Theoretical considerations

Because of the intimate involvement of caspases in apoptosis, there has been considerable interest in studying their activation and activity under a variety

Table 2. Polypeptides cleaved by caspases in apoptotic cells' Polypeptides

Abundant cytoplasmic proteins

Gelsolin Gas-2 Fodrin ß-Catenin Cyto keratin 18

Cleavage Responsible Sizes reported (kDa) site caspase Intact Fragmentls)6

DQTD/G 3

DETD/S 3

41,39 31 150 65

29,23

Abundant nuclear proteins

Lamiri A

VEID/N

6

69

47

Lamin B1

VEVD/S

6,?3

67

45

NuMA

?

3,6

230

160,180

HnRNP proteins C1 and C2

?

3,7

40

-35

70 kDa protein of U1 snRNP

DGPD/G

3

70

40

mdm2

DVPD/C

3,6,7

100

55

Proteins involved in DNA metabolism and repair^

Proteins involved in DNA metabolism and repair^

Poly(ADP-ribose) polymerase DNA-PKcs

Replication factor C large subunit Topoisomerase I

DEVD/N 3

DEVD/G 3

DDVD/Y 3

113 460 140 100

89,24 150 87,53 70

Protein kinases

Protein kinase C8

DMQD/M

3

78

40

Protein kinase C6

DEVD/K

3

78

40

Protein kinase C-related kinase 2

DITD/C

3

130

110,1C

Calci um/cal moduli n-dependent protein

PAPD/A

3

55

38

kinase IV

p21-activated kinase 2

SHVD/G

3,8

65

36

PITSLRE kinase a2-1

YVPD/S

3

110

60,43

Mstl kinase

DEMD/S

?

63

34-36

Mst2 kinase

DELD/S

?

63

34

Focal adhesion kinase

DQTD/S

3,7

125

85,77

VSWD/S

6

MEKK-1

DTVD/G

3

200

-100,

Wee1 kinase

?

3,7,8

-80

-60

Other proteins involved in signal transduction and gene expression

Pro-interleukin-1ß

FEAD/G

1

31

28

YVHD/A

17.5

Pro-interleukin-16

SSTD/S

3

50

20

Pro-interleukin-18

LESD/Y

1,3

24

18,16,15

Ras GTPase activating protein

DTVD/G

3

120

-80, -65

D4-GDP dissociation inhibitor

DELD/S

3

28

23

Protein phosphatase 2A subunit Aa

DEQD/S

3

65

42

Cytosolic phospholipase A2

DELD/T

3

100

Polypeptides

Statl

NF-kB p65 NF-kB p50 IkB

Sterol response element binding protein-1 Sterol response element binding protein-2

Cleavage Responsible Sizes reported (kDa) site caspase Intact Fragment(s)11

DRHD/S SEPD/S DEPD/S

caspase

91,84 65 50 40

55,10 35,15 36

60-70 60-70

Proteins involved in regulation of cell cycle and proliferationd p21wafi/ciPi DHVD/L 3,7 21 1416

p27kip1 DPSD/S 3,7 27 22

Rb retinoblastoma protein DEAD/G 3 105 100

Proteins involved in human genetic diseases

Dentatorubral pallidalysian atrophy protein DSLD/G 3

Presenilin-1, C-terminal fragment ARQD/S ?

Presenilin-2, C-terminal fragment DSYD/S 3

145 10-14 20

Apoptotic regulatory proteins*

Bcl-2

DAG DA/

?

26

23

Bcl-XL

HLAD/S

Not 3

28

16

FLIPU

LEVD/G

3,8,10

55

43

BID

LQTD/G

8

25

16

BAX

FIQD/R

?

21

18

ICAD/DFF45

DEPD/S

3

45

30,11

"For a list of proposed biological functions of cleavage, as well as a comprehensive list of references, see ref. 1.

'Estimated masses of caspase-generated fragments are based on mobility in SDS-polyacrylamide gels as reported by authors of the original reports describing cleavage in intact cells. ~ sign indicates our estimate based on published gels in cases where fragment sizes were not reported. Fragments generated by other proteases (e.g. calpains) are not listed.

