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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?