BC 212 Signal transduction growth control Death receptor

BC 212 Signal transduction & growth control Death receptor signaling BC 212/week 7/2003

Lecture overview Death receptor signaling pathways: • TNF/TNFR gene-families • DISC signaling • NF-kappa. B signaling --------------------- • Discussion: FLIP: a switch between death & survival? BC 212/week 7/2003

Lecture overview Death receptor signaling pathways: • TNF/TNFR gene-families BC 212/week 7/2003

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 BC 212/week 7/2003

TNF/TNFR families Ashkenazi Nat Rev Cancer 2: 420, 2002 BC 212/week 7/2003

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 BC 212/week 7/2003

TNF/TNFR families Ashkenazi Nat Rev Cancer 2: 420, 2002 BC 212/week 7/2003

TNF/TNFR families Goldstein Science 288: 2388, 2000 BC 212/week 7/2003

TNF/TNFR families PLAD: pre-ligand assembly domain ALS (autoimmune lymphoproliferative syndrome): del 52 -96 (het) Siegel et al. Science 288: 2354, 2000 BC 212/week 7/2003

Lecture overview Death receptor signaling pathways: • TNF/TNFR gene-families • DISC signaling BC 212/week 7/2003

DISC signaling • Cell death, survival and cancer • Intrinsic versus extrinsic death pathways BC 212/week 7/2003

DISC signaling Lost track of this reference BC 212/week 7/2003

DISC signaling Death receptor ligands TRAIL (DR 4, DR 5) Fas. L (Fas) TNF (TNFR 1) TWEAK (TWEAK-R) BC 212/week 7/2003

DISC signaling BC 212/week 7/2003

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) BC 212/week 7/2003

DISC signaling Ashkenazi Nat Rev Cancer 2: 420, 2002 BC 212/week 7/2003

DISC signaling BC 212/week 7/2003

DISC signaling • Identifying components Fas screen for cytotoxic antibodies antibody crosslinks/multimerizes Fas. L cell line which killed Fas-expressing cells (only) Fas. Fc blocked killing Used biotinylated Fas. Fc to affinity purify Fas. L FADD two-hybrid screen for DD interactors BC 212/week 7/2003

DISC signaling • lpr and gld mice Lymphoproliferative phenotype Resembles SLE lpr: homozygous mutation in Fas DD gld: homozygous inactivating mutation in Fas. L Implicates Fas-Fas. L in immune cell apoptosis BC 212/week 7/2003

DISC signaling Ashkenazi Nat Rev Cancer 2: 420, 2002 BC 212/week 7/2003

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 BC 212/week 7/2003

Lecture overview Death receptor signaling pathways: • TNF/TNFR gene-families • DISC signaling • NF-kappa. B signaling BC 212/week 7/2003

NFkappa. B signaling BC 212/week 7/2003 Haefner Drug Discovery Today 7: 653 2002

NFkappa. B signaling BC 212/week 7/2003 Karin, et al Nat Rev Cancer 2: 301, 2002

NFkappa. B signaling BC 212/week 7/2003 Haefner Drug Discovery Today 7: 653 2002

NFkappa. B signaling BC 212/week 7/2003 Karin, et al Nat Rev Cancer 2: 301, 2002

NFkappa. B signaling • Evidence linking NFkappa. B to proliferation/cancer Rel. A: viral NFkappa. B homolog incrd stability; decrd susceptibility to degradation Bcl 3: chromosomal translocations in leukemia over-expression NFKB 2: chromosomal translocation in leukemia/lymphoma deletes Ikappa. B part BC 212/week 7/2003

NFkappa. B signaling Evidence establishing mechanism of NFkappa. B 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 (Ikappa. B) 5. NFk. B-Ik. B complex disrupted, EMSA induced, by PKC 6. Ik. B degradation: proteosome inhs, Ub’n, etc. BC 212/week 7/2003

NFkappa. B signaling BC 212/week 7/2003

Lecture overview Death receptor signaling pathways: • TNF/TNFR gene-families • DISC signaling • NF-kappa. B signaling --------------------- • BC 212/week 7/2003 Discussion: FLIP: a switch between death & survival?

FLIP as a switch? BC 212/week 7/2003