"See also the HnRNP particle proteins C1 and C2 as well as the U1 snRNP particle 70 kDa polypeptide in other parts of this table.

dSee also Wee1 kinase, mdm2 protein, and replication factor C in other parts of this table. eln addition, all of the procaspases are caspase substrates.

"For a list of proposed biological functions of cleavage, as well as a comprehensive list of references, see ref. 1.

'Estimated masses of caspase-generated fragments are based on mobility in SDS-polyacrylamide gels as reported by authors of the original reports describing cleavage in intact cells. ~ sign indicates our estimate based on published gels in cases where fragment sizes were not reported. Fragments generated by other proteases (e.g. calpains) are not listed.

"See also the HnRNP particle proteins C1 and C2 as well as the U1 snRNP particle 70 kDa polypeptide in other parts of this table.

dSee also Wee1 kinase, mdm2 protein, and replication factor C in other parts of this table. eln addition, all of the procaspases are caspase substrates.

of conditions. Immunoblotting provides one potential method for detecting active caspases within cells. In principle, antibodies generated against neo-epitopes (i.e. epitopes that are generated during the proteolytic activation of caspases) could be utilized to selectively detect active caspases on immuno-blots. Such antibodies are not generally available. Instead, most commercially available anti-caspase antibodies (Table 1) have been raised against epitopes present in either the large or small subunits of the active enzymes. These antibodies should theoretically recognize the corresponding zymogen and active caspase with equal affinities.

In applying these antibodies to the study of cells undergoing apoptosis, one might expect that decreases in zymogen levels would be accompanicd by stoichiometric increases in active caspase species. This is not usually observed. Although the signals for certain procaspases decrease or disappear during apoptosis, suggesting that these zymogens have been activated, a corresponding increase in the large or small subunit is not always detected by immuno-blotting. One possible explanation for this phenomenon is a rapid degradation of caspase species once they are activated. Although this explanation remains to be rigorously tested, the detection of active caspases by immunobiotting remains difficult in many model systems. Accordingly, the disappearance of the procaspase signal is commonly accepted as evidence of caspase activation.

Despite the widespread use of anti-caspase antibodies to study caspase activation, published data do not indicate whether the caspase zymogens are abundant or rare polypeptide species. Data in Figure 2 indicate that 3 x 103 K562 human leukaemia cells contain —10 ng of procaspasc-3 and procaspase -8, with 10-fold lower levels of procaspase-6. This corresponds to ~6 x 10s, 4 X 105, and 6 X 104 molecules of procaspasc-3, -8, and -6, respectively, per cell. Based on a cell diameter of 15 |xm, this translates into a concentration of

Caspase-8 K562

Caspase-8 K562

Figure?. Estimation of procaspase levels in K562 human leukaemia cells. Samples containing 20, 10, or 5 ng of the indicated purified recombinant caspase (lanes 1-3, respectively) or 3 x TO5, 1.5 X 10E, and 0.75 x 106 K562 cells (lanes 4-6, respectively) were subjected to SDS-PAGE, followed by immunobiotting with antibodies raised against the indicated caspase. Closed arrowhead, caspase zymogen. Two alternatively spliced variants of caspase-8 are present in K562 cells. Open arrowhead, large subunit of active caspase.

Figure?. Estimation of procaspase levels in K562 human leukaemia cells. Samples containing 20, 10, or 5 ng of the indicated purified recombinant caspase (lanes 1-3, respectively) or 3 x TO5, 1.5 X 10E, and 0.75 x 106 K562 cells (lanes 4-6, respectively) were subjected to SDS-PAGE, followed by immunobiotting with antibodies raised against the indicated caspase. Closed arrowhead, caspase zymogen. Two alternatively spliced variants of caspase-8 are present in K562 cells. Open arrowhead, large subunit of active caspase.

~3 |xg/ml (100 nM) for procaspase-3 if the zymogen is uniformly distributed throughout the cell. Additional experiments in our laboratory have revealed that procaspase-3 levels in a panel of 60 different human cancer cell lines range from undetectable (in a cell line with a caspase-3 gene rearrangement) to four times as high as K562 cells. Procaspase-8 levels in the same cell lines range from 0.5 to six times as high as K562 cells.

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