Ikappa B-alpha , the NF-kappa B Inhibitory Subunit, Interacts with ANT, the Mitochondrial ATP/ADP Translocator*

Virginie Bottero, Franca Rossi, Michel SamsonDagger , Mireille Mari§, Paul HofmanDagger §, and Jean-François Peyron

From INSERM U526 "Activation des Cellules Hématopoïétiques," IFR50, Faculté de Médecine Pasteur, 06107 Nice cedex 2, France, Dagger  INSERM U364, Faculté de Médecine Pasteur, 06107 Nice cedex 2, France, § Laboratoire d'Anatomo-Pathologie, Hôpital Pasteur, 06107 Nice cedex 2, France

Received for publication, July 5, 2000, and in revised form, March 16, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The transcription factor NF-kappa B regulates a wide set of genes involved in the establishment of many cellular processes that control cell activation, proliferation, and apoptosis. Ikappa B inhibitory subunits integrate NF-kappa B activation signals through phosphorylation and ubiquitination of its N-terminal domain. Using the two-hybrid system in yeast, we searched for Ikappa B-alpha N-terminal domain interactors and therefore potential NF-kappa B regulators. An interaction of Ikappa B-alpha with the mitochondrial ATP/ADP translocator ANT was detected in yeast and confirmed in glutathione S-transferase pull-down assays and co-precipitation experiments in transfected cells. Subcellular cell fractionation, resistance to proteinase K treatment, and electron microscopy experiments demonstrated the presence of Ikappa B-alpha and associated p65 NF-kappa B in the mitochondrial intermembrane space. Ikappa B-alpha ·NF-kappa B appeared to be released from mitochondria upon the induction of apoptosis by engagement of the Fas receptor. These data suggest that the mitochondrial Ikappa B-alpha ·NF-kappa B pool participates in the regulation of apoptosis.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Rel/NF-kappa B transcription factors are ubiquitously expressed and respond to more than 150 stimuli to regulate an equally wide array of genes (1). While NF-kappa B was well known to participate in the control of immune (2) and inflammatory responses (3), it recently appeared as an important player in regulating the balance between cell survival and apoptosis (4, 5). NF-kappa B is maintained inactive by inhibitory subunits of the Ikappa B family such as Ikappa B-alpha . Ikappa B-alpha interacts with NF-kappa B via its ankyrin motifs, masking nuclear localization signals on NF-kappa B subunits. The release of transcriptionally competent NF-kappa B dimers is achieved after phosphorylation-induced degradation or dissociation of Ikappa B-alpha molecules. At least two kinases (IKKalpha and IKKbeta ) that are specific for the conserved tandem serines in Ikappa B-alpha molecules have been identified. They show an extensive homology and form homo- as well as heterodimers within the cell. IKKs are localized in the signalsome, a multiprotein complex (700-900 kDa). This complex also contains NEMO/IKKgamma , (6) a structural component that is crucial for the correct assembly and functionality of the signalsome (for a review, see Refs. 7 and 8). An alternative mechanism that links tyrosine kinases to NF-kappa B activation has been described that uses phosphorylation of tyrosine 42 of Ikappa B-alpha to dissociate Ikappa B-alpha ·NF-kappa B complexes (9).

Phosphorylation of Ikappa B-alpha on serines 32 and 36 allows its specific recognition by the E3RS ubiquitin protein ligase, which then transfers ubiquitin to lysines 20 and 21. The polyubiquitinated Ikappa B-alpha molecule is thereafter recognized and degraded in situ by the proteasome 26 S complex (7). The N-terminal domain of Ikappa B-alpha thus appears to integrate NF-kappa B activation signals. We used this regulatory domain as a bait in a yeast two-hybrid screening of a Jurkat cDNA library to search for protein interactors and therefore potential regulators of NF-kappa B activation. In this report, we describe and characterize the interaction between Ikappa B-alpha and ANT, the mitochondrial ATP/ADP translocator (10). ANT is a central component of the mitochondria permeability transition pore (11). The opening of this pore, during induction of apoptosis allows the release of molecules, such as caspases or apoptosis-inducing factor, which are important to amplify the cell suicide response. The possible functional importance of the Ikappa B-alpha /ANT interaction in the context of regulation of apoptosis is discussed.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Biological Reagents and Cell Culture-- Anti-Ikappa B-alpha antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit antiserum anti-Ikappa B-alpha , anti-Myc epitope, and anti-F1beta ATPase monoclonal antibody were kindly provided by Jean Imbert (U119, Marseille, France), Jean-François Tanti (INSERM, EPI E99-11, Nice, France), and Joël Lunardi (Commissariat à l'Energie Atomique, Grenoble, France), respectively.

Human embryonic kidney 293 cells were maintained in Dulbecco's modified Eagle's medium containing 2 mM glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, 1 mM pyruvate, and 10% fetal calf serum (Life Technologies, Inc.). Cells were transfected by the calcium phosphate method as previously described (12).

