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J. Biol. Chem., Vol. 276, Issue 18, 14537-14540, May 4, 2001

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ACCELERATED PUBLICATION
The RING Heterodimer BRCA1-BARD1 Is a Ubiquitin Ligase Inactivated by a Breast Cancer-derived Mutation^*

Rintaro Hashizume, Mamoru Fukuda, Ichiro Maeda, Hiroyuki Nishikawa, Daisuke Oyake, Yukari Yabuki, Haruki Ogata, and Tomohiko Ohta

From the Division of Breast and Endocrine Surgery, St. Marianna University School of Medicine, Kawasaki, 216-8511 Japan

Received for publication, December 14, 2000, and in revised form, February 20, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

BRCA1-BARD1 constitutes a heterodimeric RING finger complex associated through its N-terminal regions. Here we demonstrate that the BRCA1-BARD1 heterodimeric RING finger complex contains significant ubiquitin ligase activity that can be disrupted by a breast cancer-derived RING finger mutation in BRCA1. Whereas individually BRCA1 and BARD1 have very low ubiquitin ligase activities in vitro, BRCA1 combined with BARD1 exhibits dramatically higher activity. Bacterially purified RING finger domains comprising residues 1-304 of BRCA1 and residues 25-189 of BARD1 are capable of polymerizing ubiquitin. The steady-state level of transfected BRCA1 in vivo was increased by co-transfection of BARD1, and reciprocally that of transfected BARD1 was increased by BRCA1 in a dose-dependent manner. The breast cancer-derived BARD1-interaction-deficient mutant, BRCA1^C61G, does not exhibit ubiquitin ligase activity in vitro. These results suggest that the BRCA1-BARD1 complex contains a ubiquitin ligase activity that is important in prevention of breast and ovarian cancer development.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Germline mutations of BRCA1 predispose women to breast and ovarian cancers (1). BRCA1 contains several domains that interact with a variety of molecules and is potentially responsible for multiple functions in DNA damage repair, transcription, and cell-cycle regulation (2-4). BARD1 was identified in a yeast two-hybrid screen as a protein that interacts with BRCA1 (5). Both BRCA1 and BARD1 proteins contain a RING finger (5) and exist as homodimers or preferentially form stable heterodimers (6). The heterodimeric interaction is mediated by the flanking regions of the RING finger motif of the two molecules (6). Although a transcriptional function in the C terminus of BRCA1 has been recently reported (3), the biochemical function of the heterodimeric RING finger constituted from the N termini of BRCA1 and BARD1 is not known.

Previously, we and others identified a highly conserved small RING finger protein, ROC1 (also called Rbx1 and Hrt1), as an essential subunit of the SCF Ub¹ ligase (7-10). The Ub ligase (E3) catalyzes the formation of polyubiquitin chains onto substrate proteins via isopeptide bonds utilizing the Ubs that have been sequentially activated by enzymes E1 and E2. Polyubiquitinated substrates are then rapidly degraded by the 26 S proteasome (11). The SCF and the APC are the two major Ub ligase complexes that regulate ubiquitin-mediated proteolysis during G₁/S and anaphase (12), and contain the small RING finger proteins ROC1 and APC11, respectively (7-10). Point mutations in the RING finger domain of ROC1 completely disrupted the Ub ligase activity, suggesting an essential role of the domain for its activity (7). APC11 also contains Ub ligase activity in vitro (7). More recently, several large RING finger proteins, such as MDM2, c-Cbl, IAP, and AO7, with otherwise diverse structures and functions were linked to ubiquitination (13-16), suggesting a potentially broad and general function for RING fingers in activating Ub ligase activity. One of these RING proteins, BRCA1, has been closely scrutinized for Ub ligase activity. However, the ability of BRCA1 by itself to promote ubiquitin polymerization was found to be limited (16).

