Neurotrophin Receptor-interacting Mage Homologue Is an Inducible Inhibitor of Apoptosis Protein-interacting Protein That Augments Cell Death

Abstract

The inhibitor of apoptosis proteins (IAPs) have been shown to interact with a growing number of intracellular proteins and pathways to fulfil their anti-apoptotic role. In the search for novel IAP-interacting proteins we identified the neurotrophin receptor-interacting MAGE homologue (NRAGE) as being able to bind to the avian IAP homologue ITA. This interaction requires the RING domain of ITA. NRAGE additionally coimmunoprecipitates with XIAP. When overexpressed in 32D cells NRAGE augments interleukin-3 withdrawal induced apoptosis, possibly through binding endogenous XIAP. Moreover, NRAGE is able to overcome the anti-apoptotic effect of Bcl-2. The inhibitor of apoptosis proteins (IAPs) have been shown to interact with a growing number of intracellular proteins and pathways to fulfil their anti-apoptotic role. In the search for novel IAP-interacting proteins we identified the neurotrophin receptor-interacting MAGE homologue (NRAGE) as being able to bind to the avian IAP homologue ITA. This interaction requires the RING domain of ITA. NRAGE additionally coimmunoprecipitates with XIAP. When overexpressed in 32D cells NRAGE augments interleukin-3 withdrawal induced apoptosis, possibly through binding endogenous XIAP. Moreover, NRAGE is able to overcome the anti-apoptotic effect of Bcl-2. inhibitor of apoptosis protein neurotrophin receptor-interacting Mage homologue inhibitor of T cell apoptosis baculovirus IAP repeat tumor necrosis factor receptor TNFR-associated factor nerve growth factor p75 neurotrophin receptor interleukin-3 polymerase chain reaction glutathione S-transferase fetal calf serum X-linked inhibitor of apoptosis polyacrylamide gel electrophoresis hemagglutinin neuronal apoptosis inhibitory protein The inhibitor of apoptosis proteins (IAPs)1 are encoded by a recently identified family of highly conserved genes, playing a crucial role in the regulation of apoptosis by interfering with the process of caspase activation (1Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2270) Google Scholar). The mammalian IAPs consist of the X-linked inhibitor of apoptosis (XIAP, MIHA, ILP), cellular IAP1 (cIAP1, HIAP2, MIHC), cIAP2 (HIAP1, MIHB), NAIP, and survivin (2Roy N. Mahadevan M.S. McLean M. Shutler G. Yaraghi Z. Farahani R. Baird S. Besner-Johnston A. Lefebvre C. Kang X. et al.Cell. 1995; 80: 167-178Abstract Full Text PDF PubMed Scopus (871) Google Scholar, 3Rothe M. Pan M.G. Henzel W.J. Ayres T.M. Goeddel D.V. Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1053) Google Scholar, 4Duckett C.S. Nava V.E. Gedrich R.W. Clem R.J. Van Dongen J.L. Gilfillan M.C. Shiels H. Hardwick J.M. Thompson C.B. EMBO J. 1996; 15: 2685-2694Crossref PubMed Scopus (519) Google Scholar, 5Uren A.G. Pakusch M. Hawkins C.J. Puls K.L. Vaux D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4974-4978Crossref PubMed Scopus (445) Google Scholar, 6Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3000) Google Scholar). The inhibitor of T cell apoptosis (ITA) is an avian IAP homologue (7Digby M.R. Kimpton W.G. York J.J. Connick T.E. Lowenthal J.W. DNA Cell Biol. 1996; 15: 981-988Crossref PubMed Scopus (28) Google Scholar). Members of this family must contain at least one NH2-terminal cysteine-rich baculovirus IAP repeat (BIR) domain, which is required for pro-survival activity, and may also possess a carboxyl-terminal RING zinc finger. cIAP1 and cIAP2 were originally identified as part of the TNFR2-TRAF signaling complex, with the BIR domains being responsible for the interaction with TRAFs 1 and 2 (3Rothe M. Pan M.G. Henzel W.J. Ayres T.M. Goeddel D.V. Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1053) Google Scholar). XIAP on the other hand was shown to act as a mediator of bone morphogenic protein signaling linking the receptors to the signaling modulators TAB1 (TAK1-binding protein) and TAK1. The RING domain of XIAP binds to the receptor, while the BIR domains are important for recruiting TAB1 (8Yamaguchi K. Nagai S. Ninomiya-Tsuji J. Nishita M. Tamai K. Irie K. Ueno N. Nishida E. Shibuya H. Matsumoto K. EMBO J. 1999; 18: 179-187Crossref PubMed Scopus (324) Google Scholar). XIAP was also shown to interact with nuclear proteins such as XAF1 (9Liston P. Fong W.G. Kelly N.L. Toji S. Miyazaki T. Conte D. Tamai K. Craig C.G. McBurney M.W. Korneluk R.G. Nat. Cell Biol. 2001; 3: 128-133Crossref PubMed Scopus (390) Google Scholar) and can be diverted away from caspase inhibition by Smac/DIABLO (10Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2902) Google Scholar, 11Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1966) Google Scholar), as well as being involved in other signaling processes such as JNK1 (c-Jun NH2-terminal kinase) (12Sanna M.G. Duckett C.S. Richter B.W. Thompson C.B. Ulevitch R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6015-6020Crossref PubMed Scopus (116) Google Scholar) and NF-κB (8Yamaguchi K. Nagai S. Ninomiya-Tsuji J. Nishita M. Tamai K. Irie K. Ueno N. Nishida E. Shibuya H. Matsumoto K. EMBO J. 1999; 18: 179-187Crossref PubMed Scopus (324) Google Scholar) activation. Another group of proteins important in the regulation of apoptosis is the Bcl-2 family (13Reed J.C. Oncogene. 1998; 17: 3225-3236Crossref PubMed Scopus (936) Google Scholar). Bcl-2 itself has been shown to play a crucial role in the prevention of apoptosis at the mitochondrial surface impeding the function of the pro-death family members or by obstructing the release of apoptogenic molecules (13Reed J.C. Oncogene. 1998; 17: 3225-3236Crossref PubMed Scopus (936) Google Scholar). NRAGE is a very recently cloned neurotrophinreceptor-interacting MAGE homologue, uncovered in a two-hybrid search for proteins interacting with the intracellular domain of the p75 neurotrophin receptor (p75NTR). This protein was shown to be involved in the apoptotic response after nerve growth factor (NGF) binding in neuronal cells (14Salehi A.H. Roux P.P. Kubu C.J. Zeindler C. Bhakar A. Tannis L.L. Verdi J.M. Barker P.A. Neuron. 2000; 27: 279-288Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). NGF can mediate a variety of cellular responses, such as survival, differentiation, growth, and apoptosis in neurons, by binding to two types of cell surface receptors, the Trk receptor tyrosine kinases and the p75NTR (15Kaplan D.R. Miller F.D. Curr. Opin. Neurobiol. 2000; 10: 381-391Crossref PubMed Scopus (1647) Google Scholar). Interestingly, NRAGE shows a high homology in its carboxyl-terminal with the melanoma associated antigen (MAGE) family, which codes for antigens that present on many human tumors, although their intracellular function is mostly unknown. One exception is necdin, a postmitotic neuron-specific growth suppressor that can interact with both intracellular proteins such as E2F1 and p53 and viral transforming proteins such as adenovirus E1A (16Taniura H. Taniguchi N. Hara M. Yoshikawa K. J. Biol. Chem. 1998; 273: 720-728Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In this study we identified the pro-apoptotic protein NRAGE as a novel IAP-interacting protein in the yeast two-hybrid system. Furthermore, we show that NRAGE coprecipitates with ITA and XIAP, via the RING zinc finger domain of the IAPs. NRAGE can augment cell death upon growth factor withdrawal in the IL-3-dependent promyeloid leukemic 32D cell line, even in the presence of Bcl-2. Molecular cloning of ITA was described previously (7Digby M.R. Kimpton W.G. York J.J. Connick T.E. Lowenthal J.W. DNA Cell Biol. 1996; 15: 981-988Crossref PubMed Scopus (28) Google Scholar). The full-length cDNA was inserted into theApaI and EcoRI sites of pcDNA3 (Invitrogen). A 5′ PCR product was amplified using the primers 5′-tccagcaacagttgtcgactatgaacataatg-3′ and 3′-tgggtctcgaggaaaactg-5′, with pCDNA3-ITA as a template and Pfu polymerase (Stratagene). The PCR product was digested with SalI andSmaI, ligated with pCDNA3-ITASmaI/EcoRI, and inserted first into the two-hybrid vector pPC86 at SalI/EcoRI then into pPC97 at SalI/AaT2(17). pCDNA3HA-ITA construction was described previously (18Wiese S. Digby M.R. Gunnersen J.M. Gotz R. Pei G. Holtmann B. Lowenthal J. Sendtner M. Nat. Neurosci. 