Cloning and Characterization of a Novel RING-B-box-Coiled-coil Protein with Apoptotic Function
2003; Elsevier BV; Volume: 278; Issue: 27 Linguagem: Inglês
10.1074/jbc.m303438200
ISSN1083-351X
AutoresFumihiko Kimura, Shinya Suzu, Yukitsugu Nakamura, Yukiko Nakata, Muneo Yamada, Naruo Kuwada, Takuya Matsumura, Takuya Yamashita, Takashi Ikeda, Ken Sato, Kazuo Motoyoshi,
Tópico(s)RNA Interference and Gene Delivery
ResumoWe have identified a novel RING-B-box-coiled-coil (RBCC) protein (MAIR for macrophage-derived apoptosis-inducing RBCC protein) that consists of an N-terminal RING finger, followed by a B-box zinc finger, a coiled-coil domain, and a B30.2 domain. MAIR mRNA was expressed widely in mouse tissues and was induced by macrophage colony-stimulating factor in murine peritoneal and bone marrow macrophages. MAIR protein initially showed a granular distribution predominantly in the cytoplasm. The addition of zinc to transfectants containing MAIR cDNA as part of a heavy metal-inducible vector caused apoptosis of the cells characterized by cell fragmentation; a reduction in mitochondrial membrane potential; activation of caspase-7, -8, and -9, but not caspase-3; and DNA degradation. We also found that the RING finger and coiled-coil domains were required for MAIR activity by analysis with deletion mutants. We have identified a novel RING-B-box-coiled-coil (RBCC) protein (MAIR for macrophage-derived apoptosis-inducing RBCC protein) that consists of an N-terminal RING finger, followed by a B-box zinc finger, a coiled-coil domain, and a B30.2 domain. MAIR mRNA was expressed widely in mouse tissues and was induced by macrophage colony-stimulating factor in murine peritoneal and bone marrow macrophages. MAIR protein initially showed a granular distribution predominantly in the cytoplasm. The addition of zinc to transfectants containing MAIR cDNA as part of a heavy metal-inducible vector caused apoptosis of the cells characterized by cell fragmentation; a reduction in mitochondrial membrane potential; activation of caspase-7, -8, and -9, but not caspase-3; and DNA degradation. We also found that the RING finger and coiled-coil domains were required for MAIR activity by analysis with deletion mutants. The RING-B-box-coiled-coil (RBCC) 1The abbreviations used are: RBCC, RING-B-box-coiled-coil; BERP, Brain-expressed RING finger protein; PML, promyelocytic leukemia; RFP, ret finger protein; MAIR, macrophage-derived apoptosis-inducing RBCC protein; M-CSF and GM-CSF, macrophage and granulocyte/macrophage colony-stimulating factor, respectively; 5′-RACE, 5′-rapid amplification of cDNA ends; HA, hemagglutinin; GFP, green fluorescent protein; Z-, benzyloxycarbonyl; fmk, fluoromethyl ketone; DCB, dichlorobenzene. proteins are a subgroup of the RING finger family characterized by an N-terminal RING finger, followed by one or two additional cysteine-rich zinc fingers (B-box) and a leucine coiled-coil domain forming the RBCC or tripartite motif (1Reddy B.A. Etkin L.D. Freemont P.S. Trends Biochem. Sci. 1992; 17: 344-345Google Scholar, 2Borden K.L. Biochem. Cell Biol. 1998; 76: 351-358Google Scholar). The core members of the RBCC family possess a B30.2 domain at their C terminus in addition to the RBCC motif (3Henry J. Mather I. McDermott M. Pontarotti P. Mol. Biol. Evol. 1998; 15: 1696-1705Google Scholar). However, despite their structural similarity, RBCC proteins show varied subcellular localization and diverse cellular function (4Borden K.L. J. Mol. Biol. 2000; 295: 1103-1112Google Scholar). Some members are known to be putative transcription factors and are developmentally regulated or expressed in a tissue-specific manner (5Torok M. Etkin L.D. Differentiation. 