Many molecules are inducibly localized in lipid rafts, and their alteration inhibits early activation events, supporting a critical role for these domains in signaling. Using confocal microscopy and cellular fractionation, we have shown that the pool of Bad, attached to lipid rafts in proliferating cells, is released when cells undergo apoptosis. Kinetic studies indicate that rafts alteration is a consequence of an intracellular signal triggered by interleukin-4 deprivation. Growth factor deprivation in turn induces PP1α phosphatase activation, responsible for cytoplasmic Bad dephosphorylation as well as caspase-9 and caspase-3 activation. Caspases translocate to rafts and induce their modification followed by translocation of Bad from rafts to mitochondria, which correlates with apoptosis. Taken together, our results suggest that alteration of lipid rafts is an early event in the apoptotic cascade indirectly induced by interleukin-4 deprivation via PP1α activation, dephosphorylation of cytoplasmic Bad, and caspase activation.
Apoptosis is a genetically regulated process that allows removal of abnormal growing cells and controls homeostasis. Apoptosis may be induced by drugs, radiations, infections, or growth factor deprivation as well as by signals transmitted through membrane receptors. Apoptosis eliminates cells in an ordered manner, and deregulation of the apoptotic machinery can lead to many diverse diseases (1-4).
The Bcl-2 family proteins act as a decision point in the apoptotic pathway by regulating the permeabilization of the outer mitochondrial membrane. This family comprises both proapoptotic and antiapoptotic members, including the BH3-only member Bad, which promotes apoptosis. Thus far, 10 distinct BH3-only proteins have been described in mammals. The reason for this redundancy remains unexplained. It has been suggested that BH3-only members function as death sensors where various death stimuli seem to activate different BH3-only proteins (5, 6).
The serine/threonine protein phosphatases are usually classified as type 1 (PP1) or type 2 (PP2) depending on their substrate specificity and sensitivity to inhibitors. PP1 and PP2 are abundant serine/threonine phosphatases expressed in mammalian cells (7). PP1 represents a family of holoenzymes generated by specific interactions between catalytic subunits and a wide variety of regulatory or anchoring proteins involved in targeting as well as in controlling phosphatase activity (8, 9). PP2A enzymatic activity is reported to be more potently inhibited by okadaic acid (10, 11), and controlled concentrations of okadaic acid can selectively inhibit PP2A without affecting PP1 activity (12). We have shown that Bad is an in vitro and in vivo substrate for PP1α and that growth factor deprivation induced apoptosis operate by regulating cytoplasmic Bad dephosphorylation through PP1α phosphatase (13). We have also shown that growth factor deprivation of TS1αβ cells induces a mitochondrial-mediated apoptotic pathway that leads to cytochrome c release, thus activating caspase-9 and caspase-3 (14).
Localization of proteins to distinct subcellular compartments, including membranes, is a critical event in multiple cellular pathways, such as apoptosis. Plasma membranes of many cell types contain microdomains called lipid rafts, which are biochemically different from bulk plasma membrane (15-17). These domains are enriched in sphingolipids and cholesterol and show resistance to Triton X-100 solubilization and can be isolated by density gradient ultracentrifugation. It has also recently been shown using in vitro experiments that ceramide lipid, which is involved in important biological processes, reside and function within rafts (18). Rafts may be visualized in intact cells by confocal microscopy using fluorescently labeled cholera toxin subunit B, which binds to the ganglioside GM1 (19).