1999; 2: 978-983Crossref PubMed Scopus (61) Google Scholar). The expression vectors for truncated ITA isoforms were generated by PCR using HF DNA polymerase (CLONTECH) on the pCDNA3HA-ITA template. For ITAΔBIR, the KpnI site containing forward primer 5′-gcgcggtacccctgcttcgtgtgaaaggaggag-3′ together with the reverse primer 5′-tagctgaaagtaaactcccaagg-3′ were used, the PCR product digested at the KpnI and MroI sites, and cloned into a MroI- and KpnI-digested pcDNA3HA-ITA. For ITAΔRING, the forward primer 5′-ggtttccgaggtgtgagtatctg-3′ together with the EcoRI containing reverse primer 5′-gcgcgaattcttatgttctctcttcttgcaatctcc-3′ were used and the PCR product digested at the MroI and EcoRI sites and cloned into MroI- and EcoRI-digested pcDNA3HA-ITA. c-IAP1, c-IAP2 (29Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1131) Google Scholar) and XIAP (27Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1712) Google Scholar) were cloned into the blunt-endedSalI site of pPC97 using EcoRI- andXhoI-digested and Klenow-treated inserts of pCDNA3 constructs (courtesy J. Reed, Burnham Institute) inserted into the Klenow-treated SalI site in pPC97. pCDNA3myc-XIAP (J. Reed) was first subcloned into the GST fusion vector pGEX-4T-1 using the EcoRI and XhoI sites, before being cloned in frame into the eukaryotic GST fusion vector pEBG. pPC97-NAIP was constructed by inserting theEcoRV/SacII-flanked pBluescript NAIP (19Gotz R. Karch C. Digby M.R. Troppmair J. Rapp U.R. Sendtner M. Hum. Mol. Genet. 2000; 9: 2479-2489Crossref PubMed Scopus (57) Google Scholar) into Klenow-treated SalI/SacII sites of pPC97. The largest two-hybrid insert was labeled with [32P]dCTP using therediprimeTM random labeling system (Amersham Pharmacia Biotech) according to manufacturer's instructions and used to screen a bacterially expressed S194 murine plasmacytoma cDNA library as described previously (20Pfisterer P. Zwilling S. Hess J. Wirth T. J. Biol. Chem. 1995; 270: 29870-29880Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The full-length NRAGE cDNA was amplified with Pfu polymerase from a resultant 2.8-kilobase pair putative full-length clone with the forward primer 5′-gcgaattctgccaccatggactacaagaaggacgatgagatgatgacaaactggaaaccatccag-3′, incorporating an EcoRI site and a flag tag, and the reverse primer 5′-gcgcctcgagttactcaacccagaagaagccaatg-3′, and inserted into pCFG5-IEGZ (provided by D. Lindemann) at the Klenow-treatedEcoRI site. This allowed expression of both the gene of interest from the retroviral long terminal repeat and the green fluorescent protein and Zeocin resistance genes from an internal ribosomal entry site. The yeast two-hybrid screen was performed using a PC12 cDNA library cloned into pPC86HA3 (17Kalmes A. Hagemann C. Weber C.K. Wixler L. Schuster T. Rapp U.R. Cancer Res. 1998; 58: 2986-2990PubMed Google Scholar). The yeast strain Y190 was manipulated according to the MATCHMAKER Library User Manual (CLONTECH). A sequential transformation protocol was used with pPC97-ITA as bait. Positive clones were identified by growth on SD/-TRP/-LEU/-HIS/ + 25 mm 3-amino-1,2,4-triazole (Sigma) plates and activity of the lacZ reporter gene in filter assays. Direct two-hybrid tests were performed according to standard protocols. The human embryonic kidney cell line HEK293 and PhoenixTM packaging cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 100 units of penicillin and streptomycin/ml, and 2 mml-glutamine, at 37 °C in humidified air with 6% CO2. The 32D cells were obtained from Dr. J. S. Greenberger and grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% FCS, 100 units of penicillin and streptomycin/ml, 2 mml-glutamine, and 15% of WEHI-conditioned medium at 37 °C, 5% CO2, and 90% humidity. 32D cells expressing a human Bcl-2 protein were obtained from J. Reed (21Wang H.G. Rapp U.R. Reed J.C. Cell. 1996; 87: 629-638Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). Cells were transfected by a modified calcium phosphate coprecipitation method (22Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4816) Google Scholar). HEK293 cells were starved in Dulbecco's modified Eagle's medium with 0.3% (v/v) FCS for 48 h after transfection. Transfected HEK293 cells and 32D cells were lysed at 4 °C in Nonidet P-40 buffer (10 mm Hepes, pH 7.4, 145 mm KCl, 5 mm MgCl2, 1 mm EGTA, 0.2% IGEPAL, and a protease/phosphatase inhibitor mix containing 1 mm Pefabloc, 1 mmsodium orthovanadate, 5 mm benzamidine, 5 µg/ml aprotinin, 5 µg/ml leupeptin). Cell debris was removed by centrifugation, and 500 µg of protein supernatants were precleared with 10 µl of protein-A/G-agarose (Amersham Pharmacia Biotech). The lysates were then incubated with the appropriate