2001; 67: 63-71Google Scholar). Xnf7 was first detected in the Xenopus oocyte nucleus and is released to the cytoplasm during oocyte maturation (6Li X. Shou W. Kloc M. Reddy B.A. Etkin L.D. J. Cell Biol. 1994; 124: 7-17Google Scholar). At the mid-blastula stage, Xnf7 re-enters the nuclei and is involved in regulating the expression of genes required for axial patterning (7El-Hodiri H.M. Shou W. Etkin L.D. Dev. Biol. 1997; 190: 1-17Google Scholar). PwA33 was cloned as a nuclear protein on the loops of amphibian lampbrush chromosomes and is suggested to have a role in the synthesis or processing of pre-mRNA during oogenesis (8Bellini M. Lacroix J.C. Gall J.G. EMBO J. 1993; 12: 107-114Google Scholar). Transcriptional intermediary factor-1α and -1β (KAP-1/KRIP-1) bind to the KRAB (Krüppel-associated box) domain of human zinc finger factors and enhance transcriptional repression exerted by the KRAB domain (9Friedman J.R. Fredericks W.J. Jensen D.E. Speicher D.W. Huang X.P. Neilson E.G. Rauscher 3rd, F.J. Genes Dev. 1996; 10: 2067-2078Google Scholar, 10Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Google Scholar). Some RBCC proteins show localization in the cytoplasm. SS-A/Ro is an autoantigen in Sjögrens's syndrome and binds to a specific small RNA (11Chan E.K. Hamel J.C. Buyon J.P. Tan E.M. J. Clin. Invest. 1991; 87: 68-76Google Scholar, 12Itoh K. Itoh Y. Frank M.B. J. Clin. Invest. 1991; 87: 177-186Google Scholar). FXY/MID1, the gene responsible for X-linked Opitz syndrome (13Quaderi N.A. Schweiger S. Gaudenz K. Franco B. Rugarli E.I. Berger W. Feldman G.J. Volta M. Andolfi G. Gilgenkrantz S. Marion R.W. Hennekam R.C. Opitz J.M. Muenke M. Ropers H.H. Ballabio A. Nat. Genet. 1997; 17: 285-291Google Scholar), is confined to the cytoplasm and is associated with microtubules (14Schweiger S. Foerster J. Lehmann T. Suckow V. Muller Y.A. Walter G. Davies T. Porter H. van Bokhoven H. Lunt P.W. Traub P. Ropers H.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2794-2799Google Scholar). Its mutations in the C terminus in patients with Opitz syndrome completely abolish microtubule association (14Schweiger S. Foerster J. Lehmann T. Suckow V. Muller Y.A. Walter G. Davies T. Porter H. van Bokhoven H. Lunt P.W. Traub P. Ropers H.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2794-2799Google Scholar). The Brain-expressed RING finger protein BERP is associated with myosin V and α-actin-4 (15El-Husseini A.E. Vincent S.R. J. Biol. Chem. 1999; 274: 19771-19777Google Scholar, 16El-Husseini A.E. Kwasnicka D. Yamada T. Hirohashi S. Vincent S.R. Biochem. Biophys. Res. Commun. 2000; 267: 906-911Google Scholar). The Estrogen-responsive finger protein EFP is induced in response to 17β-estradiol (17Inoue S. Orimo A. Hosoi T. Kondo S. Toyoshima H. Kondo T. Ikegami A. Ouchi Y. Orimo H. Muramatsu M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11117-11121Google Scholar) and is essential in estrogen-induced cell proliferation (18Orimo A. Inoue S. Minowa O. Tominaga N. Tomioka Y. Sato M. Kuno J. Hiroi H. Shimizu Y. Suzuki M. Noda T. Muramatsu M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12027-12032Google Scholar). HERF1 was cloned as a downstream target of the acute myeloid leukemia 1/core-binding factor-β transcription factor and is required for the terminal differentiation of erythroid cells, although the precise localization of HERF1 remains to be elucidated (19Harada H. Harada Y. O'Brien D.P. Rice D.S. Naeve C.W. Downing J.R. Mol. Cell. Biol. 1999; 19: 3808-3815Google Scholar). In contrast to the RBCC proteins localized in the nucleus, the exact function of most cytoplasmic RBCC proteins remains unknown. The RBCC domain was found to be involved in protein-protein interaction, and some RBCC proteins were discovered in macromolecular complexes, suggesting a role of the RBCC domain in connecting