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J Biol Chem, Vol. 275, Issue 4, 2647-2653, January 28, 2000

Molecular Cloning and Characterization of Amida, a Novel Protein Which Interacts with a Neuron-specific Immediate Early Gene Product Arc, Contains Novel Nuclear Localization Signals, and Causes Cell Death in Cultured Cells^*

Yasuyuki Irie, Kanato Yamagata, Yehua Gan, Kaoru Miyamoto, Eunju Do, Che-Hui Kuo, Eiichi Taira, and Naomasa Miki^§

From the Department of Pharmacology, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan and the  Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu 183-0042, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Amida was isolated by the yeast two-hybrid system as a novel protein which associated with Arc, a non-transcriptional immediate early gene specific to the brain. Amida was confirmed to be associated with Arc in vitro and in vivo. Amida shows no homology to known proteins. Amida is ubiquitously expressed, although it is abundant in the brain. A transfection study revealed that Amida was localized in the nucleus and after 72 h the transfected cells underwent apoptosis. Furthermore, we found two nuclear localization signals and a domain needed for interacting with Arc was encompassed by two nuclear localization signals. Co-transfection experiment with Amida and Arc suggested that Amida transported Arc into the nucleus and negatively regulated Amida-induced cell death. These results indicate that Arc together with Amida may modulate cell death in the brain.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Immediate early genes are induced rapidly and transiently in response to various stimuli including neuronal activities, growth factors, and long term potentiation, and are thought to play roles in mediating stimulus-induced neuronal plasticity (1-4) as well as programmed cell death (PCD)¹ or apoptosis (5, 6). These genes can be classified into two groups, one consists of genes that encode transcription factors, such as Egr3/Pilot (7), and the other "effector" proteins that directly affect cellular function, such as tPA (8), rheb (9), cox-2 (10), and Arc (11, 12). Immediate early genes are also induced in pathologic states. Kindling in the hippocampus is a typical model of temporal lobe epilepsy, which induces many immediate early genes including Arc (18). In the case of ischemia (6), the mRNAs of immediate early genes are increased in regions of the brain known to be most susceptible to ischemia.

Recently, surprising advances have been achieved in the study of PCD or apoptosis (13). In the central nervous system, PCD has been shown to play an important role in the normal course of development and in pathologic states such as ischemia, Alzheimer's disease, Huntington's disease, and epilepsy (14-16). Neuronal plasticity, which can be thought of as a kind of differentiation, has been suggested to share a signal transduction pathway with PCD, but the details are yet to be determined (17).

Arc is thought to be an immediate early gene encoding an effector protein, which is proposed to mediate cytoskeletal changes underlying stimulus-induced neuronal plasticity (11). In order to investigate the function of Arc, we searched for proteins with which it interacts using the yeast two-hybrid system. Such interacting proteins may constitute a signal transduction pathway including Arc in neuronal plasticity or PCD. Here, we report the identification of a novel protein designated Amida (named after Amida, a Buddhist god who is believed to come over the dying and give them a comfortable death), which is associated with Arc, localized in the nucleus and induced apoptosis when overexpressed in cultured cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals-- Adult male rats (Harlan Sprague-Dawley) were purchased from Nihon-Dohbutsu Co. (Japan) and cared for in accordance with the guideline of Experimental Animal Care issued from the Prime Minister's Office of Japan.

Yeast Two-hybrid Screening-- Two-hybrid screening (19) of a Gal4 activation domain-tagged rat hippocampus subtracted library, using the cDNA sequence corresponding to Ser⁶⁷-Glu³⁹⁶ of Arc as "bait," was performed as described previously (20). Transformation of yeast strain PCY2 with bait constructs and, subsequently, with the library DNA fusion constructs, was performed as described. Transformants were grown on -Trp -Leu selective plate, before -galactosidase selection. -Galactosidase expression was tested on nylon membrane (Amersham Pharmacia Biotech, Uppsala, Sweden).

Screening and DNA Sequencing-- The subtracted library (10) was screened under high stringency condition (three time washing in 0.1 × SSC, 0.1% SDS at 60 °C for 20 min) with Amida cDNA labeled by random priming (Amersham Pharmacia Biotech, Uppsala Sweden) using [-³²P]dCTP. Both strands of the full-length cDNA of Amida were sequenced.

