Alix and ALG-2 Are Involved in Tumor Necrosis Factor Receptor 1-induced Cell Death
2008; Elsevier BV; Volume: 283; Issue: 50 Linguagem: Inglês
10.1074/jbc.m803140200
ISSN1083-351X
AutoresAnne-Laure Mahul-Mellier, Flavie Strappazzon, Anne Petiot, Christine Chatellard-Causse, Sakina Torch, Béatrice Blot, Kimberley Freeman, Lauriane Kühn, Jérôme Garin, Jean‐Marc Verna, Sandrine Fraboulet, Rémy Sadoul,
Tópico(s)Cell death mechanisms and regulation
ResumoAlix/AIP1 regulates cell death in a way involving interactions with the calcium-binding protein ALG-2 and with proteins of ESCRT (endosomal sorting complex required for transport). Using mass spectrometry we identified caspase-8 among proteins co-immunoprecipitating with Alix in dying neurons. We next demonstrated that Alix and ALG-2 interact with pro-caspase-8 and that Alix forms a complex with the TNFα receptor-1 (TNF-R1), depending on its capacity to bind ESCRT proteins. Thus, Alix and ALG-2 may allow the recruitment of pro-caspase-8 onto endosomes containing TNF-R1, a step thought to be necessary for activation of the apical caspase. In line with this, expression of Alix deleted of its ALG-2-binding site (AlixΔALG-2) significantly reduced TNF-R1-induced cell death, without affecting endocytosis of the receptor. In a more physiological setting, we found that programmed cell death of motoneurons, which can be inhibited by AlixΔALG-2, is regulated by TNF-R1. Taken together, these results highlight Alix and ALG-2 as new actors of the TNF-R1 pathway. Alix/AIP1 regulates cell death in a way involving interactions with the calcium-binding protein ALG-2 and with proteins of ESCRT (endosomal sorting complex required for transport). Using mass spectrometry we identified caspase-8 among proteins co-immunoprecipitating with Alix in dying neurons. We next demonstrated that Alix and ALG-2 interact with pro-caspase-8 and that Alix forms a complex with the TNFα receptor-1 (TNF-R1), depending on its capacity to bind ESCRT proteins. Thus, Alix and ALG-2 may allow the recruitment of pro-caspase-8 onto endosomes containing TNF-R1, a step thought to be necessary for activation of the apical caspase. In line with this, expression of Alix deleted of its ALG-2-binding site (AlixΔALG-2) significantly reduced TNF-R1-induced cell death, without affecting endocytosis of the receptor. In a more physiological setting, we found that programmed cell death of motoneurons, which can be inhibited by AlixΔALG-2, is regulated by TNF-R1. Taken together, these results highlight Alix and ALG-2 as new actors of the TNF-R1 pathway. Endocytosis of cell surface receptors has long been described as an effective way of switching off extracellularly induced signals. Endocytosed activated receptors traffic through early endosomes and are sorted into intralumenal vesicles accumulating inside endosomes known as multivesicular bodies (MVBs). 5The abbreviations used are: MVB, multivesicular body; DD, death domain; DED, death effector domain; HH, Hamburger-Hamilton; TNF, tumor necrosis factor; TNF-R1, TNF receptor 1; TUNEL, terminal transferase dUTP nick end labeling; HA, hemagglutinin; wt, wild type; RIPA, radioimmune precipitation assay; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; MTN, motoneurons; DISC, death-inducing signaling complex; CaRIPA, RIPA buffer containing 1 mm CaCl2; IP, immunoprecipitation; FADD, Fas-associated protein with death domain.5The abbreviations used are: MVB, multivesicular body; DD, death domain; DED, death effector domain; HH, Hamburger-Hamilton; TNF, tumor necrosis factor; TNF-R1, TNF receptor 1; TUNEL, terminal transferase dUTP nick end labeling; HA, hemagglutinin; wt, wild type; RIPA, radioimmune