other proteins to form large multiprotein complexes. Borden (4Borden K.L. J. Mol. Biol. 2000; 295: 1103-1112Google Scholar) reported that RING proteins have a common characteristic in that they mediate protein-protein interactions involved in forming large molecular scaffolds. Apoptosis is a physiological cell suicide process that is indispensable in development, and its malfunction is involved in tumorigenesis. Two RBCC proteins, the promyelocytic leukemia protein PML and the ret finger protein RFP, form PML nuclear bodies, which play crucial roles in apoptosis (20Matera A.G. Trends Cell Biol. 1999; 9: 302-309Google Scholar, 21Zhong S. Salomoni P. Pandolfi P.P. Nat. Cell Biol. 2000; 2: E85-E90Google Scholar, 22Doucas V. Biochem. Pharmacol. 2000; 60: 1197-1201Google Scholar). Several reports indicate that PML is a growth suppressor and that the disturbance of PML functions provides a growth advantage to the leukemic cells (23Rogaia D. Grignani F. Nicoletti I. Pelicci P.G. Leukemia (Baltimore). 1995; 9: 1467-1472Google Scholar, 24Borden K.L. CampbellDwyer E.J. Salvato M.S. FEBS Lett. 1997; 418: 30-34Google Scholar). Indeed, the overexpression of PML induces apoptosis, and analysis of PML knockout mice has shown that PML is essential in the induction of apoptosis by various stimuli such as DNA damage, Fas, tumor necrosis factor, ceramide, and interferons (25Quignon F. De Bels F. Koken M. Feunteun J. Ameisen J.C. de The H. Nat. Genet. 1998; 20: 259-265Google Scholar, 26Wang Z.G. Ruggero D. Ronchetti S. Zhong S. Gaboli M. Rivi R. Pandolfi P.P. Nat. Genet. 1998; 20: 266-272Google Scholar). In this work, we report on the identification of a novel member of the RBCC group of RING finger proteins, referred to as MAIR for macrophage-derived apoptosis-inducing RBCC protein. This gene was identified by cDNA library subtraction in which we screened genes up-regulated in bone marrow macrophages by a hematopoietic growth factor, macrophage colony-stimulating factor (M-CSF). We also show the subcellular localization of the novel RBCC protein and its apoptosis-inducing function. Library Subtraction and Cloning of MAIR—Library subtraction was performed as reported previously (27Suzu S. Tanaka-Douzono M. Nomaguchi K. Yamada M. Hayasawa H. Kimura F. Motoyoshi K. EMBO J. 2000; 19: 5114-5122Google Scholar). Briefly, murine bone marrow macrophages were prepared by culturing femoral bone marrow cells from C57BL/6 mice (Charles River Japan, Yokohama, Japan) with 100 ng/ml M-CSF for 7 days (28Suzu S. Kimura F. Ota J. Motoyoshi K. Itoh T. Mishima Y. Yamada M. Shimamura S. J. Immunol. 1997; 159: 1860-1867Google Scholar). Cells were factor-depleted for 12 h in RPMI 1640 medium containing 10% fetal calf serum and then treated with 100 ng/ml M-CSF for 3 h. Poly(A) RNA from untreated cells or from those treated with M-CSF was prepared using an mRNA separator kit (Clontech, Palo Alto, CA). cDNA library construction and library subtraction were performed with a PCR-Select cDNA subtraction kit (Clontech). The cDNA fragments of the subtracted cDNA library were cloned into the pCR2.1 vector (Invitrogen). Randomly isolated clones were further analyzed by direct sequencing and by Northern hybridization using total RNA from unstimulated and M-CSF-stimulated bone marrow macrophages. A 3.4-kbp cDNA as a cDNA probe for mouse MAIR was isolated by screening a peritoneal macrophage cDNA library with the cDNA fragment obtained by the subtraction approach. 5′-Rapid amplification of cDNA ends (5′-RACE) was performed using a Marathon-Ready cDNA amplification kit (Clontech). 