Cloning and Characterization of a Novel RING-B-box-Coiled-coil Protein with Apoptotic Function*

Fumihiko Kimura {ddagger}, Shinya Suzu §, Yukitsugu Nakamura {ddagger}, Yukiko Nakata {ddagger}, Muneo Yamada §, Naruo Kuwada {ddagger}, Takuya Matsumura {ddagger}, Takuya Yamashita {ddagger}, Takashi Ikeda {ddagger}, Ken Sato {ddagger} and Kazuo Motoyoshi {ddagger} 

From the {ddagger}Third Department of Internal Medicine, National Defense Medical College, Namiki, Tokorozawa, Saitama 359-8513 and the §Biochemical Research Laboratory, Morinaga Milk Industry Company Limited, Higashihara, Zama, Kanagawa 228-8583, Japan

Received for publication, April 3, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The RING-B-box-coiled-coil (RBCC)1 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 (1, 2). The core members of the RBCC family possess a B30.2 domain at their C terminus in addition to the RBCC motif (3). However, despite their structural similarity, RBCC proteins show varied subcellular localization and diverse cellular function (4).

Some members are known to be putative transcription factors and are developmentally regulated or expressed in a tissue-specific manner (5). Xnf7 was first detected in the Xenopus oocyte nucleus and is released to the cytoplasm during oocyte maturation (6). At the mid-blastula stage, Xnf7 re-enters the nuclei and is involved in regulating the expression of genes required for axial patterning (7). 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 (8). Transcriptional intermediary factor-1{alpha} and -1{beta} (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 (9, 10).

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 (11, 12). FXY/MID1, the gene responsible for X-linked Opitz syndrome (13), is confined to the cytoplasm and is associated with microtubules (14). Its mutations in the C terminus in patients with Opitz syndrome completely abolish microtubule association (14). The Brain-expressed RING finger protein BERP is associated with myosin V and {alpha}-actin-4 (15, 16). The Estrogen-responsive finger protein EFP is induced in response to 17{beta}-estradiol (17) and is essential in estrogen-induced cell proliferation (18). HERF1 was cloned as a downstream target of the acute myeloid leukemia 1/core-binding factor-{beta} transcription factor and is required for the terminal differentiation of erythroid cells, although the precise localization of HERF1 remains to be elucidated (19). 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 (4) 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 (2022). Several reports indicate that PML is a growth suppressor and that the disturbance of PML functions provides a growth advantage to the leukemic cells (23, 24). 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 (25, 26).

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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Library Subtraction and Cloning of MAIR—Library subtraction was performed as reported previously (27). 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 (28). 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) (29). 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 (30). 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 ({Delta}{Psi}m), MitoTracker CMX-Ros (Molecular Probes, Inc., Eugene, OR) dissolved in Me2SO was added to the culture to a final concentration of 100 nM (31). After a 15-min incubation, the cells were harvested and stained with propidium iodide for dye exclusion analysis (32). 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) (33). 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 (34). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (27). A number of genes induced by M-CSF stimulation were obtained.2 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) (35), followed by a B-box zinc finger (solid line b) (36), a coiled-coil domain (solid line c), and a C-terminal region referred to as the B30.2 domain (dashed lines d–f) (3).



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FIG. 1A.
Structure, tissue distribution, and M-CSF-induced expression of MAIR. A, nucleotide sequence and predicted amino acid sequence of MAIR. The 3'-untranslated region is omitted. The RBCC motif is underlined (solid line a, RING finger; solid line b, B-box zinc finger; solid line c, coiled-coil region). The B30.2 domain consists of an LDP motif (dashed line d), a WEVE motif (dashed line e), and an LDYE motif (dashed line f). The putative nuclear localization sequences are boxed. B, schematic representation of the MAIR protein and other members of RBCC protein family. The percentage of homology to MAIR is shown. C, distribution of the MAIR transcript in mouse tissues. The blot was probed with the MAIR or {beta}-actin cDNA probe. D, M-CSF-induced expression of MAIR in bone marrow macrophages. Mouse bone marrow macrophages were factor-depleted and then stimulated with M-CSF for the time periods indicated. Total RNA was blotted onto a membrane and hybridized with the MAIR or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe.