In this report, we have provided evidence demonstrating that the RING finger of BRCA1, in concert with BARD1, exhibits significant ubiquitin ligase activity. This activity can be disrupted by a breast cancer-derived RING finger mutation of BRCA1, suggesting a direct relationship between the ubiquitin ligase function of BRCA1 and breast cancer development.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmids-- cDNAs for full-length human BARD1 and CstF-50 were amplified by polymerase chain reaction from a Hela cell cDNA library with pfu polymerase (Stratagene) and subcloned into the pcDNA3 vector inframe with appropriate tags. pGEX-BARD1-(1-111) was created by digesting the full-length BARD1 with HindIII, and subcloning into the pGEX-KG vector. pET-His₆-BARD1-(14-189) and pET-His₆-BARD1-(25-189) were created by digesting the full-length BARD1 with BamHI and PstI, and NcoI and PstI, respectively and subcloned into the pET-3E-His₆ vector. Full-length BRCA1 cDNA was a generous gift from Dr. Wen-Hwa Lee. The N-terminal fragment (1) of BRCA1 was generated by digesting the full-length BRCA1 at its KpnI restriction site and subcloning into either the pGEX vector or the pcDNA3 vector with the appropriate tag. pGEX-BRCA1-(1-342) was created by self-ligation of BglII-digested pGEX-BRCA1-(1-772). pET-His₆-BRCA1-(1-304) was created by digesting BRCA1-(1-772) with EcoRI and subcloning into the pET-3E-His₆ vector. BRCA1 or BARD1 point mutations were introduced by site-directed mutagenesis (Stratagene). cDNA for each human E2/Ubc was amplified by polymerase chain reaction from a HeLa cell cDNA library and subcloned into the pET-3E-His₆ vector. cDNA for human E2F1 and cyclin B1 in the pcDNA3 expression vector were gifts from Dr. Yue Xiong. All the constructs used were verified by DNA sequencing.

Cell Culture, Transfection, and Immunoprecipitation-- Cells (293T) were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and 1% antibiotic-antimycotic agent (Life Technologies, Inc.) in a 37 °C incubator with 5% CO₂. DNA was transfected using the standard calcium phosphate precipitation method. For each transfection, the total plasmid DNA was adjusted to 15 µg per 100-mm dish by adding the parental pcDNA3 vector. For immunoprecipitation, cells were harvested 36 h after transfection and lysed by incubating at 4 °C for 1 h with 0.6 ml per 100 mm dish of buffer A containing 15 mM Tris-HCl pH 7.5, 0.5 M NaCl, 0.35% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 10 µg/ml trypsin inhibitor, and 150 µg/ml benzamidine. Lysed cells were then clarified by centrifugation at 100,000 × g at 4 °C for 1 h. The supernatants (0.3 ml) were mixed with 3 µg of anti-Myc (9E10) or anti-HA (12CA5) antibody, and then the antibody-bound proteins were precipitated with protein A-agarose beads (7.5 µl). The proteins bound to the beads were used either for the Ub ligation assay or immunoblotting. For straight immunoblotting to analyze the steady-state levels of Myc-BRCA1 or HA-BARD1, the transfected cells were lysed and clarified as described above, and 50 µg of each sample was resolved by SDS-PAGE on a 7.5% gel, followed by immunoblotting.

Recombinant Proteins-- Rabbit E1 was purchased from Affiniti Research Products. His₆-tagged Ub with a protein kinase C recognition site, His₆-tagged E2 proteins, and His₆-E2F1 were purified as previously described (7, 17). His₆-BARD1-(14-189), His₆-BARD1-(25-189), His₆-BRCA1-(1-304), GST-BRCA1-(1-342) and GST-BARD1-(1-111) were produced in BL21/DE3 bacteria by induction with 0.4 mM IPTG for 12 h at 25 °C. Cells were lysed in buffer containing 50 mM Tris-HCl, pH 8.0, 0.5% NP-40, 1% Triton X-100, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 10% glycerol, and protease inhibitors, and the proteins were purified either with nickel beads (Qiagen) or glutathione-agarose beads (Sigma) according to the manufacturer's instructions.