Northern Analysis-- Twenty micrograms of total RNA from rat hippocampus was electrophoresed and blotted on a nylon membrane. This membrane or a Multiple Northern blot membrane (Origene, Rockville, MD) was hybridized as described (21). The probe used for Northern analysis was a 700-base pair fragment from the 3' end of the Amida cDNA. The cDNA fragment was labeled by random priming (Amersham Pharmacia Biotech) using [-³²P]dCTP.

In Vitro Interaction Assay-- Using the TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI), pBluescript-Amida was translated with [³⁵S]methionine. PC12 cells grown on a 9-cm Petri dish were washed with PBS and lysed by sonication in IP buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 2 mM Na₂VO₄, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40). After centrifugation at 15,000 × g for 20 min, the supernatant was used as cell lysate. The in vitro translation reaction mixture was incubated at 4 °C with continuous rotation for 16 h with the lysate of PC12 cells which express Arc protein abundantly. Then the mixture was incubated with 10 ml of protein A-agarose suspension (Santa Cruz, Santa Cruz, CA) for another 1 h. After eight washes, immunoprecipitated proteins were separated by 10% SDS-PAGE.

In Vivo Interaction Assay-- For the in vivo interaction assay, COS-7 cells were cultured on 9-cm Petri dishes and transiently transfected with 16 µg of pCS2+c-myc-tag-Amida and the same amount of pEGFP-C1-Arc (pEGFP-C1; CLONTECH, Palo Alto, CA) using LipofectAMINE reagent (Life Technologies, Inc.). After 48 h, the cells were washed with PBS and lysed by sonication in IP buffer. After centrifugation at 15,000 × g for 20 min, the supernatant was used as cell lysate. The cell lysate was incubated with 10 µg of anti-Myc monoclonal antibody (Oncogene Science, Cambridge, MA) at 4 °C with continuous rotation for 16 h. Then the mixture was incubated with 20 µl of protein A-agarose suspension for another 1 h. After eight washes, immunoprecipitated proteins were separated by 10% SDS-PAGE. Immunoblotting was performed using anti-Arc-specific antiserum (11) at 1000-fold dilution and detected by the ECL immunodetection system (Amersham Pharmacia Biotech). An identical procedure was performed for analysis of Amida deletion mutants except that the transfected plasmids were the pEGFP-C1 derived vectors which code EGFP-Amida (wild type), EGFP-D1, EGFP-D2, or EGFP-D3 with pCS2+c-myc-tag-Arc and that the antibody used for immunoblotting was a polyclonal antibody against GFP (CLONTECH, Palo Alto, CA).

Morphological Analysis-- COS-7 or NG108-15 cells were grown on poly-D-lysine-coated chamber slides and apoptosis was assessed by the use of the DNA-staining dye DAPI. TUNEL staining was also performed using the Apoptosis Detection System (Promega, Madison, WI) except that staining was carried out with propidium iodide. Then, anti-Myc monoclonal antibody was added in 2 mg/ml dilution with 10% bovine serum albumin, 0.2% Triton X-100 in PBS. After 16 h incubation at 4 °C, cells were washed in PBS 3 times and incubated with rhodamine-conjugated goat F(ab')₂ fragment to mouse IgG (Cappel, West Chester, PA) in 1:500 dilution with the same buffer at 4 °C for 16 h. Finally, cells were washed in PBS three times, mounted with Slow Fade Antifade (Molecular Probe, Eugene, OR) and observed by fluorescent microscopy.

