The Carboxyl-terminal Fragment of Pro-HB-EGF Reverses Bcl6-mediated Gene Repression
2007; Elsevier BV; Volume: 282; Issue: 20 Linguagem: Inglês
10.1074/jbc.m611036200
ISSN1083-351X
AutoresYumi Kinugasa, Miki Hieda, Masatsugu Hori, Shigeki Higashiyama,
Tópico(s)Virus-based gene therapy research
ResumoHeparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family, is synthesized as a type I transmembrane precursor (pro-HB-EGF). Ectodomain shedding of pro-HB-EGF yields an amino-terminal soluble ligand of EGF receptor (HB-EGF) and a carboxyl-terminal fragment (HB-EGF-CTF) consisting of the transmembrane and cytoplasmic domains. We previously showed that the HB-EGF-CTF translocates from the plasma membrane to the nucleus and plays a role as a signaling molecule. Immunoprecipitation showed that HB-EGF-CTF can associate with Bcl6, a transcriptional repressor in mammalian cells. A glutathione S-transferase pulldown assay revealed that HB-EGF-CTF interacted efficiently with zinc fingers 4–6 of Bcl6. A luciferase reporter assay showed that the nuclear translocation of HB-EGF-CTF following shedding reversed transcriptional repression of cyclin D2 by Bcl6. Additionally, the level of cyclin D2 protein increased and Bcl6 interaction with the cyclin D2 promoter decreased in parallel with the shedding of pro-HB-EGF at all endogenous levels. These findings suggest that HB-EGF-CTF is a potent regulator of gene expression via its interaction with the transcriptional repressor Bcl6. Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family, is synthesized as a type I transmembrane precursor (pro-HB-EGF). Ectodomain shedding of pro-HB-EGF yields an amino-terminal soluble ligand of EGF receptor (HB-EGF) and a carboxyl-terminal fragment (HB-EGF-CTF) consisting of the transmembrane and cytoplasmic domains. We previously showed that the HB-EGF-CTF translocates from the plasma membrane to the nucleus and plays a role as a signaling molecule. Immunoprecipitation showed that HB-EGF-CTF can associate with Bcl6, a transcriptional repressor in mammalian cells. A glutathione S-transferase pulldown assay revealed that HB-EGF-CTF interacted efficiently with zinc fingers 4–6 of Bcl6. A luciferase reporter assay showed that the nuclear translocation of HB-EGF-CTF following shedding reversed transcriptional repression of cyclin D2 by Bcl6. Additionally, the level of cyclin D2 protein increased and Bcl6 interaction with the cyclin D2 promoter decreased in parallel with the shedding of pro-HB-EGF at all endogenous levels. These findings suggest that HB-EGF-CTF is a potent regulator of gene expression via its interaction with the transcriptional repressor Bcl6. Heparin-binding EGF 2The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; HB-EGF, heparin-binding EGF-like growth factor; CTF, carboxyl-terminal fragment; GST, glutathione S-transferase; TPA, phorbol ester 12-O-tetradecanoylphorbol-13-acetate; ADAM, a disintegrin and metalloprotease; PLZF, promyelocytic leukemia zinc finger; Bcl6, B-cell lymphoma 6; ZnF, zinc finger; CFP, cyan fluorescent protein; AP, alkaline phosphatase; PBS, phosphate-buffered saline; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; consensus binding site, consensus binding site; RT, reverse transcription; m1, -2, -3, mutants 1–3; wt, wild type. 2The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; HB-EGF, heparin-binding EGF-like growth factor; CTF, carboxyl-terminal fragment; GST, glutathione S-transferase; TPA, phorbol ester 12-O-tetradecanoylphorbol-13-acetate; ADAM, a disintegrin and metalloprotease; PLZF, promyelocytic leukemia zinc finger; Bcl6, B-cell lymphoma 6; ZnF, zinc finger; CFP, cyan fluorescent protein; AP, alkaline phosphatase; PBS, phosphate-buffered saline; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; consensus binding site, consensus binding site; RT, reverse transcription; m1, -2, -3, mutants 1–3; wt, wild type.