Ub Ligation Assay-- The procedure for the in vitro Ub ligation assay was essentially the same as previously described (7, 18). The BRCA1-BARD1 immunocomplexes immobilized on protein A-agarose beads were washed three times with buffer A and two times with buffer B containing 25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.01% Nonidet P-40, 10% glycerol, and 1 mM EDTA, and added to a ubiquitin ligation reaction mixture (30 µl) that contained 50 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, 2 mM NaF, 10 nM okadaic acid, 2 mM ATP, 0.6 mM DTT, 0.75 µg of ³²P-Ub, 40 ng of E1, and 0.3 µg of E2 protein. After incubation for 30 min at 37 °C with shaking, the reactions were terminated by boiling in Laemmli SDS-loading buffer with 0.1 M DTT, and half of the sample was resolved by SDS-PAGE followed by autoradiography. For the in vitro Ub ligation assay using the purified RING finger domain of BRCA1 or BARD1, GST-fused proteins bound to glutathione-agarose beads, and eluted His₆-tagged proteins were used instead of immunocomplexes. The activity of E2 was analyzed by the same procedure without E3.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ub Ligase Activities of the BRCA1 or BARD1 Immunocomplexes in Collaboration with E2/UbcH5c-- The discovery that the small RING finger proteins, ROC1 and ROC2, are ubiquitin ligases prompted us to determine whether BRCA1 and BARD1, two of the most important RING finger proteins implicated in breast cancer, also function as ubiquitin ligases. We first determined which E2/Ubc could be activated by BRCA1 or BARD1. Five representative mammalian E2s (UbcH1, UbcH2, Cdc34, UbcH5c and UbcH7) were purified from bacteria (Fig. 1A). Individual E2s were incubated with E1 and ³²P-Ub in the presence of ATP to verify their Ub binding capacity. Four of the five E2s (UbcH2, Cdc34, UbcH5c, and UbcH7) were determined to be active enzymes by judging the ability to bind to ³²P-Ub (Fig. 1B, lanes 3-6, lower panel), and was dissociated by addition of DTT (lanes 3-6, upper panel). When the anti-Myc immunocomplex derived from Myc-BRCA1-(1-772) or Myc-BARD1-transfected 293T cells was added to the reaction, only UbcH5c among the five E2s was capable of promoting ubiquitin polymerization with the immunocomplex as determined by the appearance of a high molecular weight ³²P smear (Fig. 1C, lanes 4 and 9). Although a crystal structure study revealed the interaction between the RING finger of c-Cbl and UbcH7 (19), UbcH7 was deficient in collaborating with the RING finger of BRCA1 or BARD1 to promote Ub polymerization (lanes 5 and 10). These results suggest that the BRCA1- and BARD1-associated protein complexes contain Ub ligase activities.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   E2/UbcH5c collaborates with the Myc-BRCA1 or Myc-BARD1 immunocomplex to promote Ub polymerization. A, individual purified His6-tagged E2 proteins (2 µg) were resolved by SDS-PAGE followed by Coomassie Blue staining. B, the purified E2s were mixed with E1, 32P-labeled Ub, and ATP. After incubation, the reactions were terminated by boiling in Laemmli SDS loading buffer with (upper panel) or without (lower panel) 0.1 M DTT and were resolved by SDS-PAGE followed by autoradiography. C, cell lysates from 293T cells transfected with Myc-BRCA1-(1-772) (left panel) or Myc-BARD1 (right panel) were immunoprecipitated with anti-Myc antibody. Immunocomplexes immobilized on the beads were subjected to Ub ligation assay with the indicated EZ (overnight exposure).

Ub Ligase Activity of the BRCA1-BARD1 Immunocomplex and Its Inactivation by a RING Finger Mutation of BRCA1-- We noted that the Ub ligase activities of the BRCA1 and BARD1 immunocomplexes, which were only detectable after long exposure of the film (Fig. 1C), were very low and sought conditions for higher activity. Because it was reported that BRCA1 and BARD1 proteins preferentially formed stable heterodimers (6), we next tested whether the BRCA1-BARD1 heterodimer complex formation affects Ub ligase activity. Surprisingly, when Myc-BRCA1-(1-772) was co-transfected with HA-BARD1, the anti-Myc immunocomplex exhibited significantly higher Ub ligase activity (Fig. 2A, lane 5) than that of either the Myc-BARD1 (lane 3) or Myc-BRCA1-(1-772) (lane 4) single transfection. It is possible that the low Ub ligase activity detected with the immunocomplexes from single transfections of BRCA1 or BARD1 may be caused by contamination with the endogenous partner RING finger protein. Similarly, addition of Myc-BRCA1-(1-772) dramatically enhanced the Ub ligase activity of the HA-BARD1 immunocomplex (lane 8). E2F1 and cyclin B1, the proteins known to interact with the N terminus of BRCA1 (20), as well as CstF50, a protein that binds to BARD1 (21) do not have a stimulatory effect on Ub ligase activity of BRCA1 or BARD1 (Fig. 2B, lanes 1-6), arguing against the possibility that the stimulatory effect observed with BRCA1-BARD1 is mediated by stabilizing the structure of each protein by its partners nonspecifically. Coupled immunoprecipitation and Western blotting verified the interactions between these proteins (lane 7). To verify the importance of the RING finger in activity and to explore the relationship between the BRCA1 tumor suppressor function and the Ub ligase activity, we made constructs containing BRCA1 with a tumor-derived mutation at Cys-61 (BRCA1^C61G) (22). The mutation completely abolished Ub ligase activity (Fig. 2C, lane 3) when compared with the anti-Myc immunocomplex precipitated from cells cotransfected with wild-type Myc-BRCA1-(1-772) and HA-BARD1 (lane 2) and reduced the activity of the HA-BARD1 immunocomplex to the level of the corresponding HA-BARD1 single transfection (lanes 7 versus 5). An artificial mutation in the RING finger domain, BRCA1^C39A/H41A, also disrupted the activity to the same degree as BRCA1^C61G (lanes 4 versus 3 and 8 versus 7). On the other hand, BARD1^C83G and BARD1^C66A/H68A, the RING mutations corresponding to those of BRCA1^C61G and BRCA1^C39A/H41A, respectively, did not abolish the Ub ligase activity, although a detectable reduction of the activity was observed (Fig. 2D, lanes 3 and 4).