precipitation assay; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; MTN, motoneurons; DISC, death-inducing signaling complex; CaRIPA, RIPA buffer containing 1 mm CaCl2; IP, immunoprecipitation; FADD, Fas-associated protein with death domain. These MVBs fuse with lysosomes where the receptors meet their end by acid hydrolysis (1.van der Goot F.G. Gruenberg J. Trends Cell Biol. 2006; 16: 514-521Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar).In some cases however, such as for neurotrophin-bound Trk receptors, activated receptors continue signaling inside endosomes (2.Barker P.A. Hussain N.K. McPherson P.S. Trends Neurosci. 2002; 25: 379-381Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Also, in the case of death receptors, Schütze and co-workers (3.Schneider-Brachert W. Tchikov V. Neumeyer J. Jakob M. Winoto-Morbach S. Held-Feindt J. Heinrich M. Merkel O. Ehrenschwender M. Adam D. Mentlein R. Kabelitz D. Schütze S. Immunity. 2004; 21: 415-428Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar) showed that endocytosis of TNF-R1, which occurs after binding to TNFα, is a necessary step for activation of caspases and consequently apoptosis. They found that the apical procaspase-8 is recruited and thereby activated on the surface of multivesicular endosomes containing activated TNF-R1.Biogenesis of MVBs is under tight control by a set of proteins, making the so-called ESCRT-0 to -III (endosomal sorting complex required for transport), which sequentially associate on the cytosolic surface of endosomes (4.Williams R.L. Urbe S. Nat. Rev. Mol. Cell. Biol. 2007; 8: 355-368Crossref PubMed Scopus (551) Google Scholar). A partner of ESCRT proteins, which also regulates the making of MVBs, is the protein Alix/AIP1, first characterized as an interactor of the calcium-binding protein ALG-2 (apoptosis-linked gene-2) (5.Missotten M. Nichols A. Rieger K. Sadoul R. Cell Death Differ. 1999; 6: 124-129Crossref PubMed Scopus (210) Google Scholar, 6.Vito P. Lacana E. D'Adamio L. Science. 1996; 271: 521-525Crossref PubMed Scopus (453) Google Scholar, 7.Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Enveloped viruses, like human immunodeficiency virus, type 1, use Alix to recruit the ESCRT machinery to deform membranes and allow fission during viral budding (8.Strack B. Calistri A. Craig S. Popova E. Gottlinger H.G. Cell. 2003; 114: 689-699Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar, 9.von Schwedler U.K. Stuchell M. Muller B. Ward D.M. Chung H.Y. Morita E. Wang H.E. Davis T. He G.P. Cimbora D.M. Scott A. Krausslich H.G. Kaplan J. Morham S.G. Sundquist W.I. Cell. 2003; 114: 701-713Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar). Furthermore, two recent reports have claimed that Alix together with ESCRT proteins might also be involved in the abscission stage of cytokinesis (10.Carlton J.G. Martin-Serrano J. Science. 2007; 316: 1908-1912Crossref PubMed Scopus (565) Google Scholar, 11.Morita E. Sandrin V. Chung H.Y. Morham S.G. Gygi S.P. Rodesch C.K. Sundquist W.I. EMBO J. 2007; 13: 13Google Scholar).Besides Tsg101 and CHMP-4B of ESCRT-I and –III, respectively, Alix interacts with lysobisphosphatidic acid, a phospholipid involved in intralumenal vesiculation of endosomes (12.Matsuo H. Chevallier J. Mayran N. Le Blanc I. Ferguson C. Faure J. Blanc N.S. Matile S. Dubochet J. Sadoul R. Parton R.G. Vilbois F. Gruenberg J. Science. 