Post-translational Modification of Bid Has Differential Effects on Its Susceptibility to Cleavage by Caspase 8 or Caspase 3*

Mauro Degli EspostiDagger §, Gilles Ferry, Peggy MasdehorsDagger §, Jean A. Boutin, John A. Hickman, and Caroline DiveDagger ||

From the Dagger  Cancer Research UK Cellular and Molecular Pharmacology Group, School of Biological Sciences, University of Manchester, Stopford Bldg., Oxford Road, Manchester M13 9PT, United Kingdom and the  Institut de Recherches Servier, 125 chemin de Ronde, Croissy sur Seine 78290, France

Received for publication, September 9, 2002, and in revised form, February 12, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bid is instrumental in death receptor-mediated apoptosis where it is cleaved by caspase 8 at aspartate 60 and aspartate 75 to generate truncated Bid (tBID) forms that facilitate release of mitochondrial cytochrome c. Bid is also cleaved at these sites by caspase 3 that is activated downstream of cytochrome c release after diverse apoptotic stimuli. In this context, tBid may amplify the apoptotic process. Bid is phosphorylated in vitro by casein kinases that regulate its cleavage by caspase 8 (Desagher, S., Osen-Sand, A., Montessuit, S., Magnenat, E., Vilbois, F., Hochmann, A., Journot, L. Antonsson, A., and Martinou, J.-C. (2001) Mol. Cell 8, 601-611). Using a Bid decapeptide substrate, we observed that phosphorylation at threonine 59 inhibited cleavage by caspase 8. This was also seen when recombinant Bid (rBid) and Bid isolated from murine kidney were incubated with casein kinase II. However, there were differences in the susceptibility of rBid and isolated Bid to cleavage by caspases 3 and 8. Caspase 8 cleaved rBid to generate two C-terminal products, p15 and p13 tBid, but produced only p15 tBid from isolated Bid. Contrary to rBid, isolated Bid was resistant to cleavage by caspase 3, yet was readily cleaved within the cytosolic milieu. Our data suggest that one or more distinct cellular mechanisms regulate Bid cleavage by caspases 8 and 3 in situ.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Proteins of the Bcl-2 family are crucially involved in the control of apoptosis (1-3). This family includes anti-apoptotic proteins such as Bcl-2 itself and an increasing number of pro-apoptotic proteins like Bax and Bid that translocate to mitochondria and facilitate the release of cytochrome c during the induction of apoptosis (2-7). The pro-apoptotic members of the Bcl-2 family can be further divided in multidomain proteins such as Bax and Bak, which share three domains of sequence homology with Bcl-2, and the so-called "BH3-only" proteins such as Bid and Bad, which share a single region of sequence homology with Bcl-2, the BH3 domain (1, 3, 5-10).

A key aspect of BH-3-only pro-apoptotic proteins of the Bcl-2 family is their conversion from latent to active state in response to apoptotic stimuli or, more broadly, to cellular stress. Two general mechanisms of activation are known: (a) enhanced expression due to transcriptional up-regulation and (b) post-translational modification of the protein by phosphorylation or cleavage. Bad provides the best characterized example of pro-apoptotic regulation via protein phosphorylation (2, 9, 10): multiple serine residues of Bad are phosphorylated as a consequence of several survival signaling pathways, promoting its cytosolic sequestration by 14-3-3 and thus preventing its interaction with mitochondrial anti-apoptotic proteins such as Bcl-2 and Bcl-xL (1, 9, 10). More recently, the phosphorylation of Bik (11) and Bid (12) have been reported, suggesting that phosphorylation may be a common mechanism to control the action of pro-apoptotic proteins related to Bcl-2.

The current consensus assigns a primary role to proteolytic cleavage in the activation of Bid during death receptor signaling (3, 5, 6). Ligation of death receptors such as Fas results in the assembly of an intracellular death-inducing signaling complex (DISC)1 that activates the initiator caspase 8 (13). Whereas in type I cells DISC formation is very efficient, leading to rapid activation of caspase 8 and then of effector caspases such as caspase 3, in type II cells DISC formation proceeds slowly, and the activation of effector caspases requires amplification via the mitochondrial pathway (13, 14). Bid cleavage by DISC-activated caspase 8 provides the major mechanism of engaging the mitochondrial pathway during Fas-induced apoptosis (3, 5, 6). Caspase 8 cleaves human Bid at aspartate 60 to generate 15-kDa tBid that translocates to the mitochondrial outer membrane. tBid is thought to promote the oligomerization of Bax or Bak at the mitochondrial surface to facilitates the release into the cytosol of cytochrome c and other apoptogenic proteins (3, 5-8, 15). However, in vitro tBid oligomers are also capable of facilitating cytochrome c release in the absence of interaction with Bax or Bak (16).

Whatever the mitochondrial role of tBid, it is noteworthy that recombinant full-length Bid (rBid) is nearly as potent as tBid in the cell-free assay of cytochrome c release (15, 16-18). Furthermore, the action of various cellular kinases, including ERK, JNK, and protein kinase C can attenuate Fas-induced apoptosis (12, 19-22). These observations suggested that mechanisms in addition to, or upstream of, caspase cleavage might regulate the mitochondrial action of Bid (12, 18). A recent study has shown that this is indeed the case (12), because the phosphorylation status of Bid regulates its susceptibility to caspase 8-mediated cleavage. Specifically, treatment of murine Bid with casein kinases (CKs) affects its cleavage (12). Bid phosphorylation may thus block cleavage by caspase 8, producing a safeguard mechanism to prevent the activation of this potent pro-apoptotic molecule in situations of basal caspase 8 activity (12).

