APCL, a Central Nervous System-specific Homologue of ... · pAS2-1 vector. We performed...
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[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 Perinucleus 1 Hidewaki Nakagawa, Kumiko Koyama, Yoji Murata, Monden Morito, Tetsu Akiyama, and Yusuke Nakamura 2 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 APCL, a central nervous system-specific sequence homologue of the adenomatous polyposis coli tumor suppressor, can regulate the cytoplas- mic level of b-catenin as the adenomatous polyposis coli tumor suppressor does, but its overall biological function remains unclear. Using a yeast two-hybrid system, we attempted to isolate proteins that might associate with the unique COOH-terminus of APCL. Among 166 cDNA clones isolated from a human fetal-brain cDNA library as candidates for inter- action with APCL, 32 encoded parts of p53-binding protein 2 (53BP2), a 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. Confocal microscopy showed that APCL and 53BP2 proteins were local- ized together in the perinuclei of normal mammalian cells, but this was not the case in cells that expressed truncated APCL and 53BP2 proteins. These findings suggested that binding of the COOH-terminus of APCL to 53BP2 regulates the cytoplasmic location of 53BP2. Because 53BP2 also interacts with p53 and Bcl2 and regulates p53 function, our results suggest that APCL might be involved in the p53/Bcl2-linked pathway of cell-cycle progression and cell death. INTRODUCTION APCL, a protein encoded on chromosome 19p13.3 that bears sig- nificant sequence homology to the APC 1 tumor suppressor (1, 2) is expressed strongly and specifically in the central nervous system (3). The expression pattern indicates a specific role in proliferation and differentiation in the central nervous system, but the overall biological functions of APCL remain unclear. The heptad-repeat domain found in the APC protein is well conserved in APCL (45% of the amino acids are identical); therefore APCL likely also forms homo- or heterodimers (4). Moreover, because the Armadillo domain is also well conserved (76% identical), both proteins may interact with the same or similar molecular entities (5). The central portion of APCL consists of five copies of a 20-amino acid motif (FXVEXTPXCFS- RXSSLSSLS; Refs. 6, 7), and we showed earlier that through this domain, APCL can interact with b-catenin and deplete its intracellular concentration (3). Because the COOH-terminus of APC protein binds to EB1 (8), microtubules (9), and hDLG (10), it appears that APC is involved in cell-cycle progression and/or growth control. However, because the COOH-terminus of the APCL protein bears little simi- larity to that of APC (only 13% identical amino acids), that part of the molecule can be presumed to interact with different proteins; if so, APCL would possess at least some functions distinct from those of APC. To clarify those functions, we used a yeast two-hybrid system to search for proteins that might associate with the COOH-terminus of APCL. We found that this portion of the molecule indeed bound to a p53-binding protein, 53BP2. In vivo studies confirmed that this asso- ciation regulated the cytoplasmic localization of 53BP2 by translo- cating it to the perinuclear region. MATERIALS AND METHODS Yeast Two-Hybrid Screening. To generate a fusion protein containing a GAL4-binding domain, we excised a 2.6-kb NotI fragment (residues 1520 – 2303) of APCL cDNA, filled in the ends, and subcloned the fragment into a SmaI site of pAS2-1 vector (Clontech). After confirmation of the DNA sequences in the plasmid, we used it as “bait” to screen a human fetal-brain cDNA library using the pACT2 vector (Clontech). Briefly, the plasmids were transformed into Y190 yeast cells, and positive clones were selected on Trp(2), Leu(2), His(2) plates containing 25 mM 3-aminotriazole, and assayed for b-galactosidase activity by colony-lift filter assay. The positive clones were cotransformed with either the bait vector or the original pAS2-1 vector into yeast to confirm the interaction. Quantitative b-Galactosidase Assays. A fusion plasmid containing the GAL4-binding domain and residues 458-1008 of 53BP2 was isolated from the yeast two-hybrid screening as the prey of the smallest fragment of 53BP2. Various fusion plasmids containing parts of 53BP2, i.e., 53BP2-A (residues 458 –796), 53BP2-B (residues 756 –921), 53BP2-C (residues 919-1008), or 53BP2-D (residues 756-1008), were subcloned into pACT2 vector as PCR- amplified fragments. A mutant 53BP2 plasmid, P53BP2-DM (W974K) was generated from pACT2-53BP2-D (residues 756-1008) by PCR-based site- directed mutagenesis. Misincorporations of nucleotides during PCR amplifi- cation were checked by DNA sequencing. The bait and each 53BP2-fused plasmid were transformed into Y190 yeast cells, and five separate colonies were selected from the positive clones growing on Trp(2), Leu(2), His(2) plates containing 25 mM 3-aminotriazole. To confirm the interaction, positive clones were cotransformed into yeast with either the bait vector or the original pAS2-1 vector. We performed liquid-culture assays for b-galactosidase in triplicate, using ONPG as a substrate according to the manufacturer’s instruc- tions (Clontech). Production of GST Fusion Proteins. cDNA fragments that contained parts of the coding sequence of 53BP2 were excised from the various pACT2- 53BP2 plasmids by digestion with EcoRI and XhoI. Each fragment was ligated in-frame into pGEX5-2 (Pharmacia). Overnight cultures of XL1Blue MRF9, transformed with the plasmids encoding GST fusion proteins, were diluted and cultured with 0.1 mM isopropyl-1-thio-b-D-galactopyranoside for 6 h at 30°C. Cells were lysed by sonication in PBS containing 1% Triton X-100, and the lysates were clarified by centrifugation. Each GST-53BP2 fusion protein was purified on glutathione-agarose beads in PBS containing 1% Triton X-100. In Vitro Binding Assay. We generated a [ 35 S]Met-labeled COOH-termi- nus peptide of APCL from a template containing nucleotides 4120 – 6912 (stop) of the APCL cDNA sequence, using TNT rabbit reticulocyte lysate (Promega) according to the manufacturer’s instructions. The immobilized GST fusion proteins were mixed with the in vitro-translated APCL proteins in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA containing 0.1% Triton Received 5/3/99; accepted 10/28/99. 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. 1 This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan, and by “Research for the Future” Program Grant 96L00102 of The Japan Society for the Promotion of Science. 2 To whom requests for reprints should be addressed, at Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato, Tokyo 108-8639, Japan. Phone: 81-3-5449-5372; Fax: 81-3-5449-5433; E-mail: [email protected]. 3 The abbreviations used are: APC, adenomatous polyposis coli; GST, glutathione-S- transferase. 101 Research. on November 9, 2020. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from
Transcript of APCL, a Central Nervous System-specific Homologue of ... · pAS2-1 vector. We performed...
[CANCER RESEARCH 60, 101–105, January 1, 2000]
APCL, a Central Nervous System-specific Homologue of Adenomatous PolyposisColi Tumor Suppressor, Binds to p53-binding Protein 2 and Translocates it tothe 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, CancerChemotherapy Center, Japanese Foundation for Cancer Research [K. K.] and Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, TheUniversity of Tokyo, Tokyo 108-8639, Japan [K. K., Y. N.]
ABSTRACT
APCL, a central nervous system-specific sequence homologue of theadenomatous polyposis coli tumor suppressor, can regulate the cytoplas-mic level ofb-catenin as the adenomatous polyposis coli tumor suppressordoes, but its overall biological function remains unclear. Using a yeasttwo-hybrid system, we attempted to isolate proteins that might associatewith the unique COOH-terminus of APCL. Among 166 cDNA clonesisolated from a human fetal-brain cDNA library as candidates for inter-action with APCL, 32 encoded parts of p53-binding protein 2 (53BP2), amolecule that interacts with p53 and Bcl2. An in vitro binding assayindicated that the Src-homology-3 domain and the ankyrin-repeat domainof 53BP2 were both required for binding to the COOH-terminus of APCL.Confocal microscopy showed that APCL and 53BP2 proteins were local-ized together in the perinuclei of normal mammalian cells, but this was notthe case in cells that expressed truncated APCL and 53BP2 proteins.These findings suggested that binding of the COOH-terminus of APCL to53BP2 regulates the cytoplasmic location of 53BP2. Because 53BP2 alsointeracts with p53 and Bcl2 and regulates p53 function, our results suggestthat APCL might be involved in the p53/Bcl2-linked pathway of cell-cycleprogression and cell death.