2004; 303: 531-534Crossref PubMed Scopus (522) Google Scholar), and with regulators of endocytosis (CIN85 and endophilins) (13.Chatellard-Causse C. Blot B. Cristina N. Torch S. Missotten M. Sadoul R. J. Biol. Chem. 2002; 277: 29108-29115Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 14.Chen B. Borinstein S.C. Gillis J. Sykes V.W. Bogler O. J. Biol. Chem. 2000; 275: 19275-19281Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). However, the precise role of Alix on endosomes remains largely unclear because neither we nor other laboratories have found any striking effect of Alix on endocytosis and degradation of EGF or transferrin receptors (15.Cabezas A. Bache K.G. Brech A. Stenmark H. J. Cell Sci. 2005; 118: 2625-2635Crossref PubMed Scopus (90) Google Scholar, 16.Schmidt M.H. Hoeller D. Yu J. Furnari F.B. Cavenee W.K. Dikic I. Bogler O. Mol. Cell. Biol. 2004; 24: 8981-8993Crossref PubMed Scopus (102) Google Scholar).We and others have gathered evidence that Alix plays a role in cell death. In particular, expression of a mutant lacking the N-terminal part (Alix-CT) blocks death of HeLa cells induced by serum starvation (7.Vito P. Pellegrini L. Guiet C. D'Adamio L. J. Biol. Chem. 1999; 274: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) and of cerebellar neurons deprived of potassium (17.Trioulier Y. Torch S. Blot B. Cristina N. Chatellard-Causse C. Verna J.M. Sadoul R. J. Biol. Chem. 2004; 279: 2046-2052Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In this latter paradigm, Alix-CT, whose protecting activity was strictly correlated with its capacity to bind ALG-2, accumulated inside cytoplasmic aggregates together with ALG-2 and caspases. We also demonstrated, using electroporation in the chick embryo, that Alix mutants block programmed cell death of motoneurons during normal development, depending on binding to ALG-2 and ESCRT-I and -III. Our interpretation of these results is that some truncated forms of Alix behave as dominant negative mutants, blocking the formation of an ALG-2-Alix-ESCRT complex necessary for cell death (18.Mahul-Mellier A.-L. Hemming F.J. Blot B. Fraboulet S. Sadoul R. J. Neurosci. 2006; 26: 542-549Crossref PubMed Scopus (71) Google Scholar). Therefore the Alix-ALG-2 complex could make a link between endosomes and a signaling or an execution step of neuronal death (19.Sadoul R. Biol. Cell. 2006; 98: 69-77Crossref PubMed Scopus (52) Google Scholar).We have undertaken the present study to characterize this link and found that Alix and ALG-2 form a complex with endocytosed TNF-R1 and pro-caspase-8. The physiological relevance of these interactions was revealed by the demonstration that several Alix mutants inhibit TNF-R1-induced cell death both in vitro and in vivo.EXPERIMENTAL PROCEDURESReagents and Antibodies—Human recombinant TNFα-FLAG was from Alexis Biochemicals (Covalab). Mouse monoclonal anti-hemagglutinin (HA) antibody was from Cell Signaling (Ozyme); polyclonal antibodies against Myc, TNF-R1, and FADD were from Santa Cruz Biotechnology; polyclonal antibodies against LAMP1 and EEA1 were from AbCam; anti-FLAG monoclonal (M1 and M2) and polyclonal antibodies were from Sigma-Aldrich; GM130 and monoclonal anti-AIP1/Alix were from BD Transduction; anti-ALG-2 was from Swant; HSP70 mitochondria was from Affinity Bioreagents (Ozyme); anti-caspase 8 was from Biovision (Clinisciences); horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit Alexa594 antibody were from Jackson Laboratories; and biotinylated goat anti-rabbit antibody was from Vector Laboratories.DNA Constructs—For expression in the chick embryo, human TNF-R1 (wt or mutants), a catalytically dead version of human pro-caspase-8 (mutation C360A), and baculovirus p35 were inserted into the pCAGGS expression vector (a gift of Tsuyoshi Momose, Nara Institute of Science and Technology). Mammalian expression vectors coding for DN-caspase-8-HA-tagged and p35 were a gift of P. Mehlen (INSERM, Lyon, France). Those coding for wt or mutated human TNF-R1 were kind gifts from W. Schneider-Brachert (University of Regensburg, Regensburg, Germany). Mutated ALG-2ΔEF1,3 was a gift of M. Maki (Nagoya University, Nagoya, Japan).In Ovo Electroporation—Fertilized Isa Brown eggs (Société Française de Production Avicole, St. Marcellin, France) were electroporated at Hamburger-Hamilton (HH) stage 16. Plasmid DNA was electroporated as described in Ref. 18.Mahul-Mellier A.