HTLV-I1-infected MT4 cells (13) and cells of the human leukemic T-cell line Jurkat were maintained in RPMI 1640 medium containing 50 units/ml penicillin, 50 µg/ml streptomycin, 1 mM pyruvate, and 5% fetal calf serum.

Yeast Two-hybrid and cDNA Cloning-- Ikappa B-alpha cDNA encoding amino acids 2-72 was subcloned into the GAL4 DNA-binding vector pAS2.1 (CLONTECH, Palo Alto, CA). The resulting plasmid, pAS-Ikappa B2-72, was used as the bait in a two-hybrid screening of a human Jurkat T cell cDNA library (CLONTECH) in the S. cerevisiae HF7c strain according to the Matchmaker Two-Hybrid System II Protocol (CLONTECH). Positive yeast clones were selected for prototrophy for histidine and expression of beta -galactosidase. Yeast DNA was recovered. Sequencing of positive clones have been performed using the T7 Amersham Pharmacia Biotech sequencing kit. Full-length ANT cDNA was amplified by PCR from the human Jurkat T cell cDNA library.

Expression Vectors-- cDNAs encoding full-length ANT or its C-terminal domain were subcloned downstream and in frame with glutathione S-transferase (GST) open reading frame in the pGex-4T-2 plasmid.

Full-length ANT cDNA was also subcloned in pcDNA-Myc plasmid (gift of Dr. J-F. Tanti, INSERM E99-11).

Expression and Purification of Recombinant Proteins from Escherichia coli Pull-down Assays-- Plasmids coding for the GST fused proteins were transformed in E. coli strain DH5alpha and induced with 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside for 2 h at 30 °C. GST-fused proteins were purified by incubating precleared bacterial lysates (phosphate-buffered saline (PBS)-1% Nonidet P-40 sonication) with glutathione-Sepharose beads (Amersham Pharmacia Biotech). The absorbed proteins were incubated with recombinant hexahistidine-Ikappa B-alpha purified for 1 h at 4 °C in binding buffer (25 mM Hepes, pH 7.4, 50 mM NaCl, 2.5 mM CaCl2, 1 mM MgCl2, 0.5% Nonidet P-40, 1% bovine serum albumin) supplemented with Complete protease inhibitor mixture (Roche Molecular Biochemicals, Mannheim, Germany). After extensive washes in the same buffer without bovine serum albumin, Ikappa B-alpha was detected by immunoblotting with monoclonal anti-Ikappa B-alpha antibodies.

Immunoprecipitation and Immunoblotting-- Transfected 293 cells were incubated for 30 min at 4 °C in lysis buffer (20 mM Hepes, pH 7.4, 50 mM NaCl, 0.5% Nonidet P40, 1 mM CaCl2, 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture). Lysates were cleared at 10,000 × g for 10 min at 4 °C, and supernatants were incubated with antisera against Ikappa B-alpha or Myc epitope that were preabsorbed on Protein A-Sepharose and Protein G-Sepharose, respectively. The immunocomplexes were washed three times in lysis buffer. Proteins were resolved by SDS-PAGE and transferred to Immobilon membranes (Millipore, Bedford, MA) by standard procedures. Immunoblot analysis was performed with the indicated antibodies. Bound antibodies were revealed with horseradish peroxidase-coupled immunoglobulins (Dako, Trappes, France) using the ECL Western blotting detection system (Amersham Pharmacia Biotech) according to the manufacturer's instructions.

Mitochondrial Purification-- Subcellular fractionation was performed according to Ref. 14. 108 Jurkat cells were washed with PBS and resuspended in buffer A (250 mM sucrose, 20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, and protease inhibitor mixture). Cells were Dounce homogenized (2-cm diameter, 100 strokes, 4 °C). Unbroken cells and nuclei were pelleted by centrifugation at 2500 rpm for 5 min. Supernatants were further centrifuged at 10,000 × g for 30 min. The resultant supernatants and pellets were designated as cytosolic and mitochondrial fractions, respectively.

Mitochondria were highly purified according to Ref. 15. Briefly, after Dounce homogenization, cell extracts were laid over the top of a Percoll/metrizamide discontinuous gradient. After centrifugation at 20,000 rpm for 15 min, mitochondria were collected at the 17/35% interface. They were then diluted in buffer A, centrifuged for 30 min at 10,000 × g, and recovered in the pellet.

Cross-contamination with cytosol was assessed using a specific enzyme marker: lactate dehydrogenase. The level of cross-contamination was calculated as the percentage of the total cellular enzyme activity in the different fractions.

Proteinase K Treatment-- Mitochondria or whole cell extracts were incubated in buffer A without protease inhibitors in the presence of 5-15 ng/ml proteinase K (Sigma) for 10 min on ice. Protease inhibitors were added to stop the reaction, and proteins were analyzed by Western blotting, as described below.

