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BC212

Signal transduction & growth control

Death receptor signaling

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Lecture overview

Death receptor signaling pathways:

• TNF/TNFR gene-families

• DISC signaling

• NF-kappaB signaling

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• Discussion: FLIP: a switch between death & survival?

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Lecture overview

Death receptor signaling pathways:

• TNF/TNFR gene-families

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TNF/TNFR families

• Ligands = typically membrane bound; at least 18

• Receptors = one subunit; at least 28

• Receptor-ligand promiscuity

• Decoy receptors

• Trimerization

PLAD: pre-ligand assembly domain

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TNF/TNFR families

Ashkenazi Nat Rev Cancer 2:420, 2002

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TNF/TNFR families

• Ligands = typically membrane bound; at Ligands = typically membrane bound; at least 18least 18

• Receptors = one subunit; at least 28Receptors = one subunit; at least 28

• Receptor-ligand promiscuityReceptor-ligand promiscuity

• Decoy receptors Decoy receptors

• Trimerization

PLAD: pre-ligand assembly domain

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TNF/TNFR families

Ashkenazi Nat Rev Cancer 2:420, 2002

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TNF/TNFR families

Goldstein Science 288:2388, 2000

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TNF/TNFR families

PLAD: pre-ligand assembly domain

ALS (autoimmune lymphoproliferative syndrome): del 52-96 (het)

Siegel et al. Science 288:2354, 2000

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Lecture overview

Death receptor signaling pathways:

• TNF/TNFR gene-families TNF/TNFR gene-families

• DISC signaling

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DISC signaling

• Cell death, survival and cancer

• Intrinsic versus extrinsic death pathways

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DISC signaling

Lost track of this reference

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DISC signaling

Death receptor ligands

TRAIL (DR4, DR5)FasL (Fas)TNF (TNFR1)TWEAK (TWEAK-R)

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DISC signaling

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DISC signaling

Death receptor pathway components:FADD

DD & DEDCaspase-8, -10

DED & caspase (protease)FLIP

Dominant inhibitor caspase relative

Caspase-3 (etc.)

Extrinsic pathway (via Bid)

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DISC signaling

Ashkenazi Nat Rev Cancer 2:420, 2002

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DISC signaling

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DISC signaling

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DISC signaling

• Identifying components

Fasscreen for cytotoxic antibodiesantibody crosslinks/multimerizes Fas

FasLcell line which killed Fas-expressing cells (only)FasFc blocked killingUsed biotinylated FasFc to affinity purify FasL

FADDtwo-hybrid screen for DD interactors

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DISC signaling

• lpr and gld mice

Lymphoproliferative phenotype

Resembles SLE

lpr: homozygous mutation in Fas DD

gld: homozygous inactivating mutation in FasL

Implicates Fas-FasL in immune cell apoptosis

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DISC signaling

Ashkenazi Nat Rev Cancer 2:420, 2002

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DISC signaling

Induced proximity model of caspase activation

Parallels with kinase activation

Evidence:

i) artificial dimerization activates caspase

ii) zymogen has low, but detectable, activity

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Lecture overview

Death receptor signaling pathways:

• TNF/TNFR gene-families TNF/TNFR gene-families

• DISC signalingDISC signaling

• NF-kappaB signaling

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NFkappaB signaling

Haefner Drug Discovery Today 7:653 2002

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NFkappaB signaling

Karin, et al Nat Rev Cancer 2:301, 2002

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NFkappaB signaling

Haefner Drug Discovery Today 7:653 2002

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NFkappaB signaling

Karin, et al Nat Rev Cancer 2:301, 2002

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NFkappaB signaling

Karin, et al Nat Rev Cancer 2:301, 2002

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NFkappaB signaling

• Evidence linking NFkappaB to proliferation/cancer

RelA: viral NFkappaB homolog

incrd stability; decrd susceptibility to degradation

Bcl3: chromosomal translocations in leukemia

over-expression

NFKB2: chromosomal translocation in leukemia/lymphoma

deletes IkappaB part

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NFkappaB signaling

Evidence establishing mechanism of NFkappaB signaling

1. Isolation via enhancer affinity chromatography

2. PMA (cells) & DOC (extracts) induces EMSA activity

3. PMA stimulates transport to the nucleus

4. DOC-dissociable inhibitor (IkappaB)

5. NFkB-IkB complex disrupted, EMSA induced, by PKC

6. IkB degradation: proteosome inhs, Ub’n, etc.

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NFkappaB signaling

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NFkappaB signaling

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Lecture overview

Death receptor signaling pathways:

• TNF/TNFR gene-families TNF/TNFR gene-families

• DISC signalingDISC signaling

• NF-kappaB signalingNF-kappaB signaling

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• Discussion: FLIP: a switch between death & survival?

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FLIP as a switch?