Proapoptotic BAX and BAK control multiple initiator caspases Lab meeting; Antonio Ruiz-Vela 9/23/03

Bax and Bak Double-Knockout MEFs #1 Resistant to BH3-only-induced apoptosis .BIM .t-Bid .BAD .Noxa #2 Resistant to DNA-damaging agents .Etoposide .UV light .Cisplatinum .Doxorubicin #3 Resistant to chronic UPR-induced apoptosis .Tunicamycin .Brefeldin A

BAX and BAK gateway? ? ? ER stress-induced apoptosis ER membrane “BH3-only” CED-4 like CASP-12 ? Misfolded proteins ER stress-induced apoptosis “BH3-only” CED-4 like CASP-2 ? Mito. membrane DSS DNA damage-induced apoptosis CASP-3, -7 IRE-1 TRAF2 CASP-12 CASP-3, -7 RAIDD CASP-2 BAX and BAK gateway?

#1 BCL-2 function upstream of BAX and BAK gateway #2 Caspases upstream of BAX and BAK gateway #3 Caspase activation always requires APAF-1/ CASP-9

“DNA damage-induced apoptosis” “ER stress-induced apoptosis” DKO/ GFP MEFs “DNA damage-induced apoptosis” CED-4 like CASP -2 ? WT/ GFP DKO/ GFP WT/ hBCL-2 DKO/ hBCL-2 hBCL-2 Actin “ER stress-induced apoptosis” CED-4 like CASP-12 ?

“DNA damage-induced apoptosis” “ER stress-induced apoptosis” WT/ GFP DKO/ GFP WT/ hBCL-2 DKO/ hBCL-2 hBCL-2 Actin DKO/ BCL-2 MEFs “DNA damage-induced apoptosis” CED-4 like CASP -2 ? “ER stress-induced apoptosis” CASP-12

Etoposide (5 mM) proCASP-7 p20 p17 CASP-3 WT DKO Etoposide (mM) - 5 50 5 50 - 5 50 5 50 WT DKO Etoposide (mM) 12h 24h

WT CASP-9-/- P19 (Cleaved CASP-3) proCASP-7 p2O (Cleaved CASP-7) ETO-24 ETO-24 P19 (Cleaved CASP-3) proCASP-7 p2O (Cleaved CASP-7) proCASP-9 P37 (Cleaved CASP-9)

WT DKO FL5.12 ETO-24 ETO-24 +IL-3 -IL-3 proCASP-2 p32 (Cleaved CASP-2) P19 (Cleaved CASP-2)

- WT DKO - 4h 12h 24h - 4h 12h 24h CHOP/ GADD153 28S 18S P17 (Cleaved CASP-3)

Brefeldin A (0.5 mg/ml) proCASP-7 p20 p17 CASP-3 WT DKO - 0.5 5 0.5 5 - 0.5 5 0.5 5 WT DKO Brefeldin A (mg/ml) 12h 24h

WT CASP-9-/- P19 (Cleaved CASP-3) proCASP-7 proCASP-9 BA-24 BA-24 P19 (Cleaved CASP-3) proCASP-7 proCASP-9 P37 (Cleaved CASP-9)

WT DKO Untreated BA-24h Untreated BA-24h proCASP-12 HM LM S-100 HM LM S-100 HM LM S-100 HM LM S-100 proCASP-12 P42 (Cleaved CASP-12) p35 (Cleaved CASP-12) proCASP-9 P37 (Cleaved CASP-9) COX IV

Calpain CASP-12 CASP-7 and CASP-9 CASP-12

Late cell death Early cell death (CASP-9- APAF-1- and BAX-BAK-independent) Early cell death (CASP-9- APAF-1- and BAX-BAK-dependent)

WT CASP-9-/- P19 (Cleaved CASP-3) proCASP-7 p2O (Cleaved CASP-7) UV-24 UV-24 P19 (Cleaved CASP-3) proCASP-7 p2O (Cleaved CASP-7) proCASP-9 P37 (Cleaved CASP-9)

DKO/ UV-light (100 mJ/cm2) Annexin V (%) VEID-ase activity (a.u) DEVD-ase activity (a.u) Time (h) 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96

DKO Time (h) proCASP-7 P19 (Cleaved CASP-3) p16 (Cleaved CASP-6) UV-light 100 mJ/cm2 P19 (Cleaved CASP-3) DKO proCASP-7 p16 (Cleaved CASP-6) 24 48 72 96 Time (h) proCASP-9

