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• W2047368106 abstract "The breast and ovarian cancer-specific tumor suppressor RING finger protein BRCA1 has been identified as an E3 ubiquitin (Ub) ligase through in vitro studies, which demonstrated that its RING finger domain can autoubiquitylate and monoubiquitylate histone H2A when supplied with Ub, E1, and UBC4 (E2). Here we report that the E3 ligase activity of the N-terminal 110 amino acid residues of BRCA1, which encodes a stable domain containing the RING finger, as well as that of the full-length BRCA1, was significantly enhanced by the BARD1 protein (residues 8–142), whose RING finger domain itself lacked Ub ligase activity in vitro. The results of mutagenesis studies indicate that the enhancement of BRCA1 E3 ligase activity by BARD1 depends on direct interaction between the two proteins. Using K48A and K63A Ub mutants, we found that BARD1 stimulated the formation of both Lys48- and Lys63-linked poly-Ub chains. However, the enhancement of BRCA1 autoubiquitylation by BARD1 mostly resulted in poly-Ub chains linked through Lys63, which could potentially activate biological pathways other than BRCA1 degradation. We also found that co-expression of BRCA1 and BARD1 in living cells increased the abundance and stability of both proteins and that this depended on their ability to heterodimerize. The breast and ovarian cancer-specific tumor suppressor RING finger protein BRCA1 has been identified as an E3 ubiquitin (Ub) ligase through in vitro studies, which demonstrated that its RING finger domain can autoubiquitylate and monoubiquitylate histone H2A when supplied with Ub, E1, and UBC4 (E2). Here we report that the E3 ligase activity of the N-terminal 110 amino acid residues of BRCA1, which encodes a stable domain containing the RING finger, as well as that of the full-length BRCA1, was significantly enhanced by the BARD1 protein (residues 8–142), whose RING finger domain itself lacked Ub ligase activity in vitro. The results of mutagenesis studies indicate that the enhancement of BRCA1 E3 ligase activity by BARD1 depends on direct interaction between the two proteins. Using K48A and K63A Ub mutants, we found that BARD1 stimulated the formation of both Lys48- and Lys63-linked poly-Ub chains. However, the enhancement of BRCA1 autoubiquitylation by BARD1 mostly resulted in poly-Ub chains linked through Lys63, which could potentially activate biological pathways other than BRCA1 degradation. We also found that co-expression of BRCA1 and BARD1 in living cells increased the abundance and stability of both proteins and that this depended on their ability to heterodimerize. BRCA1 is a tumor suppressor gene that is mutated in 50–90% of hereditary breast and ovarian cancers (1Futreal P.A. Liu Q. Shattuck-Eidens D. Cochran C. Harshman K. Tavtigian S. Bennett L.M. Haugen-Strano A. Swensen J. Miki Y. Eddington K. McClure M. Frye C. Weaver-Feldhaus J. Ding W. et al.Science. 1994; 266: 120-122Crossref PubMed Scopus (1137) Google Scholar, 2Alberg A.J. Helzlsouer K.J. Curr. Opin. Oncol. 1997; 9: 505-511Crossref PubMed Scopus (50) Google Scholar). The humanBRCA1 gene encodes a large protein of 1863 amino acids, which contains an N-terminal RING-finger domain and two C-terminal BRCT domains (3Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshman K. Tavtigian S. Liu Q. Cochran C. Bennett L.M. Ding W. Rosenthal J. Hussey C. Tran T. McClure M. et al.Science. 1994; 266: 66-71Crossref PubMed Scopus (5289) Google Scholar, 4Bork P. Hofmann K. Bucher P. Neuwald A.F. Altschul S.F. Koonin E.V. FASEB J. 1997; 11: 68-76Crossref PubMed Scopus (661) Google Scholar). To date, BRCA1 has been implicated in interactions with more than 20 proteins and involved in a remarkable range of cellular processes from transcriptional regulation to DNA damage repair (5Deng C.X. Brodie S.G. Bioessays. 