Immunoelectron Microscopy Study-- For immunoelectron microscopy, cell pellets from control or Fas-stimulated cells (6 h, 100 ng/ml) were fixed in 3.7% paraformaldehyde and embedded at low temperature into LR White resin (Hard LR White, London, UK). Ultrathin sections were laid to 300-mesh nickel grids, washed with PBS, and then incubated for 60 min at room temperature with Ikappa B-alpha antibody (1:200) or with anti-p65 antibody (1:500). After washing with PBS, the grids were incubated for 60 min with 10-nm colloidal gold-conjugated rabbit anti-mouse secondary antibody (British BioCell International, Cardiff, UK). The grids were washed with PBS and then with distilled water and stained with uranyl acetate. Sections were examined with a JEOL 1200 EXII electron microscope.

Electrophoretic Mobility Shift Assays-- The cytosolic and mitochondrial fractions from indicated cells were prepared as described above. Mitochondria were lysed in totex buffer (20 mM Hepes, pH 7.9, 350 mM NaCl, 20% glycerol, 1% Nonidet P-40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin). 30 µg of protein from total mitochondria extract or 50 µg of protein from digitonin supernatant were subjected to an electrophoretic mobility shift assay analysis. Ikappa B-alpha and NF-kappa B complexes were dissociated with a 0.6% deoxycholate treatment during 10 min at room temperature.

The NF-kappa B probe used was a synthetic double-stranded oligonucleotide containing the NF-kappa B binding site of the interleukin-2 gene promoter (5'-GATCCAAGGGACTTTCCATG-3'). The end-labeled probe was incubated with extract samples for 20 min at 37 °C. Complexes were separated by electrophoresis on a 5% nondenaturing polyacrylamide gel in 0.5× TBE. The dried gels were subjected to autoradiography (X-Omat; Eastman Kodak Co.).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of ANT as a Potential Ikappa B-alpha -binding Protein-- Activation of the transcription factor NF-kappa B is regulated by phosphorylation of the N-terminal domain of the inhibitory subunit Ikappa B-alpha . We used this N-terminal regulatory domain (Ikappa B-alpha amino acids 2-72) as the bait in a yeast two-hybrid-based approach to identify new Ikappa B-alpha interactors and therefore potential NF-kappa B regulators. By the screening of a commercial human Jurkat cell library, ~6 × 105 double yeast transformants were obtained, among which 232 grew on selective medium and only 88 turned blue when tested in a filter lift beta -galactosidase assay. Fifty-six clones were further characterized by sequencing.

Primary nucleotide sequencing revealed one cDNA clone encoding a protein not previously described in GenBankTM and two clones corresponding to two isoforms of the adenine nucleotide translocator: ANT1-(171-297) and ANT2-(152-298).

The interaction between ANT and Ikappa B-alpha was further characterized in the yeast two-hybrid system. Full-length Ikappa B-alpha could not be used in the two-hybrid system because of a constitutive activity of reporter genes (not shown; see Ref. 16). As shown in Table I, the DB-Ikappa B-alpha -(2-72) fused protein cannot act as activator of transcription when coexpressed in yeast with GAL4 AD nor in combination with AD-T Ag (large T antigen) fusion protein (lanes 1 and 2). The AD-ANT fused protein cannot act as activator of transcription when coexpressed in yeast with GAL4 DB nor in combination with the DB-p53 fusion protein (lanes 7 and 8). On the contrary, co-expression of Ikappa B-alpha -(2-72) with the portion of ANT1 or ANT2 isolated during the screening (lanes 4 and 5) or with the full-length ANT1 cDNA (lane 6) induced expression of the two reporter genes, thus demonstrating a direct and specific interaction between the two partners. We performed in vitro binding assay to confirm, in an independent experimental setting, the physical interaction between ANT and Ikappa B-alpha proteins. The C-terminal part of ANT isolated during the screening as well as the full-length ANT protein sequences were fused to GST and used in pull-down experiments with recombinant Ikappa B-alpha . The size of the recombinant proteins was checked by SDS-PAGE and Coomassie staining (Fig. 1A, upper panel). The GST protein or a nonrelevant fusion protein could not interact with Ikappa B-alpha (Fig. 1A, lower panel, lanes 2-5). By contrast, the C-terminal domain of ANT1 (lanes 6 and 7) as well as the full-length protein (lanes 8 and 9) interact specifically and in a dose-dependent manner with Ikappa B-alpha .

                              
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Table I
Specific testing of ANT/Ikappa B-alpha interactions in the yeast two-hybrid system
Yeast HF7c cells were cotransfected with expression vectors encoding the indicated GAL4 DNA-binding domain (DB)-fused and GAL4 transcription activation domain (AD)-fused proteins. Colonies were tested for their ability to grow on medium lacking histidine, tryptophan, and leucine, and beta -galactosidase activity was monitored by a standard filter assay. Growth on selective medium and beta -galactosidase activity are scored as a range from no growth and no beta -galactosidase activity (-) to activity generated by the positive control (+).


