Proapoptotic BAX and BAK control multiple initiator caspases

Lab meeting; Antonio Ruiz-Vela9/23/03

Bax and Bak Double-Knockout MEFs

#3 Resistant to chronic UPR-induced apoptosis.Tunicamycin.Brefeldin A

#2 Resistant to DNA-damaging agents.Etoposide.UV light.Cisplatinum.Doxorubicin

#1 Resistant to BH3-only-induced apoptosis.BIM.t-Bid.BAD.Noxa

CASP-3, -7

IRE-1IRE-1

TRAF2

CASP-12CASP-12

“BH3-only”

CED-4 like

CASP-2

?Mito. membrane

DSS

DNA damage-induced apoptosis

ER membrane

“BH3-only”

CED-4 like

CASP-12

?

Misfolded proteins

ER stress-induced apoptosis

CASP-3, -7

RAIDD

CASP-2CASP-2

BAX and BAK gateway?

#3 Caspase activation always requires APAF-1/ CASP-9

#2 Caspases upstream of BAX and BAK gateway

#1 BCL-2 function upstream of BAX and BAK gateway

DKO/ GFP MEFs

“DNA damage-induced apoptosis”

CED-4 like

CASP -2

?

WT/

GFP

DK

O/

GFP

WT/

hB

CL-

2

DK

O/

hB

CL-

2

hBCL-2

Actin “ER stress-induced apoptosis”

CED-4 like

CASP-12

?

WT/

GFP

DK

O/

GFP

WT/

hB

CL-

2

DK

O/

hB

CL-

2

hBCL-2

Actin

DKO/ BCL-2 MEFs

“DNA damage-induced apoptosis”

CED-4 like

CASP -2

?

“ER stress-induced apoptosis”

CED-4 like

CASP-12

?

Etoposide (5 M)

proCASP-7

p20

p17 CASP-3

- 5 50 5 50 - 5 50 5 50

WT DKO

Etoposide (M)

12h 24h 12h 24h 12h 24h 12h 24h

ETO-24ETO-24

WT

P19 (Cleaved CASP-3)

CASP-9-/-

proCASP-7

p2O (Cleaved CASP-7)

P37 (Cleaved CASP-9)

proCASP-9

ETO-24 -IL-3ETO-24 +IL-3

WT DKO FL5.12

proCASP-2

p32 (Cleaved CASP-2)

P19 (Cleaved CASP-2)

CHOP/ GADD153

28S

18S

P17 (Cleaved CASP-3)

- - 4h 12h 24h - 4h 12h 24h

WT DKO

Brefeldin A (0.5 g/ml)

proCASP-7

p20

p17 CASP-3

- 0.5 5 0.5 5 - 0.5 5 0.5 5

WT DKO

Brefeldin A (g/ml)

12h 24h 12h 24h 12h 24h 12h 24h

P19 (Cleaved CASP-3)

BA-24BA-24

WT CASP-9-/-

proCASP-7

P37 (Cleaved CASP-9)

proCASP-9

HM LM S-100 HM LM S-100 HM LM S-100 HM LM S-100

Untreated

p35 (Cleaved CASP-12)P42 (Cleaved CASP-12)

proCASP-12

P37 (Cleaved CASP-9)

proCASP-9

COX IV

UntreatedBA-24h BA-24h

WT DKO

Calpain

CASP-12

CASP-7 and CASP-9

CASP-12

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

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

P19 (Cleaved CASP-3)

UV-24UV-24

WT CASP-9-/-

proCASP-7

P37 (Cleaved CASP-9)

proCASP-9

p2O (Cleaved CASP-7)

0

10

20

30

40

50

60

70

80

90

100

1/1/00 1/2/00 1/3/00

DKO/ UV-light (100 mJ/cm2)

Annexin V (%)

DEVD-ase activity (a.u)

VEID-ase activity (a.u)

0 24 48 72 96 0 24 48 72 96 0 24 48 72 96Time (h)

UV-light 100 mJ/cm2

P19 (Cleaved CASP-3)

DKO

proCASP-7

p16 (Cleaved CASP-6)