2000; 22: 728-737Crossref PubMed Scopus (275) Google Scholar, 6Deng C.X. Scott F. Oncogene. 2000; 19: 1059-1064Crossref PubMed Scopus (133) Google Scholar, 7Scully R. Livingston D.M. Nature. 2000; 408: 429-432Crossref PubMed Scopus (554) Google Scholar, 8Welcsh P.L. Owens K.N. King M.C. Trends Genet. 2000; 16: 69-74Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 9Deng C.X. Oncogene. 2002; 21: 6222-6227Crossref PubMed Scopus (99) Google Scholar, 10Hartman A.R. Ford J.M. Nat. Genet. 2002; 32: 180-184Crossref PubMed Scopus (199) Google Scholar). It has been suggested that BRCA1 may regulate various biological pathways via a common mechanism such as chromatin remodeling (11Hu Y.F. Hao Z.L. Li R. Genes Dev. 1999; 13: 637-642Crossref PubMed Scopus (107) Google Scholar, 12Bochar D.A. Wang L. Beniya H. Kinev A. Xue Y. Lane W.S. Wang W. Kashanchi F. Shiekhattar R. Cell. 2000; 102: 257-265Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, 13Miyake T. Hu Y.F. Yu D.S. Li R. J. Biol. Chem. 2000; 275: 40169-40173Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 14Ye Q. Hu Y.F. Zhong H. Nye A.C. Belmont A.S. Li R. J. Cell Biol. 2001; 155: 911-921Crossref PubMed Scopus (176) Google Scholar). 20% of the clinically relevant mutations of BRCA1 occur within the N-terminal 100 residues, which contain the RING motif (residues 23–76) (15Brzovic P.S. Rajagopal P. Hoyt D.W. King M.C. Klevit R.E. Nat. Struct. Biol. 2001; 8: 833-837Crossref PubMed Scopus (387) Google Scholar). Recently, RING domains have been documented to have E3 ubiquitin ligase activity (16Freemont P.S. Curr. Biol. 2000; 10: R84-R87Abstract Full Text Full Text PDF PubMed Google Scholar, 17Joazeiro C.A. Weissman A.M. Cell. 2000; 102: 549-552Abstract Full Text Full Text PDF PubMed Scopus (1044) Google Scholar). The RING E3s function as adaptors to recruit substrates and a Ub-conjugating enzyme (E2), 1The abbreviations used are: E2, ubiquitin conjugating enzyme; E1, ubiquitin activating enzyme; E3, ubiquitin-protein isopeptide ligase; Ub, ubiquitin; WT, wild type; aa, amino acid(s); GST, glutathione S-transferase 1The abbreviations used are: E2, ubiquitin conjugating enzyme; E1, ubiquitin activating enzyme; E3, ubiquitin-protein isopeptide ligase; Ub, ubiquitin; WT, wild type; aa, amino acid(s); GST, glutathione S-transferase and to mediate the transfer of Ub from E2 to substrate proteins (16Freemont P.S. Curr. Biol. 2000; 10: R84-R87Abstract Full Text Full Text PDF PubMed Google Scholar, 17Joazeiro C.A. Weissman A.M. Cell. 2000; 102: 549-552Abstract Full Text Full Text PDF PubMed Scopus (1044) Google Scholar, 18Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar). Ub conjugation (ubiquitylation) is well known as a signal for protein degradation and is involved in multiple biological pathways (19Pickart C.M. Mol. Cell. 2001; 8: 499-504Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). The RING domain of BRCA1 exhibits E3 ligase activity in vitro (20Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (943) Google Scholar, 21Ruffner H. Joazeiro C.A. Hemmati D. Hunter T. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5134-5139Crossref PubMed Scopus (307) Google Scholar, 22Hashizume R. Fukuda M. Maeda I. Nishikawa H. Oyake D. Yabuki Y. Ogata H. Ohta T. J. Biol. Chem. 2001; 276: 14537-14540Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar), and all of the cancer predisposing mutations in the RING domain that have been tested inactivate BRCA1 E3 Ub ligase activity (21Ruffner H. Joazeiro C.A. Hemmati D. Hunter T. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5134-5139Crossref PubMed Scopus (307) Google Scholar, 22Hashizume R. Fukuda M. Maeda I. Nishikawa H. Oyake D. Yabuki Y. Ogata H. Ohta T. J. Biol. Chem. 2001; 276: 14537-14540Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar). Human BARD1 (BRCA1-associatedRING domain 1) encodes a protein of 777 amino acids and contains an N-terminal RING domain (residues 49–100) and two C-terminal BRCT motifs (15Brzovic P.S. Rajagopal P. Hoyt D.W. King M.C. Klevit R.E. Nat. Struct. Biol. 2001; 8: 833-837Crossref PubMed Scopus (387) Google Scholar, 23Wu L.C. Wang Z.W. Tsan J.T. Spillman M.A. Phung A. Xu X.L. Yang M.C. Hwang L.Y. Bowcock A.M. Baer R. Nat. Genet. 1996; 14: 430-440Crossref PubMed Scopus (624) Google Scholar). In living cells, BRCA1 exists mostly as a heterodimeric complex with BARD1 (23Wu L.C. Wang Z.W. Tsan J.T. Spillman M.A. Phung A. Xu X.L. Yang M.C. Hwang L.Y. Bowcock A.M. Baer R. Nat. Genet. 1996; 14: 430-440Crossref PubMed Scopus (624) Google Scholar, 24Yu X. Baer R. J. Biol. Chem. 2000; 275: 18541-18549Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The recently reported NMR structure of a BRCA1-BARD1 heterodimeric complex reveals that the α-helices flanking the central RING motif of BRCA1 and BARD1 form a stable four-helix bundle that acts as the major heterodimerization interface between the two proteins (15Brzovic P.S. Rajagopal P. Hoyt D.W. King M.C. Klevit R.E. Nat. Struct. Biol. 2001; 8: 833-837Crossref PubMed Scopus (387) Google Scholar). Several lines of evidence suggest that BARD1 is involved in BRCA1-mediated tumor suppression. BARD1 mutations have been detected in breast, ovarian, and uterine tumors (25Thai T.H. Du F. Tsan J.T. Jin Y. Phung A. Spillman M.A. Massa H.F. Muller C.Y. Ashfaq R. Mathis J.M. Miller D.S. Trask B.J. Baer R. Bowcock A.M. Hum. Mol. Genet. 1998; 7: 195-202Crossref PubMed Scopus (160) Google Scholar), and inhibition of BARD1 expression in cultured cells results in a premalignant phenotype (26Irminger-Finger I. Soriano J.V. Vaudan G. Montesano R. Sappino A.P. J. Cell Biol. 1998; 143: 1329-1339Crossref PubMed Scopus (89) Google Scholar). The BRCA1-BARD1 complex has been shown to interact with the polyadenylation factor CstF-50 (27Kleiman F.E. Manley J.L. Science. 1999; 285: 1576-1579Crossref PubMed Scopus (143) Google Scholar), presumably to inhibit mRNA processing at sites of DNA damage (28Kleiman F.E. Manley J.L. Cell. 2001; 104: 743-753Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). BRCA1-BARD1 co-localize with DNA replication and repair factors in response to DNA damage (29Jin Y. Xu X.L. Yang M.C. Wei F. Ayi T.C. Bowcock A.M. Baer R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12075-12080Crossref PubMed Scopus (157) Google Scholar, 30Scully R. Chen J. Ochs R.L. Keegan K. Hoekstra M. Feunteun J. Livingston D.M. Cell. 1997; 90: 425-435Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar, 31Bhattacharyya A. Ear U.S. Koller B.H. Weichselbaum R.R. Bishop D.K. J. Biol. Chem. 2000; 275: 23899-23903Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar, 32Fabbro M. Rodriguez J.A. Baer R. Henderson B.R. J. Biol. Chem. 2002; 277: 21315-21324Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Importantly, it has been reported that BRCA1-BARD1 heterodimers exhibit significant E3 Ub ligase activity and that the BARD1 RING finger domain greatly potentiates the ligase activity of the BRCA1 RING finger (22Hashizume R. Fukuda M. Maeda I. Nishikawa H. Oyake D. Yabuki Y. Ogata H. Ohta T. J. Biol. Chem. 2001; 276: 14537-14540Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, 33Chen A. Kleiman F.E. Manley J.L. Ouchi T. Pan Z.Q. J. Biol. Chem. 2002; 277: 