 

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 (6), PwA33 (8), human 52-kDa SS-A/Ro autoantigen (11, 12), acid finger protein (AFP) (37), human RFP (38), and HERF1 (19) (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 (6). 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 (39) 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 GenBankTM/EBI Data Bank by the Kazusa DNA Research Institute (KIAA1098, accession no. AB029021 [GenBank] ) as a new cDNA clone (40). These molecules are 78% identical and 85% similar at the amino acid level (Fig. 1B).



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FIG. 1B.
Structure, tissue distribution, and M-CSF-induced expression of MAIR. A, nucleotide sequence and predicted amino acid sequence of MAIR. The 3'-untranslated region is omitted. The RBCC motif is underlined (solid line a, RING finger; solid line b, B-box zinc finger; solid line c, coiled-coil region). The B30.2 domain consists of an LDP motif (dashed line d), a WEVE motif (dashed line e), and an LDYE motif (dashed line f). The putative nuclear localization sequences are boxed. B, schematic representation of the MAIR protein and other members of RBCC protein family. The percentage of homology to MAIR is shown. C, distribution of the MAIR transcript in mouse tissues. The blot was probed with the MAIR or {beta}-actin cDNA probe. D, M-CSF-induced expression of MAIR in bone marrow macrophages. Mouse bone marrow macrophages were factor-depleted and then stimulated with M-CSF for the time periods indicated. Total RNA was blotted onto a membrane and hybridized with the MAIR or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe.

 

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,3 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).



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FIG. 2.
Analysis of subcellular localization of MAIR and propidium iodide-positive cells in transiently transfected NIH3T3 cells. A, NIH3T3 cells were transfected with pEGFP-MAIR and subjected to fluorescence microscopy after 12 h (panel a) or 24 h (panel b). The cells were also transfected with a control pEGFP vector (panel c). The arrowheads indicate fragmented cells. The magnifications are shown. B, NIH3T3 cells were transfected with pEGFP-MAIR and analyzed by confocal microscopy at 12 h (panel a). Cell mitochondria were stained with MitoTracker CMX-Ros (panel b). Panel c is a merged image. C, NIH3T3 cells were transfected with pEGFP or pEGFP-MAIR and subjected to flow cytometric analysis for propidium iodide-positive cells of GFP-positive cells after 72 h.

 

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 (30). 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).



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FIG. 3.
Establishment and analysis of TF-1 cells expressing HA-tagged MAIR in response to zinc treatment. A, three clones (TF-1-HA-MAIR clones 18, 19, and 24) were treated with ZnCl2 (100 µM) for 2 h and analyzed for the expression of the HA-tagged MAIR protein by immunoblotting with anti-HA antibody. To verify the amount of protein loaded, immunoblotting with anti-{beta}-actin antibody was performed. B, the mock-transfected TF-1 cells (white bars) and TF-1-HA-MAIR clones 18 (hatched bars), 19 (black bars), and 24 (gray bars) were treated with ZnCl2 at a final concentration of 75 or 100 µM for 24 h and analyzed for cell viability by flow cytometry. Error bars from triplicate assays are shown. The data shown are representative of three independent experiments. C, presented are the results from phase-contrast microscopy showing the presence of apoptotic bodies. TF-1-HA-MAIR clone 24 was treated with ZnCl2 (100 µM) for 24 h. Magnification is x200.

 

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).



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FIG. 4.
Establishment and analysis of TF-1 cells expressing GFP-fused MAIR in response to zinc treatment. A, TF-1-GFP-MAIR clone 12 was treated with the indicated concentrations of ZnCl2 for 2 h, and the expression of GFP-fused MAIR was determined by flow cytometry. The fluorescence of parental TF-1 cells is shown by solid lines. B, the parental TF-1 cells (white bars) and TF-1-GFP-MAIR clones 12 (hatched bars) and 25 (black bars) were treated with ZnCl2 at a final concentration of 75 or 100 µM for 24 h and analyzed for cell viability by flow cytometry. Error bars from triplicate assays are shown. The data shown are representative of three independent experiments.

 

Using this clone (clone 12), we analyzed the time course relationship between the expression of MAIR and {Delta}{Psi}m. 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 (41). 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.



