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• W2170291310 abstract "LMO4 belongs to the LIM-only (LMO) group of transcriptional regulators that appear to function as molecular adaptors for protein-protein interactions. Expression of the LMO4 gene is developmentally regulated in the mammary gland and is up-regulated in primary breast cancers. Using LMO4 in a yeast two-hybrid screen, we have identified the cofactor CtIP as an LMO4-binding protein. Interaction with CtIP appeared to be specific for the LMO subclass of LIM domain proteins and could be mediated by a single LIM motif of LMO4. We further identified the breast tumor suppressor BRCA1 as an LMO4-associated protein. The C-terminal BRCT domains of BRCA1, previously shown to bind CtIP, also mediated interaction with LMO4. Tumor-associated mutations within the BRCT repeats that abolish interaction between BRCA1 and CtIP had no effect on the association of BRCA1 with LMO4. A stable complex comprising LMO4, BRCA1, and CtIP was demonstrated in vivo. The LIM domain binding-protein Ldb1 also participated in this multiprotein complex. In functional assays, LMO4 was shown to repress BRCA1-mediated transcriptional activation in both yeast and mammalian cells. These findings reveal a novel complex between BRCA1, LMO4, and CtIP and indicate a role for LMO4 as a repressor of BRCA1 activity in breast tissue. LMO4 belongs to the LIM-only (LMO) group of transcriptional regulators that appear to function as molecular adaptors for protein-protein interactions. Expression of the LMO4 gene is developmentally regulated in the mammary gland and is up-regulated in primary breast cancers. Using LMO4 in a yeast two-hybrid screen, we have identified the cofactor CtIP as an LMO4-binding protein. Interaction with CtIP appeared to be specific for the LMO subclass of LIM domain proteins and could be mediated by a single LIM motif of LMO4. We further identified the breast tumor suppressor BRCA1 as an LMO4-associated protein. The C-terminal BRCT domains of BRCA1, previously shown to bind CtIP, also mediated interaction with LMO4. Tumor-associated mutations within the BRCT repeats that abolish interaction between BRCA1 and CtIP had no effect on the association of BRCA1 with LMO4. A stable complex comprising LMO4, BRCA1, and CtIP was demonstrated in vivo. The LIM domain binding-protein Ldb1 also participated in this multiprotein complex. In functional assays, LMO4 was shown to repress BRCA1-mediated transcriptional activation in both yeast and mammalian cells. These findings reveal a novel complex between BRCA1, LMO4, and CtIP and indicate a role for LMO4 as a repressor of BRCA1 activity in breast tissue. LIM-only hemagglutinin activation domain DNA-binding domain glutathione S-transferase chloramphenicol acetyltransferase BRCA1 C-terminal The LIM domain is characterized by a double zinc finger structure found in proteins that have critical functions in cell fate determination, growth control, and cytoskeleton organization (reviewed in Refs. 1Sanchez-Garcia I. Rabbitts T.H. Trends Genet. 1994; 10: 315-320Abstract Full Text PDF PubMed Scopus (323) Google Scholar, 2Dawid I.B. Breen J.J. Toyama R. Trends Genet. 1998; 14: 156-162Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar, 3Bach I. Mech. Dev. 2000; 91: 5-17Crossref PubMed Scopus (486) Google Scholar, 4Jurata L.W. Gill G.N. Curr. Top. Microbiol. Immunol. 1998; 228: 75-113PubMed Google Scholar). This motif was originally identified in LIM homeodomain transcription factors and subsequently found in a variety of nuclear and cytoplasmic proteins including LIM-only (LMO),1 LIM kinase, and focal adhesion proteins. In these proteins, there are usually two or more LIM domains, which may occur in association with functionally divergent domains or by themselves, where they constitute the majority of the protein (1Sanchez-Garcia I. Rabbitts T.H. Trends Genet. 1994; 10: 315-320Abstract Full Text PDF PubMed Scopus (323) Google Scholar, 2Dawid I.B. Breen J.J. Toyama R. Trends Genet. 