antiserum coupled to 30 µl of protein-A/G-agarose overnight at 4 °C. The immune complexes were washed twice in Nonidet P-40 buffer containing 500 mm NaCl. After resuspension in Laemmli buffer and SDS-PAGE, proteins were detected by immunoblot. Anti-FLAG M2 monoclonal antibody (Eastman Kodak Co.) was a generous gift from S. Feller (Institut für Medizinische Strahlenkunde und Zellforschung). Goat polyclonal anti-FLAG raised against the OctA probe and rabbit polyclonal ERK2 antibody were purchased from Santa Cruz. HA-tagged proteins were detected with a monoclonal anti-HA antibody (12CA5). Anti-XIAP rabbit polyclonal antibody was purchased from R & D systems anti-mouse or anti-rabbit horseradish peroxidase-linked F(ab′)2 fragments (Amersham Pharmacia Biotech), and anti-goat IgG horseradish peroxidase (Santa Cruz) was also employed. The protocol for transfection of PhoenixTM virus packaging cells and establishment of stably transduced 32D cells has been described previously (23von Gise A. Lorenz P. Wellbrock C. Hemmings B. Berberich-Siebelt F. Rapp U.R. Troppmair J. Mol. Cell. Biol. 2001; 21: 2324-2336Crossref PubMed Scopus (160) Google Scholar). Retroviral expressed proteins were confirmed after selection with Zeocin by Western blotting. For the analysis of cell survival, cells were washed three times in tissue culture medium containing 10% FCS but without WEHI cell supernatant. 0.5 in a of 1 were into a well of a tissue culture Cell was by the cells in For protein the cell number and were by In the search for IAP-interacting a yeast two-hybrid screen was performed with the full-length ITA cDNA as using a PC12 cDNA The fusion protein expressed in yeast cells reporter genes and was clones were obtained from this of which were and after of clones analysis of clones that three clones from the cDNA homology to previously described an gene from The of show a high of was identified as NRAGE (14Salehi A.H. Roux P.P. Kubu C.J. Zeindler C. Bhakar A. Tannis L.L. Verdi J.M. Barker P.A. Neuron. 2000; 27: 279-288Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). NRAGE interact with other IAP family yeast two-hybrid tests were the interacting clone and c-IAP1, XIAP, and XIAP with with an of activity with ITA XIAP also to and c-IAP1, and NAIP interact with of the clones in the two-hybrid interaction we performed in a eukaryotic cell system. The of NRAGE was cloned into a mammalian expression vector as described and HEK293 cells were with HA-tagged ITA and NRAGE was and using a anti-FLAG antibody 1 shown in NRAGE was after with the antibody using lysates from lysates 1 but from cells 1 Furthermore, NRAGE with an ITA containing the RING domain 1 but with the containing the BIR domains of ITA 1 were able to high with 500 mm NaCl. In 1 the presence of the ITA proteins used in the coprecipitation was confirmed by and the with the monoclonal In that ITA with NRAGE with high and that this interaction via its carboxyl-terminal RING zinc finger The was to the HEK293 cells were transfected with vectors expressing and or and GST In 1 NRAGE is with protein was detected in the NRAGE was and as a The was and using the rabbit polyclonal XIAP shown in the In 1 the reverse was was with NRAGE 2 this interaction was to high mm and was used as the while GST containing was used as a in this The were and using the antibody to shown in the NRAGE as a novel with a binding IL-3-dependent 32D cells have been well with to the process of apoptosis induced by growth factor withdrawal C.J. R.J. Proc. Natl. Acad. Sci. U. S. A. 80: PubMed Scopus Google Scholar, J.L. Troppmair J. G. P. M. G. Rapp U.R. Oncogene. 9: Google Scholar). For this we NRAGE and vector stably transduced cell to the of NRAGE to this NRAGE expression was by Western The of cells was by the of the retroviral vector or NRAGE protein expression Cell after withdrawal was by shown in the cells to the In expression of NRAGE a effect on the of cell death in 32D The for NRAGE and the very on and this the the three times as many cells were with the cell This that NRAGE the cell death process upon growth factor withdrawal in this IL-3-dependent promyeloid leukemic cell a we the interaction of NRAGE with endogenous XIAP in 32D shows the binding of NRAGE with XIAP is in 32D h after withdrawal we an of and at as in the of interaction in 32D