-like growth factor (HB-EGF), a member of the EGF family, directly binds to and activates the EGFR (ErbB1/HER1) and can indirectly transactivate ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4) by forming a heterodimer with the EGFR (1Higashiyama S. 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Blood. 1995; 86: 28-37Crossref PubMed Google Scholar). Bcl6 is highly conserved among vertebrate species, and the mouse and human Bcl6 proteins are 94% conserved (27Allman D. Jain A. Dent A. Maile R.R. Selvaggi T. Kehry M.R. Staudt L.M. Blood. 1996; 87: 5257-5268Crossref PubMed Google Scholar). Bcl6 contains a BTB/POZ domain in the amino terminus and six Kruöppel-type (C2H2) zinc finger (ZnF) motifs in the carboxyl terminus. The BTB domain of Bcl6 autonomously represses transcription by recruiting various co-repressors, including SMRT, NCoR, BCoR, and class I or II histone deacetylases (28Huynh K.D. Bardwell V.J. Oncogene. 1998; 17: 2473-2484Crossref PubMed Scopus (246) Google Scholar, 29Huynh K.D. Fischle W. Verdin E. Bardwell V.J. Genes Dev. 2000; 14: 1810-1823PubMed Google Scholar). The ZnF motifs of Bcl6 bind to the consensus core sequence TTCCT(A/C)GAA found in several Bcl6 target genes (30Dent A.L. Vasanwala F.H. Toney L.M. Crit. Rev. Oncol. Hematol. 2002; 41: 1-9Crossref PubMed Scopus (77) Google Scholar). These target genes are cyclin D2, CD69, and MIP-1α (31Shaffer A.L. Yu X. He Y. Boldrick J. Chan E.P. Staudt L.M. Immunity. 2000; 13: 199-212Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar). PLZF contains two functional domains: (i) an amino-terminal self-interacting BTB/POZ domain and (ii) a carboxyl-terminal domain containing multiple Kruöppel-like C2H2 ZnFs, which mediate binding DNA and HB-EGF-CTF binding. Bcl6 and PLZF have similar domain structures and amino acid sequences, suggesting that Bcl6 may functionally interact with HB-EGF-CTF. Our study was, therefore, directed at whether HB-EGF-CTF interacts with and regulates Bcl6. To accomplish this, we characterized the properties of HB-EGF-CTF as a transcriptional regulator. Our results indicate that HB-EGF-CTF interacts with Bcl6 and reverses the transcriptional repression of the cyclin D2 gene expression by Bcl6. Our results also suggest that HB-EGF-CTF acts as a regulator of cell growth.EXPERIMENTAL PROCEDURESAntibodies—The mouse anti-FLAG monoclonal antibody (M2), the anti-cyclin D2 monoclonal antibody (DCS-3), and the anti-β-actin monoclonal antibody (AC-15) were purchased from Sigma-Aldrich. The mouse anti-Bcl6 monoclonal antibodies (D-8 and C-19) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-mouse IgG antibody was obtained from Promega (Madison, WI). Rabbit polyclonal antibodies against synthetic peptides corresponding to the cytoplasmic region of pro-HB-EGF (#H1) and the extracellular region of pro-HB-EGF (#H6) have been described previously (32Miyagawa J. Higashiyama S. Kawata S. Inui Y. Tamura S. Yamamoto K. Nishida M. Nakamura T. Yamashita S. Matsuzawa Y. Taniguchi N. J. Clin. Invest. 1995; 95: 404-411Crossref PubMed Google Scholar). Rabbit and mouse normal IgGs were obtained from IBL.Expression Vectors—The expression vector for the cyan fluorescent protein (CFP)-tagged Bcl6 (CFP-Bcl6) was constructed by inserting human Bcl6 cDNA into the BglII and SalI sites of the pECFP-C1 vector (Clontech). The full-length Bcl6 and a series of their truncated regions were cloned into the BglII and XhoI sites of modified pME18S vector (pME18S-FLAG) (17Nanba D. Mammoto A. Hashimoto K. Higashiyama S. J. Cell Biol. 2003; 163: 489-502Crossref PubMed Scopus (138) Google