1998; 14: 156-162Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar, 3Bach I. Mech. Dev. 2000; 91: 5-17Crossref PubMed Scopus (486) Google Scholar, 4Jurata L.W. Gill G.N. Curr. Top. Microbiol. Immunol. 1998; 228: 75-113PubMed Google Scholar). The LIM domain functions primarily as a module for the assembly of protein complexes. There is no evidence to suggest that the LIM domain binds DNA, despite possessing similarity to the GATA-1 zinc finger motifs. The LMO subclass of LIM proteins comprises four members (LMO1–4), each of which is defined by two tandem LIM domains (1Sanchez-Garcia I. Rabbitts T.H. Trends Genet. 1994; 10: 315-320Abstract Full Text PDF PubMed Scopus (323) Google Scholar, 5Rabbitts T.H. Genes Dev. 1998; 12: 2651-2657Crossref PubMed Scopus (145) Google Scholar). These regulatory molecules appear to have essential functions in cell proliferation and lineage determination. LMO1 and LMO2, both translocated in acute T cell leukemia (T-ALL), are oncogenic within T cells (5Rabbitts T.H. Genes Dev. 1998; 12: 2651-2657Crossref PubMed Scopus (145) Google Scholar). LMO2 has been demonstrated to have a central role in hematopoiesis where it is required for the development of all cell lineages (6Yamada Y. Warren A.J. Dobson C. Forster A. Pannell R. Rabbitts T.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3890-3895Crossref PubMed Scopus (266) Google Scholar). Furthermore, LMO2 has been established to form a multiprotein complex with the hematopoietic transcription factors SCL/TAL-1 and GATA-1 (7Valge-Archer V.E. Osada H. Warren A.J. Forster A. Li J. Baer R. Rabbitts T.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8617-8621Crossref PubMed Scopus (206) Google Scholar, 8Wadman I. Li J. Bash R.O. Forster A. Osada H. Rabbitts T.H. Baer R. EMBO J. 1994; 13: 4831-4839Crossref PubMed Scopus (219) Google Scholar, 9Wadman I.A. Osada H. Grutz G.G. Agulnick A.D. Westphal H. Forster A. Rabbitts T.H. EMBO J. 1997; 16: 3145-3157Crossref PubMed Scopus (738) Google Scholar). These findings indicate a close functional relationship between LMO proteins and DNA-binding factors in blood cells. LMO4, the most recently described member, was isolated by virtue of its interaction with the ubiquitous adaptor protein Ldb1/NL1/CLIM2 (10Agulnick A.D. Taira M. Breen J.J. Tanaka T. Dawid I.B. Westphal H. Nature. 1996; 384: 270-272Crossref PubMed Scopus (294) Google Scholar, 11Jurata L.W. Kenny D.A. Gill G.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11693-11698Crossref PubMed Scopus (212) Google Scholar, 12Bach I. Carriere C. Ostendorff H.P. Andersen B. Rosenfeld M.G. Genes Dev. 1997; 11: 1370-1380Crossref PubMed Scopus (269) Google Scholar, 13Visvader J.E. Mao X. Fujiwara Y. Hahm K. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13707-13712Crossref PubMed Scopus (138) Google Scholar) and in an expression screen with autologous serum from a breast cancer patient (14Kenny D.A. Jurata L.W. Saga Y. Gill G.N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11257-11262Crossref PubMed Scopus (86) Google Scholar, 15Sugihara T.M. Bach I. Kioussi C. Rosenfeld M.G. Andersen B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15418-15423Crossref PubMed Scopus (85) Google Scholar, 16Grutz G. Forster A. Rabbitts T.H. Oncogene. 1998; 17: 2799-2803Crossref PubMed Scopus (65) Google Scholar, 17Racevskis J. Dill A. Sparano J.A. Ruan H. Biochim. Biophys. Acta. 1999; 1445: 148-153Crossref PubMed Scopus (43) Google Scholar). LMO4 is the most divergent member of the LMO subfamily, sharing only 50% homology with the LIM domains of other LMO proteins. The LMO4 gene is widely expressed in embryonic and adult tissues, but high levels are restricted to specific cell types (14Kenny D.A. Jurata L.W. Saga Y. Gill G.N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11257-11262Crossref PubMed Scopus (86) Google Scholar, 15Sugihara T.M. Bach I. Kioussi C. Rosenfeld M.G. Andersen B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15418-15423Crossref PubMed Scopus (85) Google Scholar,17Racevskis J. Dill A. Sparano J.A. Ruan H. Biochim. Biophys. Acta. 1999; 1445: 148-153Crossref PubMed Scopus (43) Google Scholar). We have recently established that the LMO4 gene is highly expressed in the proliferating mammary gland during pregnancy and that it is overexpressed in greater than 50% of primary breast cancers (18Visvader J.E. Venter D. Hahm K. Santamaria M. Sum E.Y.M. O'Reilly L. White D. Williams R. Armes J. Lindeman G.