Quantification of Ability to Induce Apoptosis-- COS-7 cells cultured on poly-D-lysine-coated 2-well culture slides were transiently transfected with pSV40NLS-EGFP-EGFP-C1 (encoding tandem repeated EGFP fused with the nuclear localization signal of SV40 large T antigen), pCS2+c-myc-tag-Amida, pCS2+c-myc-tag-Arc, and pCS2+c-myc-tag in various combinations. The amount of pSV40NLS-EGFP-EGFP-C1 was always 1/4 of total DNA transfected using Superfect reagent (Qiagen, Germany). After 72 h, cells were fixed in 4% paraformaldehyde in PBS for 20 min, washed three times with PBS for 5 min each, and stained with DAPI. After washing three times in PBS for 10 min, cells were mounted and observed by fluorescent microscopy. Pictures of cells were analyzed by the following methods. First, EGFP-positive cells were identified and then pyknotic nuclei observed by DAPI staining of those cells were counted. Each graph represents at least three independent experiments performed in duplicate in which about 150 cells were individually examined. The ratio of pyknotic to total cells was calculated and statistically analyzed using the t test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation of Arc-associated Protein using the Yeast Two-hybrid Screening System-- Yeast two-hybrid screening was employed to investigate the function of Arc. A complementary DNA encoding Ser⁶⁷-Glu³⁹⁶ of Arc, which was used as bait, was ligated to the GAL 4-binding domain in plasmid vector pPC97 (20) for screening of a rat hippocampus cDNA library. This region contains nearly the full-length amino acid sequence of Arc including the -spectrin homologous domain. Screening of approximately 200,000 colonies resulted in 15 positive clones. One clone (Amida) had an insert of 700 base pairs with no homology to known genes.

The cDNA insert of the Amida clone was used to screen a rat hippocampus cDNA libraries to obtain a full-length cDNA sequence. Screening over 300,000 clones resulted in three independent positive clones. One of them contained the start consensus sequence (22) with an upstream stop codon in an identical frame with the Amida-Gal4 activation domain fusion protein. This 900-base pair cDNA contains the full-length coding region of Amida as shown in Fig. 1.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   cDNA and amino acid sequences of Amida. The numbers to the right refer to the last amino acid in each line. The predicted amino acid sequence is displayed under the corresponding nucleotide sequence. Underlined regions are novel nuclear localization signals.

Amida has no homology with known proteins, but showed significant homology to a clone (AA895828) from mouse expressed sequence tag (EST) library. The protein of highest amino acid homology found in a FASTA data base search was Homo sapiens hepatitis  antigen interacting protein A, which has 28.8% identity in a 177-amino acid overlap.

mRNA Expression of Amida-- The expression of Amida mRNA was investigated by Northern blot. Analysis with 2 mg of poly(A) RNA revealed that expression of Amida is ubiquitous, but abundant in the brain (Fig. 2A). Transcripts of approximately 1.2 and 1.0 kilobases were found in Northern blot. Maximal electroconvulsive seizure with pretreatment with cycloheximide, which superinduce immediate early genes in the hippocampus, did not increase the mRNA (Fig. 2B). Amida mRNA was mainly detected in the hippocampus and dentate gyrus by in situ hybridization (data not shown).

View larger version (54K): [in this window] [in a new window]   Fig. 2.   mRNA expression of Amida. A, Northern analysis of 2 µg of poly(A) RNA per lane prepared from the brain (lane 1), heart (lane 2), kidney (lane 3), spleen (lane 4), thymus (lane 5), or liver (lane 6). B, Northern analysis of 20 µg of total RNA per lane prepared from the control hippocampus (lane 1) and the hippocampus (lane 2) from the maximal electroconvulsive seizure rat stimulated at 30 min after pretreatment of 20 mg/kg cycloheximide.

Amida Protein Is Associated to Arc in Vitro-- To confirm that Amida is associated to Arc, a reconstruction experiment in vitro was performed. Amida protein was translated in vitro and [³⁵S]methionine-labeled using the reticulocyte lysate system. The predicted molecular mass of Amida protein is about 30 kDa, but the in vitro translated product migrated as 46 kDa in SDS-PAGE. This discrepancy may be due to the acidity. The mixture was incubated with the lysate of PC12 cells which abundantly express Arc protein. Then, immunoprecipitation with anti-Arc-specific antiserum was performed. Labeled Amida protein was specifically co-precipitated, while the in vitro translated products of other clones were not (Fig. 3A).