Scholar). Glu-627, Glu-655, and both in the linker region of Bcl6 ZnF 4–6 were mutated to Ala to construct three mutants, E627A (m1), E655A (m2), and E627A/E655A (m3). The plasmid for the recombinant expression of glutathione S-transferase (GST)-fused HB-EGF-CTF (pGEX6p1-HB-EGF-CTF) has been described previously (17Nanba D. Mammoto A. Hashimoto K. Higashiyama S. J. Cell Biol. 2003; 163: 489-502Crossref PubMed Scopus (138) Google Scholar). All of the cDNA constructs were verified by DNA sequencing using a CEQ 8000 DNA Analysis System (Beckman Coulter).Cell Culture, cDNA Transfection, and TPA Treatment—Stable transfectants of human fibrosarcoma HT1080 expressing human placental alkaline phosphatase (AP)-tagged pro-HB-EGF (HT1080/AP-HB-EGF), both AP-HB-EGF, and a mutant of ADAM12 lacking the metalloprotease domain (HT1080/AP-HB-EGF/ΔMP-ADAM12), wild-type pro-HB-EGF (HT1080/wt HB-EGF), an uncleavable form of pro-HB-EGF (HT1080/uc HB-EGF), and a mutant of pro-HB-EGF lacking the cytoplasmic domain (HT1080/ΔC HB-EGF) have been described previously (6Asakura M. Kitakaze M. Takashima S. Liao Y. Ishikura F. Yoshinaka T. Ohmoto H. Node K. Yoshino K. Ishiguro H. Asanuma H. Sanada S. Matsumura Y. Takeda H. Beppu S. Tada M. Hori M. Higashiyama S. Nat. Med. 2002; 8: 35-40Crossref PubMed Scopus (638) Google Scholar, 8Tokumaru S. Higashiyama S. Endo T. Nakagawa T. Miyagawa J.I. Yamamori K. Hanakawa Y. Ohmoto H. Yoshino K. Shirakata Y. Matsuzawa Y. Hashimoto K. Taniguchi N. J. Cell Biol. 2000; 151: 209-220Crossref PubMed Scopus (262) Google Scholar, 14Chen J.K. Capdevila J. Harris R.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6029-6034Crossref PubMed Scopus (82) Google Scholar, 17Nanba D. Mammoto A. Hashimoto K. Higashiyama S. J. Cell Biol. 2003; 163: 489-502Crossref PubMed Scopus (138) Google Scholar). Wild-type and stably transfected HT1080 cells were grown in Eagle's minimum essential medium (Nikken Biomedical Laboratory) supplemented with 0.1 mm nonessential amino acids (Invitrogen), 10% fetal bovine serum (HyClone, Lot AGN19241), penicillin, and streptomycin sulfate. Human colorectal adenocarcinoma LoVo cells were grown in Dulbecco's modified Eagle's medium (Nikken Biomedical Laboratory) containing 10% fetal bovine serum, penicillin, and streptomycin sulfate. Transfections were performed using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's instructions. The cells were treated with 100 nm TPA in each culture medium for each individual time.Preparation of Cell Extracts—Cells were washed with ice-cold phosphate-buffered saline (PBS(–)) and lysed in ice-cold lysis buffer (PBS(–) containing 5 mm EDTA, 1% Triton X-100, 0.2 mm p-amidinophenyl methanesulfonyl fluoride hydrochloride, and 2 μg/ml aprotinin). Cell lysates were mixed by end-over-end rotation for 30 min at 4 °C and then centrifuged at 9100 × g for 10 min. The resulting supernatants were used as cell extracts.Immunoprecipitation—Cell extracts of LoVo cells and HT1080/AP-HB-EGF cells transfected with FLAG-tagged full-length Bcl6 (amino acid residues 1–706) were incubated with an antibody to cyclin D2 (DCS-3), the cytoplasmic region of pro-HB-EGF (#H1), or Bcl6 (D-8) for 2 h at 4°C with end-over-end rotation. Protein G-Sepharose 4 Fast Flow beads (Amersham Biosciences) were then added to the mixture. After a 2-h incubation at 4 °C with mixing, the suspension was centrifuged, and the collected protein G-Sepharose beads were washed three times with lysis buffer. The bound proteins were analyzed by Western blotting using an anti-cyclin D2 antibody (DCS-3), an anti-FLAG monoclonal antibody (M2), or an anti-Bcl6 antibody (D-8), followed by a horseradish peroxidase-conjugated anti-mouse IgG antibody. Immunoreactive proteins were visualized using an AEC