Activation of membrane-associated procaspase-3 is regulated by Bcl-2

Abstract

The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is unknown. Here we characterize an intracellular, membrane-associated form of procaspase-3 whose activation is controlled by Bcl-2. Heavy membranes isolated from control cells contained a spontaneously activatable caspase-3 zymogen. In contrast, in Bcl-2 overexpressing cells, although the caspase-3 zymogen was still associated with heavy membranes, its spontaneous activation was blocked. However, Bcl-2 expression had little effect on the levels of cytoplasmic caspase activity in unstimulated cells. Furthermore, the membrane-associated caspase-3 differed from cytosolic caspase-3 in its responsiveness to activation by exogenous cytochrome c. Our results demonstrate that intracellular membranes can generate active caspase-3 by a Bcl-2-inhibitable mechanism, and that control of caspase activation in membranes is distinct from that observed in the cytoplasm. These data suggest that Bcl-2 may control cytoplasmic events in part by blocking the activation of membrane-associated procaspases.

Figure 1

Immunoblots of subcellular fractions from 697-neo and 697-Bcl-2 cells using antibodies to PARP, cytochrome oxidase (subunit IV), D4-GDI and Bcl-2. The immunoblots were visualized on film by chemiluminescence, except the cytochrome oxidase immunoblot which was visualized by chemifluorescence. Nuc, nuclear fraction; HM, heavy membrane fraction; LM, light membrane fraction; S100, cytosolic fraction. Arrows indicate the specific immunoreactive band. The diffuse signal present in the nuclear fractions probed with the D4-GDI antibody represents a non-specific background band.

Figure 2

acDEVD-amc cleavage activity in subcellular fractions of 697 cells. 697 cells transfected with neo control or Bcl-2 expression vectors were fractionated and the caspase activity of each subcellular fraction was assayed using acDEVD-amc as substrate. The observed cleavage activity values in the histogram are normalized for constant number of cells (A and B) or mg protein (C and D). The values listed for each column in A and B indicate the percent of total cleavage activity present in each fraction. The error bars indicate the range of observed values for two independent 697 cell preparations.

Figure 3

Spontaneous activation of membrane-associated procaspase-3. (A) Spontaneous activation of caspase activity in heavy membranes from 697-neo and Bcl-2 cells. The acDEVD-amc cleavage activity of was measured by adding 20 μg of freshly prepared membranes into hypotonic buffer containing 20 μM acDEVD-amc (final concentration). The evolution of amc product was measured by the change in fluorescence (ex, 360 nm; em, 460 nm) at room temperature. (B) Generation of soluble caspase activity from neo-membranes. Neo-membranes were added to hypotonic buffer containing acDEVD-amc. At the indicated time points from 0 to 90 min (right hand box), the sample was centrifuged for 10 min at 14,000 g at 10°C to remove the membranes. The acDEVD-amc cleavage activity of the supernatant was measured and is plotted as the increase in fluorescence over the subsequent 30-min period after centrifugation for each time point.

Figure 3

Spontaneous activation of membrane-associated procaspase-3. (A) Spontaneous activation of caspase activity in heavy membranes from 697-neo and Bcl-2 cells. The acDEVD-amc cleavage activity of was measured by adding 20 μg of freshly prepared membranes into hypotonic buffer containing 20 μM acDEVD-amc (final concentration). The evolution of amc product was measured by the change in fluorescence (ex, 360 nm; em, 460 nm) at room temperature. (B) Generation of soluble caspase activity from neo-membranes. Neo-membranes were added to hypotonic buffer containing acDEVD-amc. At the indicated time points from 0 to 90 min (right hand box), the sample was centrifuged for 10 min at 14,000 g at 10°C to remove the membranes. The acDEVD-amc cleavage activity of the supernatant was measured and is plotted as the increase in fluorescence over the subsequent 30-min period after centrifugation for each time point.

Figure 4

Neo-membranes and Bcl-2-membranes contain similar amounts of procaspase-3. (A) Immunoblot of heavy membrane and cytosolic fractions from 697-neo and 697-Bcl-2 cells using an affinity-purified rabbit polyclonal antibody to caspase-3. The arrowheads indicate the migration of protein size markers (Rainbow Markers; Novex); the arrow indicates procaspase-3. HM, heavy membrane fractions; S100, cytosolic fraction. Note: The immunoreactive procaspase-3 band in heavy membrane fractions migrates more slowly than the cytosolic form of the protein. (B) Activation of membrane-associated acDEVD-amc cleavage activity by exogenous caspase-1. Heavy membrane fractions (containing 50 μg total protein) from 697-Bcl-2 and 697-neo cells were resuspended and treated with murine caspase-1 or buffer for 1 h at room temperature. After centrifugation, the acDEVD-amc cleavage activity of the resulting supernatant was measured. The acDEVD-amc cleavage activity of caspase-1–treated samples was corrected for exogenous caspase-1 activity by subtracting the fluorescence of control samples containing only caspase-1 from the observed fluorescence. The error bars represent the standard deviation of the observed values in three independent experiments.

