APCL, a Central Nervous System-Specific Homologue Of

[CANCER RESEARCH 60, 101–105, January 1, 2000] APCL, a Central Nervous System-specific Homologue of Adenomatous Polyposis Coli Tumor Suppressor, Binds to p53-binding Protein 2 and Translocates it to the Perinucleus1

Hidewaki Nakagawa, Kumiko Koyama, Yoji Murata, Monden Morito, Tetsu Akiyama, and Yusuke Nakamura2 Division of Clinical Genetics, Biomedical Research Center [H. N., K. K., Y. N.] and the Second Department of Surgery [H. N., M. M.], Osaka University Medical School, Osaka, Japan; Department of Oncogene Research, Institute of Microbial Disease, Osaka University, Osaka, Japan [Y. M., T. A.]; Department of Human Genome Analysis, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research [K. K.] and Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan [K. K., Y. N.]

ABSTRACT molecule can be presumed to interact with different proteins; if so, APCL would possess at least some functions distinct from those of APCL, a central nervous system-specific sequence homologue of the APC. adenomatous polyposis coli tumor suppressor, can regulate the cytoplas- To clarify those functions, we used a yeast two-hybrid system to mic level of ␤-catenin as the adenomatous polyposis coli tumor suppressor does, but its overall biological function remains unclear. Using a yeast search for proteins that might associate with the COOH-terminus of two-hybrid system, we attempted to isolate proteins that might associate APCL. We found that this portion of the molecule indeed bound to a with the unique COOH-terminus of APCL. Among 166 cDNA clones p53-binding protein, 53BP2. In vivo studies confirmed that this asso- isolated from a human fetal-brain cDNA library as candidates for inter- ciation regulated the cytoplasmic localization of 53BP2 by translo- action with APCL, 32 encoded parts of p53-binding protein 2 (53BP2), a cating it to the perinuclear region. molecule that interacts with p53 and Bcl2. An in vitro binding assay indicated that the Src-homology-3 domain and the ankyrin-repeat domain of 53BP2 were both required for binding to the COOH-terminus of APCL. MATERIALS AND METHODS Confocal microscopy showed that APCL and 53BP2 proteins were local- ized together in the perinuclei of normal mammalian cells, but this was not Yeast Two-Hybrid Screening. To generate a fusion protein containing a the case in cells that expressed truncated APCL and 53BP2 proteins. GAL4-binding domain, we excised a 2.6-kb NotI fragment (residues 1520– These findings suggested that binding of the COOH-terminus of APCL to 2303) of APCL cDNA, filled in the ends, and subcloned the fragment into a 53BP2 regulates the cytoplasmic location of 53BP2. Because 53BP2 also SmaI site of pAS2-1 vector (Clontech). After confirmation of the DNA interacts with p53 and Bcl2 and regulates p53 function, our results suggest sequences in the plasmid, we used it as “bait” to screen a human fetal-brain that APCL might be involved in the p53/Bcl2-linked pathway of cell-cycle cDNA library using the pACT2 vector (Clontech). Briefly, the plasmids were progression and cell death. transformed into Y190 yeast cells, and positive clones were selected on Trp(Ϫ), Leu(Ϫ), His(Ϫ) plates containing 25 mM 3-aminotriazole, and assayed for ␤-galactosidase activity by colony-lift filter assay. The positive clones were INTRODUCTION cotransformed with either the bait vector or the original pAS2-1 vector into APCL, a protein encoded on chromosome 19p13.3 that bears sig- yeast to confirm the interaction. ␤ nificant sequence homology to the APC1 tumor suppressor (1, 2) is Quantitative -Galactosidase Assays. A fusion plasmid containing the GAL4-binding domain and residues 458-1008 of 53BP2 was isolated from the expressed strongly and specifically in the central nervous system (3). yeast two-hybrid screening as the prey of the smallest fragment of 53BP2. The expression pattern indicates a specific role in