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Full Text (PDF) [CANCER RESEARCH 63, 4204–4210, July 15, 2003] Menin Associates with FANCD2, a Protein Involved in Repair of DNA Damage1 Shenghao Jin,2 Hua Mao,2 Robert W. Schnepp, Stephen M. Sykes, Albert C. Silva, Alan D. D’Andrea, and Xianxin Hua3 Abramson Family Cancer Research Institute, Department of Cancer Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6160 [S. J., H. M., R. W. S., S. M. S., A. C. S., X. H.], and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, 02115 [A. D. D.] ABSTRACT protein that binds single strand DNA, has been shown recently to interact with menin (12). Replication protein A is involved in DNA Multiple endocrine neoplasia type I (MEN1) is an inherited tumor replication, DNA repair, DNA recombination, and potentially gene syndrome characterized by tumors in multiple endocrine organs including transcription. However, it is not clear what role these menin-interact- the parathyroids, pancreatic islets, and the pituitary. The gene mutated in MEN1 patients, Men1, encodes a protein of 610 amino acid residues, ing proteins play in the development of MEN1. menin, and mutations in the Men1 gene lead to the MEN1 syndrome. Several previous reports show increased chromosome breakage in Although the chromosomal instability in the peripheral lymphocytes from lymphocytes from MEN1 patients (13, 14). Peripheral blood lympho- the MEN1 patients has been reported previously, it is not clear whether cytes from MEN1 patients undergo extensive chromosomal breakage, menin is involved in repair of DNA damage. Here we show that menin as compared with normal lymphocytes, after treatment with DEB, an specifically interacts with FANCD2, a protein encoded by a gene involved agent cross-linking double-strand DNA (15, 16). A genome-wide in DNA repair and mutated in patients with an inherited cancer-prone LOH screening of 13 MEN1 patients indicates that MEN1 pancreatic syndrome, Fanconi anemia. The interaction between menin and FANCD2 tumors fail to maintain DNA integrity and chromosomal stability (17). ␥ is enhanced by -irradiation. Moreover, loss of menin expression in mouse These findings implicate that Men1 may be involved in the mainte- embryonic fibroblasts leads to increased sensitivity to DNA damage. nance of genomic stability (15, 18). However, it is not clear whether Furthermore, menin is localized to chromatin and nuclear matrix, and the association with nuclear matrix is enhanced by ␥-irradiation. Together, menin is involved in the maintenance of genomic stability and, if it is, these results suggest that menin plays a critical role in repair of DNA how menin carries out this function. Here we show that menin damage in concert with FANCD2. interacts with FANCD2, a protein involved in a BRCA1-mediated DNA repair pathway (19), and the interaction between menin and FANCD2 is enhanced by ␥-irradiation. Moreover, targeted disruption INTRODUCTION of Men1 results in increased sensitivity to DNA damage. Furthermore, MEN14 is an inherited tumor syndrome characterized by tumors in menin is localized to chromatin and NM, and the association with NM ␥ multiple endocrine organs including the parathyroids, pancreatic is- is enhanced by -irradiation. Thus, menin may play a critical role in lets, and the pituitary (1–4). The gene mutated in MEN1 patients, repair of DNA damage in concert with FANCD2. Men1, was identified by positional cloning and mapped to chromo- some 11 (5). The product of Men1, menin, encodes a protein of 610 MATERIALS AND METHODS amino acid residues (5). However, there are no paralogs throughout Plasmid Construction. Oligonucleotides were synthesized by Integrated Ͼ various genomes. Thus far, 300 individual mutations in Men1 have DNA Technologies, Inc. To construct pcDNA3-F-menin and other constructs been identified (4). The tumors from patients carrying a germ-line expressing various fragments of menin, human cDNA (20) was amplified by mutation in Men1 often lose the normal allele of Men1. Mice het- PCR and cloned into the BamHI/NotI site of pcDNA3-Flag with two repeats of erozygous for Men1 knockout have been found recently to develop a Flag epitope (21). Human menin cDNA was also cloned into the BamHI and tumors in the parathyroids, ␤-islets, and the pituitary (6), closely NotI sites of retroviral vector pMX-puro to generate retroviruses expressing mimicking the human MEN1 syndrome. Ectopic expression of menin menin as described previously (22). inhibits proliferation of Ras-transformed NIH-3T3 cells (7). Menin is Cell Lines, Tissue Culture, and Transfection. TGP61 cells, a murine a nuclear protein containing two nuclear localization signals in the insulinoma cell line, HeLa cells, and NIH 3T3 cells were purchased from American Type Culture Collection. HEK 293 and HeLa S3 cells were cultured COOH terminus (8), but its subnuclear distribution is not yet clear. in DMEM supplemented with 10% (v/v) FCS, penicillin (100 units/ml), and Menin