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FIG. 5.
Effect of ZnCl2 on TF-1 cells expressing GFP-fused MAIR. A, TF-1-GFP-MAIR clone 12 (Fig. 4) was treated with 100 µM ZnCl2 for the indicated periods. The cells were then analyzed by flow cytometry for forward and side scatter (upper panels) or for the uptake of MitoTracker CMX-Ros and GFP-MAIR expression (lower panels). B, the cells (parental TF-1 cells, TF-1 cells transfected with the control pEGFP vector, and TF-1-GFP-MAIR clone 12) were left untreated or were treated with 75 µM ZnCl2 for 24 h and subjected to flow cytometric analysis of apoptotic cells as determined by the percentage of sub-G1 cells. The data shown are representative of two independent experiments with similar results.

 

The emerging view of apoptosis is that this complex biochemical event is carried out by a family of cysteine proteases called caspases (42). 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 {Delta}{Psi}m 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 (43). Consistent with the decrease in {Delta}{Psi}m 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).



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FIG. 6.
Effect of pan-caspase inhibitors on MAIR-induced cell death in TF-1-GFP-MAIR cells and immunoblot analysis of caspase activation. A, TF-1-GFP-MAIR clone 12 (Fig. 4) was pretreated with dimethyl sulfoxide (vehicle for the caspase inhibitors), Z-VAD-fmk (zVAD; 100 µM), or Z-Asp-CH2-DCB (zAsp; 100 µM) and then treated with 75 µM ZnCl2 (Zn75) for 12 h. The cells were analyzed by flow cytometry for the uptake of MitoTracker CMX-Ros. B, whole cell lysates from TF-1-GFP-MAIR cells were analyzed by immunoblotting for caspase-3, -7, -8, or -9 or poly(ADP-ribose) polymerase (PARP) after the following treatments: 0 lane, untreated; UV lane, cultured for 12 h after UV irradiation (25 J/m2); GM lane, GM-CSF-depleted for 24 h; MAIR 12h lane, treated with 75 µM ZnCl2 for 12 h; MAIR 24 h lane, treated with 75 µM ZnCl2 for 24 h. The arrowheads indicate the cleaved form of each protein. Immunoblotting with anti-actin antibody confirmed comparable protein loading in these lanes. The data shown are representative of three independent experiments with similar results.

 

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 ({Delta}BB) or with the B30.2 domain deleted ({Delta}B30.2) 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 ({Delta}RF) or with the C-terminal region containing the coiled-coil and B30.2 domains deleted ({Delta}CC-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.



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FIG. 7.
MAIR deletion mutants and their apoptotic activity. A, shown is a diagram of the MAIR deletion mutants used in this study. See details under "Experimental Procedures." {Delta}RF, RING finger domain-deleted mutant; {Delta}BB, B-box domain-deleted mutant; {Delta}CC-B30.2, mutant with the C-terminal region containing the coiled-coil and B30.2 domains deleted; {Delta}B30.2, B30.2 domain-deleted mutant. B, the deletion mutants were cloned into the pEGFP vector as a fusion protein to the C terminus of GFP. NIH3T3 cells were transfected with the pEGFP vector, pEGFP-MAIR, or pEGFP-mutant MAIR. The cells were analyzed for the expression of GFP-fused MAIR proteins (left panels) and for the uptake of MitoTracker CMX-Ros (right panels) by flow cytometry at the indicated times. C, the transfected NIH3T3 cells were subjected to fluorescence microscopy to detect GFP-fused MAIR proteins.

 