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Amida is associated with Arc in vitro and in vivo. A, in vitro association of Amida and Arc. In vitro translated and 35S-labeled Amida was incubated with lysate from PC12 cells which express Arc protein abundantly. Then immunoprecipitation using anti-Arc antibody and SDS-PAGE were performed. Lane 1 represents Amida. Lanes 2 and 3 show the other two-hybrid positive clones which could not bind Arc protein under these conditions. B, in vivo association of Amida and Arc. COS-7 cells were co-transfected with pEGFP-C1-Arc and pCS2+c-myc-tag-Amida. After 48 h transfection, the lysate was immunoblotted with anti-Arc antibody (lane 1). Lane 2, the lysate was immumoprecipitated with anti-Myc monoclonal antibody and then EGFP-Arc was detected with anti-Arc antibody. Lane 3, pCS2+c-myc and pEGFP-C1-Arc were co-transfected as a control. C, no association of Amida with EGFP. Lane 1, Myc-tag-Amida (Amida-MT) and EGFP were co-expressed in COS-7 cells and the lysate was immunoblotted with anti-Myc antibody (lane 1) or with anti-EGFP antibody (lanes 2-4). The lysate (lane 2) was also immunoprecipitated with anti-Myc antibody, and the resultant supernatant (lane 3) and precipitate (lane 4) were immunoblotted with anti-EGFP antibody.

Amida Protein Is Associated to Arc in Vivo-- For the in vivo interaction assay, COS-7 cells were transiently transfected with both Myc-tagged Amida and EGFP-Arc fusion proteins. After 48 h, cells were lysed and immunoprecipitated with anti-Myc monoclonal antibody. The immunoprecipitated proteins were analyzed by SDS-PAGE. Immunoblotting was performed using anti-EGFP-specific antiserum (Fig. 3B). EGFP-Arc fusion protein of 80 kDa was specifically precipitated with Myc-tagged Amida protein, while no signal was seen when control Myc-tag peptide was co-expressed.

Furthermore, we tried to examine whether Amida interacts with the other proteins nonspecifically or not. When Myc-tagged (MT) Amida and EGFP were co-transfected in COS-7 cells and then the cell lysate was immunoprecipitated with anti-Myc antibody, Amida did not associate with EGFP (Fig. 3C).

Amida Protein Is Localized in the Nucleus and Is Involved in Apoptosis-- To investigate the function of Amida in cells, a mammalian expression vector pEGFP-C1-Amida which encodes an EGFP-Amida fusion protein was transiently transfected into COS-7 cells. An intense fluorescent signal was exclusively detected inside the nucleus (Fig. 4, B and C), while the signal was seen diffusely throughout the cell which expresses only EGFP (Fig. 4A). Three days after transfection, most of the COS-7 cells expressing the EGFP-Amida fusion protein showed a small and round morphology which is characteristic of cells undergoing apoptosis. Fluorescent microscopic observation by DAPI staining revealed that only EGFP-Amida positive cells exhibited nuclear condensation and fragmentation (Fig. 4, D and E). Similar changes were seen in other cell lines such as NG108-15 (data not shown).

View larger version (90K): [in this window] [in a new window]   Fig. 4.   Amida protein localizes in the nucleus and induces apoptosis. A mammalian expression vector encoding EGFP (A) or EGFP-Amida fusion protein (B) was transiently transfected into COS-7 cells. DAPI staining of the same field as B is shown to allow visualization of the nucleus (C). Arrows indicate the same positions in B and C. The morphology of COS-7 cells 72 h after transfection (D) and the same cells stained with DAPI (E). Condensation and fragmentation of the nuclei of Amida-transfected cells were seen. NG108-15 cells were also transfected with the same expression vector containing the Myc-tagged Amida (F-H). F shows immunocytochemistry using anti-Myc monoclonal antibody with rhodamine. TUNEL staining using fluorescein isothiocyanate is shown in G. Superimpose of fluorescein isothiocyanate and rhodamine staining indicates that the TUNEL positive cell is expressing Amida (H). Scale bar, 10 µm.

To confirm that the Amida expressing cells were dying by apoptosis, a double staining study using the TUNEL reaction and immunocytochemistry against Myc-tag was performed. Most of the NG108-15 cells expressing Myc-tagged Amida were TUNEL positive (Fig. 4, F, G, and H), while few cells transfected with vector alone showed a positive TUNEL response.