In addition to caspase 8-mediated cleavage at Asp-60 and Asp-75, rBid can also be cleaved by caspase 3 at both of these sites (6, 23). Given the greater catalytic capacity of caspase 3 compared with caspase 8 and its activation by wider range of apoptotic stimuli, it seems critical that Bid should be protected from activation by basal caspase 3 activity in healthy cells. We have thus tested whether endogenous Bid in primary tissues presents modifications that affect its cleavage by caspase 8 and/or caspase 3 and provide biochemical evidence for both common and different mechanisms that regulate Bid cleavage by these two caspases.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Western Blotting-- Western blotting was performed as previously reported (18) after protein separation with SDS-PAGE gels, which were routinely run until the dye front migrated out of the gel. The acrylamide content of the gels was normally set at 15% to achieve optimum resolution of tBid bands (5, 12, 18) but was reduced to 13% for enhanced resolution of closely migrating bands of full-length Bid. Primary antibodies for Bid were obtained from R&D Systems (#AF860, raised against the whole recombinant mouse protein, Abingdon, UK) and from Santa Cruz Biotechnology Inc. (C-20, raised against the C terminus of Bid, and N-19 raised against the N terminus of Bid, Santa Cruz Biotechnology, Santa Cruz, CA). Polyclonal antibodies to Bid provided by colleagues (J. C. Reed and X. Wang) confirmed the specificity of our blotting. To accurately evaluate the size of the Bid-reactive bands, various molecular weight markers were used, including Bio-Rad prestained Precision protein standards (Bio-Rad, Hemel Hempstead, UK). Quantitative loading of proteins was determined by re-blots of reference proteins such as actin and India ink staining of the proteins.

Other Reagents-- Phosphate-buffered saline was obtained from Oxoid (Basingstoke, UK), and cell culture media was from Invitrogen (Paisley, UK). DNA restriction and modification enzymes were from Roche Molecular Biochemicals (Mannheim, Germany), fluorescent probes were from Molecular Probes (Eugene, OR), caspase substrates and inhibitors were from Pharmingen (Oxford, UK), and Alexis (Nottingham, UK), and electrophoresis reagents were from Bio-Rad. The Bid-derived peptides, dodecapeptides corresponding to residues 55-67 (substrates), 55-60, and 61-67 (products) of human Bid, were custom synthesized and analyzed by Neosystem (Strasbourg, France) and dissolved in distilled water. Other reagents were purchased from Sigma (Poole, UK) and were of high analytical grade.

Cell Culture-- Human T lymphoma CEM C7A and Jurkat T cells were grown in RPMI 1640 medium supplemented with fetal bovine serum (10% v/v) and L-glutamine (2 mM) as described previously (18).

Fractionation and Fas Activation of Primary Tissues-- Fas treatment and fractionation of mouse kidney and liver were carried out as described earlier (18). The mitochondrial pellets were resuspended and stored in assay buffer (20 mM K-HEPES, 0.12 M mannitol, 0.08 M KCl, 1 mM EDTA, pH 7.4) containing a mixture of protease inhibitors (Sigma). After centrifugation of the post-nuclear extract at 10,000 × g, the supernatant was taken apart as cytosolic extract (fraction S10) while the mitochondrial pellet was washed as describe earlier (18). The supernatant of the first mitochondrial wash, fraction S3, was kept for further analysis. The protein content of the various fractions was determined using the Bio-Rad Bradford mini-assay in the presence of non-ionic detergents to solubilize membrane proteins; bovine serum albumin was used as a standard.

Isolated Proteins-- rBid (murine) was obtained in purified and active form from R&D Systems. Recombinant human caspases were purchased from Pharmingen or Calbiochem (Cambridge, MA). Endogenous Bid was isolated from cytosolic extracts of mouse kidney by the procedure described previously (18). Briefly, frozen cytosolic extracts were thawed and clarified by extensive centrifugation at 12,000 × g, then diluted with assay buffer containing 2 mM DTT and protease inhibitors. Subsequently, the extracts were heated at 70 °C for 15 min, followed by centrifugation at 4 °C for 40 min at 12,000 × g. The supernatant containing the majority of endogenous Bid was filtered through Sartorius 100-kDa filters (Fisher Scientific, Loughborough, UK) by extensive centrifugation. The filtrates were further fractionated and then concentrated by ultrafiltration with 30- and 5-kDa filters (Sartorius or Millipore, Watford, UK) and gel filtration chromatography. Bid produced by the above process is referred to throughout as "isolated Bid." Importantly, when this isolation process was applied to recombinant Bid, it had no effect upon its phosphorylation by casein kinase II (data not shown). The crude Bid preparations obtained as in a previous study (18) could be used without further manipulation, because they did not contain other proteins of the Bcl-2 family, although in some cases the preparations of endogenous Bid were further purified by ammonium sulfate fractionation, gel filtration, and immunoprecipitation.