22085-22092Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). However, BARD1 may also have BRCA1-independent functions, since it can act as an apoptosis inducer in a BRCA1-independent manner (34Irminger-Finger I. Leung W.C. Li J. Dubois-Dauphin M. Harb J. Feki A. Jefford C.E. Soriano J.V. Jaconi M. Montesano R. Krause K.H. Mol. Cell. 2001; 8: 1255-1266Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Here we report that BARD1 can significantly enhance BRCA1 E3 Ub ligase activity by directly binding BRCA1, although BARD1 itself does not exhibit E3 ligase activity in vitro. BARD1 and BRCA1 form heterodimers in living cells and mutually control each other's abundance and stability. The enhancement of BRCA1 autoubiquitylation by BARD1 mostly results in poly-Ub chains linked through Lys63, which could be involved in pathways related to DNA damage response and repair rather than signaling BRCA1 degradation. A DNA fragment containing the N-terminal 110 amino acids of BRCA1 was PCR-amplified from pBluescript II SK(+) 73.1 (35Chapman M.S. Verma I.M. Nature. 1996; 382: 678-679Crossref PubMed Scopus (436) Google Scholar) using the primers (5′-GCCGGATCCATGGATTTATCTGCTCTTCGC-3′) and (5′-GGCGAATTCCTTACTTTTTTGCAAAATTATAGC-3′). The fragment was cloned using BamHI and EcoRI into pGEX-KG and pHis8 to create pYN122 and pYN131, respectively. A DNA fragment containing the N-terminal 308 amino acids of BRCA1 was PCR-amplified from pBluescript II SK(+) 73.1 using the primers (5′-GCCCACTAGTATGGATTTATCTGCTCTTCGC-3′) and (5′-GAAATGCGGCCGCTCAGAATTCAGCCTTTTCTACAT-3′) and cloned usingSpeI and NotI into pFLAG to generate pYN146. Based on the plasmid B230AE/pGEX, which contains WT GST-tagged N-terminal 8–142 aa of BARD1 (a gift from Dr. Richard Baer), plasmids pYN124 (C83G), pYN128 (C50G), pYN129 (R58A), pYN130 (I69A), pYN132 (H68A), pYN141 (H68A/C83G), pYN142 (C50G/H68A), pYN143 (C50G/C83G), pYN156 (L44R), and pYN158 (I105D) were created using the QuikChange site-directed mutagenesis kit (Stratagene). A DNA fragment containing the N-terminal 8–142 aa of BARD1 was PCR-amplified from B230AE/pGEX using primers (5′-GCCCACTAGTCCTCGAGGCCACGAAG-3′) and (5′-GAAATGCGGCCGCTCACGATGAATTCTTCTTG-3′) and cloned usingSpeI and NotI into pFLAG to generate pYN125. DNA fragment containing full-length BARD1 was PCR-amplified from BARD1-m1/pSP6 using primers (5′-GAAATGCGGCCGCTCAGCTGTCAAGAGGAAGC-3′) and (5′-GCCCACTAGTATGCCGGATAATCGGC-3′) and cloned usingSpeI and NotI into pFLAG to generate pYN147. Based on pYN147, pYN148 (C50G), pYN149 (R58A), pYN150 (H68A), pYN151 (I69A), pYN152 (C83G), pYN153 (C50G/C83G), pYN154 (C50G/H68A), pYN155 (H68A/C83G), pYN159 (L44R), and pYN161 (I105D) were generated using the QuikChange site-directed mutagenesis kit. The plasmid expressing GST-BRCA1-(1–78) has been described previously (21Ruffner H. Joazeiro C.A. Hemmati D. Hunter T. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5134-5139Crossref PubMed Scopus (307) Google Scholar). The plasmid pHIV CSC was constructed by introducing the cytomegalovirus promoter from pEGFP-C1 (Invitrogen) into the pHIV CS vector (36Miyoshi H. Blomer U. Takahashi M. Gage F.H. Verma I.M. J. Virol. 1998; 72: 8150-8157Crossref PubMed Google Scholar). Subsequently, the full-length BRCA1 cDNA was introduced into the pHIV CSC vector from pBluescript 73.1, giving rise to pHIV CSC BRCA1. The baculovirus plasmid expressing full-length BRCA1 was generated by inserting BRCA1 cDNA into pAcSG2 (Pharmingen) with FLAG sequence tagged at the N terminus of BRCA1. GST-tagged and His-tagged proteins were expressed and purified as described by Leverson et al. (37Leverson J.D. Joazeiro C.A. Page A.M. Huang H. Hieter P. Hunter T. Mol. Biol. Cell. 2000; 11: 2315-2325Crossref