Finally, we investigated how the difference in the apoptotic activity of the mutants corresponds to their subcellular localization (Fig. 7C). The B-box-deleted ({Delta}BB panel) and B30.2 domain-deleted ({Delta}B30.2 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 ({Delta}RF panel) and the mutant in which the C-terminal region containing the coiled-coil domain was deleted ({Delta}CC-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 the subcellular distribution appears to correlate with apoptotic activity.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have identified a novel RBCC protein involved in the apoptotic pathway. The subcellular localization of proteins provides valuable information on their functions. Many RBCC proteins are localized in the nucleus and are involved in transcriptional regulation (5). For example, Xnf7, a maternally expressed cytoplasmic protein, moves to the nucleus during mid-blastula transition (7). The intracellular distribution of some RBCC proteins was reported to be dependent on the cell types. Endogenous RFP is located predominantly in the cytoplasm with low levels in the nucleus in NIH3T3 and mouse A9 cells (44). In contrast, the protein was shown to be a nuclear protein in HepG2, HeLa, and mouse fetal heart cells (44). When HeLa cells were transfected with wild-type RFP, the exogenous protein showed the same distribution pattern as the endogenous protein (44). MAIR possesses three putative sites of nuclear localization signals (Fig. 1A). However, upon examining the subcellular localization of exogenous MAIR using NIH3T3 cells (Fig. 2A) and HeLa and TF-1 cells (data not shown), we found MAIR to be predominantly localized in the cytoplasm. We also found that MAIR was distributed as granules in the cytoplasm (Fig. 2A). This is in good accordance with the hypothesis that the RBCC domain mediates protein oligomerization and the fact that RBCC proteins are often found in large complexes, possibly acting as scaffolding elements (4). However, we cannot rule out the possibility that MAIR moves to the nucleus during the apoptotic process (Fig. 2A, panel b).

SS-A/Ro, FXY/MID1, and BERP were reported to be cytoplasmic RBCC proteins (14, 15, 45). The gene for BERP is mapped to chromosome 11p15, which is frequently deleted in several human cancers and is speculated to be a tumor suppressor gene (46). However, the exact molecular functions of these cytoplasmic RBCC proteins remain to be elucidated. In this study, we demonstrated that MAIR was involved in programmed cell death. The induction of the MAIR protein in TF-1 cells caused apoptosis of the cells characterized by cell fragmentation (Fig. 3C); a reduction in mitochondrial membrane potential (Fig. 5A); and activation of caspase-7, -8, and -9 (Fig. 6B). The caspase cascade and mitochondria play central roles in the cytoplasmic process of apoptosis (4749). Some apoptotic stimuli such as growth factor starvation and reactive oxygen species cause mitochondrial membrane permeabilization and release of cytochrome c, apoptosis-inducing factor, and some caspases (48). Released cytochrome c forms complexes with Apaf1 and procaspase-9 and accordingly activates caspase-9, which then processes and activates other caspases (42). Apoptosis induced by MAIR was found to be partially dependent on caspases because the pan-caspase inhibitor Z-Asp-CH2-DCB prevented a reduction in the mitochondrial membrane potential in the cells (Fig. 6A). Dissipation of the mitochondrial membrane potential in the early phase and activation of caspase-9 and -7 imply that MAIR triggers the event upstream or in the vicinity of the mitochondria. In fact, confocal microscopic examination showed partial colocalization of MAIR with the mitochondria (Fig. 2B). Furthermore, KIAA1098, the putative human homolog of MAIR, has been mapped to chromosome 8p21, which is frequently deleted in human cancers (Mitelman Database of Chromosome Aberrations in Cancer).4

MAIR is an RBCC family member that has a B30.2 domain in the C-terminal portion in addition to the RBCC motif. The apoptotic activity of PML appears to be mediated through its RING domain (24). In addition, deletion of the coiled-coil motif in PML yields a diffuse nuclear localization pattern and results in no growth suppression (50). Although MAIR is a cytoplasmic protein rather than a nuclear protein, corresponding regions of MAIR appear to be required for the apoptosis-inducing activity. The deletion of the RING finger domain and the coiled-coil region of MAIR, but not the B-box or B30.2 domain, partially abolished the MAIR activity (Fig. 7B). Of importance, the less potent mutants showed a distinct cytoplasmic distribution pattern from the wild type (Fig. 7C). Therefore, the RING finger domain and the coiled-coil region of MAIR might be important in forming a molecular scaffold with cytoplasmic proteins. Interestingly, stable transformants obtained by transfection with the RING finger domain-deleted MAIR mutant showed detectable MAIR expression upon immunoblotting even when the cells were not treated with ZnCl2,2 raising the possibility that the RING finger domain is involved in the stability of MAIR itself. There is recent evidence that the RING finger domain can act as a ubiquitin-protein isopeptide ligase (E3) to target proteins for degradation (51). Ubiquitin-protein isopeptide ligase (E3) proteins are responsible for providing specificity to ubiquitin conjugation by acting as links or bridges between ubiquitin-conjugating enzyme and substrate. Therefore, ubiquitination may control the stability or degradation of MAIR through its RING finger.