INTRODUCTION
APCL, a protein encoded on chromosome 19p13.3 that bears sig-nificant sequence homology to the APC1 tumor suppressor (1, 2) isexpressed strongly and specifically in the central nervous system (3).The expression pattern indicates a specific role in proliferation anddifferentiation in the central nervous system, but the overall biologicalfunctions of APCL remain unclear. The heptad-repeat domain foundin the APC protein is well conserved in APCL (45% of the aminoacids are identical); therefore APCL likely also forms homo- orheterodimers (4). Moreover, because the Armadillo domain is alsowell conserved (76% identical), both proteins may interact with thesame or similar molecular entities (5). The central portion of APCLconsists of five copies of a 20-amino acid motif (FXVEXTPXCFS-RXSSLSSLS; Refs. 6, 7), and we showed earlier that through thisdomain, APCL can interact withb-catenin and deplete its intracellularconcentration (3). Because the COOH-terminus of APC protein bindsto EB1 (8), microtubules (9), and hDLG (10), it appears that APC isinvolved in cell-cycle progression and/or growth control. However,because the COOH-terminus of the APCL protein bears little simi-larity to that of APC (only 13% identical amino acids), that part of the
molecule can be presumed to interact with different proteins; if so,APCL would possess at least some functions distinct from those ofAPC.
To clarify those functions, we used a yeast two-hybrid system tosearch for proteins that might associate with the COOH-terminus ofAPCL. We found that this portion of the molecule indeed bound to ap53-binding protein, 53BP2.In vivo studies confirmed that this asso-ciation regulated the cytoplasmic localization of 53BP2 by translo-cating it to the perinuclear region.
MATERIALS AND METHODS
Yeast Two-Hybrid Screening. To generate a fusion protein containing aGAL4-binding domain, we excised a 2.6-kbNotI fragment (residues 1520–2303) of APCL cDNA, filled in the ends, and subcloned the fragment into aSmaI site of pAS2-1 vector (Clontech). After confirmation of the DNAsequences in the plasmid, we used it as “bait” to screen a human fetal-braincDNA library using the pACT2 vector (Clontech). Briefly, the plasmids weretransformed into Y190 yeast cells, and positive clones were selected onTrp(2), Leu(2), His(2) plates containing 25 mM 3-aminotriazole, and assayedfor b-galactosidase activity by colony-lift filter assay. The positive clones werecotransformed with either the bait vector or the original pAS2-1 vector intoyeast to confirm the interaction.
Quantitative b-Galactosidase Assays.A fusion plasmid containing theGAL4-binding domain and residues 458-1008 of 53BP2 was isolated from theyeast two-hybrid screening as the prey of the smallest fragment of 53BP2.Various fusion plasmids containing parts of 53BP2,i.e., 53BP2-A (residues458–796), 53BP2-B (residues 756–921), 53BP2-C (residues 919-1008), or53BP2-D (residues 756-1008), were subcloned into pACT2 vector as PCR-amplified fragments. A mutant 53BP2 plasmid, P53BP2-DM (W974K) wasgenerated from pACT2-53BP2-D (residues 756-1008) by PCR-based site-directed mutagenesis. Misincorporations of nucleotides during PCR amplifi-cation were checked by DNA sequencing. The bait and each 53BP2-fusedplasmid were transformed into Y190 yeast cells, and five separate colonieswere selected from the positive clones growing on Trp(2), Leu(2), His(2)plates containing 25 mM 3-aminotriazole. To confirm the interaction, positiveclones were cotransformed into yeast with either the bait vector or the originalpAS2-1 vector. We performed liquid-culture assays forb-galactosidase intriplicate, using ONPG as a substrate according to the manufacturer’s instruc-tions (Clontech).
Production of GST Fusion Proteins. cDNA fragments that containedparts of the coding sequence of 53BP2 were excised from the various pACT2-53BP2 plasmids by digestion withEcoRI andXhoI. Each fragment was ligatedin-frame into pGEX5-2 (Pharmacia). Overnight cultures of XL1Blue MRF9,transformed with the plasmids encoding GST fusion proteins, were diluted andcultured with 0.1 mM isopropyl-1-thio-b-D-galactopyranoside for 6 h at 30°C.Cells were lysed by sonication in PBS containing 1% Triton X-100, and thelysates were clarified by centrifugation. Each GST-53BP2 fusion protein waspurified on glutathione-agarose beads in PBS containing 1% Triton X-100.