RNAi Caspase-2 RS-CASP-2 #101 RS-CASP-2 #102 RS-CASP-2 #103 TGGGAGGCTGGGGGAGGGATGCGGGGGCTGGCGCGGCCCAGCGTTTGTTTGTTTTGTGGG TGGTGCGTGTGCAGCCCGGGAAAAGCTGGAAATGGCGGCGCCGAGCGGGAGGTCGCAGTC CTCCCTGCACAGGAAGGGGCTGATGGCGGCTGACAGGAGGAGCAGGATTTTGGCAGTGTG TGGAATGCATCCTGACCACCAGGAAACACTGAAAAAGAATCGAGTGGTGCTGGCCAAGCA GCTGCTGCTGAGCGAGCTGTTAGAACACCTCCTAGAGAAGGACATTATCACTTTGGAAAT GAGGGAGCTCATCCAGGCCAAAGGGGGCAGTTTCAGCCAGAATGTGGAACTCCTCAACCT GCTGCCAAAGAGAGGACCCCAGGCTTTTGATGCCTTCTGTGAAGCCCTGCGGGAGACCAG GCAGGGTCACTTGGAAGACTTACTGCTCACAACCCTCTCAGATATTCAGCACGTACTCCC ACCGTTGAGCTGTGACTATGACACAAGTCTCCCTTTCTCGGTGTGTGAGTCCTGCCCTCC TCACAAGCAGCTCCGCCTATCCACAGATGCTACGGAACACTCCTTAGATAATGGTGATGG TCCTCCCTGTCTTCTGGTGAAGCCATGCACTCCTGAGTTTTACCAGGCACACTACCAGCT GGCCTATAGGTTGCAATCTCAGCCCCGTGGCTTGGCACTGGTGCTGAGCAATGTGCACTT CACTGGAGAGAAAGACCTGGAATTCCGCTCTGGAGGGGATGTGGACCACACTACTCTAGT CACCCTCTTCAAGCTTTTGGGCTACAATGTCCATGTGCTACATGACCAGACCGCACAGGA AATGCAAGAGAAACTTCAGAATTTTGCACAGTTACCTGCACACCGGGTCACAGACTCCTG CGTAGTGGCACTCCTCTCACATGGTGTGGAAGGTGGCATCTATGGTGTAGATGGCAAACT GCTTCAGCTCCAAGAGGTTTTTCGACTTTTTGACAATGCTAACTGTCCAAGTCTACAGAA CAAGCCAAAAATGTTCTTCATCCAAGCATGTCGTGGAGATGAGACAGATAGAGGTGTCGA CCAGCAAGATGGAAAGAACCACACACAATCCCCTGGATGTGAGGAGAGTGATGCTGGCAA AGAGGAGTTGATGAAGATGAGACTGCCTACTCGCTCAGACATGATATGTGGCTATGCTTG RS-CASP-2 #101 RS-CASP-2 #102 RS-CASP-2 #103 Retro-Super CASP-2 Actin DKO

proCASP-3 P19 (Cleaved CASP-3) Annexin V (%) Retro-S RS-C2#101 UV-48 UV-48 UV-48 UV-48 proCASP-3 P19 (Cleaved CASP-3) 2.3 37.3 4.4 38.8 2.7 39 3.8 36.2 Annexin V (%)

RNAi Caspase-6 Retro-Super Actin CASP-6 DKO APAF-1 null CASP-9 null CGGAGGCCTCAGGGCTAGGACACCGGTGGGAGCTCGCGGGCAGGTGAAAGTAAAACATGA CAGAAACCGATGGCTTCTACAAAAGTAGGGAAGTGTTCGATCCAGCCGAGCAGTACAAGA TGGACCACAAGAGGAGAGGAGTTGCCCTCATCTTCAATCACGAGAGGTTCTTTTGGCACC TGACCCTCCCAGAGAGGCGGGGCACCAACGCAGACAGAGACAACCTGACTCGCAGGTTTT CAGATCTAGGATTTGAAGTGAAATGCTTTAACGACCTCAGAGCAGAAGAACTCCTGCTCA AAATTCACGAGGTGTCGACTTCAAGCCACATAGATGCCGATTGCTTCATCTGTGTCTTCC TGAGCCATGGGGAAGGCAACCACGTTTACGCATACGACGCCAAAATTGAAATTCAGACGT TGACTGGCTTGTTCAAAGGAGACAAGTGTCAGAGCCTGGTTGGAAAACCCAAGATATTTA TCATCCAGGCCTGTCGGGGTAGCCAGCATGACGTACCCGTGGTTCCCCTGGACATGGTGG ATCACCAGACAGACAAGCTGGACAACGTGACCCAGGTGGATGCTGCGTCCGTGTACACGC TGCCGGCAGGGGCAGACTTCCTCATGTGCTACTCTGTCGCAGAAGGGTATTACTCTCACC GAGAAACTGTGAATGGCTCCTGGTACATTCAGGATTTGTGTGAGATGCTGGCGAGGTACG GCAGTTCCCTGGAGTTCACGGAGCTGCTCACGCTGGTGAACAGAAAGGTCTCTCAGCGCC GCGTGGACTTCTGCAAAGACCCGGATGCAATCGGCAAGAAGCAGGTGCCCTGCTTCGCCT CAATGCTGACCAAAAAGCTGCATTTCTGTCCCAAACCTAGCAAGTAGGGCCATCTGTCTT GCTACATATATTTCACACACATTTCCCTTTTTATACAAAGGCCAGCTGGTGGAAGACTTT Retro-Super Actin CASP-6 DKO APAF-1 null CASP-9 null RS-CASP-6 #104

proCASP-3 P19 (Cleaved CASP-3) UV-light (10 mJ/cm2) CASP-9 null UV-48 Retro-S proCASP-3 RS-C6#104 CASP-9 null Annexin V (%) RS-CASP-6 #104 Retro-Super 0 24 36 48

UV-light (10 mJ/cm2) APAF-1 null 0 24 36 48 Annexin V (%) DKO 0 24 36 48 RS-CASP-6 #104 Retro-Super Annexin V (%) DKO 0 24 48 72 Retro-Super RS-CASP-6 #104 Annexin V (%)

Ac-Tyr-Val-Lys-(Biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone (Potent and irreversible inhibitor of caspases) UV-light 100mJ/cm2 72h 24h M M p35 p17 DKO Wt

p17

p35

Acknowledgements Stan Korsmeyer Joe Opferman