MAIR was originally isolated by cDNA subtraction, in which genes that were up-regulated in bone marrow macrophages by M-CSF stimulation were screened (Fig. 1D). Thus, the finding that MAIR possesses apoptosis-inducing activity seems to be contradictory to the fact that M-CSF is a growth factor for macrophages. Differentiated macrophages are known to be resistant to various death stimuli such as death receptor ligation, antineoplastic agents, and ionizing irradiation, whereas monocytes are susceptible to Fas-mediated apoptosis (52). Four factors have been reported to date to be involved in the resistance of differentiated macrophages to apoptosis inhibition: Flip, BclxL, A1, and cytoplasmic p21Cip1/WAF1 (5356). From the view point of M-CSF stimulation, M-CSF induces Ets2 expression, and in turn, Ets2 up-regulates Bcl-xL expression in the BAC1.2F5 macrophage cell line (57). In NIH3T3 cells expressing the M-CSF receptor, M-CSF promotes cell survival through Akt-induced inhibition of caspase-9 activation (58). Akt mediates I{kappa}B kinase-{alpha} phosphorylation, leading to NF{kappa}B activation, which is essential for tumor necrosis factor signaling (59). M-CSF up-regulates XIAP (X-linked inhibitor of apoptosis) upon activation of NF{kappa}B possibly through activated Akt (60). However, at the same time, M-CSF was reported to induce the expression of pro-apoptotic factors procaspase-3 and -9 and Bax in M-CSF-induced bone marrow macrophages (60). An increased expression of procaspase-3 and -8 was also observed in phorbol 12-myristate 13-acetate-stimulated U937 monocytic cells (61). These observations imply that M-CSF is able to up-regulate both pro-apoptotic and anti-apoptotic proteins and tilts the balance to an anti-apoptotic state. We recently demonstrated that p56dok-2, a molecule that negatively regulates signal transduction and cell proliferation mediated by cytokines, is up-regulated in macrophages by M-CSF stimulation (27). Thus, the up-regulation of the gene for MAIR in macrophages by M-CSF is also likely to be a negative feedback mechanism.

Based on the results of our transient and inducible expression experiments with the cell lines, we conclude that the novel RBCC protein MAIR possesses an apoptosis-inducing function. Our data presented here suggest that the cell death induced by MAIR is at least in part dependent on the mitochondria. However, the precise mechanism of MAIR-induced apoptosis is a subject of future investigation. Many RBCC proteins interact with other proteins and seem to construct macromolecular complexes as molecular blocks. Accordingly, identification of MAIR-associated proteins will reveal the role of MAIR in the apoptotic pathway more precisely as well as the structure of the molecular complexes in which MAIR is involved. We are now screening MAIR-associated proteins in a bone marrow cDNA library using a yeast two-hybrid system.


    FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB060155 [GenBank] .

Note Added in Proof—Following the acceptance of this article, we became aware of an article describing the RBCC protein family. MAIR was not investigated minutely in this study, but the coiled-coil region was essential for both homo-oligomerization and proper subcellular localization of RBCC proteins as a common character (62). The human homologue KIAA1098 was renamed TRIM35, a HUGO (Human Genome Organisation) nomenclature committee-approved gene symbol.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Third Dept. of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. Tel.: 81-42-995-1617; Fax: 81-42-996-5202; E-mail: motoyosi{at}me.ndmc.ac.jp.