5′-RACE products were cloned into the pCR2.1 vector and sequenced. The following primer was used to amplify the 5′-region: 5′-GCCTCGGTCTGTTCTGCTGCTGCTTCA-3′. Northern Blot Analysis—Total RNAs from bone marrow macrophages were isolated using RNAzol B reagent (Tel-Test, Friendswood, TX), electrophoresed on agarose gels, and transferred to a nylon membrane (Hybond N+, Amersham Biosciences). The membrane was hybridized with a radiolabeled MAIR cDNA probe or glyceraldehyde-3-phosphate dehydrogenase cDNA (Clontech) (29Suzu S. Hatake K. Ota J. Mishima Y. Yamada M. Shimamura S. Kimura F. Motoyoshi K. Biochem. Biophys. Res. Commun. 1998; 245: 120-126Google Scholar). A probe for MAIR was prepared by PCR using primers 5′-CCTTCGCGCTCCTTCAAAGAG-3′ and 5′-GGAGACACGCAGGTGGCAGAT-3′. Mouse multiple-tissue Northern blots (OriGene, Rockville, MD) were hybridized with the radiolabeled MAIR cDNA or actin cDNA (Clontech). Expression Constructs—MAIR cDNA was subcloned into an epitope tag expression vector, pHM6 or pMH (Roche Applied Science, Mann-heim, Germany), to introduce a hemagglutinin (HA) tag at the N or C terminus of MAIR, respectively. The cDNA was also cloned into the pEGFP vector (Clontech) to express a fusion protein of MAIR with the C terminus of green fluorescent protein (GFP). In selected experiments, the HA-tagged or GFP-fused MAIR cDNA was subsequently inserted into the zinc-inducible expression vector pMEP4 (Invitrogen). The RING finger and B-box zinc finger domains were deleted by a PCR-based technique using the following primer pairs: 5′-GAACGAGCGGTGCCCGGGGAG-3′ and 5′-CAGCTCCTCTTTGAAGGAGCG-3′ for deletion of the RING finger domain and 5′-CGTGTGCAGCCCATCAAGGAC-3′ and 5′-GGGGCGCGGGGACCGGCGACC-3′ for deletion of the B-box domain. The C-terminal region-deleted mutants were generated using the ApaI or BamHI restriction enzyme. Cell Culture and Transfection—The GM-CSF-dependent TF-1 cells (gift from T. Kitamura, Tokyo University, Tokyo, Japan) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and 2 ng/ml GM-CSF (30Kitamura T. Tange T. Terasawa T. Chiba S. Kuwaki T. Miyagawa K. Piao Y.F. Miyazono K. Urabe A. Takaku F. J. Cell. Physiol. 1989; 140: 323-334Google Scholar). NIH3T3 fibroblasts were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum. The plasmid vectors described above were transfected into TF-1 cells using Lipofectin reagent (Invitrogen). The transfected cells were selected in 96-well plates with medium containing 400 μg/ml hygromycin B (Wako Pure Chemicals, Osaka, Japan) and GM-CSF and were screened by immunoblotting using anti-HA antibody (F-7; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-GFP antibody (1E4; Medical & Biological Laboratories, Nagoya, Japan). Transfection into NIH3T3 cells was performed with LipofectAMINE (Invitrogen). Assays for Apoptosis—Harvested cells were analyzed by flow cytometry after zinc exposure for the indicated times. The parental cells without zinc exposure were also analyzed to set the live cell gate on the forward and side light scatter. The cell number in the gate was counted for cell viability. To measure the mitochondrial transmembrane potential (ΔΨm), MitoTracker CMX-Ros (Molecular Probes, Inc., Eugene, OR) dissolved in Me2SO was added to the culture to a final concentration of 100 nm (31Poot M. Gibson L.L. Singer V.L. Cytometry. 1997; 27: 358-364Google Scholar). After a 15-min incubation, the cells were harvested and stained with propidium iodide for dye exclusion analysis (32Darzynkiewicz Z. Juan G. Li X. Gorczyca W. Murakami T. Traganos F. Cytometry. 1997; 27: 1-20Google Scholar). For cell cycle analysis, cells were prepared in lysis buffer (phosphate-buffered saline containing 0.2% Triton X-100 and 50 mg/ml propidium iodide) (33Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. J. Immunol. Methods. 