substrate kit (Vector Laboratories, Inc.) or the ECL Plus Western blotting Detection system (Amersham Biosciences).Imaging of CFP Fusion Proteins—Transiently transfected cells expressing CFP-Bcl6 cDNA were cultured for at least 12 h in serum-free medium to decrease endogenous HB-EGF and its shedding. The cells were then used to examine the subcellular localization of CFP-Bcl6 proteins by fluorescence microscopy (IX70, Olympus).Expression and Purification of Recombinant Proteins—GST and GST-HB-EGF-CTF were produced in and purified from Escherichia coli strain BL21. Protein expression was induced by treatment with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 4 h at 30 °C. The cells were then transferred to a centrifuge tube, collected by centrifugation, washed once with ice-cold PBS(–), frozen at –80 °C for at least 1 h, resuspended in 10 ml of lysis buffer (50 mm Tris-HCl, pH 8.0, 1 mm MgCl2, and 100 μg/ml lysozyme) by repeated pipetting, and incubated on ice for 10 min. The mixture was then adjusted to 5 mm dithiothreitol, and protease inhibitors (Pefablock SC (Roche Applied Science) and aprotinin) were added. The solution was adjusted to 1% N-laurylsarcosine and then vortexed and sonicated on ice using a UR-20P sonicator (Tomy Seiko Co., Ltd.). After sonication, the lysates were clarified by centrifugation at 10,000 × g for 10 min at 4 °C. The supernatants were transferred to new tubes, and the solution was adjusted to 2% Triton X-100. The lysates were vortexed, and glutathione-Sepharose 4B beads (Amersham Biosciences) were added. After mixing by end-over-end rotation for 30 min at 4 °C, the beads were washed five times with PBS(–) and resuspended in storage buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 5 mm dithiothreitol, and 10% v/v glycerol).GST Pulldown Assay—Extracts from HT1080 cells expressing various FLAG-tagged Bcl6 mutants were mixed with 2 μg of recombinant GST or GST-HB-EGF-CTF immobilized on glutathione-Sepharose beads for 2 h at 4°C by end-over-end rotation. After washing the beads, the bound proteins were analyzed by SDS-PAGE, followed by Western blotting using an anti-FLAG antibody (M2), followed by a horseradish peroxidase-conjugated anti-mouse IgG antibody. Immunoreactive proteins were visualized using the ECL Plus Western blotting detection system (Amersham Biosciences). The binding abilities of the Bcl6 ZnF 4–6 E/A mutants (m1–3) were estimated by the efficiency of pulldown, which was calculated by using the ratio of each band intensity (lane 2) in a pulldown assay panel to the corresponding band intensity in a cell lysate panel.Luciferase Reporter Assay—Plasmids encoding the human cyclin D2 promoter-driven luciferase reporter gene and its mutant (pGL3-wt cyclin D2 promoter and pGL3-mut cyclin D2 promoter) were constructed by inserting the PCR-amplified promoter fragment of cyclin D2 (nucleotides –1560 to –4) or its mutated fragment (three base mutations; see Fig. 4A and Ref. 31Shaffer A.L. Yu X. He Y. Boldrick J. Chan E.P. Staudt L.M. Immunity. 2000; 13: 199-212Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar) into the pGL3-Control Vector (Promega). Vector pRL-TK (Promega) was used as an internal control. Wild-type and stably transfected HT1080 cells (5 × 104) were transiently transfected with luciferase reporter plasmids using Lipofectamine™ 2000 (Invitrogen). After a 24-h culture, cells were treated with 100 nm TPA, and promoter activities were analyzed using the DualGlo™ luciferase assay system (Promega). These assays were repeated at least three times, and firefly luciferase (cyclin D2 promoter) activities were normalized by the Renilla luciferase (internal control) activities.siRNA—The siRNAs for the knockdown of Bcl6 (siBcl6-1 and siBcl6-2, CUGCGUCAUGCUUGUGUUAUA