Many receptors are inducibly localized in lipid rafts, which have been shown to function as platforms coordinating the induction of signaling pathways. Disruption of lipid rafts inhibits early activation events, supporting a critical role for these domains in signaling. Recent publications have proposed that rafts are involved in the control of apoptosis via positive interactions involving Fas or via negative interactions involving Bad (20-23). It has been shown that interaction of Fas with rafts induces apoptosis in HL-60 and Jurkat cell lines (23), and disruption of membrane rafts integrity inhibits Fas-dependent apoptosis in leukemic cells as well as in cell lines. In addition to Fas, other apoptotic molecules, such as Fas-associated death domain protein, pro-caspase-8, pro-caspase-10, c-Jun NH2-terminal kinase, and Bid, are recruited into rafts, linking Fas and mitochondria signaling pathways (24). Moreover, caspase-3 is also a component of the Fas-inducing signaling complex in lipid rafts, and its activity is required for caspase-8 activation in Fas-induced cell death (25). This translocation of Fas into rafts may provide a mechanism for amplifying Fas signaling through reorganization of membrane microdomains. In resveratrol-induced apoptosis, Fas is triggered to membrane rafts in SW480 human colon cancer cells together with Fas-associated death domain protein and pro-caspase-8. This redistribution is associated with the formation of the death-inducing signaling complex (26). In addition, Akt2, phosphatidylinositol 3′ -kinase, protein kinase C, and PTEN are also localized in lipid rafts (27, 28). In the same direction, recruitment of tumor necrosis factor receptor to rafts is essential for tumor necrosis factor-α–mediated nuclear factor-κB activation (29). Other signaling molecules, such as the Lck kinase and interleukin (IL)-2 and IL-15α subunit receptor, are also associated to lipid rafts (30, 31). The translocation of Lck to rafts regulates Fyn kinase activity. Finally,tyrosine-phosphorylated CD28 is recruited to lipid rafts and is associated to phosphatidylinositol 3′-kinase in both Jurkat and peripheral blood T lymphocytes (32). Bad resides in rafts in proliferating T cell lines and thymocytes while associated to mitochondria in apoptotic cells. Changes of rafts integrity induce segregation of Bad from rafts followed by its translocation to mitochondria, which correlates with apoptosis induction (22, 33). Here, we show that alteration of rafts is a consequence of an intracellular signal initiated by cytoplasmic Bad dephosphorylation and represents an early biochemical hallmark in the apoptotic process induced by IL-4 deprivation.
IL-4 Deprivation Induces Alteration of Lipid Rafts
TS1αβ is a lymphokine-dependent murine T cell line. When IL-4-maintained cells are deprived of lymphokine, they undergo apoptosis (Table 1).. As early as 4 hours after IL-4 deprivation, 14% of the cells were apoptotic, progressively increasing and reaching 47% on 24 hours of IL-4 starvation. Control IL-4-stimulated cells showed no significant level of apoptosis.
|Apoptotic Cells (%) .||.||.|
|Time (h) .||IL-4 .||Apoptosis .|
|0||+||5 ± 2|
|4||−||14 ± 3|
|8||−||22 ± 2|
|12||−||30 ± 4|
|16||−||36 ± 3|
|24||−||47 ± 5|
|Apoptotic Cells (%) .||.||.|
|Time (h) .||IL-4 .||Apoptosis .|
|0||+||5 ± 2|
|4||−||14 ± 3|
|8||−||22 ± 2|
|12||−||30 ± 4|
|16||−||36 ± 3|
|24||−||47 ± 5|
NOTE: Cells were cultured in the presence or absence of IL-4 for the indicated times, harvested, diluted in ice-cold binding buffer, stained with Annexin and propidium iodide, and analyzed by flow cytometry. Percentages ± SD of apoptotic cells in each sample.
Given that 4 hours after IL-4 deprivation we already were able to distinguish apoptosis signals, we analyzed whether alteration of rafts was detected on this short period of lymphokine deprivation. For this purpose, IL-4-stimulated or IL-4-deprived cells were incubated with cholera toxin subunit B coupled to FITC, which binds to ganglioside GM1, followed by fluorescence-activated cell sorting analysis. As shown in Fig.1A, a significant diminution of the signal intensity with the rafts marker cholera toxin-FITC was observed 4 hours after IL-4 deprivation. The level of staining progressively decreases as the IL-4 starvation period increases (Fig. 1A), reaching the minimum level of signal intensity 24 hours after lymphokine withdrawal. Figure 1B illustrates the mean fluorescence of cholera toxin-FITC labeling of control and IL-4-deprived cells. This result suggests an early alteration of rafts on growth factor deprivation that may trigger subsequent apoptotic events.