24 48 72 96Time (h)

proCASP-9

TGGGAGGCTGGGGGAGGGATGCGGGGGCTGGCGCGGCCCAGCGTTTGTTTGTTTTGTGGGTGGTGCGTGTGCAGCCCGGGAAAAGCTGGAAATGGCGGCGCCGAGCGGGAGGTCGCAGTCCTCCCTGCACAGGAAGGGGCTGATGGCGGCTGACAGGAGGAGCAGGATTTTGGCAGTGTGTGGAATGCATCCTGACCACCAGGAAACACTGAAAAAGAATCGAGTGGTGCTGGCCAAGCAGCTGCTGCTGAGCGAGCTGTTAGAACACCTCCTAGAGAAGGACATTATCACTTTGGAAATGAGGGAGCTCATCCAGGCCAAAGGGGGCAGTTTCAGCCAGAATGTGGAACTCCTCAACCTGCTGCCAAAGAGAGGACCCCAGGCTTTTGATGCCTTCTGTGAAGCCCTGCGGGAGACCAGGCAGGGTCACTTGGAAGACTTACTGCTCACAACCCTCTCAGATATTCAGCACGTACTCCCACCGTTGAGCTGTGACTATGACACAAGTCTCCCTTTCTCGGTGTGTGAGTCCTGCCCTCCTCACAAGCAGCTCCGCCTATCCACAGATGCTACGGAACACTCCTTAGATAATGGTGATGGTCCTCCCTGTCTTCTGGTGAAGCCATGCACTCCTGAGTTTTACCAGGCACACTACCAGCTGGCCTATAGGTTGCAATCTCAGCCCCGTGGCTTGGCACTGGTGCTGAGCAATGTGCACTTCACTGGAGAGAAAGACCTGGAATTCCGCTCTGGAGGGGATGTGGACCACACTACTCTAGTCACCCTCTTCAAGCTTTTGGGCTACAATGTCCATGTGCTACATGACCAGACCGCACAGGAAATGCAAGAGAAACTTCAGAATTTTGCACAGTTACCTGCACACCGGGTCACAGACTCCTGCGTAGTGGCACTCCTCTCACATGGTGTGGAAGGTGGCATCTATGGTGTAGATGGCAAACTGCTTCAGCTCCAAGAGGTTTTTCGACTTTTTGACAATGCTAACTGTCCAAGTCTACAGAACAAGCCAAAAATGTTCTTCATCCAAGCATGTCGTGGAGATGAGACAGATAGAGGTGTCGACCAGCAAGATGGAAAGAACCACACACAATCCCCTGGATGTGAGGAGAGTGATGCTGGCAAAGAGGAGTTGATGAAGATGAGACTGCCTACTCGCTCAGACATGATATGTGGCTATGCTTG

RNAi Caspase-2

Actin

CASP-2

Retro-S

uper

RS-CASP-2 #101

RS-CASP-2 #102

RS-CASP-2 #103

DKO

P19 (Cleaved CASP-3)

UV-48

Retro-S

proCASP-3

UV-48 UV-48 UV-48

RS-C2#101 RS-C2#102 RS-C2#103

Annexin V (%) 2.3 37.3 4.4 38.8 2.7 39 3.8 36.2

CGGAGGCCTCAGGGCTAGGACACCGGTGGGAGCTCGCGGGCAGGTGAAAGTAAAACATGACAGAAACCGATGGCTTCTACAAAAGTAGGGAAGTGTTCGATCCAGCCGAGCAGTACAAGATGGACCACAAGAGGAGAGGAGTTGCCCTCATCTTCAATCACGAGAGGTTCTTTTGGCACCTGACCCTCCCAGAGAGGCGGGGCACCAACGCAGACAGAGACAACCTGACTCGCAGGTTTTCAGATCTAGGATTTGAAGTGAAATGCTTTAACGACCTCAGAGCAGAAGAACTCCTGCTCAAAATTCACGAGGTGTCGACTTCAAGCCACATAGATGCCGATTGCTTCATCTGTGTCTTCCTGAGCCATGGGGAAGGCAACCACGTTTACGCATACGACGCCAAAATTGAAATTCAGACGTTGACTGGCTTGTTCAAAGGAGACAAGTGTCAGAGCCTGGTTGGAAAACCCAAGATATTTATCATCCAGGCCTGTCGGGGTAGCCAGCATGACGTACCCGTGGTTCCCCTGGACATGGTGGATCACCAGACAGACAAGCTGGACAACGTGACCCAGGTGGATGCTGCGTCCGTGTACACGCTGCCGGCAGGGGCAGACTTCCTCATGTGCTACTCTGTCGCAGAAGGGTATTACTCTCACCGAGAAACTGTGAATGGCTCCTGGTACATTCAGGATTTGTGTGAGATGCTGGCGAGGTACGGCAGTTCCCTGGAGTTCACGGAGCTGCTCACGCTGGTGAACAGAAAGGTCTCTCAGCGCCGCGTGGACTTCTGCAAAGACCCGGATGCAATCGGCAAGAAGCAGGTGCCCTGCTTCGCCTCAATGCTGACCAAAAAGCTGCATTTCTGTCCCAAACCTAGCAAGTAGGGCCATCTGTCTTGCTACATATATTTCACACACATTTCCCTTTTTATACAAAGGCCAGCTGGTGGAAGACTTT