and AACCUUAGUGUGAUCAAUCUA) were designed by and obtained from Qiagen. The siRNAs for the knockdown of HB-EGF (siHB-EGF, AGCUCUUUCUGGCUGCAGU) and a scrambled siRNA (GCGCGCUUUGUAGGAUUCG), used as a negative control, were obtained from Dharmacon Research, Inc. The scrambled sequence did not correspond to any mammalian sequence in the NCBI date base. HT1080 and LoVo cells were transfected with 100 nm siRNA, after which the mRNA and protein levels were examined by RT-PCR and Western blotting, respectively.RT-PCR—Total RNA was extracted from the cells using TRIzol® (Invitrogen), and first-strand cDNA was synthesized using Superscript II (Invitrogen). PCRs for Bcl6 were performed using the following primer pair: 5′-GATGAGATTGCCCTGCATTT-3′ (sense) and 5′-TTCTTCCAGTTGCAGGCTTT-3′ (antisense).Chromatin Immunoprecipitation Assay—LoVo cells cultured at 5 × 106 cells per 100-mm culture dish containing 10 ml of growth media were either treated with 100 nm TPA for 1 h or not treated. After adjusting to a final formaldehyde concentration of 1% (w/v), the cells were incubated for 10 min at 37 °C, followed by the addition of glycine to 0.136 m and incubated for 10 min. After washing with cold PBS(–), the cells were sonicated in lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris-HCl, pH 8.1, 1× protease inhibitor mixture (Roche Applied Science)). The supernatant was cleared by centrifugation at 15,000 × g and diluted nine times with dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA, 16.7 mm Tris-HCl, pH 8.1, 167 mm NaCl). DNA shearing was controlled by running part of the sonicated chromatin on an agarose gel to ensure that the fragmented DNA is 200–100 bp. To preclear the chromatin solution, 50 μl of a 50% (v/v) slurry of Protein G-agarose/salmon sperm DNA (Upstate) was added to the rest of the sample. After incubation with agitation for 30 min at room temperature, the beads were removed by centrifugation at 5,000 × g, and the supernatant, of which 1/100th was retained as an input control, was collected. The retrieved supernatant was incubated with either an anti-Bcl6 antibody, C-19 (Santa Cruz Biotechnology), or an isotype control antibody (IBL) for 2 h at room temperature with rotation in the presence of 50 μl ofa 50% (v/v) slurry of Protein G-agarose/salmon sperm DNA (Upstate). The beads were then sequentially washed with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.1, 150 mm NaCl), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.1, 500 mm NaCl), LiCl buffer (0.25 m LiCl, 1% IGEPAL-CA630, 1% sodium deoxycholate, 1 mm EDTA, 10 mm Tris-HCl, pH 8.1), and then twice with TE buffer (10 mm Tris-HCl, 1 mm EDTA). The immunoprecipitated DNA (or the input control) was extracted with 200 μl of elution buffer (1% SDS, 0.1 m NaHCO3), and then 8 μl of 5 m NaCl was added, followed by incubation at 65 °C overnight to reverse the cross-links. The DNA was purified with a Qiagen Nucleotide Removal kit according to the manufacturer's protocol and then used in a PCR reaction. PCRs were then performed on the purified DNA with BIOTAQ (BIOLINE) in the presence of 2.5 mm MgCl2 at 60 °C for 30 cycles. The sequences of the primers used for the PCR were as follows: 5′-ATGCCTCCGGGGAAAGG-3′ (sense) and 5′-AAAAACCCGCTTCCTCG-3′ (antisense). Analysis of the PCR products was performed on a standard 2% agarose gel by electrophoresis in Tris acetate-EDTA buffer.RESULTSHB-EGF-CTF Binds the ZnF Region of Bcl6—To analyze the interaction between HB-EGF-CTF and Bcl6 in mammalian cells, FLAG-tagged Bcl6 (FLAG-Bcl6 as shown in Fig. 1B) were transfected into HT1080 cells stably expressing AP-tagged pro-HB-EGF (HT1080/AP-HB-EGF). Antibodies against the cytoplasmic region of pro-HB-EGF (#H1) immunoprecipitated AP-tagged