The alteration of lipid rafts on IL-4 deprivation was also analyzed in intact cells by confocal microscopy (Fig. 1C). Control IL-4-stimulated or IL-4-deprived cells for different times were incubated with FITC-labeled cholera toxin subunit B. We observed a homogeneous distribution of green fluorescence in the membrane of IL-4-stimulated cells. In contrast, we detected a nonhomogeneous distribution of fluorescence with interruption of the signal at the cell surface as well as a reduction in the fluorescence intensity, which proves that the rafts structure is already altered in 4-hour IL-4-deprived cells. Green fluorescence labeling on the cell surface progressively disappears as the IL-4 deprivation period increases. These confocal microscopy studies suggest an early alteration of lipid rafts in the course of apoptotic process.
Segregation of Bad from Lipid Rafts Is an Event Induced by IL-4 Deprivation
We have shown previously colocalization of a pool of Bad and lipid rafts in proliferating cells, whereas rafts were altered in apoptotic cells, and as a consequence, Bad translocates to mitochondria (13). To dissect whether alteration of rafts and, as a result, segregation of Bad is the first phenomenon occurring in the apoptotic process triggered by growth factor deprivation or vice versa, we analyzed the subcellular distribution of Bad in intact cells on distinct IL-4 deprivation periods (Fig. 2A). Immunofluorescence analyses show homogeneous distribution of Bad in plasma membrane and in the cytoplasm in both control and 4-hour IL-4-deprived cells. Interestingly, 8 hours on IL-4 withdrawal, we observed a decrease in the amount of Bad associated to the plasma membrane, reaching the minimum level 24 hours after lymphokine deprivation (Fig. 2A). The distribution of Bad was also analyzed by subcellular fractionation. Rafts and mitochondria were isolated from IL-4-stimulated or IL-4-deprived cells for different times. Bad was found to be associated to rafts in control IL-4-stimulated cells. No difference in the quantity of attached Bad to rafts was detected 4 hours after IL-4 deprivation.The amount of Bad associated to rafts decreased on 8 hours of IL-4 starvation, and only traces of Bad were detected associated to rafts on 24 hours of IL-4 deprivation (Fig. 2B). In addition, the pool of Bad associated to rafts is dephosphorylated. To validate the fractionation protocol, rafts and the cytosolic fraction were immunoblotted with cholera toxin-biotin and α-tubulin antibody.
The subcellular localization of Bad was also examined in mitochondrial fraction of IL-4-stimulated or IL-4-deprived cells (Fig. 2C). Low amount of Bad was detected associated to mitochondrial fraction of both IL-4-stimulated and 4-hour IL-4-deprived cells. An increase in the amount of Bad localized to mitochondria was observed 8 hours after IL-4 deprivation, reaching the maximum 24 hours after lymphokine deprivation. At that time, only traces of Bad are associated to rafts (Fig.2B).The purity of the mitochondrial and cytosolic fraction was confirmed by reimmunoblotting with α-VDAC and α-caspase-3 antibodies. Figure 2D shows that total Bad expression was not modified on IL-4 deprivation. Given that alteration of lipid rafts is detectable 4 hours after lymphokine deprivation, whereas Bad is still associated to lipid rafts, this result strongly suggests that alteration of rafts precedes Bad redistribution. Translocation of Bad to mitochondria was also confirmed in intact cells by confocal microscopy (Fig. 3). Double immunofluorescence analyses with anti-Bad and anti-mitochondria antibody show very weak association of Bad to mitochondria in IL-4-stimulated cells, whereas there is a significant fraction of Bad associated to mitochondria in 12-hour IL-4-deprived cells (Fig. 3).
Because the integrity of lipid rafts is necessary for association of Bad, we analyzed whether other proteins known to be associated to rafts, such as Lck, are able to segregate on alteration of rafts. Rafts were isolated from IL-4-stimulated orIL-4-deprived cells and immunoblotted with anti-Lck antibody (Fig.4). Lck was detected in the rafts fraction of control IL-4-stimulated and 4-hour IL-4-deprived cells, progressively decreasing on 8 hours of IL-4 deprivation. No Lck was detected on 12 hours of IL-4 starvation (Fig. 4), demonstrating that alteration of rafts induces segregation of attached proteins. Taken together, these results provide evidence that rafts alteration is an early event in the apoptotic process and its integrity is necessary for protein association.