PubMed Scopus (158) Google Scholar). GST-tagged proteins were expressed in Escherichia coli strain BL21 (DE3) by induction with 0.4 mmisopropyl-1-thio-β-d-galactopyranoside for about 3 h at 30 °C. The cell pellets were then lysed in lysis buffer (50 mm Tris-Cl, pH 8.0, 120 mm NaCl, 1 mm dithiothreitol, plus protease inhibitors). Proteins bound to glutathione-agarose (Sigma) were eluted with phosphate-buffered saline buffer containing 20 mmglutathione (pH 7.1–7.5) and dialyzed against 20 mmTris-Cl, pH 8.0, 50 mm NaCl, 10% glycerol, and 1 mm dithiothreitol. GST pull-down assays were performed as described previously (38Leverson J.D. Ness S.A. Mol. Cell. 1998; 1: 203-211Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Equal amounts of WT or mutant GST-BARD1-(8–142) were mixed together with His-BRCA1-(1–110) on ice for 30 min, in buffer containing 20 mm HEPES, pH 7.0, 1 mm EDTA, 10% glycerol, and protease inhibitors. The mixtures were then incubated with glutathione-agarose for another 30 min with rolling at 4 °C, and the beads were washed extensively with the same buffer supplemented with 150 mm NaCl and 0.1% Nonidet P-40. Bound proteins were eluted with phosphate-buffered saline buffer containing 20 mm glutathione (pH 7.1–7.5). His-tagged proteins were expressed as described above and purified by using Talon metal affinity resin (Clontech). Cells were lysed in binding buffer (20 mm Tris-Cl, pH 7.5, 100 mm NaCl, 10% glycerol, 10 μmZnSO4, and 1 mm imidazole) and bound to a 1-ml bed volume of Talon resin. After washing with 10 bed volumes of binding buffer plus 10 mm imidazole, the bound proteins were eluted with binding buffer plus 100 mm imidazole. Baculovirus FLAG-tagged full-length BRCA1 was generated and purified essentially as described (39Chen H. Lin R.J. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar). The concentration of purified BRCA1 was ∼0.1 mg/ml and judged to be 95% pure as evidenced by a single Coomassie-stained band after resolution by Tris acetate 3–8% SDS-PAGE (Novex/Invitrogen). In vitroubiquitylation assays were carried out as previously described (40Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (912) Google Scholar), using purified bacterially expressed His-E1 and His-Ubc4. About 1 μg of purified GST-BRCA1-(1–110) was incubated with 50–500 nm His-E1, 0.5–5 μm His-Ubc4, 10 μm bovine ubiquitin or GST-Ub (WT or mutant), and 2 mm ATP in reaction buffer (50 mm Tris-Cl, pH 7.5, 2.5 mm MgCl2, and 0.5 mmdithiothreitol). Purified GST-BARD1-(8–142) (WT or mutant) was added to the reactions as indicated. After a 90-min incubation at room temperature, reactions were stopped with 2 × SDS buffer, separated by SDS-PAGE, and analyzed by immunoblotting with anti-Ub monoclonal antibody (Zymed Laboratories Inc.), anti-GST monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-His5 monoclonal antibody (Qiagen), or affinity-purified anti-BRCA1 (A) polyclonal antibodies (41Ruffner H. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7138-7143Crossref PubMed Scopus (179) Google Scholar). Immunoblots to detect histone ubiquitylation were performed with a monoclonal anti-Ub antibody (Zymed Laboratories Inc.) and rabbit polyclonal anti-H2A, anti-H2B, anti-H3, and anti-H4 antibodies (Upstate Biotechnology). 3 μg of Drosophilacore histones (a gift from Joaquin Espinosa and Beverly Emerson, Regulatory Biology Laboratory, The Salk Institute for Biological Studies) and 0.25 μg of full-length FLAG-BRCA1 were added to each reaction in the absence or presence of 0.5 μg of GST-BARD1-(8–142). 293T human embryonic kidney cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. DNA transfections were carried out by a standard calcium phosphate precipitation protocol. In all of the transfections, the total amount of DNA was equalized. 