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14452-14457Crossref PubMed Scopus (113) Google Scholar). Moreover, high levels of LMO4 were found to inhibit mammary differentiation (18Visvader J.E. Venter D. Hahm K. Santamaria M. Sum E.Y.M. O'Reilly L. White D. Williams R. Armes J. Lindeman G.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14452-14457Crossref PubMed Scopus (113) Google Scholar). To gain insight into the mechanism by which LMO4 functions in breast epithelium, we searched for partners of LMO4 in these cells. Two interacting proteins were identified, the cofactor CtIP (CtBP-interacting protein) and the breast and ovarian tumor suppressor protein BRCA1, which has previously been shown to associate with CtIP (19Yu X. Wu L.C. Bowcock A.M. Aronheim A. Baer R. J. Biol. Chem. 1998; 273: 25388-25392Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 20Wong A.K. Ormonde P.A. Pero R. Chen Y. Lian L. Salada G. Berry S. Lawrence Q. Dayananth P. Ha P. Tavtigian S.V. Teng D.H. Bartel P.L. Oncogene. 1998; 17: 2279-2285Crossref PubMed Scopus (137) Google Scholar, 21Li S. Chen P.L. Subramanian T. Chinnadurai G. Tomlinson G. Osborne C.K. Sharp Z.D. Lee W.H. J. Biol. Chem. 1999; 274: 11334-11338Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). A multiprotein complex involving LMO4, CtIP, and BRCA1 could be demonstrated in vivo. LMO4 was found to be a repressor of BRCA1-mediated transcriptional activity, invoking a potential role for LMO4 as a negative regulator of BRCA1 function in sporadic breast cancers. The pGBT9-LMO4 bait plasmid was generated by PCR amplification of mouse LMO4 in pSP72 using the following primers: forward, 5′-CGCGGATCCCCGGCTCCCTCTCCTGGAAGCGCTGC-3′ and reverse, 5′-CGCGGATCCTCAGCAGACCTTCTGGTCTGCCAG-3′; the resultant product was inserted into the BamHI site of pGBT9 (CLONTECH). The first LIM domain (residues 1–82) of LMO4 was PCR-amplified using the forward primer (5′-CGCGGATCCTGAATCCGGGCAGCAGCTCGC-3′) and the reverse primer (5′-CGCGGATCCTCACCCAAATAACCTAATGTAGTCATT-3′), then cloned into theBamHI site of the FLAG-pEF1α-puro vector (22Huang D.C. Cory S. Strasser A. Oncogene. 1997; 14: 405-414Crossref PubMed Scopus (231) Google Scholar). The second LIM domain (residues 79–165) of LMO4 was PCR-amplified using the forward primer (5′-CGCGGATCCGGTTATTTGGGAATAGCGGTGCTTG-3′) and the reverse primer (5′-CGCGGATCCTCAGCAGACCTTCTGGTCTGCCAG-3′) and subsequently cloned into the BamHI site of the FLAG-pEF1α-puro vector. Expression vectors encoding Lhx1 or Lhx3 (pSV-sport-FLAG) and LMK1 were kindly provided by A. Agulnick and O. Bernard, respectively. Full-length cDNAs corresponding to the coding region of mouse or human LMO4 were cloned into the FLAG-pEF1α-puro expression vector, and a 0.7-kb mouse LMO4 cDNA fragment was cloned into pEF1α-puro vector. The expression plasmids encoding residues 45–897 of human CtIP (pCMV-HA-ns311), human BRCA1 (pcDNA3-HA-BRCA1), and mouse LMO2 (pEF1α-FLAG-LMO2) have been described previously (13Visvader J.E. Mao X. Fujiwara Y. Hahm K. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13707-13712Crossref PubMed Scopus (138) Google Scholar, 19Yu X. Wu L.C. Bowcock A.M. Aronheim A. Baer R. J. Biol. Chem. 1998; 273: 25388-25392Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 23Scully R. Chen J. Plug A. Xiao Y. Weaver D. Feunteun J. Ashley T. Livingston D.M. Cell. 1997; 88: 265-275Abstract Full Text Full Text PDF PubMed Scopus (1329) Google Scholar). HA-tagged CtIP deletion mutants were generated either by PCR amplification or subcloning from plasmids (19Yu X. Wu L.C. Bowcock A.M. Aronheim A. Baer R. J. Biol. Chem. 1998; 273: 25388-25392Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar), into the expression vectors HA-pcDNA3.1 or HA-EF1α-puro. The SZ fragment of BRCA1 (residues 1528–1863) was recloned from pCMV-Gal4b-BR-SZ (19Yu X. Wu L.C. Bowcock A.M. Aronheim A. Baer R. J. Biol. Chem. 1998; 273: 25388-25392Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar) into HA-pcDNA3.1. Myc-tagged derivatives of wild-type and mutant BRCA1 were cloned into pcDNA3.0. 