Indirect Effect of IL-4 Deprivation on Rafts Alteration
We further examined whether the absence of IL-4 was responsible for rafts alteration or whether IL-4 deprivation gives an intracellular signal that triggers, as a final consequence, modification of rafts. For this purpose, IL-4-stimulated or IL-4-deprived cells for different times were treated with or without z-VAD-fmk to block caspase-dependent apoptosis and then incubated with cholera toxin-FITC followed by fluorescence-activated cell sorting analysis. As shown in Fig.5A, we observed a significant diminution of the signal intensity with cholera toxin-FITC 4 hours after IL-4 deprivation. The level of staining progressively decreases as the IL-4 deprivation period increases (8 hours), confirming previous results (Fig. 1). Interestingly, z-VAD-fmk addition causes no reduction in signal intensity of the rafts marker cholera toxin-FITC on IL-4 deprivation (Fig.5A), suggesting an indirect effect of IL-4 deprivation on rafts organization and that rafts alteration is dependent on caspase activation. To validate z-VAD-fmk action on the cells, apoptosis and caspase inhibition were analyzed. z-VAD-fmk treatment blocks caspase-9 activation and apoptosis in IL-4-deprived cells as shown in Fig. 5B and C. Nontreated cells show levels of apoptosis comparable with that shown in Table 1.
IL-4 Deprivation Induces Bad-Associated PP1α Phosphatase as Well as Caspase-9 and Caspase-3 Activation
We have shown previously that Bad is an in vitro and in vivo substrate for PP1α phosphatase and that growth factor deprivation–induced apoptosis operate by regulating cytoplasmic Bad dephosphorylation through PP1α phosphatase (13). Figure 6A shows phosphatase activity in cytoplasmic Bad immunoprecipitates of IL-4-stimulated or IL-4-deprived cells for different times. Phosphatase activity in the immunoprecipitates was measured using [32P]phosphorylase a as substrate. Enzymatic activity was detected in Bad immunoprecipitates of IL-4-stimulated cells increasing 2 hours after IL-4 deprivation. The enzymatic activity progressively augments as the IL-4 deprivation period enlarges. No phosphatase activity was detected in control immunoprecipitates anti-c-Jun of IL-4-stimulated or IL-4-deprived cells. This result suggests that IL-4 deprivation rapidly induces Bad-associated phosphatase activation in a time-dependent manner. Figure 6B confirms that PP1α but not PP2A or PP2B was detected associated to Bad and was responsible for the enzymatic activity detected in cytoplasmic Bad immunoprecipitates. This association was also detected using radioimmunoprecipitation assay buffer (data not shown). Comparable PP1α levels were detected by Western blot in Bad immunoprecipitates from control IL-4-stimulted or IL-4-deprived cells. Reprobing the membrane with anti-Bad antibody showed similar levels of Bad in immunoprecipitates from control IL-4-stimulted or IL-4-deprived cells (Fig. 6B). This result suggests a rapid activation of PP1α phosphatase activity and, as a consequence, an immediate dephosphorylation of cytoplasmic Bad that turns Bad into a proapoptotic molecule.
We have shown previously that growth factor deprivation induces initiator caspase-9 as well as caspase-3 activation (14). To know whether caspase activation occurs upstream or downstream of Bad-associated PP1α phosphatase activation, we did kinetics of caspase-9 and caspase-3 activity on different periods of IL-4 deprivation. Figure 7A illustrates that caspase-9 and caspase-3 activation follow PP1α phosphatase activation, as the activity is detected 4 hours on IL-4 deprivation, progressively increasing with the deprivation kinetic (Fig.7A). On the contrary, PP1α phosphatase activation was already detected 2 hours on IL-4 starvation. This result argues that caspase activation is a downstream event of PP1α phosphatase activation.