In Vitro Binding Assay. We generated a [35S]Met-labeled COOH-termi-nus peptide of APCL from a template containing nucleotides 4120–6912(stop) of the APCL cDNA sequence, using TNT rabbit reticulocyte lysate(Promega) according to the manufacturer’s instructions. The immobilized GSTfusion proteins were mixed with thein vitro-translated APCL proteins in 10mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA containing 0.1% Triton
Received 5/3/99; accepted 10/28/99.The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by a grant-in-aid from the Ministry of Education,Science, Sports, and Culture of Japan, and by “Research for the Future” Program Grant96L00102 of The Japan Society for the Promotion of Science.
2 To whom requests for reprints should be addressed, at Laboratory of MolecularMedicine, Human Genome Center, Institute of Medical Science, The University of Tokyo,4-6-1, Shirokanedai, Minato, Tokyo 108-8639, Japan. Phone: 81-3-5449-5372; Fax:81-3-5449-5433; E-mail: [email protected].
3 The abbreviations used are: APC, adenomatous polyposis coli; GST, glutathione-S-transferase.
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X-100 and phenylmethylsulfonyl fluoride, incubated for 3 h at 4°C, thenwashed extensively four times with 10 mM Tris-HCl (pH 7.5), 150 mM NaCl,1 mM EDTA. The beads were boiled in sample buffer and separated on 8%SDS PAGE gels. After electrophoresis, the gels were dried and exposed toX-ray film overnight at280°C.
Construction of a Mammalian Expression Vector for Epitope-taggedAPCL and 53BP2. Full-length APCL was inserted into pcDNA3.1(1)Myc-HisA vector (Invitrogen), and full-length 53BP2 was inserted to pFLAG-CMVvector (Kodak) in frame. Truncated APCL was generated using a 5.2-kbCpoIfragment containing residues 1–1675 of APCL, which lacked the 39 region ofinteraction with 53BP2. This plasmid was subcloned into theEcoRV site ofpcDNA3.1(1)Myc-HisB vector (Invitrogen) after blunting.
Transfection to Mammalian Cells and Immunofluorescent Staining.Cos7 and HeLa cells were cultured on coverslips and cotransfected by lipo-fection with 1 mg of pCMV-FLAG-53BP2 and 1mg of either pCDNA3.1-APCL-Myc or pCDNA3.1-truncated-APCL-Myc, using Lipofectamine plus
(Life Technologies, Inc.). Cells were fixed in 4% formaldehyde 24 h aftertransfection and dehydrated with 100% cold methanol for 10 min. Afterblocking in PBS containing 10% fetal bovine serum for 3 h, cells were treatedwith a mixture of mouse anti-Myc antibody (1:100; Santa Cruz Biochemistry,Inc.) and rabbit anti-FLAG antibody (1:100; ZYMED Laboratories, Inc.) inPBS for 1 h. The coverslips were washed in PBS three times for 10 min andthen incubated in PBS for 1 h with a FITC-conjugated sheep antimouseantibody (1:100; Cappel) and a rhodamine-conjugated goat antirabbit antibody(1:100; Cappel). After the cells were washed four times in PBS, immunoflu-orescence was registered by means of a Zeiss LSM 410 confocal microscope.
Western Blotting. Cos7 cells were cultured and cotransfected with 1mg ofpCMV-FLAG-53BP2 and 1 mg of either pCDNA3.1-APCL-Myc orpCDNA3.1-truncated-APCL-Myc, using Lipofectamine plus (Life Technolo-gies, Inc.). At 24 h after the transfection, the cells were harvested in lysis buffer(10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, and 2% SDS). Thelysed 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. Quantitativeb-galactosidase assays using several fusion plasmids of 53BP2 demonstrated that only53BP2-D(756-1008), containing four ankyrin repeats and the SH3 domain, could specifically interact with the COOH-terminal region of APCL protein. In the mutant53BP2-DM(756-1008), the most conserved residue of the SH3 domain was changed from W to K (W974K).
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) servedas bait for yeast two-hybrid screening.aa, amino acid
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blotting with mouse anti-Myc antibody (Santa Cruz Biochemistry) and rabbitanti-FLAG antibody (ZYMED Laboratories) were visualized using the ECLsystem (Amersham).