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Fig. 1.   In vitro and in vivo binding of ANT to Ikappa B-alpha . A, in vitro interaction. Upper panel, GST fusion proteins were produced in E. coli and purified on glutathione-Sepharose beads. Recombinant proteins were analyzed by SDS-PAGE and Coomassie Blue staining. Lower panel, different GST-fused proteins still bound to glutathione-Sepharose beads were incubated with purified recombinant poly-His-Ikappa B-alpha . The presence of Ikappa B-alpha in pull-down complexes was visualized by Western blot analysis using Ikappa B-alpha antibodies. B, in vivo interaction. 293 cells were transiently transfected with expression vectors encoding wild type full-length Ikappa B-alpha and Myc-tagged ANT. Cell lysates were prepared 30 h after transfection and incubated with anti-Ikappa B-alpha , anti-Myc antibodies, or rabbit nonimmune serum. Co-precipitated proteins were detected by immunoblot analysis using anti-Ikappa B-alpha or anti-Myc antibodies.

These in vitro results confirm the specificity of the ANT and Ikappa B-alpha interaction.

Co-precipitation of ANT and Ikappa B-alpha in Transfected Intact Cells-- 293 cells were transiently transfected with two expression vectors encoding Myc-tagged ANT and Ikappa B-alpha . Cellular levels of Ikappa B-alpha and ANT were visualized by direct Western blot analysis of total lysates with antibodies to Ikappa B-alpha (Fig. 1B, lane 2 versus lane 1) or to Myc epitope (lane 10 versus lane 9). Cell soluble extracts were then subjected to immunoprecipitation with anti-Ikappa B-alpha (lanes 3, 4, 11, and 12) or anti-Myc (lanes 5, 6, 13, and 14) rabbit polyclonal antibodies or with a nonimmune serum (lanes 7, 8, 15, and 16). The precipitates were fractionated by SDS-PAGE and blotted with polyclonal Ikappa B-alpha (lanes 1-8) or anti-Myc (lanes 9-16) monoclonal antibodies. Anti-Ikappa B-alpha precipitates contained an anti-Myc reactive band (lane 12) that comigrates with MycANT (lane 10). Quantification of the bands lead to an estimate of 14% of total ANT associated with Ikappa B-alpha . On the contrary, Ikappa B-alpha could not be revealed after anti-Myc immunoprecipitation (lane 4). No anti-Myc or anti-Ikappa B-alpha reactive proteins were precipitated by a nonimmune serum (lanes 8 and 16). These results demonstrate that a specific interaction between ANT and Ikappa B-alpha could occur in intact cells.

Localization of Ikappa B-alpha and p65 NF-kappa B in Purified Mitochondria from Jurkat Cells-- Since ANT is a mitochondrial protein, we looked for the presence of Ikappa B-alpha in this organelle. Mitochondria preparation after subcellular fractionation exhibited minor contamination by cytosol, as revealed by assays for specific mitochondrial enzymes: lactate dehydrogenase (2.6%; data not shown). Ikappa B-alpha was analyzed in total cell extracts (Fig. 2A, upper panel, lane 1) or in cytosolic (lane 2) or mitochondrial (lane 3) extracts. Equal amounts (250 µg) of proteins from cytosol or mitochondria were loaded. Although the majority of Ikappa B-alpha was localized in the cytosolic fraction (lane 2), the protein could also be observed in the mitochondria (lane 3). The localization of p65 NF-kappa B was investigated. As shown in the lower panel, p65 was also present in the cytoplasm (lane 2) as well as in the mitochondria (lane 3).


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Fig. 2.   Ikappa B-alpha and p65 localization in subcellular fractions and purified mitochondria from Jurkat cells. Subcellular fractions and gradient-purified mitochondria were prepared as described under "Materials and Methods." A, visualization of Ikappa B-alpha and p65 NF-kappa B in mitochondrial fraction. Subcellular fractions (total (T), cytosol (C), and mitochondria (M), lanes 1-3) or gradient fractions (lanes 4 and 5) from resting Jurkat cells were subjected to Western blot analysis using anti-Ikappa B-alpha , anti-p65, and anti-cytochrome c antibodies. B, immunoelectron microscopy analysis of Ikappa B-alpha in purified mitochondria. Mitochondria were fixed in 3.7% paraformaldehyde and embedded into resin. The grids were incubated with anti-Ikappa B-alpha antibodies, and binding was revealed with gold-conjugated rabbit anti-mouse secondary antibody before examination under an electron microscope. C, mitochondrial Ikappa B-alpha and p65 NF-kappa B, but not Bcl2, are resistant to proteolysis by proteinase K. Extracts from mitochondrial fractions (lanes 1-3) or whole cell extracts (lanes 4-6) were incubated with the indicated doses of proteinase K for 10 min at 4 °C. The proteins were then visualized by Western blotting with specific antibodies. D, release of Ikappa B-alpha and p65 NF-kappa B from mitochondria after digitonin treatment. Highly purified mitochondria were treated with increasing concentrations of digitonin. After centrifugation, the mitoplasm was recovered from the pellet, whereas the supernatant contained released proteins. These fractions were subjected to a Western blot analysis using the indicated antibodies.