In Vitro Modification of Bid-- Phosphatase treatment of cytosolic extracts and Bid preparations was carried out in either 50 mM Tris-Cl buffer, pH 7.5 (for alkaline phosphatase), or 40 mM PIPES-Cl buffer, pH 6.0 (for acid phosphatase (24)), containing 1 mM DTT, 1 mM EDTA, and 1 mM EGTA. Alkaline phosphatase or potato acid phosphatase (Sigma) were added at 2-20 units/ml to the protein samples (5 µg/ml in the case of recombinant Bid) and incubated at either 37 °C for 1 h or, particularly for acid phosphatase, at 30 °C for 2-3 h (24). The suspension was then diluted 5-fold with caspase assay buffer (20 mM K-HEPES, 5% sucrose, 50 mM NaCl, 5 mM DTT, and 1 mM EDTA, pH 7.4, containing protease inhibitors) and either incubated with caspases or stopped with SDS-sample buffer.

To produce caspase cleavage of Bid, recombinant caspase 3 or caspase 8 were suspended at 1-2 µg/ml (or 1000 units/ml for the enzymes purchased from Calbiochem) in the caspase assay buffer supplemented with 0.1% v/v of protease inhibitors and mixed with rBid (1-2 µg/ml) or equivalent concentrations of endogenous Bid isolated from kidney cytosol or other subcellular fractions from mouse kidney (to a final protein concentration of 0.5-1 mg/ml). The reaction mixture was incubated at 30 °C for different times and then stopped by addition of sample buffer and boiling. Samples were frozen and subsequently evaluated by Western blotting. Treatment of Bid preparations (5 µg/ml, with casein, Calbiochem) was carried out for 2-3 h at 30 °C with kinase II (human recombinant CKII in 50 mM Tris-Cl buffer, pH 8.0), containing 10 mM MgCl2, 10 mM alpha -glycerophosphate, 2 mM DTT, and, when indicated, 0.2 mM ATP (compare with Ref. 12). Samples where then diluted 5-fold with caspase assay buffer and either treated with caspases as above or directly dissolved in sample buffer.

Immunoprecipitation of Bid-- For immunoprecipitation of Bid, cytosolic extracts from mouse kidney were first cleared with protein G beads (PerBio, Tattenhall, UK) for 1 h, followed by incubation with 4 µg of Bid antibody (from R&D Systems) overnight on ice. The antibody-antigen complexes were captured with protein G beads for 1 h and then resuspended in 0.5 ml of Triton buffer (50 mM, Tris-Cl, pH 7.5, containing 50 mM NaF, 10 mM alpha -glycerophosphate, 10 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM NaVO4, 0.2% v/v Triton X-100, and protease inhibitors). The protein G beads were then pelleted by centrifugation, washed three times with Triton buffer, resuspended in sample buffer, and then boiled. After centrifugation to separate the beads, the supernatant was analyzed by Western blotting.

Measurements of Caspase Activity-- The assay of caspase activity with the model peptide substrates was based upon reversed-phase HPLC coupled to UV detection. The reaction mixture contained 100 units of recombinant caspase 8 or 3 suspended in assay buffer (50 mM K-HEPES, pH. 7.4, containing 100 mM NaCl, 10 mM DTT, 1 mM EDTA, and 0.1% CHAPS) containing 1 mM peptide substrate to a final volume of 0.1 ml. After incubation at 30 °C for 6 h, the reaction was stopped by the addition of 20 µl of 20% trichloroacetic acid. 30 µl of the mixture were then analyzed by HPLC using a Platinum EPS C18 column (53 × 7 mm, Alltech, Carnforth, UK) and a Hewlett Packard 1100 apparatus. The column was eluted with a linear gradient of 0-90% acetonitrile in water/0.1% trifluoroacetic acid at a flow rate of 2.5 ml/min. Caspase activity of biological samples was measured with the fluorogenic substrates Ac-DEVD-AFC or Ac-IETD-AFC (where AFC is 7-amido-4-(trifluoromethyl) coumarin), in caspase assay buffer (compare with Refs. 18 and 25) using a PerkinElmer Life Sciences LS50B luminescence spectrometer with excitation at 360 nm and emission at 480 nm and disposable Kartell cuvettes.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Caspase 8-mediated Cleavage of Human Bid Peptides Is Decreased by Phosphorylation-- Multiple forms of Bid are observed in lysates from primary tissues or cultured cells (5, 12, 15, 18, 26). Fig. 1A illustrates how multiple protein bands are detected with a Bid-specific antibody in the 20-30 kDa range of cytosolic extracts from mouse tissues and from Jurkat cells, a T lymphoma line frequently used in apoptosis studies (5, 12). Similar results were obtained with different antibodies and other cell lines, including human colon carcinoma and mouse pro-myelocytic lines (data not shown). The presence of these different forms of Bid (compare with Ref. 18) prompted us to investigate the possibility that endogenous Bid may be post-translationally modified, consistent with recent findings in vitro (12).