RESULTS
Interaction of APCL and 53BP2. To isolate human proteinscapable of interacting with the COOH-terminus of the APCL protein,we screened a human fetal-brain cDNA library constructed with avector containing a GAL4-activator domain. The bait for our yeast
two-hybrid system was a fusion construct of the GAL4-binding do-main and aNotI fragment (nucleotides 4561–7132) of APCL cDNA(Fig. 1). By screening 3.03 107 yeast transformants, we isolated andsequenced 166 cDNA clones that interacted with APCL protein.Among them, 32 contained partial cDNA sequences of 53BP2; theclone containing the smallest fragment of 53BP2 included cDNAsequences corresponding to codons 458-1008. This region of 53BP2,which contains four adjacent ankyrin repeats and a Src-homology-3(SH3) domain, is known to interact with p53 and Bcl2 (11, 12).
To determine which region(s) of 53BP2 are sufficient for binding toAPCL, we constructed fusion plasmids containing a GAL4-activatingdomain and various parts of 53BP2. The 53BP2-B construct containedcodons 756–921 (corresponding to four ankyrin repeats); 53BP2-C(codons 919-1008) contained the SH3 domain; and 53BP2-D (codons756-1008) contained both the ankyrin repeats and the SH3 domain. Toabolish the function of the SH3 domain, we constructed a mutantplasmid, 53BP2-DM (codons 756-1008; W974K) in which a trypto-phan residue (W) at codon 974, the most conserved residue within theSH3 domain in 53BP2, was replaced with lysine (K). Quantitativeb-galactosidase assays using these fusion plasmids demonstrated thatonly 53BP2-D could interact with the COOH-terminal region of theAPCL protein (Fig. 2), indicating that the ankyrin-repeat region andthe SH3 domain were both necessary for 53BP2 to interact withAPCL.
In Vitro Binding Assay. To confirm the results of theb-galacto-sidase assays, several 53BP2 constructs were also tested in a GSTpull-down assay. Thein vitro-translated [35S]-labeled COOH-termi-nus of APCL was tested for binding to glutathione-agarose beadscoupled with GST alone, GST-53BP2-D, GST-53BP2-DM (W974K),GST-53BP2-B, or GST-53BP2-C (Fig. 3A). As in the yeast two-
Fig. 4. Western blotting analysis. Cells transfected with pCMV-FLAG-53BP2,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) of210 kDa (B).
Fig. 3.In vitro binding of APCL with 53BP2. In a GST pull-down assay, thein vitro-translated, [35S]-labeled COOH-terminus of APCL, coupled with the constructs indicated aboveeach 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), andGST-53BP2-C(919-1008)—were purified by glutathione-Sepharose, separated by SDS-PAGE, and stained with Coomassie blue.B, the COOH-terminal region of APCLprotein specifically interacted withGST-53BP2-D(756-1008), but notGSTalone,GST-53BP2-DM(756-1008 W974K),GST-53BP2-B(756-921), or GST-53BP2-C(919-1008).
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hybrid system, the COOH-terminal region of APCL associated spe-cifically with GST-53BP2, but not with GST alone, GST-53BP2-DM,GST-53BP2-B, or GST-53BP2-C (Fig. 3B).
Colocalization of Epitope-tagged APCL and 53BP2 in Mam-malian Cells. To examine the subcellular locations of APCL and53BP2, we cotransfected the two proteins into Cos7 or HeLa cells bymeans of expression plasmids encoding FLAG-tagged 53BP2 andeither Myc-tagged full-length APCL or Myc-tagged truncated-APCL
lacking the region that interacts with 53BP2. Western analysis oftransfected cells revealed that FLAG-53BP2 was expressed as an;150-kDa protein, APCL-Myc as a 260-kDa protein, and truncatedAPCL-Myc as a 210-kDa protein (Fig. 4).
Confocal microscopy revealed that full-length APCL was locatedmainly in the perinuclear region (Fig. 5A), but truncated APCLdisplayed a punctate staining pattern in the cytoplasm, which appar-ently was different from that of full-length APCL (Fig. 5B). 53BP2
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 53BP2alone (C). Full-length APCL and truncated APCL were detected with mouse anti-Myc antibody stained with FITC-conjugated sheep antimouse antibody (green). 53BP2 was detectedwith rabbit anti-FLAG antibody stained with rhodamine-conjugated goat antirabbit antibody (red). Subcellular locations were visualized by immunofluorescence and confocalmicroscopy.D, colocalization of exogenous full-length APCL (green) and 53BP2 (red), appearing asyellow stainin the perinuclear regions of Cos7 cells.E, truncated APCL (t-APCL;green) was not detectable in the perinuclear pattern, nor was 53BP2 (red).