In order to exclude a possible contamination of the mitochondrial fraction by cytosolic proteins or other cellular compartments such as the Golgi apparatus or the ER, mitochondria were further enriched and purified on a Percoll/metrizamide gradient. Lactate dehydrogenase activity was below 1% in the highly purified mitochondrial fraction (not shown). Western blot analysis with specific antibodies confirmed the presence of Ikappa B-alpha and p65 in mitochondria (lane 5, upper and middle panels). Using densitometry scanning of the autoradiography, we calculated that approximately 7% of cellular total Ikappa B-alpha is located in mitochondria. Cytochrome c was mainly detected in the mitochondrial fraction (lane 5, lower panel) and only faintly in the cytosol (lane 4, lower panel).

Immunoelectron microscopy analysis using anti-Ikappa B-alpha antibodies as primary antibodies was then performed on purified mitochondria (Fig. 2B). No signal could be detected when gold-conjugated secondary antibody was used alone or in combination with a nonrelevant primary antibody (see controls on Fig. 4, A and B). Ikappa B-alpha staining was associated with the membrane or the internal crests, but not with the matrix.

We next assessed the sensitivity of Ikappa B-alpha , p65, and Bcl2 in mitochondrial fractions to proteinase K (Fig. 2D, lanes 1-3, left panels). While Bcl2, which is associated with the external mitochondrial membrane, was completely degraded (lower panel, lanes 2 and 3), Ikappa B-alpha and p65 remained unaffected (upper and middle panels, lanes 2 and 3), suggesting that Ikappa B-alpha and p65 are inaccessible to proteinase K. The two proteins are not intrinsically resistant to proteinase K, since in whole cell extracts (right panels), Ikappa B-alpha and p65 were degraded by proteinase K to the same extent as Bcl2 (lanes 5 and 6). This suggests that Ikappa B-alpha and p65 are located inside the mitochondria.

Ikappa B-alpha and p65 Are Localized in the Mitochondrial Intermembrane Space-- To determine the sublocalization of Ikappa B-alpha and p65 in the organelle, gradient-purified mitochondria were treated with increasing concentrations of digitonin in order to solubilize the outer membrane (17). After centrifugation, pellets and supernatants were subjected to a Western blot analysis. As shown in Fig. 2D, increasing concentrations of digitonin (0.1-0.4%) induced a disappearance of Ikappa B-alpha and p65 from the mitochondria (lanes 3, 5, and 7 versus lane 1) concomitant to their appearance in the supernatant fractions (lanes 4, 6, and 8 compared with lane 2). As a control, digitonin was shown to extract intermembranal cytochrome c. By contrast, the release of the internal membrane-located F1 ATPase appeared to require a higher digitonin concentration (0.4%; lane 8).Therefore, Ikappa B-alpha and p65 NF-kappa B appeared to be located in the mitochondrial intermembrane space, since they can be released after lysis of the outer membrane. Surprisingly, we observed several times that Ikappa B-alpha ·NF-kappa B complexes were more easily and more rapidly solubilized by digitonin than cytochrome c. This unexpected result could suggest that cytochrome c may stick to some mitochondrial proteins or membrane lipids while Ikappa B-alpha ·NF-kappa B are more soluble proteins. Indeed, when F1beta ATPase was extracted by 0.4% digitonin, a significant amount of cytochrome c still remained associated with mitochondria. Nevertheless, this difference in behavior toward digitonin treatment does not necessarily mean that under physiological apoptotic conditions Ikappa B-alpha ·NF-kappa B would be more easily released from mitochondria than cytochrome c. Considering the respective size of these proteins, it is likely that they use different ways to leave the mitochondrial intermembrane space.

Mitochondrial Ikappa B-alpha ·NF-kappa B Complexes-- Ikappa B-alpha immunocomplexes from cytosolic or mitochondrial fractions were analyzed by Western blot for the presence of p65. As shown in Fig. 3A, no p65 was found in control precipitates (lanes 1 and 2), while p65 associated with Ikappa B-alpha in cytosolic (lane 3) as well as mitochondrial extracts (lane 4).


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Fig. 3.   A, interaction of Ikappa B-alpha with p65 NF-kappa B in mitochondrial fractions. Protein lysates from cytoplasm or isolated mitochondria were incubated with anti-Ikappa B-alpha or rabbit preimmune serum antibodies. Co-precipitated proteins were detected by immunoblot analysis using anti-p65 antibodies. B, mitochondria contain associated Ikappa B-alpha ·p65 complexes. Cytoplasmic extracts (30 µg) or digitonin extracts from mitochondria (50 µg) were subjected to an electrophoretic mobility shift assay analysis with a kappa B-specific-radiolabeled probe. 0.6% deoxycholate (Doc) was used to dissociate Ikappa B-alpha ·NF-kappa B complexes. The competition was performed with a 50-fold excess of unlabeled probe (cold). Nonspecific binding was indicated (ns).