Figure 4

Neo-membranes and Bcl-2-membranes contain similar amounts of procaspase-3. (A) Immunoblot of heavy membrane and cytosolic fractions from 697-neo and 697-Bcl-2 cells using an affinity-purified rabbit polyclonal antibody to caspase-3. The arrowheads indicate the migration of protein size markers (Rainbow Markers; Novex); the arrow indicates procaspase-3. HM, heavy membrane fractions; S100, cytosolic fraction. Note: The immunoreactive procaspase-3 band in heavy membrane fractions migrates more slowly than the cytosolic form of the protein. (B) Activation of membrane-associated acDEVD-amc cleavage activity by exogenous caspase-1. Heavy membrane fractions (containing 50 μg total protein) from 697-Bcl-2 and 697-neo cells were resuspended and treated with murine caspase-1 or buffer for 1 h at room temperature. After centrifugation, the acDEVD-amc cleavage activity of the resulting supernatant was measured. The acDEVD-amc cleavage activity of caspase-1–treated samples was corrected for exogenous caspase-1 activity by subtracting the fluorescence of control samples containing only caspase-1 from the observed fluorescence. The error bars represent the standard deviation of the observed values in three independent experiments.

Figure 5

Procaspase-3 immunoreactvity partially colocalized with mitochondria. T47D cells (a–c, h, and i) were double-labeled with antibodies to procaspase-3 and cytochrome c and visualized by confocal microscopy (0.4-μm optical sections). These cells show both diffuse and punctate staining with anti–procaspase-3 antibody (a). The punctate staining largely colocalizes with anti–cytochrome c staining (b, anti–cytochrome c; c, merged image from a and b). This staining is specific as shown by the lack of staining with control rabbit IgG (h), whereas anti–cytochrome c demonstrated the presence of several cells (i). The specificity of the procaspase-3 antibody is further demonstrated by the lack of staining of MCF7/cont cells that lack procaspase-3 (f), although anti–cytochrome c staining demonstrated the presence of several cells (g). However, MCF7/casp-3 cells that overexpress procaspase-3 show intense staining with anti–procaspase-3 (d) as well as with anti–cytochrome c (e). d–g are conventional fluorescence microscope images. Scale markings are in microns.

Figure 6

Membrane-associated caspase activation is not stimulated by exogenous cytochrome c. Subcellular fractions were prepared from 697-neo and 697-Bcl-2 cells. After the cells were lysed by Dounce homogenization, the sample was split into two tubes. One tube was processed using standard buffers, while bovine cytochrome c was added to the other (10 μg/ml final concentration), and cytochrome c was maintained in that sample throughout membrane isolation including the heavy membrane pellet wash steps. Aliquots of the cytochrome c–treated heavy membranes and cytoplasmic fractions were then incubated with hypotonic buffer containing 50 μM dATP/1 μg/ml cytochrome c for 40 min at 30°C, while the membranes and cytoplasmic samples that had not been treated with cytochrome c were incubated only with buffer. Each sample was then centrifuged and acDEVD-amc cleavage activity in the supernatant was measured. The graphs represent data from one out of three equivalent experiments. (A) Heavy membrane–derived caspase activities. (B) Cytoplasmic caspase activities.

Figure 6

Membrane-associated caspase activation is not stimulated by exogenous cytochrome c. Subcellular fractions were prepared from 697-neo and 697-Bcl-2 cells. After the cells were lysed by Dounce homogenization, the sample was split into two tubes. One tube was processed using standard buffers, while bovine cytochrome c was added to the other (10 μg/ml final concentration), and cytochrome c was maintained in that sample throughout membrane isolation including the heavy membrane pellet wash steps. Aliquots of the cytochrome c–treated heavy membranes and cytoplasmic fractions were then incubated with hypotonic buffer containing 50 μM dATP/1 μg/ml cytochrome c for 40 min at 30°C, while the membranes and cytoplasmic samples that had not been treated with cytochrome c were incubated only with buffer. Each sample was then centrifuged and acDEVD-amc cleavage activity in the supernatant was measured. The graphs represent data from one out of three equivalent experiments. (A) Heavy membrane–derived caspase activities. (B) Cytoplasmic caspase activities.