1991; 139: 271-279Google Scholar). All samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences). The broad-range caspase inhibitors Z-VAD-fmk and Z-Asp-CH2-DCB as well as the specific caspase inhibitors Ac-DEVD-CHO, Ac-IETD-CHO, Ac-YVAD-CHO, and Ac-LEHD-CHO were purchased from the Peptide Institute (Osaka). The caspase inhibitors were added 30 min prior to ZnCl2 treatment. Immunoblotting—The cells were resuspended in radioimmune precipitation assay buffer (1% Nonidet P-40, 50 mm Tris (pH 8.0), 150 mm NaCl, 0.5% deoxycholate, and 0.1% SDS) and then completely lysed by three cycles of freezing and thawing (34Ohnishi T. Wang X. Ohnishi K. Matsumoto H. Takahashi A. J. Biol. Chem. 1996; 271: 14510-14513Google Scholar). The protein contents of the supernatants obtained after centrifugation were quantified using the Bio-Rad protein assay kit. The cleared cell lysates containing equal amounts of protein were resolved by SDS-PAGE under reducing conditions, and the proteins were transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore Corp., Bedford, MA). The membrane was probed with anti-HA tag antibody F-7 or antibody to caspase-3 (Pharmingen), caspase-7 (Transduction Laboratories), caspase-8 (Immunotech, Marseilles, France), caspase-9 (Millennium Biotechnology, Romana, CA), poly(ADP-ribose) polymerase (Pharmingen), or actin (Roche Applied Science). The antibodies were visualized with horseradish peroxidase-coupled anti-immunoglobulin antibody (Bio-Rad) using Western blot chemiluminescent reagent (PerkinElmer Life Sciences) according to the manufacturer's instructions. Cloning of MAIR—To identify M-CSF-inducible genes, we prepared a cDNA library of murine bone marrow-derived macrophages cultured in cytokine-free medium and subtracted this cDNA library from that of cells stimulated with M-CSF (27Suzu S. Tanaka-Douzono M. Nomaguchi K. Yamada M. Hayasawa H. Kimura F. Motoyoshi K. EMBO J. 2000; 19: 5114-5122Google Scholar). A number of genes induced by M-CSF stimulation were obtained. 2F. Kimura, S. Suzu, Y. Nakamura, Y. Nakata, M. Yamada, N. Kuwada, T. Matsumura, T. Yamashita, T. Ikeda, K. Sato, and K. Motoyoshi, unpublished data. Among them, we found a novel cDNA fragment encoding a RING finger domain. A 3.4-kb cDNA was isolated by screening a murine peritoneal macrophage cDNA library with the cDNA fragment obtained by the subtraction approach as a probe (Fig. 1A). The cDNA encodes a 501-amino acid protein, which was designated MAIR for macrophage-derived apoptosis-inducing RBCC protein (see below). The MAIR protein consists of an N-terminal cysteine-rich C3HC4 zinc finger (Fig. 1A, solid line a) (35Freemont P.S. Hanson I.M. Trowsdale J. Cell. 1991; 64: 483-484Google Scholar), followed by a B-box zinc finger (solid line b) (36Reddy B.A. Etkin L.D. Nucleic Acids Res. 1991; 19: 6330Google Scholar), a coiled-coil domain (solid line c), and a C-terminal region referred to as the B30.2 domain (dashed lines d–f) (3Henry J. Mather I. McDermott M. Pontarotti P. Mol. Biol. Evol. 1998; 15: 1696-1705Google Scholar). Accordingly, MAIR is a new member of the family of RBCC proteins. There are three putative nuclear localization sequences (Fig. 1A, boxes). Other members of this RBCC family are Xnf7 of Xenopus (6Li X. Shou W. Kloc M. Reddy B.A. Etkin L.D. J. Cell Biol. 1994; 124: 7-17Google Scholar), PwA33 (8Bellini M. Lacroix J.C. Gall J.G. EMBO J. 1993; 12: 107-114Google Scholar), human 52-kDa SS-A/Ro autoantigen (11Chan E.K. Hamel J.C. Buyon J.P. Tan E.M. J. Clin. Invest. 1991; 87: 68-76Google Scholar, 12Itoh K. Itoh Y. Frank M.B. J. Clin. Invest. 1991; 87: 177-186Google Scholar), acid finger protein (AFP) (37Chu T.W. Capossela A. Coleman R. Goei V.L. Nallur G. Gruen J.R. Genomics. 