These data indicate the presence of specific Ikappa B-alpha ·p65 NF-kappa B complexes in the mitochondria.

To further demonstrate the Ikappa B-alpha ·NF-kappa B association, cytosolic or digitonin-treated mitochondrial extracts were treated or not with deoxycholate in order to dissociate NF-kappa B from Ikappa B-alpha . Free NF-kappa B was then visualized by electrophoretic mobility shift assay (Fig. 3B). Deoxycholate (Doc) treatment of cytosol (lane 2) or mitochondria preparation (lane 5) released NF-kappa B that was competent for specifically binding a 32P-labeled kappa B probe, as verified by competition assays with unlabeled probe (lanes 3 and 6). Taken together, these results confirm the existence of Ikappa B-alpha ·NF-kappa B complexes in the mitochondria.

Immunoelectron Microscopy Analysis of Ikappa B-alpha and p65 Subcellular Localization-- Immunoelectron microscopy analysis was then performed on intact Jurkat cells, using anti-Ikappa B-alpha or anti-p65 antibodies as primary antibodies. No signal could be detected when gold-conjugated secondary antibody was used in combination with a nonrelevant anti-cytokeratin antibody (Fig. 4A) or alone (Fig. 4B).


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Fig. 4.   Immunoelectron microscopy analysis of Ikappa B-alpha subcellular localization in Jurkat cells. Cell pellets were fixed in 3.7% paraformaldehyde and embedded into resin. The grids were incubated with specific antibodies as indicated, whose binding was revealed with gold-conjugated rabbit anti-mouse secondary antibody before examination under an electron microscope. A, cytokeratin staining; B, secondary antibody alone; C and D, anti-Ikappa B-alpha staining; E and F, anti-p65 staining. Higher magnification was used to demonstrate mitochondrial staining (right panels).

Analysis of anti-Ikappa B-alpha antibody-labeled cells revealed numerous beads distributed mainly in the cytosol with few in the nucleus (Fig. 4C). Beads were also found associated with mitochondria. No labeling could be detected within the endoplasmic reticulum or the plasma membrane, ruling out a nonspecific binding of the antibody to lipid membranes. A greater magnification of mitochondria showed that Ikappa B-alpha staining was localized mainly at the membrane level or in the internal crests (Fig. 4D).

Analysis of anti-p65 antibody labeled cells revealed a similar distribution of the molecule both in intact cells (Fig. 4E) and mitochondria (Fig. 4F).

Taken together, these results further confirm a mitochondrial localization of Ikappa B-alpha as well as of p65 NF-kappa B.

Ikappa B-alpha ·NF-kappa B Complexes Are Released from Mitochondria after Fas-induced Apoptosis-- The release of pro- or antiapoptotic molecules from the mitochondrial intermembrane space during apoptosis in several cellular systems is well documented (11). We analyzed the fate of Ikappa B-alpha , p65 NF-kappa B, and cytochrome c after induction of apoptosis upon engagement of the Fas surface molecule with the CH11 antibody. After cell treatment with increasing doses of CH11 antibody, mitochondria were isolated and analyzed for the presence of Ikappa B-alpha . As shown in Fig. 5A, engagement of the Fas receptor resulted in a dose-dependent release of Ikappa B-alpha (lanes 4-6, upper panel) as well as of p65 (lanes 4-6, middle panel). The specificity of these observations was confirmed by the parallel release of cytochrome c (lanes 4-6, lower panel) from mitochondria. In contrast, little to no degradation of these molecules could be visualized in the cytosol after CH11 treatment (lanes 1-3).


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Fig. 5.   Release of mitochondrial Ikappa B-alpha after Fas engagement of the Fas death receptor. A, cells were treated for 18 h with increasing concentrations of anti-Fas antibody (CH11), and mitochondria fractions were prepared as described under "Materials and Methods." Protein extracts were analyzed by Western blot with the indicated antibodies. B, cells were treated or not with CH11 antibody as above, followed by electron microscropy analysis after Ikappa B-alpha or p65 staining.

Electron microscopy analysis of CH11-treated Jurkat cells revealed cell shrinkage, perinuclear chromatin condensation, and alterations of the mitochondrial structures, including swelling, disappearance of cristae membranes, and destructuralizing of the surrounding membranes (data not shown). In these cells, Ikappa B-alpha staining was highly reduced in the mitochondria (Fig. 5B, compare a with b). Similarly, CH11 treatment resulted in the disappearance of p65 reactivity (compare c with d).