Our first approach to investigate post-translational modifications of Bid was based on sequence analysis. Specifically, we compared the available amino acid sequences for mammalian Bid and focused our attention on potential phosphorylation sites by computing the conservation and structural position of hydroxyl residues such as serines and threonines (Fig. 1B). Conserved serines and a single threonine were identified that, once mapped on the NMR-deduced structure of Bid (27), could be considered potential phosphorylation sites. Remarkably, three of these potential phosphorylation sites lie adjacent to known cleavage sites in human Bid. Thr-59 is proximal to the primary caspase 8 cleavage site at Asp-60 (5, 6, 26). Ser-65 is proximal to a recently identified site of cleavage by lysosomal proteases (25). Ser-76, is one residue distal to the specific site of cleavage (Asp-75) by granzyme B (6, 28, 29), a non-caspase protease present in granules of cytotoxic lymphocytes (28, 29), which also corresponds to a secondary cleavage site for caspases (6, 8, 23, 30). This peculiar location of potential phosphorylation sites suggested that post-translational modifications of Bid, if present in vivo, would indeed affect its susceptibility to proteolysis. Thr-59 and Ser-65 are the only hydroxyl amino acids that are conserved in the loop region containing the primary site of caspase 8 cleavage (Fig. 1B), which is shared by effector caspases such as caspase 3 (6, 23, 30). In principle, phosphorylation of either Thr-59 or Ser-65 could sterically hinder the proteolytic activity of caspases, because it would insert a bulky phosphate group adjacent to the cleavage site in the target sequence of the Bid protein. However, the effect on the catalytic efficiency of caspases will depend critically on the protein conformation around the substrate cleavage and the degree of steric specificity in the active cleft of the proteolytic enzyme (25, 31).


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Fig. 1.   A, immunoblotting of Bid shows multiple bands in various cellular contexts. Recombinant mouse Bid (5 ng, lane 1) was used as a reference for the immunoblotting of endogenous Bid in cytosolic extracts (fraction S10, 20 µg of protein) of mouse kidney (lane 2), mouse liver (lane 3), and human cultured Jurkat T lymphoma cells (lane 4). The results were obtained with a mixture of the C-20 and R&D antibodies to Bid, as reported previously (18). The presence of multiple bands was detected also with other Bid-specific antibodies (not shown, and Refs. 12, 18, and 26). The data are representative of three independent repeat experiments. B, alignment and comparison of sequences of mammalian Bid. The sequence of human Bid (6) was used as a template for aligning the protein or DNA-deduced sequences of Bid available from current releases of GenBankTM. They include the complete sequence of mouse and rat Bid, as well as that of bovine Bid reconstructed from multiple overlapping expressed sequence tags. The last 55 residues toward the C terminus are not shown because they do not present conserved serines. Amino acids that are conserved in all sequences are in boldface, and the arrows indicate the caspase cleavage sites. The horizontal black bar delineates the region corresponding to the dodecapeptides used in our simplified model system for Bid cleavage.

To verify the effect of phosphorylation of specific residues on the efficiency of caspase 8 cleavage of Bid, we developed a model system using recombinant caspases and synthetic dodecapeptide substrates corresponding to residues 55-67 in the human sequence of Bid (noted by the bar in Fig. 1B). These peptides could be effectively cleaved by caspase 8 at the position corresponding to Asp-60 of the Bid protein as detected by HPLC (Fig. 2). We then used equivalent peptides having the residues corresponding to Thr-59 and Ser-65 of Bid, which were individually or cumulatively phosphorylated. After incubation with caspase 8 for 6 h, the cleavage rate of the peptide with Thr-59 phosphorylated was ~4-fold slower than that with the parent non-phosphorylated peptide (Fig. 2, A and B, and see figure legend). Conversely, phosphorylation of the residue corresponding to Ser-65 did not significantly affect the rate of caspase 8 cleavage of the peptide substrates, whereas the phosphorylation of both Thr-59 and Ser-65 induced a similar, if not stronger, resistance to cleavage than that with phosphorylation of Thr-59 alone (Fig. 2C and legend).


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Fig. 2.   Simplified model system for Bid cleavage. Synthetic dodecapeptides corresponding to amino acid residues 55-67 of human Bid were used as substrates for caspase 8 as described under "Experimental Procedures." The products of the cleavage reactions were subsequently separated by HPLC, and their elution was detected by the normalized absorbance at 210 nm (A210). The thick arrows indicate the cleavage product corresponding to residues Asp-55 through Asp-60, which showed a slightly different migration when Thr-59 was phosphorylated. A, chromatogram of unmodified peptide 55-67 after 6-h incubation with caspase 8, the estimated rate of cleavage was 0.87 µM/h per enzyme unit. B, chromatogram of the peptide having Thr-59 phosphorylated after 6-h incubation with caspase 8, the estimated rate of cleavage was 0.25 µM/h per enzyme unit. C, chromatogram of the peptide having both Thr-59 and Ser-65 phosphorylated, the estimated rate of cleavage was 0.2 µM/h per enzyme unit compared with the rate of 1 µM/h per enzyme unit obtained with the parent peptide having only Ser-65 phosphorylated (not shown). The data are representative of three independent repeat experiments.