2J. Chen, unpublished data. The yeast expression construct encoding the activation domain (AD) of BRCA1 fused to the Gal4 DNA-binding domain (DBD) was generated by PCR amplification of the region spanning amino acids 1293–1863 of BRCA1 and subsequent cloning into pGBT9 (CLONTECH). The mammalian Gal4DBD-AD fusion construct was generated by cloning a cDNA fragment encoding the BRCA1-AD region into pCMV-Gal4b (19Yu X. Wu L.C. Bowcock A.M. Aronheim A. Baer R. J. Biol. Chem. 1998; 273: 25388-25392Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Full-length cDNA encoding mouse LMO4 was inserted into the yeast expression plasmid pYX212 (Ingenious). The reporter plasmid, pG5CAT, was from CLONTECH. Details of plasmid constructions are available on request. The pGBT9-LMO4 bait plasmid (residues 15–165) was used to screen 8.4 × 106transformants from a primary breast adenocarcinoma cDNA library (24Byrne J.A. Nourse C.R. Basset P. Gunning P. Oncogene. 1998; 16: 873-881Crossref PubMed Scopus (71) Google Scholar), following standard protocols (CLONTECH Matchmaker Two-Hybrid System). Full-length mouse LMO4 cDNA was amplified by PCR and subcloned into pGEX-2T (Amersham Biosciences, Inc.). The GST-LMO4 fusion protein was expressed in the bacterial strain UT5600, purified according to standard protocols (25Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5057) Google Scholar), and used to inject rabbits to produce polyclonal antisera. The generation of rat LMO4-specific monoclonal antibodies will be described elsewhere. 3E. Y. M. Sum, G. J. Lindeman, and J. E. Visvader, unpublished data. Human embryonal kidney 293T cells (10-cm plates) were transiently transfected with 4 μg of each expression construct and/or empty vector using the calcium phosphate precipitation method. Cell extracts (0.5 ml) were prepared in whole cell lysis buffer (150 mm NaCl, 5 mm EDTA, 50 mm Tris, pH 7.5, 1% Nonidet P-40, 1 mm dithiothreitol) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin) and normalized for protein concentration, as determined by Bradford analysis (Bio-Rad). Proteins were immunoprecipitated with either anti-FLAG M2 (Sigma) or anti-Myc 9E10 and protein G-Sepharose (Amersham Biosciences, Inc.) and separated by SDS-PAGE (Novex). After transfer to polyvinylidene difluoride membranes (Millipore), filters were blocked and incubated with rabbit antisera against CtIP or Ldb1 (13Visvader J.E. Mao X. Fujiwara Y. Hahm K. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13707-13712Crossref PubMed Scopus (138) Google Scholar) or mouse anti-BRCA1 monoclonal antibody MS110 (Oncogene Research Products). Filters were then incubated with horseradish peroxidase-coupled secondary antibodies and developed by ECL (Amersham Biosciences, Inc.). For detection of endogenous proteins, nuclear extracts from human HBL100 cells were prepared (26Watters D. Khanna K.K. Beamish H. Birrell G. Spring K. Kedar P. Gatei M. Stenzel D. Hobson K. Kozlov S. Zhang N. Farrell A. Ramsay J. Gatti R. Lavin M. Oncogene. 