proliferation and Various fusion plasmids containing parts of 53BP2, i.e., 53BP2-A (residues differentiation in the central nervous system, but the overall biological 458–796), 53BP2-B (residues 756–921), 53BP2-C (residues 919-1008), or functions of APCL remain unclear. The heptad-repeat domain found 53BP2-D (residues 756-1008), were subcloned into pACT2 vector as PCR- in the APC protein is well conserved in APCL (45% of the amino amplified fragments. A mutant 53BP2 plasmid, P53BP2-DM (W974K) was acids are identical); therefore APCL likely also forms homo- or generated from pACT2-53BP2-D (residues 756-1008) by PCR-based site- heterodimers (4). Moreover, because the Armadillo domain is also directed mutagenesis. Misincorporations of nucleotides during PCR amplifi- well conserved (76% identical), both proteins may interact with the cation were checked by DNA sequencing. The bait and each 53BP2-fused same or similar molecular entities (5). The central portion of APCL plasmid were transformed into Y190 yeast cells, and five separate colonies Ϫ Ϫ Ϫ consists of five copies of a 20-amino acid motif (FXVEXTPXCFS- were selected from the positive clones growing on Trp( ), Leu( ), His( ) plates containing 25 mM 3-aminotriazole. To confirm the interaction, positive RXSSLSSLS; Refs. 6, 7), and we showed earlier that through this ␤ clones were cotransformed into yeast with either the bait vector or the original domain, APCL can interact with -catenin and deplete its intracellular pAS2-1 vector. We performed liquid-culture assays for ␤-galactosidase in concentration (3). Because the COOH-terminus of APC protein binds triplicate, using ONPG as a substrate according to the manufacturer’s instruc- to EB1 (8), microtubules (9), and hDLG (10), it appears that APC is tions (Clontech). involved in cell-cycle progression and/or growth control. However, Production of GST Fusion Proteins. cDNA fragments that contained because the COOH-terminus of the APCL protein bears little simi- parts of the coding sequence of 53BP2 were excised from the various pACT2- larity to that of APC (only 13% identical amino acids), that part of the 53BP2 plasmids by digestion with EcoRI and XhoI. Each fragment was ligated in-frame into pGEX5-2 (Pharmacia). Overnight cultures of XL1Blue MRFЈ, transformed with the plasmids encoding GST fusion proteins, were diluted and Received 5/3/99; accepted 10/28/99. ␤ The costs of publication of this article were defrayed in part by the payment of page cultured with 0.1 mM isopropyl-1-thio- -D-galactopyranoside for6hat30°C. charges. This article must therefore be hereby marked advertisement in accordance with Cells were lysed by sonication in PBS containing 1% Triton X-100, and the 18 U.S.C. Section 1734 solely to indicate this fact. lysates were clarified by centrifugation. Each GST-53BP2 fusion protein was 1 This work was supported in part by a grant-in-aid from the Ministry of Education, purified on glutathione-agarose beads in PBS containing 1% Triton X-100. Science, Sports, and Culture of Japan, and by “Research for the Future” Program Grant 35 96L00102 of The Japan Society for the Promotion of Science. In Vitro Binding Assay. We generated a [ S]Met-labeled COOH-termi- 2 To whom requests for reprints should be addressed, at Laboratory of Molecular nus peptide of APCL from a template containing nucleotides 4120–6912 Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, (stop) of the APCL cDNA sequence, using TNT rabbit reticulocyte lysate 4-6-1, Shirokanedai, Minato, Tokyo 108-8639, Japan. Phone: 81-3-5449-5372; Fax: (Promega) according to the manufacturer’s instructions. The immobilized GST 81-3-5449-5433; E-mail: [email protected]. 3 The abbreviations used are: APC, adenomatous polyposis coli; GST, glutathione-S- fusion proteins were mixed with the in vitro-translated APCL proteins in 10 transferase. mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA containing 0.1% Triton 101