These data indicate that, although phosphorylation does not produce a complete block of caspase 8 cleavage, it can substantially inhibit the rate and efficiency of proteolysis of Bid in the region of the primary cleavage site, in broad agreement with previous data obtained with murine Bid (12). Our data also confirm that the relative importance of the phosphorylation sites affecting cleavage may differ between species, with Thr-59 more critical in human Bid as compared with Ser-61 and Ser-65 in murine Bid (12). Experiments equivalent to those in Fig. 2 were undertaken using caspase 3, but the synthetic peptides proved to be ineffective substrates for caspase 3 activity regardless of the presence of phosphorylated residues, consistent with the notion that Bid protein is a better substrate for caspase 8 than for caspase 3 (6).

rBid and Bid Isolated from Murine Kidney Cytosol Have a Different Susceptibility to Caspase 8 Cleavage and Phosphorylation-- The presence of multiple forms of endogenous full-length Bid in primary tissues and cell lysates (Fig. 1A, compare with Refs. 5, 12, and 18) suggested that rBid produced in bacteria, which consistently shows a single band in immunoblots, may provide an inappropriate model for studying the physiological modifications of the protein. Hence, we extended our studies to compare the susceptibility to caspases of rBid and Bid isolated from murine kidney (compare with Ref. 18). Because available Bid antibodies recognize tBid less efficiently than the uncleaved protein (5, 12, 18), we optimized Western blotting conditions for the detection of tBid bands, even if these conditions led to overexposure of the bands corresponding to uncleaved Bid. Fig. 3 shows that incubation of rBid with caspase 8 generates two tBid forms, p15 and p13, that correspond to the C-terminal polypeptides resulting from cleavage at both the primary and secondary site (Fig. 3, lanes 2, compare with Fig. 1B). In contrast, when caspase 8 was incubated with Bid isolated from murine kidney cytosol, only the p15 form of tBid was produced (Fig. 3, lane 5; see also Fig. 5A below). This suggested that Bid isolated from kidney cytosol exhibits an inherent resistance to proteolysis at the secondary cleavage site that is not seen with the recombinant protein.


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Fig. 3.   Caspase 8 cleavage of recombinant and isolated Bid. Recombinant caspase 8 (1000 units/ml, Calbiochem) was incubated for 2 h with 2 µg/ml rBid (lanes 1-3) and an equivalent concentration of Bid isolated from mouse kidney cytosol (lanes 4-6). Other conditions were as described for Fig. 1, and the data shown are representative of three independent repeat experiments. When indicated (lanes 3 and 6), the incubation contained 0.2 mM of the synthetic dodecapeptide having Thr-59 phosphorylated (compare with Fig. 2). The data shown are representative of three independent repeat experiments.

We investigated whether the Bid peptide phosphorylated at Thr-59 (T59P, see Fig. 2B) could act as a competitive inhibitor of Bid cleavage by the two caspases. This peptide hardly affected cleavage of rBid by caspase 8 (Fig. 3, lanes 2 and 3) but showed some inhibitory effect on the cleavage of rBid by caspase 3 (not shown) and on that of isolated Bid by caspase 8 (Fig. 3, lanes 5 and 6). These data, while supporting the contention that the target site in Bid has higher affinity for caspase 8 than for caspase 3 (6), provide additional evidence that phosphorylation may alter proteolysis of Bid.

To directly test the possibility that isolated Bid is phosphorylated (and/or modified) at residues that influence its cleavage by caspases 8, isolated Bid and rBid were incubated with ATP and CKII, the kinase recently reported to phosphorylate murine Bid, especially at Thr-58 (12). In the presence of ATP, CKII produced profound changes in the mobility of rBid from a single band to a triplet of closely migrating bands resolved using 13% acrylamide gels (Fig. 4A). There was no apparent decrease in intensity of any of the Bid triplet bands in the presence of CKII consistent with a lack of cleavage (compare lanes 3 and 4), although CKII treatment did not abolish the production of tBid forms by caspase 8 as detected with routine 15% acrylamide gels optimized for tBid resolution (Fig. 4B). CKII did not alter much the mobility of isolated Bid (Fig. 4A, lanes 5 and 7) but did reduce the caspase 8-mediated loss of isolated full-length Bid (Fig. 4A, lanes 6 and 8) as well as the production of tBid (not shown). These results were consistent with the concept that endogenous modifications of isolated Bid, presumably including phosphorylation that could only be partially mimicked by in vitro treatment with CKII, diminish its susceptibility to cleavage by caspase 8 (12).


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Fig. 4.   Effect of casein kinase II (CKII) on Bid immunodetection and cleavage by caspase 8. A, equivalent concentrations of murine rBid (lanes 1-4) and Bid isolated from kidney cytosol (lanes 5-8) were incubated for 3 h in the absence (lanes 1, 2, 5, and 6) and presence (lanes 3, 4, 7, and 8) of 0.8 unit/ml of recombinant CKII and 0.2 mM ATP. Subsequently, the samples were diluted 5-fold in caspase assay buffer and incubated with recombinant caspase 8 for 1 h as described for Fig. 3. To enhance the resolution of the blot of full-length Bid, the protein was separated with a 13% acrylamide gel and transferred onto a low porosity polyvinylidene difluoride membrane (Applied Biosystems) using a 10 mM sodium CAPS buffer, pH 11, instead of the usual Tris-glycine buffer. B, Western blotting was conducted under the routine conditions implemented for tBid resolution (compare with Fig. 3) with recombinant Bid treated with CKII as in A, either in the presence or absence of ATP (required for protein phosphorylation (12)). The data are representative of three independent repeat experiments.