-L. Hemming F.J. Blot B. Fraboulet S. Sadoul R. J. Neurosci. 2006; 26: 542-549Crossref PubMed Scopus (71) Google Scholar.Histological Analysis—Chick embryos were collected 48 h after electroporation processed and cryo-sectioned as described previously in Ref. 18.Mahul-Mellier A.-L. Hemming F.J. Blot B. Fraboulet S. Sadoul R. J. Neurosci. 2006; 26: 542-549Crossref PubMed Scopus (71) Google Scholar.Immunohistochemistry and Immunofluorescence—Frozen sections were incubated with polyclonal anti-TNF-R1 or monoclonal anti-HA antibodies, diluted to 1/100 in Tris-buffered saline containing 1% GS, 0.02% saponine (TBSS) for 12–24 h at 4 °C. The sections were rinsed in TBSS and treated with a secondary anti-rabbit Alexa594 antibody or a biotinylated goat anti-rabbit secondary antibody, amplified using the ABC kit (Vector Laboratories) and revealed with 3,3′-diaminobenzidine and nickel intensification. The sections were rinsed in TBS, incubated 30 min at 37 °C in Hoechst 33342, 2 μg/ml (Sigma, France) before mounting in Mowiol (Calbiochem, France).Terminal Deoxynucleotidyl Transferase-mediated dUTP-Biotin Nick End Labeling (TUNEL) Method—TUNEL analysis was performed using an in situ cell death detection kit (Roche Applied Science). Fluorescent positive cells were counted in every third section. Twelve sections/embryo were counted.Reverse Transcription-PCR—RNA were extracted from whole chick embryo using TRIzol reagent (Invitrogen). cDNAs were synthesized with Moloney murine leukemia virus reverse transcriptase (Promega) and controlled with glyceraldehyde-3-phosphate dehydrogenase. TNF-R1 expression was further analyzed by amplification of a 695-nt fragment with the following oligonucleotides: 5′-GATACTGTGTGTGGCTGT-3′ and 5′-CGTAAATGTCGATGCTCC-3′ based on the chick TNF-R1 homologue (Gallus gallus accession number AJ720473).Cell Culture and Transfection—HEK-293 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen), 2 mm glutamine, 10 μg/ml streptamycin, and 10 units/ml penicillin. BHK-21 cells were maintained in Glasgow minimum essential medium (Invitrogen) containing 5% fetal bovine serum, 2.6 g/liter tryptose phosphate broth, 2 mm glutamine, 10 μg/ml streptamycin, and 10 units/ml penicillin. HEK 293 or BHK-21 cells were transfected using JetPEI (Ozyme).Alix Knockdown in BHK Cells—The Alix small hairpin RNA was cloned downstream of the human H1 promoter in the vector pSuperGFP (Oligoengine). The sequences of the synthetic oligonucleotides (Invitrogen) used for Alix small hairpin RNA construct were the following: 5′-GATCCCCGCCGCTGGTGAAGTTCATCTTCAAGAGAGATGAACTTCACCAGCGGCTTTTTGGAAA-3′ and 5′-AGCTTTTCCAAAAAGTTCATCCAGCAGACTTACTCTCTTGAAGTAAGTCTGCTGGATGAACGGG-3′. Annealed oligonucleotides were ligated into the BglII cleavage site within the pSuperGFP vector linearized with the same restriction enzymes.BHK cells were transfected with pSuper/shAlix plasmid or pSuperGFP vector for control using the JetSi transfection reagent (Polyplus Transfection). Transfected cells were selected using 800 μg/ml of G418. After 15 days, the clones were isolated and selected for the best reduction in Alix expression. Cells (pSuper/shAlix and control) were grown in the presence of 800 μg/ml of G418.Mass Spectrometry and Protein