-κB/Rel family - Universitetet i oslo · MBV4230 Odd S. Gabrielsen The NF-κB/Rel family Characteristic feature: homo- and heterodimeric TFs, which in non-stimulated cells

The NF-kB subunit c-Rel regulates Bach2 tumour …eprint.ncl.ac.uk/file_store/production/215550/266F3FA7-2...The NF-κB subunit c-Rel regulates Bach2 tumour suppressor expression in

Structural and functional characterization of a family ...

Review Cancer Biology - OA Publishing · 2014. 6. 5. · family of transcription factors that include RelA (p65), RelB, c-Rel, NF-κB1 (p105/p50) and NF-κB2 (p100/ p52). NF-κB is

SLE Family Medical/Industrial rade

Family Violence Conference Faneromeni Gymnasium May 2015

NF- κ B Signaling

THP1-Blue NF-κB Cells · NF-kB cells allow the monitoring of NF- B activation by determining the activity of SEAP. The levels of NF-kB-induced SEAP in the cell culture supernatant

INVESTIGATING THE ROLE OF SRC FAMILY

Rel algebra set theory girija

SX-A Family FPGAs Datasheet - Microsemi

Earth-leakage Circuit Breakers Economical Series · PDF fileEarth-leakage Circuit Breakers Economical Series Frame Size (A) 30 50 60 100 225 400 Type ZE30-NF ZE50-NF ZE60-NF ZE100-NF

MC/NF TARGET SHIELDING STUDIES.

Magnolol Inhibits LPS-induced NF- κB/Rel … MH Li, et al METHODS Materials Magnolol and LPS from Salmonella thyposa was pur-chased from Sigma (St. Louis, MO). Reagents used for cell

Rel algebra selection girija

Kinetis KL15 Sub-Family - NXP Semiconductors · Kinetis KL15 Sub-Family, Rev5 08/2014.

For Research Use Only NF-M Polyclonal ANTIBODYBackground Information NEFM, also named as NEF3 and NFM, belongs to the intermediate filament family. Neurofilaments are the 10nm intermediate