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Identification of a Brain-Specific APC Homologue, APCL, and Its Interaction with /3-Catenin1

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Identification of a Brain-Specific APC Homologue, APCL, and Its Interaction with /3-Catenin1 ICANCKR RESEARCH 58. 5176-5181, November 15, I<W8| Identification of a Brain-specific APC Homologue, APCL, and Its Interaction with /3-Catenin1 Hidewaki Nakagawa, Yoji Murata, Kumiko Koyama, Asao Fujiyama, Yasuo Miyoshi, Morito Monden, Tetsu Akiyama, and Yusuke Nakamura2 Division of Clinictil Genetics. Department of Genetics. Biomédical Research Center. Osaka Universitv Medical School. Suita City. Osaka 565 ¡H.N.. K. K., Y. M., Y. N.J: Department of Oncogene Research. Institute for Microhidl Diseases. Osaka University, Osaka 565 ¡Y.M., T. A. I; Department of Genetics. National Institute of Genetics. Mishima City. Shizuttka ¡A.F.I; Department of Surgery 11, Osaka University Medicai School ¡H.N.. M.MJ; Laboratory of Molecular Medicine, Hitman Genome Center, Institute of Medical Science. The University of Tokyo, Minato-ku, Tokyo 108 [Y. NJ, Japan ABSTRACT pressing transfected APC (9). and a series of heptad repeats in its NH2-terminal domain can form homo- or heterodimers through the We isolated a novel gene, !/'(/. that showed significant homology to formation of a-helical rods (10). Oncogenes or tumor suppressor the adenomatous polyposis coli (APC} tumor suppressor gene. This novel genes often belong to families whose members show sequence simi gene, located on chromosome I9pl3.3, encodes a protein of 2303 amino larities with each other (11); for example, the mvc family of onco- acids that is expressed specifically in the brain. The predicted protein of APCL contains five copies of a 20-amino-acid motif (FXVEXTPXCFSRX- genes consists of c-myc, N-myc, and L-myc (12), and p2l/Ras genes SSLSSLS). Like APC, this domain of APCL was able to bind to ß-catenin constitute a small group within the large family of G-proteins (13). and deplete the intracellular ß-catenin pool. A reporter-gene assay re Among the products of known tumor suppressor genes, Rb shows vealed that APCL could also regulate interaction of ß-cateninwith T cell- significant similarities to pl07 (14) and pl30 (15), the pl6 tumor specific transcription factor, although less actively than APC. These re suppressor to pl5 (16), and p53 to p73 (17). sults suggest that the APCL protein may be involved in the \ViilAVmgless With this point in mind, we searched for novel genes related to signal pathway, and the identification of a novel relative of APC may APC. Here we report isolation and mapping of a gene, termed APCL, provide new insights into the function of APC. that shows significant structural and functional similarities to APC. INTRODUCTION MATERIALS AND METHODS The APC? tumor suppressor was isolated by positional cloning from chromosome 5c¡2i(1.2) when mutations were found in patients Screening of a Human Brain cDNA Library. We searched the EST carrying familial adenomatous polyposis. an autosomal dominant database for the DNA sequence similar to APC and found two ESTs (H50182 hereditary cancer syndrome characterized by numerous adenomatous and H50183) similar to the DNA sequence of APC. A lambda ZAPII human brain cDNA library (Stratagene) was screened by using as a probe a polyps in the colorectum (3). Because APC is mutated somatically in |'2P]dCTP-labeled reverse transcription-PCR product generated from brain two-thirds of sporadic colorectal carcinomas as well as in many cDNA according to the sequence of EST H50183. After hybridi/ation at 50°C adenomas, such events are considered to be crucial early steps in overnight, the membranes were washed in 2x SSC at room temperature and colorectal tumorigenesis (4). then twice for 15 min in 2 X SSC containing 0.1% SDS at 55°C.The cDNA The amino acid sequence of APC protein has little similarity to any clones obtained from the library screening were sequenced using a Perkin- other proteins that would yield clues to its biophysiological functions. Elmer-Cetus 377 automated DNA sequencer and the Dye-Terminator Cycle However, molecules that interact with separate domains of APC have Sequencing FS Ready Reaction kit (Perkin-Elmer) and assembled into a been identified. In particular, its interaction with ß-cateninis thought full-length cDNA. which we designated APCL. to be very important because APC protein depletes intracellular Northern Analysis. A multiple-tissue Northern blot (Clontech) was hy- ß-catenin when a domain of seven 20-amino-acid repeats in the bridi/.ed at 42°C with a 2-kb Noll fragment (Fig. 1C) of the assembled, full-length cDNA representing APCL. labeled with [12P]dCTP. The blot was middle portion of APC binds to this target protein (5). If normal APC is not present, or if the ß-catenin itself is mutated, ß-catenin is washed in 2x SSC at room temperature, then twice for 20 min