Identification—Cultures of mouse cerebellar granule neurons were prepared as described previously (17.Trioulier Y. Torch S. Blot B. Cristina N. Chatellard-Causse C. Verna J.M. Sadoul R. J. Biol. Chem. 2004; 279: 2046-2052Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and cultured in basal medium Eagle containing 25 mm potassium. The medium was changed for basal medium Eagle containing 5 mm potassium, and the cells were lysed 4 h later in RIPA buffer (150 mm NaCl, 50 mm Tris, pH 8.0, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS containing a protease inhibitor mixture (Roche Applied Science). Immunoprecipitations were performed using polyclonal anti-Alix antibody and protein G-coupled Sepharose beads. After separation by SDS-PAGE, discrete bands were excised from the Coomassie Blue-stained gel. In-gel digestion was performed as previously described (20.Ferro M. Seigneurin-Berny D. Rolland N. Chapel A. Salvi D. Garin J. Joyard J. Electrophoresis. 2000; 21: 3517-3526Crossref PubMed Scopus (126) Google Scholar). Gel pieces were then sequentially extracted with 5% (v/v) formic acid solution, 50% acetonitrile, 5% (v/v) formic acid, and acetonitrile. After drying, the tryptic peptides were resuspended in 0.5% aqueous trifluoroacetic acid. For MALDI-TOF mass spectrometry analyses, a 0.5-μl aliquot of peptide mixture was mixed with 0.5 μl of matrix solution (cyano-4-hydroxycinnamic acid at half-saturation in 60% acetonitrile, 0.1% trifluoroacetic acid (v/v)). The resulting solution was spotted on a MALDI-TOF target plate, dried, and rinsed with 2 μl of 0.1% trifluoroacetic acid. Peptide mixtures were then analyzed with a MALDI-TOF mass spectrometer (Autoflex, Bruker Daltonik, Germany) in reflector/delayed extraction mode over a mass range of 0–4200 Da. The spectra were annotated (XMass software), and the peptide mass fingerprints obtained were finally submitted to data base searches against the Swissprot Trembl data base with an intranet 1.9 version of MASCOT software.Immunoprecipitation and Western Blotting—Twenty-four hours after transfection, HEK-293 or BHK-21 cells were lysed in RIPA buffer. The cell lysates were cleared by a 14,000 × g centrifugation for 15 min and incubated overnight at 4 °C with anti-FLAG monoclonal M2 antibody. Immune complexes were precipitated with protein G-Sepharose (Amersham Biosciences), and the beads were washed with RIPA buffer. Immunoprecipitated proteins were separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore). The membranes were blocked with 5% milk in TBS containing 0.1% Tween and incubated with the appropriate antibodies. The bands were revealed using the ECL detection reagent (Perbio).In the case of immunoprecipitation between endogenous Alix and TNF-R1, HeLa cells were lysed in RIPA buffer. The cell lysates were cleared by a 14,000 × g centrifugation for 15 min followed by two incubations of 30 min with protein G-Sepharose beads. The cell lysates containing 5 mg of total proteins were incubated for 1 h at 4 °C with the polyclonal antibody against TNF-R1. Immune complexes were precipitated with protein G-Sepharose (Amersham Biosciences), and the beads were washed with RIPA buffer. Immunoprecipitated proteins were separated by 8% SDS-PAGE and treated as described above. Alix was detected using the monoclonal anti-AIP1/Alix from BD Transduction Laboratories.Quantification of Cell Death Induced by TNF-R1—Twenty hours after transfection, HEK-293 were washed in PBS, pH 7.4, and fixed in 4% paraformaldehyde for 20 min at 4 °C. The cells were stained with polyclonal anti-TNF-R1 antibody (1/100) and anti-rabbit Alexa594 antibody (1/500). The cells were rinsed in TBS, incubated for 30 