Amida Contains Two Novel Nuclear Localization Signals-- It was thought that Amida has two putative nuclear localization signals which abundantly contain positively charged residues such as lysine or arginine. Deletion mutants D1, D2, and D3 as shown in Fig. 5 were designed to evaluate these putative nuclear localization signals (NLS1 and NLS2) and transfected into COS-7 cells.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Constructs of Amida deletion mutants. Deletion mutants D1, D2, and D3 were designed to evaluate the putative nuclear localization signals NLS1 and NLS2. The abilities of each mutant for the association with Arc, nuclear localization and cell death are summarized. +++, high; ++, medium, +, low; ±, slight; , no activity.

EGFP-D1 and EGFP-D2 showed nuclear localization (Fig. 6, B and C) as was the case of EGFP-Amida (wild type, Fig. 6A). But EGFP-D3, which has neither of the putative NLSs, was distributed throughout the cytoplasm and a showed similar pattern to EGFP alone (Fig. 6D). As EGFP and EGFP-D3 proteins are both small (26 and 37 kDa, respectively), these proteins could be transported diffusely into the nucleus. To confirm that D3 does not localize in nucleus, an expression vector which encodes a large double-EGFP (tandem fused two EGFPs)-D3 fusion protein of 60 kDa was constructed. Double-EGFP-D3 was exclusively distributed in the cytoplasm (Fig. 7E) as was double-EGFP alone (Fig. 7A).

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Morphological analyses to determine the effects of the nuclear localization signals. Intracellular distribution of EGFP fusion proteins of each deletion construct was examined. Note the distribution of wild type (A), D1 (B), and D2 (C) fusion proteins in the nuclei and EGFP-D3 (D) fusion protein in the cytoplasm. NLS1 or NLS2 was fused to double-EGFP (tandemly fused two EGFPs) and transfected into COS-7 cells. Note that double-EGFP-NLS1 (E and F) and double-EGFP-NLS2 (G and H) are localized in the nuclei. F and H show the DAPI staining of E and G, respectively. Scale bar, 10 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 7.   Analysis of nuclear localization and cell death induction by D3. Double-EGFP (two tandemly fused EGFPs) (A) and NLS of SV40 large-T antigen tagged with double-EGFP (C) were transfected into COS-7 cells. Transfections of a double-EGFP-D3 fusion protein of 60 kDa (E) and double EGFP-SV40NLS-D3 (G). B, D, F, and H, DAPI staining as the same fields of A, C, E, and G, respectively. Arrows in D show healthy morphology of the nuclei and in H induced cell death. Scale bar, 10 µm.

Furthermore, we investigated whether NLS1 and NLS2 are really novel nuclear localization signals. cDNA sequences which encode NLS1 or NLS2 were ligated to double-EGFP and transfected into COS-7 cells. Double-EGFP-NLS1 (Fig. 6, E and F) and double-EGFP-NLS2 (Fig. 6, G and H) localized in the nucleus as did a fusion protein which consists of a NLS from SV40 large T antigen and double-EGFP (Fig. 7, C and D).

Accumulation of the D3 Region in the Nucleus Causes Cell Death-- We investigated the ability of each deletion mutant to promote cell death. COS-7 cells expressing EGFP-D1 or EGFP-D2 fusion proteins showed apoptotic morphology as assessed by DAPI staining 72 h after transfection (data not shown). But, cell death was not observed, when double-EGFP-D3 was overexpressed (Fig. 7, E and F). A fusion protein consisting of the NLS from SV40 large T antigen and double-EGFP-D3 restored nuclear localization, and exhibited death-promoting ability (Fig. 7, G and H). These results indicate that the D3 region is sufficient but nuclear localization of this region is necessary for promotion of cell death as summarized in Fig. 5.

Analysis of Interaction between Arc and the Deletion Mutants-- To investigate the domain of Amida which interacts with Arc, each deletion mutant was co-transfected with Arc and co-precipitated depicted in Fig. 8. EGFP-Amida (wild type), EGFP-D1, EGFP-D2, and EGFP-D3 were transiently expressed in COS-7 cells with Myc-tagged Arc. 48 h after transfection, the cells were lysed and immunoprecipitation using anti-Myc monoclonal antibody was performed. The immunoprecipitate was immunoblotted with anti-GFP antibody (Fig. 8, IP: D1, D2, D3). EGFP-Amida (wild type), EGFP-D1, and EGFP-D2 fusion proteins could interact with Myc peptide-tagged Arc protein strongly, while EGFP-D3 interacted with Arc only weakly. A control experiment was carried out using a vector which encodes only EGFP peptide, which had no ability to interact with Myc-tagged Arc (Data not shown).