1 The 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. Back

2 F. Kimura, S. Suzu, Y. Nakamura, Y. Nakata, M. Yamada, N. Kuwada, T. Matsumura, T. Yamashita, T. Ikeda, K. Sato, and K. Motoyoshi, unpublished data. Back

3 Available at www.kazusa.or.jp/huge. Back

4 Available at cgap.nci.nih.gov/Chromosomes/Mitelman. Back


    ACKNOWLEDGMENTS
 
We are indebted to Jesse Dallin for critical reading of the manuscript.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Reddy, B. A., Etkin, L. D., and Freemont, P. S. (1992) Trends Biochem. Sci. 17, 344–345[CrossRef][Medline] [Order article via Infotrieve]
  2. Borden, K. L. (1998) Biochem. Cell Biol. 76, 351–358[CrossRef][Medline] [Order article via Infotrieve]
  3. Henry, J., Mather, I., McDermott, M., and Pontarotti, P. (1998) Mol. Biol. Evol. 15, 1696–1705[Abstract/Free Full Text]
  4. Borden, K. L. (2000) J. Mol. Biol. 295, 1103–1112[CrossRef][Medline] [Order article via Infotrieve]
  5. Torok, M., and Etkin, L. D. (2001) Differentiation 67, 63–71[CrossRef][Medline] [Order article via Infotrieve]
  6. Li, X., Shou, W., Kloc, M., Reddy, B. A., and Etkin, L. D. (1994) J. Cell Biol. 124, 7–17[Abstract]
  7. El-Hodiri, H. M., Shou, W., and Etkin, L. D. (1997) Dev. Biol. 190, 1–17[CrossRef][Medline] [Order article via Infotrieve]
  8. Bellini, M., Lacroix, J. C., and Gall, J. G. (1993) EMBO J. 12, 107–114[Abstract]
  9. Friedman, J. R., Fredericks, W. J., Jensen, D. E., Speicher, D. W., Huang, X. P., Neilson, E. G., and Rauscher, F. J., 3rd (1996) Genes Dev. 10, 2067–2078[Abstract]
  10. Moosmann, P., Georgiev, O., Le Douarin, B., Bourquin, J. P., and Schaffner, W. (1996) Nucleic Acids Res. 24, 4859–4867[Abstract/Free Full Text]
  11. Chan, E. K., Hamel, J. C., Buyon, J. P., and Tan, E. M. (1991) J. Clin. Invest. 87, 68–76[Medline] [Order article via Infotrieve]
  12. Itoh, K., Itoh, Y., and Frank, M. B. (1991) J. Clin. Invest. 87, 177–186[Medline] [Order article via Infotrieve]
  13. Quaderi, 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., and Ballabio, A. (1997) Nat. Genet. 17, 285–291[Medline] [Order article via Infotrieve]
  14. Schweiger, S., Foerster, J., Lehmann, T., Suckow, V., Muller, Y. A., Walter, G., Davies, T., Porter, H., van Bokhoven, H., Lunt, P. W., Traub, P., and Ropers, H. H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2794–2799[Abstract/Free Full Text]
  15. El-Husseini, A. E., and Vincent, S. R. (1999) J. Biol. Chem. 274, 19771–19777[Abstract/Free Full Text]
  16. El-Husseini, A. E., Kwasnicka, D., Yamada, T., Hirohashi, S., and Vincent, S. R. (2000) Biochem. Biophys. Res. Commun. 267, 906–911[CrossRef][Medline] [Order article via Infotrieve]
  17. Inoue, S., Orimo, A., Hosoi, T., Kondo, S., Toyoshima, H., Kondo, T., Ikegami, A., Ouchi, Y., Orimo, H., and Muramatsu, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11117–11121[Abstract]
  18. Orimo, A., Inoue, S., Minowa, O., Tominaga, N., Tomioka, Y., Sato, M., Kuno, J., Hiroi, H., Shimizu, Y., Suzuki, M., Noda, T., and Muramatsu, M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12027–12032[Abstract/Free Full Text]
  19. Harada, H., Harada, Y., O'Brien, D. P., Rice, D. S., Naeve, C. W., and Downing, J. R. (1999) Mol. Cell. Biol. 19, 3808–3815[Abstract/Free Full Text]