min at 37 °C in 2 μg/ml Hoechst 33342 (Sigma), and mounted in Mowiol. Cell viability was scored on the basis of nuclear morphology, with condensed or fragmented nuclei being counted as dead.In some cases, tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) was added to cells at a final concentration of 1 mg/ml and incubated for 30 min at 37 °C. Dimethyl sulfoxide was used to dissolve the formazan product, and absorbance was measured in each well at 540 nm, using a measure at 630 nm as reference, in a Biotek Elx-800 microplate reader (Mandel Scientific Inc.).TNFα Internalization—BHK cells co-expressing TNF-R1 and the indicated proteins were incubated for 1 h at 4 °C with 1 ng of 125I-labeled human recombinant TNFα (specific activity, 2160 kBq/μg) (PerkinElmer Life Sciences). The cells were then incubated at 37 °C to allow endocytosis for the indicated times. The amount of intracellular 125I-TNF receptor complexes formed at 37 °C was estimated after washing cells for 5 min in cold acetic acid buffer (200 mm acetic acid, 500 mm NaCl, pH 2.5). After two washes in PBS, the cells were lysed in RIPA buffer. Total amount of cell-associated 125I-TNFα was determined on cells, washed only with PBS, instead of the acetic acid buffer. The amount of internalized (pH 2.5 resistant) 125I-TNF was calculated as a percentage of 125I-TNF bound at pH 7.4.Magnetic Isolation of Endosomes Containing TNF-R1 Bound to TNFα—HEK 293 cells transfected with TNF-R1 and Alix-Myc were incubated in a total volume of 1 ml of cold D-PBS (0.9 mm CaCl2, 0.493 mm MgCl2, 2.67 mm KCl, 1.47 mm KH2PO4, 138 mm NaCl, 8 mm Na2HPO4) containing 3% bovine serum albumin, 100 ng/ml TNFα-FLAG, and 10 μg/ml anti-FLAG monoclonal M1 for 1 h at 4 °C. They were then washed twice in cold D-PBS and incubated for 1 h at 4 °C in 1 ml of cold D-PBS containing 50 μl of protein G microbeads (μMACS Protein G Microbeads, MACS Molecular, Miltenyi Biotec). The cells were then washed twice in cold D-PBS and incubated in Dulbecco's modified Eagle's medium containing 10% SVF and kept at 4 °C or incubated for 30 min at 37 °C. Removal of surface-bound M1 antibody was achieved by washing the cells three times for 5 min in cold PBS containing 1 mm EDTA. A post-nuclear supernatant was prepared in 8% sucrose supplemented in 3 mm imidazole, pH 7.4, and 2× protease inhibitor mixture. The magnetic immune complex was passed over a column placed in the magnetic field of a MACS Separator. The labeled TNFα receptosome was retained within the column, whereas unbound material was washed away with 8% sucrose, 3 mm imidazole, pH 7.4. The magnetic fractions were collected by removing the column from the magnetic field and analyzed by SDS-PAGE and Western blotting. Solubilization of endosomes was performed using a solution of 8% sucrose, 3 mm imidazole, pH 7.4, containing 0.5% Triton X-100.RESULTSAlix Interacts with Pro-caspase-8 through ALG-2—To understand how Alix might control cell death, we first characterized some of the proteins interacting with it during apoptosis. For this, we used cerebellar granule cells, which survive in absence of serum when cultured in high extracellular potassium (25 mm) but undergo apoptosis soon after they are incubated in a medium containing normal extracellular potassium concentrations (5 mm). We previously observed that expression of Alix-CT blocks caspase activation in neurons deprived of potassium (17.Trioulier Y. Torch S. Blot B. Cristina N. Chatellard-Causse C. Verna J.M. Sadoul R. J. Biol. Chem. 2004; 279: 2046-2052Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). We used a polyclonal anti-Alix antibody to immunoprecipitate the endogenous protein from cell lysates made