Figure 7

Effects of permeabilizing detergent NP-40 on membrane caspase activity. (A) NP-40 has minimal effects on spontaneous and induced caspase activities in neo-membranes. 160 μl of neo-membranes were diluted with 180 μl hypotonic buffer and treated with 40 μl 10% NP-40 detergent or dH₂O (final vol = 380 μl). The diluted membranes were activated by the addition of 20 μl granzyme B or caspase-1 lysate or buffer, and incubated for 60 min at 30°C. After activation, the heavy membranes were removed by centrifugation and the acDEVD-amc cleaving activity of each sample was measured by adding 50 μl of each supernatant to 200 μl of 25 μM acDEVD-amc substrate in ICE buffer. (B) NP-40 has minimal effects on spontaneous caspase activation in Bcl-2- and neo-membranes. The effect of NP-40 on the progress curve for heavy membrane catalyzed acDEVD-amc hydrolysis was measured by adding 50 μl freshly prepared neo- or Bcl-2-membranes to 200 μl of 25 μM acDEVD-amc in hypotonic buffer pH 7.5 (containing 4 mM DTT) with or without 1% NP-40 detergent. Fluorescence was measured as in Fig. 3 a. (C) NP-40– dependent and –independent activation of procaspase-3 by granzyme B in fractions enriched in intact mitochondria. Mitochondrial fractions were prepared from lysed 697-neo and 697-Bcl-2 cells by the methods of Mancini et al. (1998) using isotonic buffers (see Materials and Methods). Diluted membranes, with or without 1% NP-40, were activated by the addition of granzyme B or buffer for 60 min, centrifuged, and assayed for acDEVD-amc cleavage activity as described in A.

Figure 7

Effects of permeabilizing detergent NP-40 on membrane caspase activity. (A) NP-40 has minimal effects on spontaneous and induced caspase activities in neo-membranes. 160 μl of neo-membranes were diluted with 180 μl hypotonic buffer and treated with 40 μl 10% NP-40 detergent or dH₂O (final vol = 380 μl). The diluted membranes were activated by the addition of 20 μl granzyme B or caspase-1 lysate or buffer, and incubated for 60 min at 30°C. After activation, the heavy membranes were removed by centrifugation and the acDEVD-amc cleaving activity of each sample was measured by adding 50 μl of each supernatant to 200 μl of 25 μM acDEVD-amc substrate in ICE buffer. (B) NP-40 has minimal effects on spontaneous caspase activation in Bcl-2- and neo-membranes. The effect of NP-40 on the progress curve for heavy membrane catalyzed acDEVD-amc hydrolysis was measured by adding 50 μl freshly prepared neo- or Bcl-2-membranes to 200 μl of 25 μM acDEVD-amc in hypotonic buffer pH 7.5 (containing 4 mM DTT) with or without 1% NP-40 detergent. Fluorescence was measured as in Fig. 3 a. (C) NP-40– dependent and –independent activation of procaspase-3 by granzyme B in fractions enriched in intact mitochondria. Mitochondrial fractions were prepared from lysed 697-neo and 697-Bcl-2 cells by the methods of Mancini et al. (1998) using isotonic buffers (see Materials and Methods). Diluted membranes, with or without 1% NP-40, were activated by the addition of granzyme B or buffer for 60 min, centrifuged, and assayed for acDEVD-amc cleavage activity as described in A.

Figure 7

Effects of permeabilizing detergent NP-40 on membrane caspase activity. (A) NP-40 has minimal effects on spontaneous and induced caspase activities in neo-membranes. 160 μl of neo-membranes were diluted with 180 μl hypotonic buffer and treated with 40 μl 10% NP-40 detergent or dH₂O (final vol = 380 μl). The diluted membranes were activated by the addition of 20 μl granzyme B or caspase-1 lysate or buffer, and incubated for 60 min at 30°C. After activation, the heavy membranes were removed by centrifugation and the acDEVD-amc cleaving activity of each sample was measured by adding 50 μl of each supernatant to 200 μl of 25 μM acDEVD-amc substrate in ICE buffer. (B) NP-40 has minimal effects on spontaneous caspase activation in Bcl-2- and neo-membranes. The effect of NP-40 on the progress curve for heavy membrane catalyzed acDEVD-amc hydrolysis was measured by adding 50 μl freshly prepared neo- or Bcl-2-membranes to 200 μl of 25 μM acDEVD-amc in hypotonic buffer pH 7.5 (containing 4 mM DTT) with or without 1% NP-40 detergent. Fluorescence was measured as in Fig. 3 a. (C) NP-40– dependent and –independent activation of procaspase-3 by granzyme B in fractions enriched in intact mitochondria. Mitochondrial fractions were prepared from lysed 697-neo and 697-Bcl-2 cells by the methods of Mancini et al. (1998) using isotonic buffers (see Materials and Methods). Diluted membranes, with or without 1% NP-40, were activated by the addition of granzyme B or buffer for 60 min, centrifuged, and assayed for acDEVD-amc cleavage activity as described in A.