These data suggest that Ikappa B-alpha and p65 complexes located in the intermembrane space of the mitochondria can be released in the cytosol following stimulation of Jurkat cells with apoptotic signals.

Mitochondrial Ikappa B-alpha Is Not Degraded in MT4 Cells-- We then performed experiments in order to determine if the mitochondrial Ikappa B-alpha ·NF-kappa B complexes were sensitive to activation signals. We used HTLV-I-infected MT4 cells that display a constitutive activation of NF-kappa B. The HTLV-I Tax protein is known to activate NF-kappa B by binding to the NEMO/IKKgamma component of the signalsome, resulting in a constitutive activation of the Ikappa B-alpha kinases (18). MT4 cells express low amounts of Ikappa B-alpha (Fig. 6, lane 1) as visualized by Western blot analysis. Cell treatment (1 h, 50 µM) with the proteasome inhibitor ALLN, shown to prevent degradation of Ikappa B-alpha , resulted in an increase in the cellular levels of Ikappa B-alpha (lane 2). By contrast, the amount of Ikappa B-alpha in the mitochondria was not affected by the ALLN treatment (compare lane 4 with lane 3). The nitrocellulose filter was then hybridized with a phosphospecific Ikappa B-alpha antibody. Despite constitutive stimulation of IKK kinases, no phosphorylated Ikappa B-alpha could be detected in cytosolic extracts (lane 6), suggesting that phosphorylated Ikappa B-alpha is immediately degraded. Indeed, ALLN treatment allowed the visualization of phospho-Ikappa B-alpha (lane 7). By contrast, no signal could be detected in the mitochondrial fraction (lane 8), even in the presence of ALLN (lane 9), suggesting that despite continuous firing of Ikappa B kinases, mitochondrial Ikappa B-alpha is protected from phosphorylation.


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Fig. 6.   Mitochondrial complexes are sequestered from NF-kappa B activation signals. MT4, HTLV-I-infected cells were pretreated for 1 h with the proteasome inhibitor ALLN (50 µM) when indicated. Cytosolic and mitochondrial extracts were resolved by SDS-PAGE and analyzed by Western blotting with Ikappa B-alpha -specific antibodies (upper panel) or phospho-Ikappa B-alpha -specific antibodies (lower panel).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this report, we show that Ikappa B-alpha ·NF-kappa B complexes are present in the mitochondrial intermembrane space via interaction of the N-terminal domain of Ikappa B-alpha with the internal membrane protein ANT (adenine nucleotide translocator).

The interaction was originally detected in a yeast two-hybrid screening and checked by in vitro pull-down experiments with GST-ANT and recombinant Ikappa B-alpha . A coprecipitation of the two molecules was observed in transfected 293 cells, demonstrating that the two proteins could interact in intact cells. Finally, immunoelectron microscopy allowed the unambiguous demonstration of Ikappa B-alpha ·NF-kappa B complexes in the mitochondrial intermembrane space. NF-kappa B and Ikappa B-alpha were unaffected by a proteinase K treatment of intact mitochondria and could be extracted by digitonin lysis of the outer membrane.

The ANT protein is localized in the inner mitochondrial membrane and exchanges cytosolic ADP for mitochondrial ATP (10). ANT interacts with several proteins of the outer membrane (peripheral benzodiazepine receptor, porin/VDAC, Bax) as well as the matrix (cyclophilin) to form the permeability transition pore (PTP) or megachannel (19). The PTP appears as an important regulator of the apoptotic process. Opening of the pore leads to loss of the mitochondrial transmembrane potential, Delta psi m, that can ultimately culminate in matrix swelling and outer membrane rupture, allowing the release of apoptogenic proteins such as cytochrome c, apoptosis-inducing factor, and procaspases (11, 20-22). The exit of cytochrome c is controlled by proteins of the Bcl-2 family that are anchored on the mitochondrial outer membrane. Antiapoptotic members of the family (Bcl-2, Bcl-xl) prevent cytochrome c release in contrast to the proapoptotic members Bax and Bak (23). Bax has been shown to interact with ANT to induce PTP opening and cytochrome c release (24).

Several pharmacological compounds interfere with PTP. For instance, bongkrekic acid and atractyloside are, respectively, blocker and inducer of apoptosis by binding two different conformational stages of ANT (20, 25). Cyclosporin A also blocks mitochondrial apoptotic signals by interacting with cyclophilin (26), while engagement of the benzodiazepine receptor facilitates apoptosis (27).

The observed interaction of Ikappa B-alpha with ANT suggests that mitochondrial Ikappa B-alpha ·NF-kappa B complexes could be involved in the regulation of the apoptotic cascade. Apart from its well known functions in coordination of immune and inflammatory responses, NF-kappa B appears to promote cell survival (4, 5). For instance, inactivation of the p65 NF-kappa B (relA) gene resulted in embryonic death due to massive liver apoptosis (28). This property could be demonstrated in various cell lines by expression of a dominant negative form of the inhibitory subunit Ikappa B-alpha . Replacement of serines 32 and 36 by alanines prevents phosphorylation/degradation of Ikappa B-alpha and thus activation of NF-kappa B. Cells expressing the Ikappa B-alpha superrepressor clearly become more susceptible to apoptosis induced by tumor necrosis factor, chemotherapeutic drugs, or gamma -rays (29-31).