pro-HB-EGF (Fig. 1A, lower) and an ∼90-kDa band from the cell lysates. This corresponds to FLAG-Bcl6, indicating that HB-EGF-CTF physically interacts with Bcl6 (Fig. 1A, upper).FIGURE 1HB-EGF-CTF interacts with Bcl6. A, FLAG-tagged full-length Bcl6 (FLAG-Bcl6) was transiently expressed in HT1080/AP-HB-EGF cells and then immunoprecipitated using an antibody against the cytoplasmic region of HB-EGF (#H1) or normal rabbit IgG. Western blotting was carried out using an anti-FLAG monoclonal antibody (upper) and #H1 (lower). B, schematic representation of Bcl6 and its deletion mutants, Bcl6 (amino acids 1–706), BTB+Center (amino acids 1–518), and ZnF (amino acids 519–706). All of the recombinant proteins were expressed as FLAG-tagged (black box) fusion proteins. C, recombinant GST-tagged HB-EGF-CTF pulled down Bcl6 and its mutants from crude extracts. Recombinant GST (lane 1) or GST-HB-EGF-CTF (lane 2) was immobilized on glutathione-Sepharose beads and incubated with crude extracts of HT1080 cells expressing FLAG-tagged Bcl6 or its mutants. Western blotting was carried out using an anti-FLAG monoclonal antibody. Left, analysis of crude extracts. Right, analysis of the precipitated proteins. D, shedding of pro-HB-EGF does not effect the localization of Bcl6. HT1080 cells (a and b) and HT1080/AP-HB-EGF cells (c and d) were transiently transfected with Bcl6 tagged with CFP (CFP-Bcl6). The subcellular localization of the CFP-Bcl6 protein was visualized by fluorescence microscopy before (a and c) or after treatment with TPA for 60 min (b and d). CFP-Bcl6 showed a discrete pattern of punctate staining in the nucleus. Treatment with TPA for 60 min did not affect the subcellular localization of CFP-Bcl6 in either HT1080 or HT1080/AP-HB-EGF cells. Bar, 5 μm. E, effect of TPA on the shedding of pro-HB-EGF in HT080/AP-HB-EGF cells. Shedding was examined by Western blotting with the #H1 antibody and by measurement of AP activity in the conditioned medium according to previously described methods (8Tokumaru S. Higashiyama S. Endo T. Nakagawa T. Miyagawa J.I. Yamamori K. Hanakawa Y. Ohmoto H. Yoshino K. Shirakata Y. Matsuzawa Y. Hashimoto K. Taniguchi N. J. Cell Biol. 2000; 151: 209-220Crossref PubMed Scopus (262) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next performed a GST pulldown assay using GST-tagged HB-EGF-CTF (GST-HB-EGF-CTF) and extracts of HT1080 cells expressing FLAG-Bcl6. Western blotting with an anti-FLAG antibody showed that GST-HB-EGF-CTF, but not GST alone, bound to Bcl6 (Fig. 1C). Bcl6 has two characteristic domains: an amino-terminal BTB domain and a carboxyl-terminal C2H2 ZnF domain. We determined which domain is responsible for the interaction with HB-EGF-CTF using two deletion mutants of Bcl6 (BTB+Center and ZnF, Fig. 1B). Extracts from cells expressing these deletion mutants were incubated with GST-HB-EGF-CTF-immobilized glutathione-Sepharose beads. A large fraction of ZnF and a detectable level of BTB+Center were pulled down by GST-HB-EGF-CTF but not by GST alone (Fig. 1C). These data indicate the ZnF domain mediates the interaction with HB-EGF-CTF.We have previously shown that some of the PLZF is translocated to the cytoplasm subsequent to the shedding of pro-HB-EGF (17Nanba D. Mammoto A. Hashimoto K. Higashiyama S. J. Cell Biol. 2003; 163: 489-502Crossref PubMed Scopus (138) Google Scholar). Here, we investigated whether the localization of Bcl6 is affected by the production of HB-EGF-CTF in HT1080 and HT1080/AP-HB-EGF cells. HT1080 cells, which express a very low level of endogenous pro-HB-EGF (26Onizuka T. Moriyama M. Yamochi T. Kuroda T. Kazama A. Kanazawa N. Sato K. Kato T. Ota H. Mori S. Blood. 1995; 86: 28-37Crossref PubMe
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