Fig. 1. Schematic representation of the APCL protein structure compared with APC. The COOH-terminal region indicated in the schema (residues 1520–2303 of APCL) served as bait for yeast two-hybrid screening. aa, amino acid

X-100 and phenylmethylsulfonyl fluoride, incubated for3hat4°C, then (Life Technologies, Inc.). Cells were fixed in 4% formaldehyde 24 h after washed extensively four times with 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, transfection and dehydrated with 100% cold methanol for 10 min. After 1mM EDTA. The beads were boiled in sample buffer and separated on 8% blocking in PBS containing 10% fetal bovine serum for 3 h, cells were treated SDS PAGE gels. After electrophoresis, the gels were dried and exposed to with a mixture of mouse anti-Myc antibody (1:100; Santa Cruz Biochemistry, X-ray film overnight at Ϫ80°C. Inc.) and rabbit anti-FLAG antibody (1:100; ZYMED Laboratories, Inc.) in Construction of a Mammalian Expression Vector for Epitope-tagged PBS for 1 h. The coverslips were washed in PBS three times for 10 min and APCL and 53BP2. Full-length APCL was inserted into pcDNA3.1(ϩ)Myc- then incubated in PBS for 1 h with a FITC-conjugated sheep antimouse HisA vector (Invitrogen), and full-length 53BP2 was inserted to pFLAG-CMV antibody (1:100; Cappel) and a rhodamine-conjugated goat antirabbit antibody vector (Kodak) in frame. Truncated APCL was generated using a 5.2-kb CpoI (1:100; Cappel). After the cells were washed four times in PBS, immunoflu- fragment containing residues 1–1675 of APCL, which lacked the 3Ј region of orescence was registered by means of a Zeiss LSM 410 confocal microscope. interaction with 53BP2. This plasmid was subcloned into the EcoRV site of Western Blotting. Cos7 cells were cultured and cotransfected with 1 ␮gof pcDNA3.1(ϩ)Myc-HisB vector (Invitrogen) after blunting. pCMV-FLAG-53BP2 and 1 ␮g of either pCDNA3.1-APCL-Myc or Transfection to Mammalian Cells and Immunofluorescent Staining. pCDNA3.1-truncated-APCL-Myc, using Lipofectamine plus (Life Technolo- Cos7 and HeLa cells were cultured on coverslips and cotransfected by lipo- gies, Inc.). At 24 h after the transfection, the cells were harvested in lysis buffer fection with 1 ␮g of pCMV-FLAG-53BP2 and 1 ␮g of either pCDNA3.1- (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, and 2% SDS). The APCL-Myc or pCDNA3.1-truncated-APCL-Myc, using Lipofectamine plus lysed cell were sonicated and separated by 5% or 7% SDS-PAGE. Western

Fig. 2. Interaction of APCL and 53BP2 in the yeast two-hybrid system. Quantitative ␤-galactosidase assays using several fusion plasmids of 53BP2 demonstrated that only 53BP2-D(756-1008), containing four ankyrin repeats and the SH3 domain, could specifically interact with the COOH-terminal region of APCL protein. In the mutant 53BP2-DM(756- 1008), the most conserved residue of the SH3 domain was changed from W to K (W974K). 102

Fig. 3. In vitro binding of APCL with 53BP2. In a GST pull-down assay, the in vitro-translated, [35S]-labeled COOH-terminus of APCL, coupled with the constructs indicated above each lane, was tested for binding to glutathione-agarose beads. A, GST-fusion proteins of 53BP2—GST-53BP2-D(756-1008), GST-53BP2-DM(756-1008 W974K), GST-53BP2-B(756- 921), and GST-53BP2-C(919-1008)—were purified by glutathione-Sepharose, separated by SDS-PAGE, and stained with Coomassie blue. B, the COOH-terminal region of APCL protein specifically interacted with GST-53BP2-D(756-1008), but not GST alone, GST-53BP2-DM(756-1008 W974K), GST-53BP2-B(756-921),orGST-53BP2-C(919-1008). blotting with mouse anti-Myc antibody (Santa Cruz Biochemistry) and rabbit two-hybrid system was a fusion construct of the GAL4-binding do- anti-FLAG antibody (ZYMED Laboratories) were visualized using the ECL main and a NotI fragment (nucleotides 4561–7132) of APCL cDNA system (Amersham). (Fig. 1). By screening 3.0 ϫ 107 yeast transformants, we isolated and sequenced 166 cDNA clones that interacted with APCL protein. RESULTS Among them, 32 contained partial cDNA sequences of 53BP2; the clone containing the smallest fragment of 53BP2 included cDNA Interaction of APCL and 53BP2. To isolate human proteins sequences corresponding to codons 458-1008. This region of 53BP2, capable of interacting with the COOH-terminus of the APCL protein, which contains four adjacent ankyrin repeats and a Src-homology-3 we screened a human fetal-brain cDNA library constructed with a (SH3) domain, is known to interact with p53 and Bcl2 (11, 12). vector containing a GAL4-activator domain. The bait for our yeast To determine which region(s) of 53BP2 are sufficient for binding to APCL, we constructed fusion plasmids containing a GAL4-activating domain and various parts of 53BP2. The 53BP2-B construct contained codons 756–921 (corresponding to four ankyrin repeats); 53BP2-C (codons 919-1008) contained the SH3 domain; and 53BP2-D (codons 756-1008) contained both the ankyrin repeats and the SH3 domain. To abolish the function of the SH3 domain, we constructed a mutant plasmid, 53BP2-DM (codons 756-1008; W974K) in which a trypto- phan residue (W) at codon 974, the most conserved residue within the SH3 domain in 53BP2, was replaced with lysine (K). Quantitative ␤-galactosidase assays using these fusion plasmids demonstrated that only 53BP2-D could interact with the COOH-terminal region of the APCL protein (Fig. 2), indicating that the ankyrin-repeat region and the SH3 domain were both necessary for 53BP2 to interact with APCL. In Vitro Binding Assay. To confirm the results of the ␤-galactosidase assays, several 53BP2 constructs were also tested in a GST pull-down assay. The in vitro-translated [35S]-labeled COOH-termi- Fig. 4. Western blotting analysis. Cells transfected with pCMV-FLAG-53BP2, nus of APCL was tested for binding to glutathione-agarose beads pCDNA3.1-APCL-Myc or pCDNA3.1-truncated-APCL-Myc expressed FLAG-53BP2 (53BP2) of 150 kDa (A), APCL-Myc of 260 kDa, or truncated APCL-Myc (t-APCL)of coupled with GST alone, GST-53BP2-D, GST-53BP2-DM (W974K), 210 kDa (B). GST-53BP2-B, or GST-53BP2-C (Fig. 3A). As in the yeast two- 103