1997; 14: 1911-1921Crossref PubMed Scopus (170) Google Scholar). Proteins were immunoprecipitated with either anti-LMO4 rabbit antisera or a rat anti-LMO4 monoclonal antibody and protein G-Sepharose and fractionated by SDS-PAGE. After transfer, filters were blocked and incubated with mouse monoclonal antibodies to either CtIP (14.1) (27Yu X. Baer R. J. Biol. Chem. 2000; 275: 18541-18549Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) or BRCA1 (MS110, Oncogene Research Products), followed by incubation with horseradish peroxidase-coupled secondary antibodies, and developed by ECL (Amersham Biosciences, Inc.). In vitro synthesis of35S-labeled BRCA1 (C-terminal residues 1528–1863) was performed by in vitro transcription/translation of HA-pCDNA3.1-BRCA1-SZ using the TNT T7-coupled reticulocyte lysate system (Promega). Binding assays were carried out with a 10-μl aliquot of 35S-labeled BRCA1-SZ primed lysate and 100 μl (50% slurry of GST-Sepharose beads; Amersham Pharmacia) of GST-LMO4 or GST-only protein in GST interaction buffer (150 mm NaCl, 10 mm Tris, pH 8, 0.3% Nonidet P-40, 1 mmdithiothreitol, 0.25% bovine serum albumin, and 0.5 mmphenylmethylsulfonyl fluoride) for 2 h at 4 °C. The beads were subsequently washed twice in GST interaction buffer containing bovine serum albumin, followed by two more washes in GST interaction buffer without bovine serum albumin. Finally, the bound BRCA1 C-terminal polypeptide was eluted by boiling the beads for 5 min in 30 μl of loading buffer and analyzed by SDS-PAGE. Poly(A)+ RNA was isolated from human and mouse breast epithelial cell lines cited in previous studies (28Douglas A.M. Grant S.L. Goss G.A. Clouston D.R. Sutherland R.L. Begley C.G. Int. J. Cancer. 1998; 75: 64-73Crossref PubMed Scopus (62) Google Scholar), and Northern analysis was performed (29Visvader J.E. Elefanty A.G. Strasser A. Adams J.M. EMBO J. 1992; 11: 4557-4564Crossref PubMed Scopus (163) Google Scholar). The yeast transcription assay was performed in the yeast strain BJ5462; this was cotransformed with the LacZ reporter plasmid, YepΔ62 (generously provided by P. Vaughan), and pGBT9-BRCA1(AD), and colonies selected on media deficient in Leu and Trp. These transformants were then additionally transformed with either the pYX212-LMO4 expression plasmid or empty vector and were selected on media lacking Ura, Trp, and Leu. β-Galactosidase activities were determined using theo-nitrophenyl-β-d-galactoside liquid culture assay following standard protocol (CLONTECH Yeast Protocols Handbook). Kidney embryonal 293T cells were transiently transfected (six-well plates) with the indicated plasmids: 0.5 μg of pG5CAT (CLONTECH), 1 μg of pCMV-Gal4b-BRCA1-AD, or 1 μg of Gal4b parental vector, and either 2 μg of FLAG-EF1α-LMO4 or 2 μg of empty control vector using the calcium phosphate precipitation method. CAT activity was determined using the CAT enzyme-linked immunosorbent assay system (Roche Molecular Biochemicals) and was normalized against protein concentration, as determined by the Bradford assay (Bio-Rad). We used the yeast two-hybrid system to identify LMO4-interacting proteins in breast epithelium. A screen of 8.4 × 106transformants of a primary breast adenocarcinoma cDNA library yielded more than 800 His+ colonies. Six-hundred and fifty-nine β-galactosidase-positive clones were isolated and sequentially screened by yeast colony hybridization using cDNA probes representing the known LMO4-associated proteins, Ldb1 and deformed epidermal autoregulatory factor (DEAF1) (14Kenny D.A. Jurata L.W. Saga Y. Gill G.N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11257-11262Crossref PubMed Scopus (86) Google Scholar, 15Sugihara T.M. Bach I. Kioussi C. Rosenfeld M.G. Andersen B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15418-15423Crossref PubMed Scopus (85) Google Scholar, 16Grutz G. Forster A. Rabbitts T.H. Oncogene. 1998; 17: 2799-2803Crossref PubMed Scopus (65) Google Scholar), and known false positives. Approximately 60% of these clones corresponded to Ldb1/Ldb2 (30%) or DEAF1 (30%). Of 150 cDNA clones sequenced, 22 were found to correspond to either Ldb1 or Ldb2, 20 encoded DEAF1, at least 70 corresponded to false positives (e.g. ribosomal, mitochondrial, and extracellular matrix proteins), while the remaining 38 cDNAs represented 9 distinct genes or expressed sequence tags. One of these clones corresponded to the complete coding sequence of CtIP (CtBP-interacting protein), which encodes a cofactor originally identified on the basis of its