1995; 29: 229-239Google Scholar), human RFP (38Takahashi M. Inaguma Y. Hiai H. Hirose F. Mol. Cell. Biol. 1988; 8: 1853-1856Google Scholar), and HERF1 (19Harada H. Harada Y. O'Brien D.P. Rice D.S. Naeve C.W. Downing J.R. Mol. Cell. Biol. 1999; 19: 3808-3815Google Scholar) (Fig. 1B). The MAIR protein shows 24–29% identity and 40–47% similarity to these RBCC proteins. Among these proteins, Xnf7 has a cytoplasmic retention domain that controls its subcellular localization and that precedes the RING finger domain (6Li X. Shou W. Kloc M. Reddy B.A. Etkin L.D. J. Cell Biol. 1994; 124: 7-17Google Scholar). The open reading frame of MAIR cDNA is preceded by a 19-bp upstream sequence, although we were unable to identify an in-frame and upstream stop codon (Fig. 1A). However, analysis of 5′-RACE products suggested that the cDNA has no missing 5′-end (data not shown). That the designated ATG codon is the true translated initiation codon is supported by its context within a Kozak consensus sequence (39Kozak M. Nucleic Acids Res. 1984; 12: 857-872Google Scholar) and alignment of the MAIR protein with SS-A/Ro, RFP, acid finger protein, and HERF1 (Fig. 1B). After our cloning, a cDNA sequence of the putative human homolog of MAIR was submitted to the GenBank™/EBI Data Bank by the Kazusa DNA Research Institute (KIAA1098, accession no. AB029021) as a new cDNA clone (40Kikuno R. Nagase T. Ishikawa K. Hirosawa M. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1999; 6: 197-205Google Scholar). These molecules are 78% identical and 85% similar at the amino acid level (Fig. 1B). Tissue Distribution and M-CSF-induced Expression of MAIR—Northern analysis showed that the MAIR mRNA was ubiquitously expressed in the mouse tissues examined (Fig. 1C). The mRNA was 3.4–3.6 kb, suggesting that our clone (3.4 kb) was a nearly full-length cDNA. It was relatively highly expressed in the brain, lung, spleen, thymus, heart, and muscle (Fig. 1C). According to the HUGE Database provided by the Kazusa DNA Research Institute, 3Available at www.kazusa.or.jp/huge. the human homolog (KIAA1098) is relatively highly expressed in the brain, liver, spleen, ovary, testis, and heart. MAIR cDNA was originally isolated from M-CSF-stimulated macrophages, but there was a relatively low level of MAIR expression in bone marrow cells. We therefore certified MAIR expression in primary macrophages and several macrophage cell lines. Bone marrow macrophages expressed the MAIR transcript at a level detectable by Northern blotting (Fig. 1D). Of note, MAIR expression decreased after M-CSF starvation, and M-CSF stimulation resulted in an increase in MAIR expression with a peak at 1.5 h (Fig. 1D). We also observed M-CSF-elevated MAIR expression in the M-CSF-dependent cell line M-NFS-60 and in the M-CSF-responsive macrophage cell line J774A.1 (data not shown). Transient MAIR Expression Promotes Cell Death—To analyze the function of MAIR, we initially attempted to express HA-tagged MAIR in mouse NIH3T3 cells. When we transiently transfected NIH3T3 cells with the HA-tagged MAIR expression vector, we could easily detect the HA-tagged protein by immunoblot analysis using anti-HA antibody as a band with a molecular mass of 55 kDa (data not shown). However, none of the G418-resistant stable clones showed a detectable level of HA-tagged MAIR protein (data not shown). To investigate the fate of MAIR-transfected cells and the subcellular localization of MAIR, we transiently expressed MAIR as a GFP fusion protein in NIH3T3 cells (Fig. 2A). Despite the presence of putative nuclear localization signals (Fig. 1A), fluorescence microscopic analysis revealed a granular distribution of GFP-MAIR predominantly in the cytoplasm at 12 h post-transfection (Fig. 2A, panel a). The fact that MAIR localized in the cytoplasm was confirmed using HeLa cells (data not shown). Confocal microscopic examination showed partial colocalization of MAIR with mitochondria (Fig. 2B). At 24 h post-transfection, GFP-MAIR-expressing