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protein appeared in a punctate vesicular pattern in the cells transfectedwith 53BP2 alone (Fig. 5C), but in cells expressing both proteins,full-length APCL and 53BP2 were colocalized in the perinuclearregion (Fig. 5D). However, in cells expressing truncated APCL and53BP2, this was not the case; truncated 53BP2 displayed the samestaining pattern as that in cells transfected with 53BP2 alone (Fig. 5E).
DISCUSSION
A brain-specific 2303-amino acid protein, APCL (3), possesses ahigh degree of sequence homology to APC in its NH2-terminal andmiddle regions. However, its COOH-terminal region is different fromthat of APC, and because APCL is expressed specifically in brain, itis likely to play an important role in the central nervous system. Tofurther elucidate the function(s) of APCL, we searched for proteinsthat might interact with its COOH-terminal region, using that portionof APCL as bait in a yeast two-hybrid screening system. This proce-dure detected interaction of 53BP2 with the COOH-terminal ofAPCL.
53BP2 binds wild-type p53 but not mutant p53 (13). Its amino acidsequence contains four ankyrin repeats and an SH3 domain in itsCOOH-terminus, and structural analysis has indicated that 53BP2binds to wild-type p53 through those domains (12). 53BP2 alsointeracts with Bcl2, an inhibitor of apoptosis, and may therefore beinvolved in cell-cycle progression and cell death (11). Furthermore,53BP2 seems to function in the signal transduction pathway bystimulating p53-mediated transcriptional activation (14). However,because this protein is present only in the cytoplasm (4), it probablycannot interact with nuclear p53. Hence, its functions remain unclear.
We have demonstrated here, using a yeast two-hybrid system andan in vitro binding assay, that the COOH-terminal region of APCLprotein is able to interact with the four ankyrin repeats and the SH3domain of 53BP2. Both APCL and 53BP2 appeared to be present inan insoluble fraction; they could be extracted only with bufferscontaining an ionic detergent such as SDS and sodium deoxycholate.We attempted to immunoprecipitate 53BP2 and APCL together inmammalian cells that overexpressed both proteins, but buffers con-taining such a strong detergent rendered our attempts unsuccessful.
Confocal microscopy revealed colocalization of APCL and 53BP2in the perinuclear region of mammalian cells overexpressing bothproteins, but 53BP2 was widely distributed over cytoplasmic struc-tures in cells expressing 53BP2 alone or in cells expressing 53BP2and truncated APCL proteins. These findings implied that the twoproteins form a complexin vivo and that APCL can concentrate53BP2 protein in the perinuclear region through attachment at itsCOOH-terminal region. When 53BP2 is overexpressed in mammaliancells, it also colocalizes with Bcl2 in the perinucleus (11). Therefore,when it is present in the perinuclear region, 53BP2 may function in thetransduction pathway of Bcl2 or p53.
Regulation of the subcellular locations of components involved invarious signaling pathways is crucial to effective initiation and main-tenance of signaling cascades (15, 16). 53BP2 can promote p53activity by stimulating p53-mediated transcriptional activation,
thereby inducing cell-cycle arrest at the G2-M stage (11). Theseobservations and the data presented here suggest that APCL mightmodulate the 53BP2-related signaling pathway by regulating thecytoplasmic localization of 53BP2, and also that APCL might beinvolved in mechanisms of cell-cycle progression and cell deathlinked to p53 and Bcl2. The clarification of this novel function ofAPCL may provide us new insights into the multiple functions ofmembers of the APC family.
ACKNOWLEDGMENTS
We gratefully acknowledge the technical assistance of Misae Nishijima andEiji Ouki.
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APCL BINDS TO 53BP2 AND TRANSLATES IT TO PERINUCLEUS
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2000;60:101-105. Cancer Res Hidewaki Nakagawa, Kumiko Koyama, Yoji Murata, et al. p53-binding Protein 2 and Translocates it to the PerinucleusAdenomatous Polyposis Coli Tumor Suppressor, Binds to APCL, a Central Nervous System-specific Homologue of
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