interaction with the transcriptional corepressor CtBP (adenovirus E1A C-terminal-binding protein) (30Schaeper U. Subramanian T. Lim L. Boyd J.M. Chinnadurai G. J. Biol. Chem. 1998; 273: 8549-8552Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). A specific interaction between CtIP and LMO4 was confirmed in mammalian cells. Expression vectors encoding LMO4, LMO2, or heterologous LIM domain proteins, each carrying a FLAG or Myc epitope at the N terminus, together with an expression vector harboring CtIP (residues 45–897), were transiently transfected into 293T cells. Whole cell extracts were analyzed using a coupled coimmunoprecipitation/Western blot assay. As shown in Fig. 1 A, FLAG-LMO4 and CtIP were found to specifically associate in vivo, in reciprocal coimmunoprecipitation experiments (lanes 1 and4). CtIP was not detected in immunoprecipitates from cells expressing FLAG-LMO4 alone (lane 3) or in those using an isotype-matched control antibody. A single LIM domain of LMO4 (amino acids 1–82) was found to be sufficient to mediate interaction with CtIP in mammalian cells (Fig. 1 B, lane 1). Only the first LIM domain of LMO4, but not the second LIM domain (amino acids 79–165), could associate with CtIP (lane 2). CtIP also coprecipitated with the related LIM domain protein, LMO2 (5Rabbitts T.H. Genes Dev. 1998; 12: 2651-2657Crossref PubMed Scopus (145) Google Scholar) (Fig.1 A, lane 2), but not with the nuclear LIM homeodomain proteins Lhx1 and Lhx3 (Fig. 1 C, lanes 2 and 3), nor with LIM kinase (LMK1) (Fig.1 C, lane 4). To test the interaction between endogenous proteins in breast epithelial cells, we generated a rabbit polyclonal antiserum against full-length mouse LMO4. This antiserum recognizes a protein of about 17 kDa in breast epithelial cells and in cells transfected with an LMO4 expression vector. Nuclear extracts from HBL100 cells were immunoprecipitated with anti-LMO4 antiserum (Fig. 1 D,lane 2) or preimmune serum (lane 1). Immunoblotting with a mouse anti-CtIP monoclonal antibody revealed a specific band of 125 kDa (Fig. 1 D), thus confirming thein vivo association between native CtIP and LMO4 proteins. The function of CtIP is not known but it appears to serve as a cofactor for several nuclear regulatory proteins, including BRCA1, adenovirus E1A C-terminal-binding protein (CtBP), retinoblastoma, and p130 pocket proteins (19Yu X. Wu L.C. Bowcock A.M. Aronheim A. Baer R. J. Biol. Chem. 1998; 273: 25388-25392Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 20Wong A.K. Ormonde P.A. Pero R. Chen Y. Lian L. Salada G. Berry S. Lawrence Q. Dayananth P. Ha P. Tavtigian S.V. Teng D.H. Bartel P.L. Oncogene. 1998; 17: 2279-2285Crossref PubMed Scopus (137) Google Scholar, 21Li S. Chen P.L. Subramanian T. Chinnadurai G. Tomlinson G. Osborne C.K. Sharp Z.D. Lee W.H. J. Biol. Chem. 1999; 274: 11334-11338Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 30Schaeper U. Subramanian T. Lim L. Boyd J.M. Chinnadurai G. J. Biol. Chem. 1998; 273: 8549-8552Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 31Fusco C. Reymond A. Zervos A.S. Genomics. 1998; 51: 351-358Crossref PubMed Scopus (59) Google Scholar, 32Meloni A.R. Smith E.J. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9574-9579Crossref PubMed Scopus (167) Google Scholar). In addition to the defined regions that interact with these proteins, CtIP contains two potential leucine zipper domains (amino acids 120–141 and 740–761) (31Fusco C. Reymond A. Zervos A.S. Genomics. 