from neurons incubated for 4 h in 5 mm potassium. Using peptide mass fingerprinting (see "Experimental Procedures"), we found caspase-8 among the proteins co-immunoprecipitated with Alix, suggesting that the protease may physically associate with Alix. We further showed that in BHK-21 cells, FLAG-tagged Alix (FLAG-Alix) and an HA-tagged, catalytically inactive version of pro-caspase-8 (HA-DN-pro-caspase-8) (Fig. 1) co-immunoprecipitated. This demonstrates that the two proteins exist in a complex and that activity of the caspase is not required for this interaction (Fig. 2A). Furthermore, deletion of the prodomain of pro-caspase-8 containing the two death effector domains (DEDs) abolished the capacity of the caspase to interact with Alix (Fig. 2B). The addition of 1 mm Ca2+ in the lysates strikingly enhanced the amount of Alix co-immunoprecipitating with pro-caspase-8 (Fig. 2A), and we detected endogenous ALG-2 in immunoprecipitates containing overexpressed Alix and pro-caspase-8 (Fig. 2C). This prompted us to test a potential interaction of pro-caspase-8 with ALG-2, whose binding to Alix depends on calcium. In the case where ALG-2 was co-expressed with DN-pro-caspase-8, ALG-2 co-immunoprecipitated with the caspase in presence of 1 mm CaCl2 (Fig. 3A). Here again, this interaction required the pro-domain of the caspase (Fig. 3B). FLAG-Alix deleted of the sequence 802PPYPTYPGYPGY813 necessary for the binding of ALG-2 (17.Trioulier Y. Torch S. Blot B. Cristina N. Chatellard-Causse C. Verna J.M. Sadoul R. J. Biol. Chem. 2004; 279: 2046-2052Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) (AlixΔALG-2) (Fig. 1) did not immunoprecipitate with DN-pro-caspase-8 (Fig. 3C). On the other hand, ALG-2 could be co-immunoprecipitated with DN-pro-caspase-8 equally well from lysates of wt BHK cells or from cells in which Alix expression had been down-regulated using a pSuper vector coding for an small hairpin RNA against Alix, indicating that ALG-2 can interact with pro-caspase-8 independently of Alix (Fig. 3D).FIGURE 2Alix co-immunoprecipitates with DN-pro-caspase-8. A, Alix/pro-caspase-8 co-IP requires calcium. BHK cells co-expressing FLAG-Alix and HA-DN-pro-caspase-8 were lysed and immunoprecipitated with anti-HA antibody in RIPA buffer supplemented or not with 1 mm CaCl2. Immunoprecipitates were blotted and analyzed with polyclonal antibodies against Alix (lower panel) and against HA (upper panel). B, co-IP of Alix with DN-pro-caspase-8 requires the pro-domain of the zymogen. BHK cells co-expressing FLAG-Alix together with HA-DN-pro-caspase-8 or HA-DN-caspase-8-ΔDED were lysed and immunoprecipitated with anti-HA in RIPA buffer containing 1 mm CaCl2 (CaRIPA) and analyzed as in A. C, endogenous ALG-2 is detected in pro-caspase-8/Alix co-immunoprecipitates. BHK cells co-expressing FLAG-Alix together with HA-DN-pro-caspase-8 were lysed and immunoprecipitated as in B; immunoprecipitates were analyzed by Western blot with polyclonal anti-HA, anti-ALG-2, and anti-FLAG antibodies (from top to bottom).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3ALG-2 co-immunoprecipitates with DN-pro-caspase-8. A, ALG-2/pro-caspase-8 co-IP requires calcium. BHK cells co-expressing FLAG-ALG-2 and HA-DN-pro-caspase-8 were lysed and immunoprecipitated with anti-HA in RIPA buffer (ϕ) or RIPA buffer containing either 3 mm EGTA or 1 mm CaCl2. Immunoprecipitates were analyzed using polyclonal antibodies against HA (upper panel) and FLAG (lower panel). B, co-IP of ALG-2 with DN-pro-caspase-8 requires the pro-domain of the zymogen. BHK cells co-expressing FLAG-ALG-2 together with HA-DN-pro-caspase-8 or HA-DN-caspase-8-ΔDED were lysed and immunoprecipitated with