Differential Cleavage of Endogenous and rBid by Caspase 8 and Caspase 3-- In previous studies, emphasis has been placed on the possible post-translational regulation of Bid cleavage by caspase 8, an event considered to be crucial in death receptor-induced apoptosis of type II cells (3, 7, 8, 12-14, 19, 22). However, Bid can also be effectively cleaved by caspase 3 and other downstream caspases (6, 23, 26, 30) that can be rapidly engaged in type I cells (13, 14), or subsequent to activation of the apoptosome by a variety of death stimuli (3, 23, 30). Given the potential relevance to the feed-forward amplification of the death cascade via tBid-induced mitochondrial damage (2, 3, 23, 30), we investigated whether the proteolytic cleavage of Bid displayed different properties with caspase 8 and an effector caspase, caspase 3. A comparable cleavage of rBid was observed with either caspase 8 or 3, which both produced the p15 and p13 forms of tBid (Fig. 5A, lanes 2 and 3). However, Bid isolated from murine kidney was completely resistant to caspase 3 cleavage (Fig. 5A, lane 6), whereas it was efficiently cleaved by caspase 8 to produce p15 tBid (Fig. 5A, lane 7).


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Fig. 5.   Differential cleavage of Bid by caspase 8 and caspase 3. A, tBid products resulting from caspase cleavage of either rBid or Bid isolated from mouse kidney were separated with enhanced resolution of the SDS-PAGE and related to the migration of recombinant tBid (lane 7) and precision molecular weight standards. Conditions were the same as those described for Fig. 3. B, effect of the isolated Bid preparation (diluted to the same final concentration as in the experiment of Bid cleavage, compare with A and B) on the kinetics of recombinant caspase 3 (0.1 µg/ml) cleavage of the fluorogenic substrate Ac-DEVD-AFC (50 µM). Data show the progressive increase of fluorescent product and are representative of triplicate repeat experiments. The solid lane is the control trace, whereas the dashed line is the trace obtained in the presence of isolated Bid. The dotted line shows the inhibitory effect of 20 µM Asp-Glu-Val-Asp-chloromethyl ketone (DEVD-CHO).

The unexpected differences in the proteolytic susceptibility of Bid (Fig. 5A) could derive from factors that exclusively influence the reactivity of isolated Bid with caspase 3. To test this possibility, we examined whether our preparations of isolated, partially purified Bid contained contaminants that could affect the catalytic efficiency of caspases and in particular alter susceptibility to caspase 3 cleavage (compare with Ref. 25). As shown in Fig. 5B, isolated Bid had a slight stimulatory effect, if any, on the enzymatic rate of DEVD-AFC cleavage by caspase 3 under the same conditions as those used in the experiments of Bid cleavage in Fig. 5A. Additionally, we verified that the cleavage of rBid by caspases was not significantly altered by the presence of isolated Bid preparations (data not shown). We thus excluded the possibility that the difference in caspase cleavage of isolated Bid (Fig. 5A) could derive from factors present in the isolated Bid preparation that inhibit caspase 8 or caspase 3.

Subcellular Localization of Bid Affects Its Cleavage by Caspase 3-- We subsequently investigated whether the differential susceptibility of isolated Bid to caspase 3 cleavage compared with the recombinant protein may derive from post-translation modifications by enzymes present in cytosol or other subcellular compartments where native Bid is present. Therefore, we compared the cleavage of endogenous Bid present within subcellular fractions of primary tissues after addition of exogenous caspase 3 using rBid as a standard. Endogenous Bid was much more susceptible to caspase 3 cleavage within cytosolic fractions such as S10 than in other fractions, including mitochondria and mitochondrial wash (fraction S3, Fig. 6A). We then isolated Bid from the same cytosolic fraction, re-assessed its susceptibility to caspase 3 cleavage and found it to be completely resistant (Fig. 6B). In contrast, the same isolated Bid was efficiently cleaved by caspase 8 (Fig. 3). Consequently, the results suggested that once removed from its cytosolic environment, Bid remained a good substrate for the initiator caspase 8 but became resistant to cleavage by the effector caspase 3 arguing that Bid cleavage by these two caspases is differentially regulated. The fact that addition of exogenous caspase 3 could elicit complete cleavage of cytosolic Bid (Fig. 3) could be interpreted as a result of proteolysis-dependent cleavage of endogenous caspase 8, a primary substrate of caspase 3 (31). This possibility was verified by following the activation of caspase 8 activity in cytosolic extracts treated with caspase 3 (data not shown).


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Fig. 6.   Bid cleavage by caspases differs in different subcellular fractions. A, subcellular fractions obtained from homogenates of murine kidney were treated at the same final concentration of 1 mg/ml with recombinant caspase 3 (1 µg/ml, from Pharmingen) for different times as described under "Experimental Procedures." Lanes 3-5 contained a cytosolic extract (fraction S10 consisting of the 10,000 × g supernatant (18)), whereas lanes 6-8 contained the mitochondrial wash, obtained after 10,000 × g centrifugation of crude mitochondria (fraction S3). Lanes 9-11 contained isolated kidney mitochondria (mitos). rBid (1 µg/ml, lanes 1 and 2) was treated under the same condition. Protein samples were blotted with the R&D antibody. B, recombinant caspase 3 was incubated for 1 h with S10 fraction of murine kidney (lanes 1 and 2), rBid (at a concentration about 3-fold lower than endogenous Bid, lanes 3 and 4), and Bid isolated from the same cytosolic S10 fraction (lanes 5 and 6). Samples were incubated for 1 h in the presence or absence of 20 µM Asp-Glu-Val-Asp-chloromethyl ketone to inhibit caspase activity. Note the absence of detectable tBid in isolated Bid (lane 6) versus rBid (lane 4) despite the much higher concentration. Data shown are representative of four replicate experiments.