1998; 51: 351-358Crossref PubMed Scopus (59) Google Scholar), depicted in Fig. 2 A. To delineate the domains within CtIP that mediate interaction with LMO4, a series of CtIP deletion mutants (Fig. 2 A), each linked to an N-terminal HA-epitope tag, were cotransfected with FLAG-LMO4 into 293T epithelial cells. As shown in Fig. 2 B, HA-CtIP (amino acids 45–371), HA-CtIP (amino acids 371–897), HA-CtIP (amino acids 45–897), and HA-CtIP (amino acids 620–897) proteins could associate with FLAG-LMO4 (lanes 2, 4, 5, and6). In contrast, neither the HA-CtIP (amino acids 59–320) nor HA-CtIP (amino acids 281–620) mutants were immunoprecipitable by an anti-FLAG antibody (lanes 1 and 3, respectively). Thus, there are apparently two regions within CtIP that can mediate interaction with LMO4: a small domain at the N terminus (residues 45–59) and a C-terminal region (residues 620–897), which encompasses a putative leucine zipper motif (Fig. 2 A). These regions are distinct from those that associate with CtBP (amino acids 392–396), retinoblastoma (amino acids 153–157), and BRCA1 (amino acids 133–369) (19Yu X. Wu L.C. Bowcock A.M. Aronheim A. Baer R. J. Biol. Chem. 1998; 273: 25388-25392Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 27Yu X. Baer R. J. Biol. Chem. 2000; 275: 18541-18549Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 30Schaeper U. Subramanian T. Lim L. Boyd J.M. Chinnadurai G. J. Biol. Chem. 1998; 273: 8549-8552Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 31Fusco C. Reymond A. Zervos A.S. Genomics. 1998; 51: 351-358Crossref PubMed Scopus (59) Google Scholar, 32Meloni A.R. Smith E.J. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9574-9579Crossref PubMed Scopus (167) Google Scholar). Both the LMO4 and CtIP genes have been reported to be expressed in a number of different tissues and cell types (14Kenny D.A. Jurata L.W. Saga Y. Gill G.N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11257-11262Crossref PubMed Scopus (86) Google Scholar, 15Sugihara T.M. Bach I. Kioussi C. Rosenfeld M.G. Andersen B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15418-15423Crossref PubMed Scopus (85) Google Scholar, 20Wong A.K. Ormonde P.A. Pero R. Chen Y. Lian L. Salada G. Berry S. Lawrence Q. Dayananth P. Ha P. Tavtigian S.V. Teng D.H. Bartel P.L. Oncogene. 1998; 17: 2279-2285Crossref PubMed Scopus (137) Google Scholar). We surveyed their expression in a panel of breast epithelial cell lines, the majority of which were derived from human breast cancers but also included immortalized human cells (184). Northern analysis revealed CtIP RNA (3.6 kb) levels were relatively uniform, while expression of the LMO4 transcripts (1.8 and 2.3 kb) varied dramatically (Fig. 3). High levels of LMO4 were apparent in a number of human breast cancer cell lines, including BT-549, BT-474, HS-578T, MDA-MB361, T-47D, and ZR-75B (Fig. 3), relative to the low levels evident in the immortalized 184 cells, as we reported recently (18Visvader J.E. Venter D. Hahm K. Santamaria M. Sum E.Y.M. O'Reilly L. White D. Williams R. Armes J. Lindeman G.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14452-14457Crossref PubMed Scopus (113) Google Scholar). In human breast cancers, overexpression of the LMO4 gene has been observed at both the RNA and protein levels (18Visvader J.E. Venter D. Hahm K. Santamaria M. Sum E.Y.M. O'Reilly L. White D. Williams R. Armes J. Lindeman G.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14452-14457Crossref PubMed Scopus (113) Google Scholar). Since CtIP was recently demonstrated to interact with the breast tumor suppressor BRCA1 (19Yu X. Wu L.C. Bowcock A.M. Aronheim A. Baer R. J. Biol. Chem. 1998; 273: 25388-25392Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 20Wong A.K. Ormonde P.A. Pero R. Chen Y. Lian L. Salada G. Berry S. Lawrence Q. Dayananth P. Ha P. Tavtigian S.V. Teng D.H. Bartel P.L. Oncogene. 1998; 17: 2279-2285Crossref PubMed Scopus (137) Google Scholar, 21Li S. Chen P.L. Subramanian T. Chinnadurai G. Tomlinson G. Osborne C.K. Sharp Z.D. Lee W.H. J. Biol. Chem. 1999; 274: 11334-11338Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), we investigated whether LMO4, CtIP, and BRCA1 could participate in a multiprotein complex. Both CtIP and LMO4 were coimmunoprecipitated with an anti-Myc antibody from 293T cells expressing Myc-tagged BRCA1, FLAG-tagged LMO4 and CtIP (Fig. 4 A, lane 2). This finding revealed that all three proteins can form a stable multiprotein complex in vivo. The presence of exogenous CtIP was not necessary for immunoprecipitation