cells after we endogenous XIAP protein shows that XIAP very in cells and to of XIAP In shows XIAP is in cells at and the of XIAP protein at a with NRAGE expressing Bcl-2 expression in 32D cells has been shown to be very in the cells from the of apoptosis by a variety of withdrawal (21Wang H.G. Rapp U.R. Reed J.C. Cell. 1996; 87: 629-638Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). to also the activity of 32D cells stably expressing Bcl-2 were with the retroviral vector or the containing and as described h after the 32D cells expressing Bcl-2 a in with the and was in the the cells expressing NRAGE a in survival, by Bcl-2 2 This that NRAGE is a pro-apoptotic protein Bcl-2 after growth factor Bcl-2 the IAP family members the largest group of pro-survival Members of this family are by the presence of a BIR domain, a novel zinc binding and the to apoptosis R.J. Miller Mol. Cell. Biol. PubMed Scopus Google Scholar). BIR domains mediate caspase binding and inhibition and are both and for the anti-apoptotic function of IAP proteins (27Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1712) Google Scholar). the anti-apoptotic function of Bcl-2 is well (13Reed J.C. Oncogene. 1998; 17: 3225-3236Crossref PubMed Scopus (936) Google Scholar), very is the that IAP In an we a yeast two-hybrid screen with the avian IAP and ITA and can that the NRAGE binds to the IAP proteins ITA and XIAP. For the interaction with NRAGE of the BIR but requires the presence of the RING domain, which is present in c-IAP1, and XIAP but is in with this we also to interaction of NAIP with Moreover, that expression of NRAGE in the IL-3-dependent promyeloid cell line 32D cell death growth factor withdrawal even in the presence of Bcl-2. XIAP was previously shown to apoptosis E. Wang A. H. Cancer Res. 2000; Google Scholar). of NRAGE to XIAP and of XIAP apoptosis in 32D while in the cells this process at a The role of the RING domain of IAPs in the process of apoptosis the RING domain of c-IAP2 has been shown to be important in NF-κB signaling L. J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar), was for caspase inhibition (29Roy N. Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. EMBO J. 1997; 16: 6914-6925Crossref PubMed Scopus (1131) Google Scholar). Furthermore, a that the RING domains of cIAP1 and XIAP possess activity upon apoptosis in of proteins Y. Fang S. A.M. 2000; PubMed Scopus Google Scholar). is to that proteins binding to this this NRAGE has been shown to apoptosis signaling by the of p75NTR with (15Kaplan D.R. Miller F.D. Curr. Opin. Neurobiol. 2000; 10: 381-391Crossref PubMed Scopus (1647) Google Scholar). in to one of the of et (14Salehi A.H. Roux P.P. Kubu C.J. Zeindler C. Bhakar A. Tannis L.L. Verdi J.M. Barker P.A. Neuron. 2000; 27: 279-288Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), we effect on by expression of which be to the cell expressing a of NRAGE to cell but able to with IAP proteins cIAP1 and cIAP2 have first been identified as part of the signaling (3Rothe M. Pan M.G. Henzel W.J. Ayres T.M. Goeddel D.V. Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1053) Google Scholar). The of NRAGE to with the cell death that is by withdrawal to a novel function of NRAGE in apoptosis is that Bcl-2 proteins with the process of caspase activation at the mitochondrial (13Reed J.C. Oncogene. 1998; 17: 3225-3236Crossref PubMed Scopus (936) Google Scholar), while the IAPs have been shown to act at the receptor (3Rothe M. Pan M.G. Henzel W.J. Ayres T.M. Goeddel D.V. Cell. 1995; 83: 1243-1252Abstract Full Text PDF PubMed Scopus (1053) Google Scholar) and in the of the G. M. EMBO J. 2001; PubMed Scopus Google Scholar). performed in caspase NRAGE and XIAP but were to an of NRAGE on XIAP activity have of which NRAGE XIAP and to be NRAGE may function on the of caspase Interestingly, we a very high of expression in has been previously described that in embryonic neurons, ITA expression is required for (18Wiese S. Digby M.R. Gunnersen J.M. Gotz R. Pei G. Holtmann B. Lowenthal J. Sendtner M. Nat. Neurosci. 1999; 2: 978-983Crossref PubMed Scopus (61) Google Scholar). to be the signaling from the NGF In that IAP activity may be by with proteins such as the that the of molecules in for of the and for D. for the pCFG5-IEGZ vector and J. Reed for the IAP also and for