In order to determine if the mitochondrial pool of Ikappa B-alpha ·NF-kappa B complexes could be reached by activation signals, we used HTLV-1-infected MT4 cells that display a constitutive activation of NF-kappa B, due to an interaction of the viral Tax protein with IKKgamma to activate the IKK complex. Inhibition of the degradation pathway by ALLN increased cytosolic Ikappa B-alpha levels and allowed detection of the serine phosphorylated form. In sharp contrast, mitochondrial Ikappa B-alpha was not affected by ALLN treatment. These results show that mitochondrial Ikappa B-alpha ·NF-kappa B complexes are sequestered from NF-kappa B-activating signals.

Induction of apoptosis after engagement of the Fas death receptor leads to the exit of Ikappa B-alpha ·NF-kappa B from mitochondria. While cytochrome c can leave mitochondria through open VDAC (diameter 2.4-3 nm) (32), the liberation of procaspases and Ikappa B-alpha ·NF-kappa B complexes requires rupture of the outer membrane. We believe that this release in the cytosol could increase the number of NF-kappa B complexes susceptible for activation.

Apoptosis should be considered as a multistep and integrative process. Before producing an appropriate outcome response, cells have to integrate pro- and anti-apoptotic influences. Moreover, cross-talks between apoptotic and survival pathways have already been demonstrated. NF-kappa B could be one of the important players at this cross-road. NF-kappa B is activated by the Akt kinase survival pathway (33, 34). The second face of the cross-talks is represented by the caspase-mediated inactivation of survival pathways. Transcription factors appear as key targets. SRF, which regulates growth factor-induced survival genes, is cleaved and inactivated during apoptosis (35). So is p65 NF-kappa B, whose cleavage produces a protein lacking its transactivation domain that has a dominant negative effect on NF-kappa B-induced survival genes (36). Ikappa B-alpha could also be cleaved by caspase 3, to an N-terminal truncated and stable inhibitor of NF-kappa B activation (37).

Apoptotic pathways have characteristics of a self-amplifying process. Procaspases are released from the mitochondria (38), and activated caspases are able to induce the exit of cytochrome c to exacerbate the response (39). In contrast, NF-kappa B promotes, in many cells, survival effects via transcription of genes such as those for cIAP1 and cIAP2 that code for caspases inhibitors (40, 41). It is thus possible that the release of Ikappa B-alpha ·NF-kappa B from the mitochondria is an additional control level. Reception of a survival signal at this point would limit or delay apoptosis, whereas continuous caspase activation will lead to NF-kappa B inactivation. Alternatively, Ikappa B-alpha ·NF-kappa B could exert a transcriptionally independent regulatory function on apoptosis. This has been demonstrated for p53, which translocates to mitochondria during p53-mediated apoptosis. Translocation precedes mitochondrial dysfunctional changes and appears independent of p53 transcriptional activity (42). It is thus conceivable that Ikappa B-alpha ·NF-kappa B could regulate opening of the PTP through its association with ANT.

The results presented here suggest that a reservoir of Ikappa B-alpha ·NF-kappa B complexes in the intermembrane mitochondrial space could be involved in modulating apoptosis responses.

    ACKNOWLEDGEMENTS

We thank Drs. Jean Imbert (INSERM U119, Marseille, France), J-F. Tanti (INSERM, E99-11, Nice, France), and Joel Lunardi (Commissariat à l'Energie Atomique, Grenoble, France) for the kind gifts of anti-Ikappa B-alpha , anti-Myc, and anti-F1beta antibodies, respectively. We appreciated helpful discussion with Dr. Antoine Galmiche (U452, Nice, France), Fredéric Luciano (U526), and Marie-Lise Lacombe (U402, Saint Antoine, Paris, France) concerning mitochondria preparation. We thank Drs. Patrick Auberger, Véronique Imbert, Valère Busuttil, and Antonia Livolsi (U526) for useful advice.

    FOOTNOTES

* This work was supported by an institutional grant from INSERM, and ARC Grant 9332, and grants from La Ligue Nationale contre le Cancer, La Ligue Comité des Bouches du Rhône, and La Fondation de France, Comité Leucémies.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.

To whom correspondence should be addressed: INSERM U526 Faculté de Médecine Pasteur, 06107 Nice cedex 2, France. Tel: 33 493 377 676; Fax: 33 493 817 852; E-mail: peyron@unice.fr.

Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M005850200

    ABBREVIATIONS

The abbreviations used are: HTLV, human T-cell lymphotrophic virus; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PTP, permeability transition pore.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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