View larger version (93K): [in this window] [in a new window]   Fig. 8.   Co-precipitation of the proteins from Amida deletion mutants and Arc. Immunoblotting with anti-EGFP antibody was carried out in the lysates of COS-7 cells which express the Amida deletion mutant-EGFP fusion proteins (lysate: WT, D1, D2, and D3). Each deletion mutant-EGFP fusion protein was co-expressed with Myc-tagged Arc protein, and the lysates were immunoprecipitated with anti-Myc peptide antibody and detected with anti-EGFP antibody (IP: WT, D1, D2, and D3). Note that only a faint band was visible in D3 mutant expressing cell lysate.

Amida Influences Subcellular Localization of Arc-- We investigated the co-operative function of Arc and Amida. When EGFP-Arc was expressed alone in COS-7 cells, it showed a punctate and perinuclear distribution in the cytoplasm (Fig. 9A). In contrast, EGFP-Arc was distributed in the nucleus when it was co-expressed with Myc-tagged Amida (Fig. 9C).

View larger version (53K): [in this window] [in a new window]   Fig. 9.   Amida changes the subcellular distribution of Arc. COS-7 cells were transfected with pEGFP-C1-Arc and EGFP fluorescence was detected (A). COS-7 cells co-transfected with pEGFP-C1-Arc and pCS2+c-myc-tag-Amida (C). B and D, DAPI staining. Scale bar, 10 µm.

Arc Inhibited Cell Death Caused by Amida-- To investigate the function of Arc on Amida-induced cell death, COS-7 cells were transiently transfected with pSV40NLS-EGFP-EGFP-C1 (encoding tandem repeated EGFP fused with the nuclear localization signal of SV40 large T antigen) in various combinations with pCS2+c-myc-tag(MT)-Amida, pCS2 + c-myc-tag-Arc, pCS2+c-myc-tag. A constant amount of pSV40NLS-EGFP-EGFP-C1 was used to be always 1/4 of the total DNA transfected. After 72 h, cells were fixed and stained with DAPI. At first, EGFP positive cells were checked and then the number of those cells that had pyknotic nuclei after staining with DAPI were counted. The proportion of cells with pyknotic nuclei from the EGFP-positive cells were calculated and analyzed with the statistical t test (Fig. 10). Overexpression of Amida significantly increased the proportion of pyknotic nuclei, while Arc had no influence on cell death compared with vector alone (Fig. 9, A and B). Co-transfection of Arc and Amida (99:1, w/w of transfected plasmids), which represents close to physiological proportions of Arc and Amida in the dentate gyrus, revealed that Arc inhibited cell death induced by Amida when compared with the result of co-transfection of vector and Amida (99:1, w/w) (Fig. 9). These results indicate that Arc has no influence on cell death alone, but inhibits cell death induced by overexpression of Amida.

View larger version (19K): [in this window] [in a new window]   Fig. 10.   Suppression by Arc of cell death caused by Amida. COS-7 cells were transiently transfected with pSV40NLS-EGFP-EGFP-C1 (encoding tandemly repeated EGFP fused with nuclear localization signal of SV40 large T antigen) in various combinations with pCS2+c-myc-tag(MT)-Amida, pCS2+c-myc-tag-Arc. After 72 h, cells were fixed and stained with DAPI. EGFP-positive cells were identified and then cells showing pyknotic nuclei after staining with DAPI were counted. The proportion of pyknotic nuclei to EGFP positive cells is represented. pCS2+MT, first column; pCS2+MT-Amida, second column; pCS2+MT-Arc and pCS2+MT-Amida, third column; pCS2+MT and pCS2+MT-Amida fourth column. *, p < 0.01.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have isolated a novel protein designated Amida which interacts with Arc by yeast two-hybrid system, and confirmed that Amida has the ability to associate with Arc in an in vitro reconstruction assay and in transfected cells. Northern blot analysis revealed that Amida has two major transcripts of 1.0 and 1.2 kilobases. As reverse transcriptase-polymerase chain reaction using primers from the 5'- and 3'-regions of the coding region produced a single band, the two transcripts may differ in the non-coding regions.