Acid Phosphatase Treatment Enhances Caspase Cleavage of Endogenous Bid-- Our previous data suggest that phosphorylation of isolated Bid reduces but does not abolish its cleavage by caspase 8. Consistent with data shown in Fig. 4B, incubation of isolated full-length Bid with CKII attenuated the decreased in full-length protein, but did not prevent caspase 8-mediated production of tBid (Fig. 7A, lanes 2 and 4). To investigate the role of Bid phosphorylation on its cleavability further, we examined the impact of incubating isolated Bid with phosphatases anticipating that this should enhance Bid cleavage by caspase 8. After unsuccessful attempts with alkaline phosphatase, we realized that the acid nature of mouse Bid would require treatment with phosphatase working at low pH to obtain effective de-phosphorylation. Indeed, treatment with potato acid phosphatase (PAP as in Ref. 24) significantly enhanced the cleavage by caspase 8 of isolated Bid with or without incubation with CKII with barely detectable full-length Bid remaining (Fig. 7, A and B). Fig. 7B compares the effects of CKII and PAP on isolated Bid cleavage by caspases 8 and 3. Although CK II treatment prevented the production of tBid cleavage by caspase 3 (Fig. 7B, lane 3), resistance to caspase 3 cleavage was abrogated by PAP treatment that promoted the production of p15 tBid (Fig. 7B, lane 6). Lastly, we examined the effect of PAP on the cleavage of Bid left within kidney cytosol (Fig. 7C). Here PAP produced p15 and p13 tBid. This suggested that de-phosphorylation had enhanced the capacity of endogenous cytosolic proteases to cleave Bid at both the primary and secondary site, as in the case of the recombinant protein treated with caspase 8. Comparable results were obtained using extracts of human lymphoma cells.2


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Fig. 7.   Phosphatase treatment of endogenous Bid facilitates its cleavage by caspases. A, Bid isolated from murine kidney was untreated (lanes 1 and 2) or treated with CKII as in Fig. 4 (lanes 3-5) and with potato acid phosphatase (PAP, lane 6) as described under "Experimental Procedures." At the end of the incubation, samples were diluted 5-fold with caspase assay buffer and incubated for 1 h with caspase 8 as described in Fig. 6. B, Bid isolated from murine kidney was treated with CKII (lanes 1-3) or with PAP (lanes 4-6). At the end of the incubation, samples were diluted 5-fold with caspase assay buffer and incubated for 1 h with either caspase 8 or caspase 3. C, samples of murine kidney cytosol were treated with PAP for 1 h at 37 °C and probed directly with the R&D antibody. Equal protein loading was confirmed by subsequent reblots for actin (bottom panel) and with India ink staining (not shown). Comparable results were obtained with other antibodies. Data shown are representative of three independent repeat experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Caspase cleavage is considered fundamental to the action of pro-apoptotic Bid, especially in the pathways of apoptosis triggered by death receptor ligation (3-7, 25). However, Bid is also cleaved during drug-induced apoptosis upstream or downstream of cytochrome c release, dependent on cell type. In type II cells, Bid is cleaved by caspase 3 or by caspase 8 acting as an amplifying executioner caspase in drug-induced apoptosis (32). In type I cells where drug treatment can induce formation of a DISC, Bid is cleaved by caspase 8 upstream of caspase 3 (33). Moreover, in certain scenarios, such as in the neuronal cell death that occurs in Huntington's disease, pro-caspase 8 can be activated independently of death receptor ligation (34) to promote apoptosis via components of the death receptor signaling pathway, presumably including the cleavage of Bid. Thus, the events that regulate the susceptibility of Bid cleavage are important for apoptotic pathways engaged by a variety of stimuli. Given the potent action of tBid on mitochondria it would seem mandatory that constitutive mechanisms prevent tBid formation by basal caspase activities in cells that are responding appropriately to survival signals. The intracellular location and caspase cleavage of Bid are likely to be regulated by converging pathways of cell survival, as indicated by increasing, although indirect evidence (12, 14, 19-23, 35). In this regard, the ability of the tumor suppressor PTEN (phosphatase and tensin homolog (3-phosphoinositide phosphatase)) to sensitize to drug-induced apoptosis via a pathway dependent on the DISC component FADD (Fas-associated death domain-containing protein) provides another interesting link between the interruption of a survival signaling pathway and the cleavage of Bid to the promotion of apoptosis (36).

Although it has been claimed that Bid is not phosphorylated in cultured cells (37), more recently it has been reported that Bid can be phosphorylated both in vitro and in vivo, particularly by enzymes of the casein kinase family (12). Given the limited information on the possible modification of endogenous Bid within primary tissues, we have focused our work on a comparison of the cleavage of endogenous Bid (either within or isolated from mouse kidney cytosol) and rBid by caspases 8 and 3.

Our data provide novel evidence that endogenous Bid is phosphorylated (or otherwise modified) at sites influencing its cleavage by caspases. First, we demonstrated in a model system of Bid cleavage that phosphorylation of conserved residues such as Thr-59 (in the human Bid sequence), which surround the primary site of caspase cleavage (Fig. 1B), substantially diminished the proteolytic capacity of caspase 8 (Fig. 2). Second, we found that the Bid that was constitutively located (albeit at low levels) in kidney fractions other than cytosol (but including mitochondrial Bid) was resistant to proteolytic cleavage by caspase 3 (Fig. 6B). This resistance was intrinsic to the protein in situ, because Bid isolated from the cytosolic extracts, contrary to rBid, was also resistant to caspase 3 cleavage (Figs. 5 and 6). Third, we found that dephosphorylation of endogenous Bid with PAP greatly facilitated its cleavage by caspases 3 and 8 (Fig. 7).