Caspase-9 and Caspase-3 Associate with Lipid Rafts in IL-4-Deprived Cells
It is known that localization of proteins to distinct subcellular compartments, including cellular membrane, is a critical event in multiple cellular pathways, such as apoptosis. Given that an increase in Bad-associated phosphatase activity is detected very early in the apoptotic process followed by caspase-9 and caspase-3 activation and that IL-4 deprivation is not directly responsible for rafts alteration, we wondered whether caspase-9 and caspase-3 could be localized and activated in lipid rafts on IL-4 starvation. For this purpose, we isolated rafts by Triton X-100 flotation gradient of control IL-4-stimulated or IL-4-deprived cells (Fig.7B). Caspase-9 could not be detected in lipid rafts of IL-4-stimulated cells, progressively increasing during the starvation period, whereas pro-caspase-9 was only identified in lipid rafts of IL-4-stimulated and 4-hour IL-4-deprived cells. Both pro-caspase-9 and pro-caspase-3 were found in total extracts. Similarly, low level of caspase-3 was detected in rafts of control IL-4-stimulated cells, increasing as the starvation period enlarges. To validate the gradient procedure, the blot was hybridized with anti-Lck (rafts marker) and anti-Tim 23 (non-rafts marker).
Given that IL-4 deprivation firstly induces Bad-associated PP1α phosphatase activation and subsequently caspase-9 and caspase-3 activation, we asked whether PP1α activity was an upstream event in the rafts alteration cascade. We hypothesized that inhibition of PP1α phosphatase activity in IL-4-deprived cells may prevent caspase activation and apoptosis. For this purpose, we treated IL-4-stimulated or IL-4-deprived cells with the phosphatase inhibitor okadaic acid using a concentration affecting only PP1 (13). Figure 8A shows that IL-4-deprived cells treated with 1 μmol/L okadaic acid for 6 hours showed an inhibition of pro-caspase-9 and pro-caspase-3 activation as detected by Western blot. On the contrary, caspase-9 and caspase-3 activation were detected in nontreated IL-4-deprived cells. The effect of okadaic acid on PP1α phosphatase activity was determined by inhibition of Bad phosphorylation as described previously (13). Table 2 shows that IL-4-deprived cells treated with okadaic acid for 6 hours showed a significant reduction in the fraction of apoptotic cells (9%) compared with untreated cells (18%).
|Apoptotic Cells (%) .||.||.||.|
|Time (h) .||IL-4 .||Okadaic Acid .||Apoptosis .|
|0||+||−||3 ± 2|
|6||−||+||9 ± 1|
|0||+||+||5 ± 3|
|6||−||−||18 ± 3|
|Apoptotic Cells (%) .||.||.||.|
|Time (h) .||IL-4 .||Okadaic Acid .||Apoptosis .|
|0||+||−||3 ± 2|
|6||−||+||9 ± 1|
|0||+||+||5 ± 3|
|6||−||−||18 ± 3|
NOTE: Cells were treated for 6 hours with or without 1 μmol/L okadaic acid in the presence or absence of IL-4. Cells were washed, stained with Annexin and propidium iodide, and analyzed by flow cytometry. SD for n = 3.
Taken together, these results strongly suggest that alteration of lipid rafts is the consequence of an intracellular signal delivered by IL-4 deprivation, which in turn triggers cytoplasmic Bad dephosphorylation via PP1α phosphatase followed by caspase-9 and caspase-3 activation. Caspases translocate to lipid rafts and promote rafts alteration probably due to its protease activity. As a consequence, Bad translocates from rafts to mitochondria, which correlates with apoptosis.
Lipid rafts have been implicated in the regulation of numerous cellular events, including signal transduction (34), membrane traffic (35), and viral entry/infection (36). The large majority of these studies have relied on the detergent insolubility of lipid rafts for their purification. The most widely used detergent in these studies is Triton X-100, although other detergents have been used for this purpose, including CHAPS, Tween 20, Triton X-114, Lubrol, Brig 96, Brig 98, and sodium deoxycholate (37-40). Among all of them, Triton X-100 has the highest solubilization strength (41). Triton X-100-insoluble domains had a marker enrichment of sphingolipids and cholesterol relative to glycerophospholipids. Lipid composition can vary in distinct protein-containing rafts, and detergent insolubility may be used as a basis to separate distinct types of rafts.