in 0.1 x SSC containing 0.1% SDS at 55°C.The membrane was exposed to X-ray film for stabilized and accumulates within the cell (5). Furthermore, an excess 4 days at -80°C. of cytoplasmic ß-cateninenhances interaction with members of the Isolation of Genomic Clones. A human cosmid library equivalent to Tcf family; the ß-catenin-Tcfcomplex appears to stimulate the Wng/ approximately five genomes was screened with a 1-kb fragment of the new Wingless signal pathway (6, 7). cDNA. labeled with (3-P]dCTP. Two clones that hybridized to this probe With respect to other activities, the COOH terminus of the APC (94G06 and 37F12) were isolated, purified, and compared with the full-length protein interacts with hDLG (human homologue of the Drosophila cDNA to identify exon-intron junctions. These cosmids were used as probes discs large tumor suppressor), a process that may regulate cell cycle for FISH. progression and neuronal function (8). Moreover, the basic region of Chromosomal Localization by FISH Analysis. FISH was performed as normal APC associates with microtubules in cells that are overex- described previously (18). Human metaphase chromosomes were prepared by the thymidine synchronization/bromodeoxyuridine release technique for the delineation of G-banding patterns. Before hybridi/ation. metaphase cells were Reeeivcd 6/19/98; accepted 9/18/98. stained with Hoechst 33258 and irradiated with UV light. Cosmids 94G06 and 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 37F12 were labeled with biotin-16-dUTP (Boehringer) by nick translation and 18 U.S.C. Section 1734 solely to indicate this fact. hybridized to denatured metaphase chromosomes. Hybridization signals were 1This work was supported in part by a Grant-in-Aid from the Ministry of Education. detected with FITC-avidin (Boehringer). Precise localization of the signals was Science. Sports and Culture of Japan, and by "Research for the Future" Program Grant determined by visualization of the replication G-bands. 96LOOI02 of The Japan Society for the Promotion of Science. 3 To whom requests for reprints should be addressed, at Laboratory Molecular Med Binding of APCL with ß-Catenin in Vitro. Predicted amino acids 627- icine. Human Genome Center. Institute of Medical Science. The University of Tokyo. 1666 of APCL were synthesized by in vitro transcription-translation in the 4-6-1 Shirokanedai, Minato. Tokyo 108-8639, Japan. Phone: 81-3-5449-5372: Fax: 81- presence of [35S]methionine using the TNT rabbit reticulocyte lysate system 3-5449-5433: E-mail: [email protected]. 1The abbreviations used are: APC. adenomatous polyposis coli; APCL. APC-like; Tcf. (Promega). GST-/3-catenin was generated by subcloning into pGEX5X-l T cell-specific transcription factor; EST, expressed sequence tag; FISH, fluorescence in (Pharmacia), a fragment of ß-catenincDNA encoding the armadillo repeats. situ hybridisation; GST. glulalhionc .V-lransferase; 0-gal. ß-galactosidase. expressing them in Escherichia coli, and isolating them by absorption to 5176 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. APC HOMOLOGUE. APCL. AND ITS INTERACTION WITH /3-CATENIN Fig. 2. Schematic representation of the genomic structure of the APCL gene. The boxen represent exons, numbered 1-14. The coding sequence is represented hy 123 45 67 8 shading. 1kb glutathione-Sepharose. The immobili/ed GST fusion protein was mixed with from EST clones H50182 and H50183. We obtained four cDNA in v/'/w-translated proteins in buffer A (10 mM Tris-HCI (pH 7.5), 150 mM clones and assembled them. The assembled cDNA, termed APCL, NaCl. 1 mM EGTA, 1 HIMNaVO4, and 50 mM NaF] containing 0.1% Triton consisted of 6912 nucleotides including an 6909-bp open reading X-100 tor l h at 4°Cand then washed extensively with buffer A. Proteins frame encoding a protein of 2303 amino acids (predicted molecular adhering to the beads were analyzed by 8% SDS-PAGE. Immunofluorescent Detection of /¡-(alenili in SW480 Cells Expressing weight, M, 240,000). The NH2-terminal half of the predicted protein the APCL Construct. SW480 cells (3 x IO5cells/well) were cotransfected revealed a high degree of similarity to APC, but the COOH-terminal with 1 jig of an effector plasmid [pCDNA3. pCDNA3-FLAG-APCL (627- half showed no similarity to any known protein (Fig. \A). The heptad 1666). or pMKITNeo-APC (1-1864)] and 1 /ng of a pCMV-ß-gal plasmid by repeat domain of APC was well conserved in this novel protein (45% lipotection, using Lipofectamine (Life Technologies, Inc.). Cells were fixed identical), indicating that APCL is also likely to form homo- or 48 h after transfection in 4% formaldehyde and dehydrated with 100% cold methanol. After blocking in PBS containing 10% fetal bovine serum, ß-catenin heterodimers. The armadillo domain was also well conserved between and ß-galwere detected with anti-ß-catenin monoclonal antibody (Transduc- APC and APCL (76% identical).
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