  20. Matera, A. G. (1999) Trends Cell Biol. 9, 302–309[CrossRef][Medline] [Order article via Infotrieve]
  21. Zhong, S., Salomoni, P., and Pandolfi, P. P. (2000) Nat. Cell Biol. 2, E85–E90[CrossRef][Medline] [Order article via Infotrieve]
  22. Doucas, V. (2000) Biochem. Pharmacol. 60, 1197–1201[CrossRef][Medline] [Order article via Infotrieve]
  23. Rogaia, D., Grignani, F., Nicoletti, I., and Pelicci, P. G. (1995) Leukemia (Baltimore) 9, 1467–1472[Medline] [Order article via Infotrieve]
  24. Borden, K. L., CampbellDwyer, E. J., and Salvato, M. S. (1997) FEBS Lett. 418, 30–34[CrossRef][Medline] [Order article via Infotrieve]
  25. Quignon, F., De Bels, F., Koken, M., Feunteun, J., Ameisen, J. C., and de The, H. (1998) Nat. Genet. 20, 259–265[CrossRef][Medline] [Order article via Infotrieve]
  26. Wang, Z. G., Ruggero, D., Ronchetti, S., Zhong, S., Gaboli, M., Rivi, R., and Pandolfi, P. P. (1998) Nat. Genet. 20, 266–272[CrossRef][Medline] [Order article via Infotrieve]
  27. Suzu, S., Tanaka-Douzono, M., Nomaguchi, K., Yamada, M., Hayasawa, H., Kimura, F., and Motoyoshi, K. (2000) EMBO J. 19, 5114–5122[Abstract/Free Full Text]
  28. Suzu, S., Kimura, F., Ota, J., Motoyoshi, K., Itoh, T., Mishima, Y., Yamada, M., and Shimamura, S. (1997) J. Immunol. 159, 1860–1867[Abstract]
  29. Suzu, S., Hatake, K., Ota, J., Mishima, Y., Yamada, M., Shimamura, S., Kimura, F., and Motoyoshi, K. (1998) Biochem. Biophys. Res. Commun. 245, 120–126[CrossRef][Medline] [Order article via Infotrieve]
  30. Kitamura, T., Tange, T., Terasawa, T., Chiba, S., Kuwaki, T., Miyagawa, K., Piao, Y. F., Miyazono, K., Urabe, A., and Takaku, F. (1989) J. Cell. Physiol. 140, 323–334[Medline] [Order article via Infotrieve]
  31. Poot, M., Gibson, L. L., and Singer, V. L. (1997) Cytometry 27, 358–364[CrossRef][Medline] [Order article via Infotrieve]
  32. Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., and Traganos, F. (1997) Cytometry 27, 1–20[CrossRef][Medline] [Order article via Infotrieve]
  33. Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F., and Riccardi, C. (1991) J. Immunol. Methods 139, 271–279[CrossRef][Medline] [Order article via Infotrieve]
  34. Ohnishi, T., Wang, X., Ohnishi, K., Matsumoto, H., and Takahashi, A. (1996) J. Biol. Chem. 271, 14510–14513[Abstract/Free Full Text]
  35. Freemont, P. S., Hanson, I. M., and Trowsdale, J. (1991) Cell 64, 483–484[Medline] [Order article via Infotrieve]
  36. Reddy, B. A., and Etkin, L. D. (1991) Nucleic Acids Res. 19, 6330[Medline] [Order article via Infotrieve]
  37. Chu, T. W., Capossela, A., Coleman, R., Goei, V. L., Nallur, G., and Gruen, J. R. (1995) Genomics 29, 229–239[CrossRef][Medline] [Order article via Infotrieve]
  38. Takahashi, M., Inaguma, Y., Hiai, H., and Hirose, F. (1988) Mol. Cell. Biol. 8, 1853–1856[Medline] [Order article via Infotrieve]
  39. Kozak, M. (1984) Nucleic Acids Res. 12, 857–872[Abstract]
  40. Kikuno, R., Nagase, T., Ishikawa, K., Hirosawa, M., Miyajima, N., Tanaka, A., Kotani, H., Nomura, N., and Ohara, O. (1999) DNA Res. 6, 197–205[Medline] [Order article via Infotrieve]
  41. Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M. A., Lassota, P., and Traganos, F. (1992) Cytometry 13, 795–808[Medline] [Order article via Infotrieve]