We have shown that Bad function is regulated by dynamic interaction with lipid rafts or mitochondria (13). The distinct Bad distribution and function are directly related to IL-4 stimulation or deprivation of the cells. This is in agreement with recent results showing that association of proteins to lipid rafts can be modulated because some proteins may be excluded from rafts by association with other proteins (42).
As early as 4 hours after lymphokine deprivation, we are able to observe apoptosis and alteration of rafts as detected by fluorescence-activated cell sorting and confocal microscopy, respectively. On the contrary, at that time, we did not observe significant modification of Bad staining. Subcellular fractionation studies confirm the presence of Bad associated to lipid rafts in control IL-4-stimulated or 4-hour IL-4-deprived cells. In fact, rafts alteration seems to be a consequence of an intracellular signal triggered by IL-4 deprivation. Growth factor deprivation induces Bad-associated PP1α activation, which dephosphorylates cytoplasmic Bad, activating caspase-9 and caspase-3. As a consequence, the Bad pool associated to rafts translocates toward the mitochondria leading to apoptotic cell death. This result strongly suggests that segregation of Bad from rafts 8 hours after IL-4 deprivation is a consequence of preceding apoptotic signals triggered by IL-4 withdrawal. Thus, translocation of Bad from rafts to mitochondria is observed in advanced stages of growth factor deprivation.
Our findings clearly show that rafts alteration represents an early event in the apoptotic process induced by IL-4 deprivation. The following question to answer was to know whether this phenomenon was a direct or indirect effect of IL-4. Blocking apoptosis via caspase inhibition strongly suggests an indirect effect of IL-4 in rafts alteration, indicating that IL-4 deprivation triggers an intracellular signal that induces initially Bad-associated PP1α phosphatase activation and subsequently caspase-9 and caspase-3 activation. It is interesting to notice the presence of two different pools of Bad: the cytoplasmic pool, which is phosphorylated, and the pool of Bad attached to rafts, which is dephosphorylated (22). The IL-4 deprivation-triggered pathway starts with the dephosphorylation of cytoplasmic Bad by PP1α, whereas the pool of Bad sequestered in rafts is already dephosphorylated. This association may be involved in steps leading to Bad inactivation because rafts do not constitute the final site of activation. This indirect effect of IL-4 withdrawal can be considered as a model for amplification of the cytoplasmic Bad dephosphorylation effect because dephosphorylation triggers its proapoptotic role and, as a consequence, the execution of the apoptotic signaling cascade: activation of caspases, segregation of Bad from rafts, and translocation to mitochondria, thus amplifying apoptotic cell death. In addition, inhibition of PP1α phosphatase or caspase activation blocks apoptosis and alteration of rafts, reinforcing our hypothesis.
It has recently been shown that in CD4 T cells the death-inducing signaling complex, Fas-associated death domain protein, and pro-caspase-8 are recruited to lipid rafts in Fas-induced apoptosis in CD4 T cells (43, 44).
Moreover, the anticancer drug resveratrol induces the redistribution of Fas receptor in membrane rafts of colon carcinoma cells (26). Similar observation has been made recently in human leukemic cells exposed to the antitumor ether lipid edelfosine (21). This translocation of Fas into membrane rafts may provide a mechanism for amplifying Fas signaling through reorganization of membrane microdomains, involving rafts in cancer chemotherapy and Fas-mediated apoptosis. In addition to Fas, it has been shown that other apoptotic molecules are recruited into lipid rafts: Fas-associated death domain protein, pro-caspase-8, pro-caspase-10, c-Jun NH2-terminal kinase, and Bid. This molecular aggregation may link Fas and mitochondrial signaling pathways (24). Other signaling molecules, such as Akt2, phosphatidylinositol 3′ -kinase, protein kinase C, PTEN, CD28, and Lck, have also been detected in lipid rafts.