NIH3T3 cells shrunk and began to detach from the dish (Fig. 1A, panel b). Some cells appeared to be fragmented (Fig. 1A, panel b, arrowheads). Consistent with the results, flow cytometric analysis showed that propidium iodide-positive dead cells increased in number 72 h after transfection of GFP-MAIR compared with mock transfection (Fig. 2C). Zinc-inducible Expression of MAIR in GM-CSF-dependent TF-1 Cells—To demonstrate more clearly that the expression of MAIR promotes cell death, we transfected TF-1 cells with a zinc-inducible pMEP4-HA-MAIR construct, in which a metallothionein promoter directs MAIR expression. TF-1 cells are leukemic cells of human origin whose proliferation is dependent on the presence of GM-CSF (30Kitamura T. Tange T. Terasawa T. Chiba S. Kuwaki T. Miyagawa K. Piao Y.F. Miyazono K. Urabe A. Takaku F. J. Cell. Physiol. 1989; 140: 323-334Google Scholar). Three independent clones (clones 18, 19, and 24) that expressed HA-tagged MAIR after zinc treatment were established (Fig. 3A). As shown in Fig. 3B, the addition of 75 or 100 μm ZnCl2 to the culture medium induced cell death in the three clones, whereas the treatment did not affect the viability of mock-transfected TF-1 cells. Among MAIR transfectants, clone 24, which expressed more HA-tagged MAIR compared with clones 18 and 19 (Fig. 3A), showed markedly reduced viability after zinc treatment (Fig. 3B). Phase-contrast microscopic examination revealed the presence of particles of fragmented TF-1-HA-MAIR cells after zinc induction (Fig. 3C). Next, we made a pMEP4-GFP-MAIR construct, in which GFP fused to the N terminus of MAIR could be monitored by flow cytometry, and introduced the construct into TF-1 cells. GFP-MAIR was dose-dependently expressed after exposure to ZnCl2 in a stable transfectant (clone 12) (Fig. 4A). A low but detectable level of MAIR expression could be found even if the cells were not treated with ZnCl2 (Fig. 4A, upper panel). ZnCl2 addition to the medium induced cell death in the transfectants (clones 12 and 25) (Fig. 4B), as shown in experiments in which HA-tagged MAIR was expressed (Fig. 3B). Using this clone (clone 12), we analyzed the time course relationship between the expression of MAIR and ΔΨm. GFP-MAIR expression (fluorescent intensity of GFP) could be detected within 2 h after zinc treatment (Fig. 5A, lower panels). The amount of MAIR increased until 8 h. The increase in the side scatter could be detected at 4 h, which was followed by a decrease in the forward scatter (Fig. 5A, upper panels). This change is consistent with certain characteristics of early apoptosis (41Darzynkiewicz Z. Bruno S. Del Bino G. Gorczyca W. Hotz M.A. Lassota P. Traganos F. Cytometry. 1992; 13: 795-808Google Scholar). Simultaneously, the uptake of MitoTracker CMX-Ros, which indicates mitochondrial membrane potential, decreased in response to zinc treatment (Fig. 5A, lower panels). Dye exclusion was conserved at 12 h, but membrane-damaged cells increased over 24 h (data not shown). We then analyzed the DNA content of TF-1 cells expressing GFP-MAIR by flow cytometry after culturing for 24 h in the presence of zinc. A large proportion of TF-1-GFP-MAIR cells (67%) contained degraded DNA, a characteristic of apoptosis (Fig. 5B). There was no increase in hypodiploid DNA in the control cell line and untreated TF-1-GFP-MAIR cells (Fig. 5B). These features in inducible MAIR overexpression suggested that the cell death observed was apoptosis. The emerging view of apoptosis is that this complex biochemical event is carried out by a family of cysteine proteases called caspases (42Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Google Scholar). To address the role of caspases, we used several kinds of caspase inhibitors. The addition of