Phosphorylation of residues around the cleavage site has been shown to prevent proteolysis by caspase 3 in other proteins, for example Ikappa B-alpha (38, 39). In this case, phosphorylation confers specific protection toward proteolytic events that are associated with the execution phase of caspase-mediated death (38, 39). Protection from caspase cleavage would affect both tBid action following the processing of pro-caspase 8 upstream of mitochondria and tBid involvement in the amplification of caspase cascades downstream of mitochondria. Our findings clearly indicate that physiological mechanisms are in place to allow efficient cleavage of Bid by upstream caspase 8 but not simultaneously by downstream caspases like caspase 3. What produces this differential specificity in Bid cleavage?

Structural properties intrinsic to the Bid protein may partially account for differential cleavage by different caspases. Previous results (compare with Ref. 6) showed that Bid is a better substrate for caspase 8 than for caspase 3, and our data indicate that this is particularly the case for the cleavage at the secondary site (Asp-75, compare with Fig. 5B). The different efficiency of rBid cleavage by caspase 8 and 3 could simply derive from the fact that the protein region around the secondary cleavage site in Bid is much less flexible than that around the primary cleavage site (27) and proteolytic enzymes like caspases prefer flexible peptides as substrates (31). Hence, even in the absence of Bid modification, local structural determinants would affect the reactivity of Asp-75 with caspase 3 but not with caspase 8. However, caspase 8 is essentially unable to produce p13 tBid when reacting with endogenous Bid isolated from kidney cytosol (Figs. 5 and 6). This implies that residues such as Ser-76, the only conserved hydroxyl amino acid lying around the secondary cleavage site (Fig. 1B), may be modified in vivo to prevent proteolysis. Indeed, phosphate labeling was reported to be associated also with a peptide comprising Ser-76 after in vitro phosphorylation and subsequent fragmentation of murine Bid (12).

Post-translational modifications must be present also around the primary cleavage site of endogenous Bid to explain its strong resistance to caspase 3 cleavage (Figs. 5 and 6). The same modifications, however, cannot prevent Bid cleavage by caspase 8 (compare with Fig. 3). Given the discussion above and the evidence that phosphorylation incompletely inhibits caspase 8 cleavage of peptide substrates (Fig. 2) and rBid (Fig. 4 and results not shown, compare with Ref. 12), this difference can be rationalized in terms of a superior catalytic efficiency and substrate specificity of caspase 8. It is increasingly evident that the exposed loop preceding the BH3 domain in Bid structure (residues 50-80, compare with Fig. 1B and Ref. 27) is susceptible to proteolysis by a variety of endogenous proteases. Besides caspase 8 (5, 6) and other caspases (6, 23, 24, 30), Bid can be cleaved by lysosomal proteases (25) and calpain (40). Hence, the (poly)phosphorylation at residues 59, 62/65, and possibly also 76, may serve as a general mechanism for reducing the spontaneous degradation of Bid by these endogenous proteases.

These modifications notwithstanding, endogenous Bid remains highly susceptible to cleavage by caspase 8, especially when compared with its resistance to cleavage by caspase 3 (Figs. 5 and 6). Consequently, phosphorylation (or other post-translational modifications around the caspase sites) could serve not only to stabilize the Bid protein but also to ensure its specific susceptibility to proteolysis by caspase 8. Our results (Figs. 6 and 7) lend support to this possibility, although other modifications, such as myristoylation (37), may also contribute to maintain a rigorous specificity for Bid cleavage toward activated caspase 8. In possible contradiction with this, our data also show that addition of exogenous (fully activated) caspase 3 leads to complete cleavage of cytosolic Bid (Fig. 6). However, this cleavage may derive from indirect reactions, including activation of cytosolic caspases like caspase 8, and caspase-mediated activation of protein phosphatases (42).

In conclusion, our study strengthens the contention that post-translational modifications alter Bid susceptibility to caspase cleavage and consequently its pro-apoptotic activation (12, 43). Further studies will determine the biochemical effects of Bid phosphorylation in its reactivity with membrane lipids (18, 44) that may be crucial in its biological action in mitochondria.

    ACKNOWLEDGEMENT

We thank E. Beaulieu for assistance.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Funded by an Alliance Grant from Institut de la Recherche Servier (Paris).

|| Supported by the Lister Insitute of Preventive Medicine. To whom correspondence should be addressed. Tel.: 44-161-275-5447; Fax: 44-161-275-5600; E-mail: cdive@man.ac.uk.

Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M209208200

2 P. Masdehors, unpublished data.

    ABBREVIATIONS

The abbreviations used are: DISC, death-inducing signaling complex; DTT, dithiothreitol; tBid, truncated Bid; rBid, recombinant Bid; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; CK, casein kinase; PIPES, 1,4-piperazinediethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAP, potato acid phosphatase; HPLC, high-performance liquid chromatography; AFC, 7-amido-4-(trifluoromethyl) coumarin.

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