Here, we have shown that rafts alteration is a consequence of an early intracellular signal triggered by IL-4 deprivation. Even if our results confirm the pivotal role of rafts in the control of apoptosis as illustrated in the model presented in Fig. 9, activation arrow;, translocation arrow, we do not exclude the possibility that IL-4 deprivation might induce changes in lipid rafts composition. According to this, it has been shown recently that ceramide can displace cholesterol from rafts using artificial lipid vesicles containing coexisting rafts domains and disordered fluid domains (18).
Materials and Methods
Cells, Lymphokines, and Reagents
Murine T cell line TS1αβ can be independently propagated in IL-2, IL-4, or IL-9 (45). Murine recombinant IL-4 or supernatant of a HeLa subline transfected with pKCRIL-4-neo was used as a source of murine IL-4. Okadaic acid, FITC-labeled cholera toxin subunit B, Optiprep, and cholera toxin-biotin were from Sigma-Aldrich (St. Louis, MO). Vectashield was from Vector Laboratories (Burlingame, CA). Anti-Bad, anti-phospho-Bad (Ser112, Ser136, and Ser155), and anti-PP1α antibodies were from Calbiochem (La Jolla, CA), Cell Signaling (Beverly, MA), New England Biolabs (Beverly, MA), and Transduction Laboratories (Lexington, KY). Anti-Tim 23, anti-PP2B, anti-p53, anti-caspase-3, and anti-Lck were from Transduction Laboratories. Cy2- and Cy3-conjugated secondary antibodies were purchased from Molecular Probes (Eugene,OR). Anti-VDAC antibody was from PharMingen (San Diego, CA). Anti-mitochondria serum (Mito 2813: α-pyruvate dehydrogenase) was a gift from Dr. A. Serrano (Centro Nacional de Biotecnologia, Madrid, Spain). Anti-caspase-9 was from MBL (Nagoya, Japan), and anti-c-Jun antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Protease and phosphatase inhibitor cocktails were obtained from Sigma-Aldrich, and caspase inhibitor z-VAD-fmk was from Bachem (Bubendorf, Switzerland). Anti-PP2A was generated at Pierre et Marie Curie University (Paris, France). Peroxidase-conjugated goat anti-rabbit and anti-mouse immunoglobulin antibody were from DAKO (Glostrup, Denmark). Enhanced chemiluminescence was from Amersham (Buckinghamshire, United Kingdom), and the Annexin kit was from Immunotech (Marseilles, France).
Cell Cycle Analysis and Cholera Toxin-FITC Labeling
A total of 2.5 × 105 TS1αβ cells were IL-4-stimulated or IL-4-deprived for different times and washed with ice-cold PBS diluted in ice-cold binding buffer as well as stained with Annexin and propidium iodide. Samples were maintained on ice for 10 minutes in the dark and then analyzed by flow cytometry. Apoptosis was measured as the percentage of cells in the sub-G1 region of the fluorescence scale having a hypodiploid DNA content. For cholera toxin-FITC labeling, IL-4-stimulated or IL-4-deprived cells were washed with chilled PBS and fixed for 5 minutes on ice with 1% paraformaldehyde. Cells were washed with PBS-bovine serum albumin and then incubated with cholera toxin-FITC (20 minutes, 6 μg/mL). After washing with PBS-bovine serum albumin, cells were analyzed by flow cytometry. Alternatively, cells were permeabilized for 2 minutes with 0.1% saponin in PBS and incubated with anti-Bad and/or anti-mitochondria antibody for 1 hour in PBS-bovine serum albumin followed by Cy3- and/or Cy2-labeled secondary antibody for 1 hour. After several washing steps, cells were incubated with methanol for 10 minutes at −20°C, mounted with Vectashield medium, and analyzed by confocal microscopy.