Z-Asp-CH2-DCB, but not Z-VAD-fmk, completely prevented a reduction in MitoTracker CMX-Ros in TF-1-GFP-MAIR cells (Fig. 6A). However, the percentage of membrane-damaged cells with Z-Asp-CH2-DCB was not greater than that without inhibitors even after 24 h of ZnCl2 induction (data not shown). These observations suggest that the inhibitor Z-Asp-CH2-DCB did not induce necrotic cell death in addition to preventing apoptotic cell death. The specific inhibitors Ac-YVAD-CHO (for caspase-1 and -4), Ac-DEVD-CHO (for caspase-3 and -7), Ac-IETD-CHO (for caspase-8 and -6), and Ac-LEHD-CHO (for caspase-9) could not effectively impede the decrease in ΔΨm when used alone (data not shown). We then performed immunoblot analysis for caspases to detect their activation. When TF-1-GFP-MAIR cells were treated with zinc (Fig. 6B), it was obvious that caspase-7, -8, and -9 were cleaved, whereas no processing of caspase-2 or -3 was observed (Fig. 6B) (data not shown for caspase-2). Caspase-9 is activated by binding to Apaf1 in the presence of cytochrome c released from the mitochondria (43Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Google Scholar). Consistent with the decrease in ΔΨm as mentioned above, this pattern of caspase activation suggests that the mitochondria are involved in MAIR-induced apoptosis. We also detected a cleaved form of poly(ADP-ribose) polymerase, a substrate of executioner caspases (Fig. 6B). The RING Finger and Coiled-coil Domains Are Important for the Apoptosis-inducing Activity of MAIR—To examine which domain of MAIR is responsible for its apoptosis-inducing activity, we generated a series of MAIR truncation mutants (Fig. 7A), performed transient transfections into NIH3T3 cells, and screened for cell death by determining the mitochondrial membrane potential (Fig. 7B). The expression of the mutant with the B-box domain deleted (ΔBB) or with the B30.2 domain deleted (ΔB30.2) was comparable to that of the wild type (Fig. 7B, left panels). However, for unknown reasons, the expression of the mutant with the RING finger domain deleted (ΔRF) or with the C-terminal region containing the coiled-coil and B30.2 domains deleted (ΔCC-B30.2) was somewhat higher than that of the wild type (Fig. 7B, left panels). The B30.2 domain-deleted and B-box-deleted mutants induced a reduction in mitochondrial membrane potential at a comparable level to the wild type (Fig. 7B, right panels). Thus, these domains might not be involved in the apoptosis-inducing function of MAIR. In contrast, the reduction in mitochondrial membrane potential by the RING finger domain-deleted MAIR mutant or the mutant in which the C-terminal region containing the coiled-coil and B30.2 domains was deleted was less severe than that of the wild type (Fig. 7B, right panels), despite the higher expression of these mutants in transfected cells (see left panels). These data indicate that the RING finger and coiled-coil domains are required for the apoptosis-inducing activity of MAIR. Finally, we investigated how the difference in the apoptotic activity of the mutants corresponds to their subcellular localization (Fig. 7C). The B-box-deleted (ΔBB panel) and B30.2 domain-deleted (ΔB30.2 panel) mutants showed a similar cellular distribution to the wild type (MAIR panel). In contrast, the less potent mutants, i.e. the RING finger domain-deleted MAIR mutant (ΔRF panel) and the mutant in which the C-terminal region containing the coiled-coil domain was deleted (ΔCC-B30.2 panel), showed a distinct distribution pattern from the wild type. The RING finger domain-deleted mutant showed a filamentous appearance in the cytoplasm, and the C-terminal region-deleted mutant spread throughout the cytoplasm. Thus, the change in t
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