48 h post-transfection, the cells were collected and lysed in radioimmune precipitation buffer (6 mm Na2HPO4, 4 mmNaH2PO4, 2 mm EDTA, 150 mm NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 1% Trasylol, 50 mm NaF, and 100 μmNa3VO4), and equal amounts of proteins were analyzed by SDS-PAGE. To examine the relative stability of BRCA1 and BARD1 proteins, fresh media containing cycloheximide (150 μg/ml final concentration) were added 48 h after transfection. The cells were collected at the times indicated, and cell lysates were subjected to ECL immunoblot analysis. The membranes were probed with anti-FLAG (Sigma) and anti-GST-Nck-α polyclonal antiserum from rabbit 5547. Transfection of full-length BRCA1 into 293T cells was carried out by the calcium phosphate BBS transfection method (42Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4820) Google Scholar). After lysis, the samples were separated using Novex/Invitrogen 3–8% Tris-acetate SDS-PAGE gels and blotted according to the manufacturer's recommendation except for transfer times, which were extended to 90 min. Primary antibodies were diluted in 3% bovine serum albumin/PBST at a 1:1000 dilution for the anti-BRCA1 MS110 (Ab-1; Oncogene Science) and 1:5000 for anti-β-galactosidase (Novus Biologicals, Inc.) antibodies. The BRCA1 central RING motif, which encompasses residues 23–76 (15Brzovic P.S. Rajagopal P. Hoyt D.W. King M.C. Klevit R.E. Nat. Struct. Biol. 2001; 8: 833-837Crossref PubMed Scopus (387) Google Scholar), is part of a larger proteolysis-resistant structural domain containing the first 110 residues of BRCA1 (43Brzovic P.S. Meza J. King M.C. Klevit R.E. J. Biol. Chem. 1998; 273: 7795-7799Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). A plasmid expressing GST-BRCA1-(1–110) was constructed, and bacterially expressed protein was purified using glutathione beads. Purified GST-BRCA1-(1–110) was assayed for its ability to mediate the transfer of Ub and stimulate the synthesis of stable Ub conjugates in anin vitro ubiquitylation assay, using blotting with an anti-Ub monoclonal antibody to detect ubiquitylated products. As shown in Fig. 1 B, the BRCA1 RING domain exhibited E3 Ub ligase activity in an E1- and E2-dependent manner, consistent with previously published results (21Ruffner H. Joazeiro C.A. Hemmati D. Hunter T. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5134-5139Crossref PubMed Scopus (307) Google Scholar). The polyubiquitylated conjugates include ubiquitylated BRCA1 and His-E1/E2 (data not shown). Purified His-BRCA1-(1–110) had E3 ligase activity similar to that obtained with GST-BRCA1-(1–110) (data not shown). Histone H2A could also be monoubiquitylated by GST-BRCA1-(1–110) (data not shown) and by full-length BRCA1 (Fig. 1 C, lane 7). The band was identified as monoubiquitylated H2A, based on its reactivity with both anti-H2A and anti-Ub antibodies. When the same membrane was probed with anti-H2B, anti-H3, and anti-H4 antibodies, no extra slower migrating bands were observed (data not shown), indicating that H2A is specifically monoubiquitylated by BRCA1 in vitro. To examine whether BARD1, which is itself a RING protein, might affect BRCA1 E3 Ub ligase activity, increasing amounts of purified GST-BARD1-(8–142) containing the RING domain (residues 49–100) were incubated together with GST-BRCA1-(1–110), His-E1, His-E2 (Ubc4), and GST-Ub. BARD1 significantly enhanced GST-BRCA1 E3 Ub ligase activity (Fig. 1 B), as had previously been reported in analogous studies by Hashizume et al. (22Hashizume R. Fukuda M. Maeda I. Nishikawa H. Oyake D. Yabuki Y. Ogata H. Ohta T. J. Biol. Chem. 2001; 276: 14537-14540Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar) and Chen et al.