Immunoprecipitation and Western Blot
Cells (1 × 107) were IL-4 stimulated or deprived and lysed for 20 minutes at 4°C in lysis buffer [50 mmol/L Tris-HCl (pH 8), 1% NP40, 137 mmol/L NaCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 10% glycerol, protease and phosphatase inhibitor mixture]. Alternatively, radioimmunoprecipitation assay buffer has been used for cell lysis. Lysates were immunoprecipitated with the corresponding antibody. Protein A-Sepharose was added for 1 hour at 4°C, and after washing, immunoprecipitates were lysed in Laemmli sample buffer. Protein extracts were separated by SDS-PAGE, transferred to nitrocellulose, blocked with 5% nonfat dry milk in TBS [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl], and incubated with the primary antibody in TBS-0.5% nonfat dry milk. Membranes were washed with TBS-0.05% Tween 20 and incubated with peroxidase-conjugated secondary antibody. After washing, membranes were developed using enhanced chemiluminescence system.
Isolation of Mitochondria and S-100 Fraction
Mitochondria were isolated using a modification of the method described by Yang et al. (46). Briefly, 20 × 106 cells were IL-4 stimulated or deprived, harvested, and washed with chilled PBS. Cell pellet was resuspended in 5 volumes of ice-cold buffer A [20 mmol/L HEPES-KOH (pH 7.5), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 0.1 mmol/L phenylmethylsulfonyl fluoride, 250 mmol/L sucrose] supplemented with protease inhibitors. Cells were disrupted in a Dounce homogenizer, the nuclei were centrifuged (1,000 × g, 10 minutes, 4°C), and the supernatant was further centrifuged (1,000 × g, 15 minutes, 4°C). The extracted mitochondrial pellet was resuspended in buffer A and stored at −80°C. The supernatant was centrifuged (100,000 × g, 1 hour, 4°C), and the resulting S-100 fraction stored at −80°C.
Triton X-100 Flotation
IL-4-stimulated or IL-4-deprived cells were lysed in TXNE buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 5 mmol/L EDTA, 2% Triton X-100] containing protease inhibitor mixture. Detergent-insoluble membranes were isolated by ultracentrifugation (17,000 × g, 4 hours, 4°C) in a 30% to 35% gradient of Optiprep as described previously (47, 22).
Enzyme Assay for Caspase Activity
Cells were washed with ice-cold PBS and resuspended in extraction buffer [50 mmol/L Tris-HCl (pH 7.6), 150 mmol/L NaCl, 0.5 mmol/L EDTA, 10 mmol/L NaH2PO4, 10 mmol/L Na2HPO4, 1% NP40, 0.4 mmol/L Na3V04, 1 mmol/L phenylmethylsulfonyl fluoride, protease inhibitor cocktail]. Cell lysate was centrifuged (20,000 × g, 30 minutes, 4°C), and proteins (5 μg) diluted in assay buffer [25 mmol/L HEPES (pH 7.5), 0.1 mmol/L CHAPS, 10% sucrose, 10 mmol/L DTT, 0.1 mg/mL ovalbumin] were incubated with fluorescent substrate sequence for caspase-9 and caspase-3. Cleaved substrate fluorescence was determined by reverse-phase HPLC.
In vitro Phosphatase Assay
Cells were lysed in lysis buffer, and supernatanys were immunoprecipitated (2 hours, 20°C) with anti-Bad antibody and incubated with protein A-Sepharose beads (45 minutes, room temperature). Immunoprecipitates were washed with phosphatase buffer [50 mmol/L Tris-HCl (pH 7.5), 0.1% 2-mercaptoethanol, 0.1 mmol/L EDTA, 1 mg/mL bovine serum albumin], mixed with [32P]phosphorylase a, and diluted in phosphatase buffer supplemented with 4 mmol/L caffeine. The reaction was incubated for 40 minutes at 30°C and stopped by adding 200 μL of 20% trichloroacetic acid followed by centrifugation. A total of 185 μL were used to estimate the generation of free phosphate liberated from [32P]phosphorylase a.
Institut Pasteur, Institut National de la Sante et de la Recherche Medicale-Avenir, and Fondation pour la Recherche Médicale.