anti-HA in CaRIPA and analyzed as in A. C, co-IP of Alix with DN-pro-caspase-8 requires its ALG-2 binding domain. BHK cells were co-transfected with either FLAG-Alix WT or FLAG-AlixΔALG-2 and HA-DN-pro-caspase-8. IP with anti-HA antibody were performed in CaRIPA. Immunoprecipitates were analyzed by Western blot using polyclonal antibodies against HA (upper panel) and Alix (lower panel). D, ALG-2 co-immunoprecipitates with DN-pro-caspase-8 in cells depleted of Alix. BHK cells expressing shAlix to down-regulate expression of the protein were transfected with FLAG-ALG-2 and HA-DN-pro-caspase-8 lysed in CaRIPA and immunoprecipitated with an anti-HA antibody. Immunoprecipitates were analyzed by Western blot using anti-FLAG (lower panel) and HA polyclonal antibodies (upper panel). Right inset, Western blot analysis of lysates using a polyclonal antibody anti-Alix and a monoclonal anti-actin shows the decrease of endogenous Alix expression in BHK shAlix cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Alix Immunoprecipitates with TNF-R1 Independently of ALG-2—The demonstration that TNF-R1 needs endocytosis to recruit pro-caspase-8 prompted us to test whether Alix and ALG-2 act as adaptors bringing procaspase-8 into close vicinity of endosomes containing TNF-R1. First, we examined whether Alix and ALG-2 can form a complex with TNF-R1.These experiments were performed in HEK 293 cells transiently transfected with expression vectors coding for TNF-R1 and Alix. As reported (21.Todd I. Radford P.M. Draper-Morgan K.A. McIntosh R. Bainbridge S. Dickinson P. Jamhawi L. Sansaridis M. Huggins M.L. Tighe P.J. Powell R.J. Immunology. 2004; 113: 65-79Crossref PubMed Scopus (77) Google Scholar), TNF-R1 overexpression was sufficient to induce apoptosis, even in the absence of TNFα. Western blot analysis of Alix or TNF-R1 immunoprecipitates (Fig. 4A and not shown) revealed the presence of TNF-R1 and Alix, respectively, suggesting the existence of a complex containing both proteins. We further proved the existence of such a complex by showing that endogenously expressed Alix is pulled down with endogenous TNF-R1 immunoprecipitated from HeLa cells (Fig. 4B). In HEK 293 cells, TNF-R1 mutant deleted of the death domain (TNF-R1ΔDD) (Fig. 1) did not co-immunoprecipitate with Alix, indicating that the interaction depends on the integrity of this region (Fig. 4C). AlixΔALG-2, which does not interact with pro-caspase-8, was capable of co-immunoprecipitating with TNF-R1 (Fig. 4C). This underscores the fact that binding of Alix to a TNF-R1 complex is independent of ALG-2 and of pro-caspase-8. Interestingly, Alix mutants lacking the Bro1 domain, required for interaction with CHMP-4B, or four amino acids necessary for binding to Tsg101, were not capable of co-precipitating with TNF-R1 (Fig. 4D). Thus, co-immunoprecipitation of Alix with TNF-R1 requires its ability to bind ESCRT proteins.FIGURE 4Alix co-immunoprecipitates with TNF-R1. A, Co-IP of overexpressed Alix and TNF-R1. RIPA lysates of HEK 293 cells co-expressing FLAG-Alix and TNF-R1 were immunoprecipitated with anti-FLAG antibodies and blots probed with anti-Alix (upper panel) and anti-TNF-R1 (lower panel). B, co-IP of endogenous Alix and TNF-R1. RIPA lysates of HeLa cells were immunoprecipitated using a polyclonal anti-TNF-R1. The IP was analyzed by Western blot using a monoclonal antibody against human Alix. C, co-IP of Alix with TNF-R1 requires the death domain of the receptor. RIPA lysates of HEK 293 cells co-expressing FLAG-Alix and TNF-R1 deleted from its death domain (TNF-R1ΔDD) were immunoprecipitated with anti-FLAG or with anti-TNF-R1, and blots w
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