RNAi Caspase-6

Retro-S

uper

Actin

CASP-6

DKO APAF-1 null CASP-9 null

Retro-S

uper

Retro-S

uper

RS-CASP-6 #104

RS-CASP-6 #104

RS-CASP-6 #104

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4

CASP-9 null

0 24 36 48

Retro-S

uper

RS-CASP-6 #104

An

ne

xin

V (

%)

UV-light (10 mJ/cm2)

P19 (Cleaved CASP-3)

UV-48

Retro-S

proCASP-3UV-48

RS-C6#104

CASP-9 null

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4

DKO

0 24 48 72

Retro-S

uper

RS-CASP-6 #104

An

ne

xin

V (

%)

UV-light (10 mJ/cm2)

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4

APAF-1 null

0 24 36 48

RS-CASP-6 #104

Retro-S

uper

An

ne

xin

V (

%)

p35

p17

M M

72h 24hUV-light 100mJ/cm2

DKO

Ac-Tyr-Val-Lys-(Biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone (Potent and irreversible inhibitor of caspases)

Wt

SW:H2B1_MOUSE P10853 mus musculus (mouse). histone h2bf (h2b 291a). 2/2003 [MASS=13805]PEPAKSAPAP KKGSKKAVTK AQKKDGKKRK RSRKESYSVY VYKVLKQVHPDTGISSKAMG IMNSFVNDIF ERIASEASRL AHYNKRSTIT SREIQTAVRLLLPGELAKHA VSEGTKAVTK YTSSKaverage mass = 13805, pI = 10.32

SW:H2AX_MOUSE P27661 mus musculus (mouse). histoneh2a.x. 2/2003 [MASS=15011]SGRGKTGGKA RAKAKSRSSR AGLQFPVGRV HRLLRKGHYA ERVGAGAPVYLAAVLEYLTA EILELAGNAA RDNKKTRIIP RHLQLAIRND EELNKLLGGVTIAQGGVLPN IQAVLLPKKS SATVGPKAPA VGKKASQASQ EYaverage mass = 15011, pI = 10.74

>SW:H2A1_MOUSE P22752 mus musculus (mouse). histoneh2a.1. 2/2003 [MASS=14050]SGRGKQGCKA RAKAKTRSSR AGLQFPVGRV HRLLRKGNYS ERVGAGAPVYLAAVLEYLTA EILELAGNAA RDNKKTRIIP RHLQLAIRND EELNKLLGRVTIAQGGVLPN IQAVLLPKKT ESHHKAKGK>average mass = 14050, pI = 10.87

p17

SW:H15_MOUSE P43276 mus musculus (mouse). histone h1.5(h1 var.5) (h1b). 2/2003 [MASS=22445]SETAPAETAA PAPVEKSPAK KKTTKKAGAA KRKATGPPVS ELITKAVSASKERGGVSLPA LKKALAAGGY DVEKNNSRIK LGLKSLVSKG TLVQTKGTGASGSFKLNKKA ASGEAKPKAK KTGAAKAKKP AGATPKKPKK TAGAKKTVKKTPKKAKKPAA AGVKKVAKSP KKAKAAAKPK KAAKSPAKPK AVKSKASKPKVTKPKTAKPK AAKAKKAVSK KK>average mass = 22445, pI = 10.91

SW:H12_MOUSE P15864 mus musculus (mouse). histone h1.2(h1 var.1) (h1c). 10/2001 [MASS=21135]SEAAPAAPAA APPAEKAPAK KKAAKKPAGV RRKASGPPVS ELITKAVAASKERSGVSLAA LKKALAAAGY DVEKNNSRIK LGLKSLVSKG ILVQTKGTGASGSFKLNKKA ASGEAKPQAK KAGAAKAKKP AGAAKKPKKA TGAATPKKAAKKTPKKAKKP AAAAVTKKVA KSPKKAKVTK PKKVKSASKA VKPKAAKPKVAKAKKVAAKK K>average mass = 21135, pI = 11.00

SW:H14_MOUSE P43274 mus musculus (mouse). histone h1.4(h1 var.2) (h1e). 10/2001 [MASS=21846]SETAPAAPAA PAPAEKTPVK KKARKAAGGA KRKTSGPPVS ELITKAVAASKERSGVSLAA LKKALAAAGY DVEKNNSRIK LGLKSLVSKG TLVQTKGTGASGSFKLNKKA ASGEAKPKAK RAGAAKAKKP AGAAKKPKKA AGTATAKKSTKKTPKKAKKP AAAAGAKKAK SPKKAKATKA KKAPKSPAKA KTVKPKAAKPKTSKPKAAKP KKTAAKKK>average mass = 21846, pI = 11.10

p35

Acknowledgements

Stan Korsmeyer

Joe Opferman