  42. Thornberry, N. A., and Lazebnik, Y. (1998) Science 281, 1312–1316[Abstract/Free Full Text]
  43. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479–489[Medline] [Order article via Infotrieve]
  44. Cao, T., Borden, K. L., Freemont, P. S., and Etkin, L. D. (1997) J. Cell Sci. 110, 1563–1571[Abstract/Free Full Text]
  45. Keech, C. L., Gordon, T. P., and McCluskey, J. (1995) J. Autoimmun. 8, 699–712[CrossRef][Medline] [Order article via Infotrieve]
  46. El-Husseini, A. E., Fretier, P., and Vincent, S. R. (2001) Genomics 71, 363–367[CrossRef][Medline] [Order article via Infotrieve]
  47. Green, D. R., and Reed, J. C. (1998) Science 281, 1309–1312[Abstract/Free Full Text]
  48. Gross, A., McDonnell, J. M., and Korsmeyer, S. J. (1999) Genes Dev. 13, 1899–1911[Free Full Text]
  49. Kroemer, G., and Reed, J. C. (2000) Nat. Med. 6, 513–519[CrossRef][Medline] [Order article via Infotrieve]
  50. Fagioli, M., Alcalay, M., Tomassoni, L., Ferrucci, P. F., Mencarelli, A., Riganelli, D., Grignani, F., Pozzan, T., Nicoletti, I., and Pelicci, P. G. (1998) Oncogene 16, 2905–2913[CrossRef][Medline] [Order article via Infotrieve]
  51. Joazeiro, C. A., and Weissman, A. M. (2000) Cell 102, 549–552[Medline] [Order article via Infotrieve]
  52. Kiener, P. A., Davis, P. M., Starling, G. C., Mehlin, C., Klebanoff, S. J., Ledbetter, J. A., and Liles, W. C. (1997) J. Exp. Med. 185, 1511–1516[Abstract/Free Full Text]
  53. Perlman, H., Pagliari, L. J., Georganas, C., Mano, T., Walsh, K., and Pope, R. M. (1999) J. Exp. Med. 190, 1679–1688[Abstract/Free Full Text]
  54. Sanz, C., Benito, A., Silva, M., Albella, B., Richard, C., Segovia, J. C., Insunza, A., Bueren, J. A., and Fernandez-Luna, J. L. (1997) Blood 89, 3199–3204[Abstract/Free Full Text]
  55. Pagliari, L. J., Perlman, H., Liu, H., and Pope, R. M. (2000) Mol. Cell. Biol. 20, 8855–8865[Abstract/Free Full Text]
  56. Asada, M., Yamada, T., Ichijo, H., Delia, D., Miyazono, K., Fukumuro, K., and Mizutani, S. (1999) EMBO J. 18, 1223–1234[Abstract/Free Full Text]
  57. Sevilla, L., Aperlo, C., Dulic, V., Chambard, J. C., Boutonnet, C., Pasquier, O., Pognonec, P., and Boulukos, K. E. (1999) Mol. Cell. Biol. 19, 2624–2634[Abstract/Free Full Text]
  58. Kelley, T. W., Graham, M. M., Doseff, A. I., Pomerantz, R. W., Lau, S. M., Ostrowski, M. C., Franke, T. F., and Marsh, C. B. (1999) J. Biol. Chem. 274, 26393–26398[Abstract/Free Full Text]
  59. Ozes, O. N., Mayo, L. D., Gustin, J. A., Pfeffer, S. R., Pfeffer, L. M., and Donner, D. B. (1999) Nature 401, 82–85[CrossRef][Medline] [Order article via Infotrieve]
  60. Lin, H., Chen, C., and Chen, B. D. (2001) Biochem. J. 353, 299–306[CrossRef][Medline] [Order article via Infotrieve]
  61. Hida, A., Kawakami, A., Nakashima, T., Yamasaki, S., Sakai, H., Urayama, S., Ida, H., Nakamura, H., Migita, K., Kawabe, Y., and Eguchi, K. (2000) Immunology 99, 553–560[CrossRef][Medline] [Order article via Infotrieve]
  62. Reymond, A., Meroni, G., Fantozzi, A., Merla, G., Cairo, S., Luzi, L., Riganelli, D., Zanaria, E., Messali, S., Cainarca, S., Guffanti, A., Minucci, S., Pelicci, P. G., and Ballabio, A. (2001) EMBO J. 20, 2140–2151[Abstract/Free Full Text]