Many peripheral tissues also contain Amida mRNA less than level of that in the brain, although Arc is expressed at low level. In the thymus a band of 4 kilobases was also detected in Northern blot analysis, but we have not analyzed it yet. Amida expression was also detected in PC12, C6 glioma, and NG108-15 cell lines. Compared with the dramatic induction of Arc after maximal electroconvulsive seizure (11), the increase of Amida expression in the dentate gyrus induced by maximal electroconvulsive seizure was not observed even after cycloheximide administration. These data indicate that Amida is expressed constitutively in the brain.

To assess the function of Amida, EGFP-Amida fusion protein was expressed in COS-7 cells and NG108-15 cells. We found that Amida protein is transported into the nucleus. Three days after transfection, most of COS-7 cells expressing EGFP-Amida fusion protein began to show a small and round morphology which is characteristic of apoptosis. We confirmed that this phenomenon is apoptosis by staining the cells with DAPI, nucleic acid fluorescent dye. A double staining study with the TUNEL reaction and immunocytochemistry against Myc-tag also confirmed that the mode of cell death in Amida expressing cells appears apoptosis. In a preliminary data, inhibition of caspase 1 did not attenuate cell death induced by overexpression of Amida.

We demonstrated that both NLS1 and NLS2 are novel NLSs (Fig. 6). These NLSs are characteristic in that they are rich in arginine residues instead of lysine residues. Since double EGFP-D3 fusion protein did not enter the nucleus, it did not cause apoptosis. However, double EGFP-D3 fusion protein conjugated with a foreign NLS (SV40 large T antigen-derived NLS) induced apoptosis. This phenomenon indicates that the location of D3 in nucleus is responsible for apoptosis. The similar finding has been reported that nuclear localization of overexpressed huntingtin mutant is necessary for apoptosis in primary cultured striatal neurons (23). Further experiments are necessary to determine the precise region of Amida responsible for the mechanisms of apoptosis.

Arc-EGFP expressed alone in COS-7 cells showed punctate and perinuclear distribution (Fig. 9). This pattern of distribution has been reported for the proteins which associate with cytoplasmic vesicles such as endocytotic compartments, exocytotic compartments (24, 25), or mitochondria (26). The cytosolic punctate pattern of Arc-EGFP distribution was, however, changed to a homogeneous nuclear one when it was co-expressed with Amida. These data indicate that Amida forms a complex with Arc and transports it into the nucleus. We also found that Arc suppressed the apoptosis triggered by overexpression of Amida, when they were co-transfected (Fig. 10). This result suggests a novel function of Arc in negatively modulating cell death, although further investigations using more physiological conditions are required. Taken together, our data suggest that Arc may interfere with programmed cell death by interacting with Amida and modulate the apoptotic function of Amida.

	ACKNOWLEDGEMENTS

We thank Dr. P. F. Worley (Dept. of Neuroscience, Neurology, Psychiatry and Behavioral Science, Johns Hopkins University, School of Medicine, Baltimore, MD) for the kind gift of rat hippocampal libraries, the yeast shuttle vectors for the two-hybrid system, and yeast strain PCY. Dr. D. Turner (Dept. of Genetics, Fred Hutchinson Cancer Research Center, Seattle, WA) for the kind gift of pCS2+MT. Y. Yoneda (Dept. of Anatomy and Cell Biology, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka, Japan) for the suggestion of the study in nuclear localization experiment.

	FOOTNOTES

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

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AB09495.

^§ To whom correspondence should be addressed: Dept. of Pharmacology, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3520; Fax: 81-6-6879-3529; E-mail: nmiki@pharma1.med.osaka-u.ac.jp.

	ABBREVIATIONS

The abbreviations used are: PCD, programmed cell death; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; DAPI, 4,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; NLS, nuclear localization signal.

	REFERENCES

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