(33Chen A. Kleiman F.E. Manley J.L. Ouchi T. Pan Z.Q. J. Biol. Chem. 2002; 277: 22085-22092Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). His-BRCA1-(1–110) E3 ligase activity was also greatly stimulated by BARD1 (data not shown). The GST-BARD1-(8–142) protein lacked E3 ligase activity in vitro, even when present in the assay at much greater levels than BRCA1 (Fig. 1 D, lanes 6–10). As shown in Fig. 1 C, the monoubiquitylation of histone H2A by full-length BRCA1 was also much stronger in the presence of BARD1 (lanes 7 and9), consistent with the result from Pan's group (33Chen A. Kleiman F.E. Manley J.L. Ouchi T. Pan Z.Q. J. Biol. Chem. 2002; 277: 22085-22092Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). In the presence of BARD1, several slowly migrating bands appeared when the membrane was probed with anti-Ub antibody, which could be polyubiquitylated products. However, when the same membrane was probed with anti-H2A antibodies, only one weak extra band migrating slower than monoubiquitylated H2A was observed (Fig. 1 C,lane 9). Therefore, we conclude that histone H2A was predominantly monoubiquitylated by BRCA1 in vitro in the presence of BARD1. To determine whether the enhancement of BRCA1 E3 Ub ligase activity by BARD1 depends on the integrity of the BARD1 RING domain and the interaction between the two proteins, the RING consensus residues Cys50, Cys83, and His68, in the BARD1 RING domain were mutated to Gly or Ala; the nonconserved Arg58 and Ile69 were also mutated to Ala (Fig. 2 A). The conserved Cys and His residues are necessary for the integrity of the BARD1 RING domain, which is in turn required for the proper orientation of the N- and C-terminal helices that form the four-helix bundle, and therefore these mutations might be expected to affect the interaction between BRCA1 and BARD1. The mutant GST-BARD1-(8–142) proteins were then purified and examined for their ability to bind BRCA1. GST protein and WT GST-BARD1-(8–142) were used as negative and positive controls, respectively. As shown in Fig. 2 B, WT GST-BARD1-(8–142) bound strongly to His-BRCA1-(1–110), whereas GST itself did not. The R58A and I69A mutant proteins bound equally well to BRCA1 when compared with WT BARD1. The C50G, H68A, and C83G single mutant proteins still bound to BRCA1 weakly, whereas the C50G/H68A, C50G/C83G, and H68A/C83G double mutant proteins only exhibited extremely low binding activity in the GST pull-down assay (Fig. 2 B). To test the importance of the hydrophobic interactions in the four-helix bundle that stabilize the BRCA1-BARD1 heterodimer, we also constructed two BARD1 mutants with mutations in critical hydrophobic residues in the N- or C-terminal α-helix, L44R (N-terminal α-helix) and I105D (C-terminal α-helix), which contain an intact RING domain but fail to bind BRCA1 in the yeast two-hybrid assay (15Brzovic P.S. Rajagopal P. Hoyt D.W. King M.C. Klevit R.E. Nat. Struct. Biol. 2001; 8: 833-837Crossref PubMed Scopus (387) Google Scholar, 44Morris J.R. Keep N.H. Solomon E. J. Biol. Chem. 2002; 277: 9382-9386Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Consistently, the L44R mutant protein showed no detectable binding, and the I105D mutant protein showed only extremely weak binding to BRCA1 in the GST pull-down assay (Fig. 2 C). Next, equal amounts of purified WT or mutant BARD1 proteins were tested for their effects on BRCA1 E3 Ub ligase activity using GST-Ub, and ubiquitylated products were detected by blotting with anti-GST monoclonal antibody. As shown in Fig. 3, WT GST-BARD1-(8–142) and the R58A and I69A mutant proteins significantly enhanced