BC212 Signal transduction

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BC212Signal transduction growth
controlDeath receptor signaling
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Lecture overview

• Death receptor signaling pathways
• TNF/TNFR gene-families
• DISC signaling
• NF-kappaB signaling
• ------------------------------------------
• 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 2420, 2002
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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 2420, 2002
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TNF/TNFR families
Goldstein Science 2882388, 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 2882354, 2000
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Lecture overview

• Death receptor signaling pathways
• 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
DED Caspase-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 2420, 2002
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DISC signaling
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DISC signaling
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DISC signaling

• Identifying components
• Fas
• screen for cytotoxic antibodies
• antibody crosslinks/multimerizes Fas
• FasL
• cell line which killed Fas-expressing cells
  (only)
• FasFc blocked killing
• Used biotinylated FasFc to affinity purify FasL
• FADD
• two-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 2420, 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
• DISC signaling
• NF-kappaB signaling

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NFkappaB signaling
Haefner Drug Discovery Today 7653 2002
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NFkappaB signaling
Karin, et al Nat Rev Cancer 2301, 2002
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NFkappaB signaling
Haefner Drug Discovery Today 7653 2002
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NFkappaB signaling
Karin, et al Nat Rev Cancer 2301, 2002
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NFkappaB signaling
Karin, et al Nat Rev Cancer 2301, 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
• Isolation via enhancer affinity chromatography
• PMA (cells) DOC (extracts) induces EMSA
  activity
• PMA stimulates transport to the nucleus
• DOC-dissociable inhibitor (IkappaB)
• NFkB-IkB complex disrupted, EMSA induced, by PKC
• IkB degradation proteosome inhs, Ubn, 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
• DISC signaling
• NF-kappaB signaling
• ------------------------------------------
• Discussion FLIP a switch between death
  survival?

34
FLIP as a switch?