interacts with a number of transcriptional factors such as streptomycin (100 ␮g/ml) as described previously (23). TGP61 cells were ␬ JunD and NF B and, based on luciferase reporter gene assays, inhibits cultured in 45% DMEM and 45% Ham’s F12 medium supplemented with 10% their activities in gene transcription (9, 10). Menin has also been (v/v) FCS, penicillin (100 units/ml), and streptomycin (100 units/ml). The cells shown to interact with Smad3, a signal transducer downstream of the were transfected using the calcium phosphate precipitation method as de- transforming growth factor ␤ signaling pathway and is involved in scribed previously (21). transforming growth factor ␤-induced gene transcription (11). Inhibi- Western Blotting and Immunoprecipitation. Proteins in cell lysates or in tion of NF␬B or activation of Smad3 by menin may lead to inhibition various cellular fractions were separated on SDS-PAGE gels, transferred to of cell proliferation. Replication protein A2, a subunit of a trimeric polyvinylidene difluoride membranes (Millipore), and blotted with various primary antibodies. The blotted proteins were visualized using the enhanced chemiluminescence detection system (Amersham-Pharmacia Biotech). To im- Received 11/1/02; accepted 5/13/03. munoprecipitate menin, cells were lysed in lysis buffer [50 mM HEPES (pH 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 7.5), 0.15 M NaCl, 0.5% NP40, 1 mM EDTA, 1 mM EGTA, 1 mM glycero- 18 U.S.C. Section 1734 solely to indicate this fact. phosphate, 0.5 mM vanadate, and 10% glycerol] supplemented with 1 mM 1 Supported in part by a Howard Temin Award (1K01CA78592) and a Burroughs phenylmethylsulfonyl fluoride and 4 ␮g/ml protease inhibitor mixture (Cal- Wellcome Career Award for Biomedical Research (#1676; to X. H.). 2 biochem). The lysates were incubated with antimenin antibody (#80) or with These authors contributed equally to this work. ␣ 3 To whom requests for reprints should be addressed, at University of Pennsylvania agarose beads coupled with -Flag M2 (Sigma) at 4°C for 2 h. For immuno- Cancer Center, 412 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6160. precipitation with the antimenin antibody, the lysates were additionally incu- Phone: (215) 746-5565; Fax: (215) 746-5525; E-mail [email protected]. bated with the antirabbit IgG secondary antibody, followed with agarose beads 4 ␬ The abbreviations used are: MEN1, multiple endocrine neoplasia type I; NF B, coupled with protein G (Amersham-Pharmacia Biotech) at 4°C for 3 h. nuclear factor ␬B; DEB, diepoxybutane; LOH, loss of heterozygosity; NM, nuclear matrix; HEK, human embryonic kidney; MEF, mouse embryonic fibroblast; MMC, Isolation of Menin-interacting Proteins. HEK 293 cells transfected with mitomycin C; NHS, N-hydroxysuccinimide; FA, Fanconi anemia. vector or pcDNA3-F-menin were lysed in the high salt lysis buffer [50 mM 4204 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2003 American Association for Cancer Research. TUMOR SUPPRESSOR MENIN INTERACTS WITH FANCD2 HEPES (pH 7.5), 420 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% NP40, 0.3% in urea buffer [8 M urea, 100 mM NaH2PO4, and 10 mM Tris (pH 8.0)] to obtain Triton X-100, 1 mM ␤-glycerophosphate, 0.5 mM vanadate, 0.5 mM phenyl- the NM fraction. methylsulfonyl fluoride, 4 ␮g/ml of aprotinin, pepstatin A, and leupeptin] by Dounce homogenization. The samples were diluted with the above lysis buffer RESULTS without NaCl to reach a final concentration of 150 mM NaCl. The diluted samples were centrifuged at 105 ϫ g, and the supernatant was incubated with Menin Interacts with FANCD2. To identify menin-interacting 100 ␮l of the equilibrated agarose beads coupled with the anti-Flag antibody proteins that might shed light on the biochemical function of menin, (M2; Sigma) at 4°C for 16 h. The beads were thoroughly washed and then we first stably transfected 293 cells with either vector DNA or a ␮ incubated with 150 l of the elution buffer (the lysis buffer with 150 mM NaCl construct expressing a Flag-tagged menin. Expression of the tagged and 0.5 mg/ml Flag peptide from Sigma) at 4°C for 20 min. The purified menin in the cells was confirmed by Western blotting analysis using proteins from the control cells and the cells expressing F-menin were separated an anti-Flag antibody (Fig. 1A). Cell lysates from the control cells and by 6% SDS-PAGE, stained with a Novex Colloid Coomassie Staining kit (Invitrogen), and excised under a sterile hood. The peptide sequences from the cells expressing the epitope-tagged menin were prepared and then these proteins were identified using mass spectrometry at the Harvard Micro- incubated with agarose beads covalently conjugated to the anti-Flag chemistry Facility. antibody. The tagged menin and its associating proteins were sepa- Isolation of Men1؊/؊ MEFs and Complementation of the Cells with rated by SDS-PAGE and stained with a sensitive silver staining Menin.
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