BRCA1 E3 Ub ligase activity (lanes 4, 6, and 8). The C50G, H68A, and C83G mutant proteins also enhanced BRCA1 E3 ligase activity, although relatively weakly (lanes 5, 7, and9). The I105D, C50G/H68A, and C50G/C83G mutant proteins had only very small stimulatory effects (lanes 12–14), and the L44R mutant protein had almost no detectable effect on BRCA1 E3 Ub ligase activity (Fig. 3,lane 11). In addition, the same amount of purified GST-BARD1-(8–142) had only an extremely weak stimulatory effect on the E3 ligase activity of GST-BRCA1-(1–78) (Fig. 3,lanes 15 and 16), which lacks the C-terminal α-helix (residues 81–96) flanking the central RING motif (15Brzovic P.S. Rajagopal P. Hoyt D.W. King M.C. Klevit R.E. Nat. Struct. Biol. 2001; 8: 833-837Crossref PubMed Scopus (387) Google Scholar), consistent with the previous report that BARD1 does not interact stably with the N-terminal 71 residues of BRCA1 (23Wu L.C. Wang Z.W. Tsan J.T. Spillman M.A. Phung A. Xu X.L. Yang M.C. Hwang L.Y. Bowcock A.M. Baer R. Nat. Genet. 1996; 14: 430-440Crossref PubMed Scopus (624) Google Scholar) and the fact that this helix is critical for the four-helix bundle (15Brzovic P.S. Rajagopal P. Hoyt D.W. King M.C. Klevit R.E. Nat. Struct. Biol. 2001; 8: 833-837Crossref PubMed Scopus (387) Google Scholar). These results indicate that a direct stable interaction between BARD1 and BRCA1 is required for BARD1 to enhance BRCA1 E3 Ub ligase activity and that the integrity of the BARD1 RING domain is not so important. To further investigate the molecular basis of BRCA1 and BARD1 cooperation, expression plasmids for FLAG-tagged human BRCA1 (full-length or N-terminal 308 amino acids) and FLAG-tagged WT or mutant human BARD1 (full-length or aa 8–142) were co-transfected into human 293T cells. As shown in Fig. 4 A, the levels of FLAG-BRCA1-(1–308) protein were dramatically increased in the presence of increasing amounts of FLAG-tagged WT BARD1 (full-length or aa 8–142). To test the effect of BARD1 on full-length BRCA1, pHIV CSC BRCA1, which expresses full-length human BRCA1 in the third generation lentiviral vector, was co-transfected with FLAG-tagged BARD1 (full-length) expression plasmid or pBluescript II (KS+) (Stratagene) control plasmid. pCMX LacZ (45Umesono K. Murakami K.K. Thompson C.C. Evans R.M. Cell. 1991; 65: 1255-1266Abstract Full Text PDF PubMed Scopus (1493) Google Scholar) and pEGFP C-2 (Clontech) were also co-transfected as controls for transfection efficiency. The level of transfected full-length BRCA1 protein was significantly elevated when full-length FLAG-BARD1 was co-expressed (Fig. 4 B). The endogenous BRCA1 protein level was also increased by transfected BARD1 (data not shown). Next, the effects of mutant BARD1 proteins on BRCA1 abundance were assessed. As shown in Fig. 4 C, the levels of FLAG-BRCA1-(1–308) protein were dramatically increased when full-length WT BARD1 or the R58A mutant was co-expressed (lanes 2, 4, and 13). In contrast, the C50G, H68A, and C83G single mutant proteins caused a relatively small increase in BRCA1 levels (lanes 3, 5, and 6), whereas the C50G/H68A, C50G/C83G, and H68A/C83G double mutant and L44R and I105D mutant proteins only had an extremely small stimulatory effect on BRCA1 abundance (Fig. 4 C, lanes 7–9,11, and 12). Mutant BARD1-(8–142) proteins behaved s" @default.
• W2047368106 created "2016-06-24" @default.
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• W2047368106 date "2003-02-01" @default.
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• W2047368106 title "Enhancement of BRCA1 E3 Ubiquitin Ligase Activity through Direct Interaction with the BARD1 Protein" @default.
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