of LMO4 by the anti-Myc antibody, as shown in Fig. 4 A (lane 1). These results raised the possibility that LMO4 might directly associate with BRCA1 (see below). To further examine this interaction in epithelial cells, a rat anti-LMO4 monoclonal antibody was used to immunoprecipitate proteins from HBL100-derived nuclear extracts. This antibody specifically recognizes a 17-kDa protein in cells transfected with a LMO4 expression vector but not in those lacking LMO4. Endogenous BRCA1 was immunoprecipitated by the anti-LMO4 monoclonal antibody (Fig.4 B, lane 2), but not with a control antibody (Fig. 4 B, lane 3). This result confirms anin vivo association between LMO4 and BRCA1 and, moreover, demonstrates that this interaction occurs between native proteins in epithelial cells. Immunoblotting of the anti-LMO4 immunoprecipitated protein with anti-CtIP monoclonal antibody yielded a faint band of 125 kDa, corresponding to CtIP (Fig. 4 B, middle panel), while blotting with anti-LMO4 antibody gave rise to the expected 17-kDa LMO4 protein (Fig. 4 B, lower panel). Thus LMO4, BRCA1, and CtIP have the potential to form a native complex in vivo. We investigated whether the nuclear adaptor protein Ldb1, which binds LMO4 and other LIM proteins with high affinity (14Kenny D.A. Jurata L.W. Saga Y. Gill G.N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11257-11262Crossref PubMed Scopus (86) Google Scholar, 15Sugihara T.M. Bach I. Kioussi C. Rosenfeld M.G. Andersen B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15418-15423Crossref PubMed Scopus (85) Google Scholar, 16Grutz G. Forster A. Rabbitts T.H. Oncogene. 1998; 17:" @default.
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• W2170291310 cites W1489499258 @default.
• W2170291310 cites W1512321076 @default.
• W2170291310 cites W1966034415 @default.
• W2170291310 cites W1966479600 @default.
• W2170291310 cites W1972710550 @default.
• W2170291310 cites W1975356481 @default.
• W2170291310 cites W1977035912 @default.
• W2170291310 cites W1977860917 @default.
• W2170291310 cites W1979251604 @default.
• W2170291310 cites W1980344333 @default.
• W2170291310 cites W1982746675 @default.
• W2170291310 cites W1986625228 @default.
• W2170291310 cites W1986926802 @default.
• W2170291310 cites W199379996 @default.
• W2170291310 cites W1996635873 @default.
• W2170291310 cites W2000054590 @default.
• W2170291310 cites W2000437208 @default.
• W2170291310 cites W2004945062 @default.
• W2170291310 cites W2006136518 @default.
• W2170291310 cites W2011360841 @default.
• W2170291310 cites W2011658104 @default.
• W2170291310 cites W2023320396 @default.
• W2170291310 cites W2027376329 @default.
• W2170291310 cites W2029676157 @default.
• W2170291310 cites W2030720512 @default.
• W2170291310 cites W2034485619 @default.
• W2170291310 cites W2036245489 @default.
• W2170291310 cites W2044713924 @default.
• W2170291310 cites W2047428236 @default.
• W2170291310 cites W2047871075 @default.
• W2170291310 cites W2048430104 @default.
• W2170291310 cites W2048738841 @default.
• W2170291310 cites W2050018959 @default.
• W2170291310 cites W2053003311 @default.
• W2170291310 cites W205967952 @default.
• W2170291310 cites W2060134332 @default.
• W2170291310 cites W2067689768 @default.
• W2170291310 cites W2068446924 @default.
• W2170291310 cites W2077743844 @default.
• W2170291310 cites W2078597493 @default.
• W2170291310 cites W2094798191 @default.
• W2170291310 cites W2099704043 @default.
• W2170291310 cites W2109465247 @default.
• W2170291310 cites W2109719547 @default.
• W2170291310 cites W2124533116 @default.
• W2170291310 cites W2128168784 @default.
• W2170291310 cites W2128325937 @default.
• W2170291310 cites W2138081366 @default.
• W2170291310 cites W2144400954 @default.
• W2170291310 cites W2164916497 @default.
• W2170291310 cites W2319218220 @default.
• W2170291310 cites W2329929261 @default.
• W2170291310 cites W2432429035 @default.
• W2170291310 cites W4297432809 @default.
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