Fig. 5. Regulation of the subcellular location of 53BP2 by APCL. A–C, Cos7 cells were transiently transfected with full-length APCL alone (A), truncated APCL alone (B), or 53BP2 alone (C). Full-length APCL and truncated APCL were detected with mouse anti-Myc antibody stained with FITC-conjugated sheep antimouse antibody (green). 53BP2 was detected with rabbit anti-FLAG antibody stained with rhodamine-conjugated goat antirabbit antibody (red). Subcellular locations were visualized by immunofluorescence and confocal microscopy. D, colocalization of exogenous full-length APCL (green) and 53BP2 (red), appearing as yellow stain in the perinuclear regions of Cos7 cells. E, truncated APCL (t-APCL; green) was not detectable in the perinuclear pattern, nor was 53BP2 (red). hybrid system, the COOH-terminal region of APCL associated spe- lacking the region that interacts with 53BP2. Western analysis of cifically with GST-53BP2, but not with GST alone, GST-53BP2-DM, transfected cells revealed that FLAG-53BP2 was expressed as an GST-53BP2-B, or GST-53BP2-C (Fig. 3B). ϳ150-kDa protein, APCL-Myc as a 260-kDa protein, and truncated Colocalization of Epitope-tagged APCL and 53BP2 in Mam- APCL-Myc as a 210-kDa protein (Fig. 4). malian Cells. To examine the subcellular locations of APCL and Confocal microscopy revealed that full-length APCL was located 53BP2, we cotransfected the two proteins into Cos7 or HeLa cells by mainly in the perinuclear region (Fig. 5A), but truncated APCL means of expression plasmids encoding FLAG-tagged 53BP2 and displayed a punctate staining pattern in the cytoplasm, which appar- either Myc-tagged full-length APCL or Myc-tagged truncated-APCL ently was different from that of full-length APCL (Fig. 5B). 53BP2 104

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2000 American Association for Cancer Research. APCL BINDS TO 53BP2 AND TRANSLATES IT TO PERINUCLEUS protein appeared in a punctate vesicular pattern in the cells transfected thereby inducing cell-cycle arrest at the G2-M stage (11). These with 53BP2 alone (Fig. 5C), but in cells expressing both proteins, observations and the data presented here suggest that APCL might full-length APCL and 53BP2 were colocalized in the perinuclear modulate the 53BP2-related signaling pathway by regulating the region (Fig. 5D). However, in cells expressing truncated APCL and cytoplasmic localization of 53BP2, and also that APCL might be 53BP2, this was not the case; truncated 53BP2 displayed the same involved in mechanisms of cell-cycle progression and cell death staining pattern as that in cells transfected with 53BP2 alone (Fig. 5E). linked to p53 and Bcl2. The clarification of this novel function of APCL may provide us new insights into the multiple functions of DISCUSSION members of the APC family. A brain-specific 2303-amino acid protein, APCL (3), possesses a ACKNOWLEDGMENTS high degree of sequence homology to APC in its NH2-terminal and middle regions. However, its COOH-terminal region is different from We gratefully acknowledge the technical assistance of Misae Nishijima and that of APC, and because APCL is expressed specifically in brain, it Eiji Ouki. is likely to play an important role in the central nervous system. To further elucidate the function(s) of APCL, we searched for proteins REFERENCES that might interact with its COOH-terminal region, using that portion of APCL as bait in a yeast two-hybrid screening system. This proce- 1. Kinzler, K. W., Nilbert, M. C., Su, L.-K., Vogelstein, B., Bryan, T. M., Levy, D. B., Smith, K. J., Preisinger, A. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2000 American Association for Cancer Research. APCL, a Central Nervous System-specific Homologue of Adenomatous Polyposis Coli Tumor Suppressor, Binds to p53-binding Protein 2 and Translocates it to the Perinucleus

Hidewaki Nakagawa, Kumiko Koyama, Yoji Murata, et al.

Cancer Res 2000;60:101-105.

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