View larger version (54K): [in this window] [in a new window]   Fig. 2.   In vitro Ub ligase activity of the BRCA1-BARD1 immunocomplex. A-D, cell lysates were obtained from 293T cells transfected with 7.5 µg of plasmid indicated at the top of each panel, followed by immunoprecipitation (IP). A substrate-nonspecific Ub ligation assay with E2/UbcH5c was performed (30-min exposure). The BRCA1 and BARD1 proteins in the immunocomplexes were verified by immunoblotting either with anti-Myc or anti-HA antibody (A, C, and D; bottom panel). The interaction between Myc-BRCA1 and either E2F1 or cyclin B1, or between Myc-BARD1 and HA-CstF50 was verified by coupled anti-Myc immunoprecipitation and immunoblotting with anti-E2F1, anti-cyclin B1, or anti-HA, respectively (B, right panel). Note that the activity of the Myc-BARD1 immunocomplex in lane 3 in A corresponds to that in lane 9 in Fig. 1C.  and  at the right of the panel indicate the positions of Myc-BRCA1-(1-772) and HA-BARD1 migration, respectively.

Ub Ligase Activity of the Purified RING Finger Domains of BRCA1 and BARD1-- To exclude the possibility that the ligase activity in the immunocomplex is caused by contaminating proteins and also to determine the core of the Ub ligase activity, both the RING finger domain of BRCA1 and of BARD1 were purified from bacteria via GST or His₆ tags (Fig. 3A). Consistent with the immunocomplex-based assay, a mixture of the purified RING finger domains of BRCA1 and BARD1 exhibited significant Ub ligase activity (Fig. 3B, lanes 3, 6, 8, and 10) whereas either the BRCA1 (lanes 1 and 5) or BARD1 (lanes 2 and 7) RING alone displayed barely detectable Ub ligase activity. Purified His₆-E2F1 did not enhance the Ub ligase activity of BRCA1 (lane 9), although it did interact with GST-BRCA1-(1-342) (data not shown). RING finger domains comprising residues 1-304 of BRCA1 and residues 25-189 of BARD1 were capable of polymerizing ubiquitin (lane 8). Importantly BRCA1^C61G, the breast cancer-derived RING finger mutant, did not collaborate with BARD1 to activate UbcH5c (lane 4). Taken together, these results indicate that BARD1 and BRCA1 collaboratively activate Ub ligase activity with the E2 UbcH5c. The reactants contained ubiquitinated products migrating at ~35 and 43 kDa (Fig. 3, ), exactly the same position as that seen in the immunocomplex-based assay (Fig. 2). These products are likely to be free ubiquitin polymers or ubiquitin polymers covalently bound to some small molecule such as UbcH5c. The majority of the polyubiquitinated products including these smaller products did not dissociate from the BRCA1-BARD1 complex when washed with buffer B (data not shown).

View larger version (57K): [in this window] [in a new window]   Fig. 3.   In vitro Ub ligase activity of the bacterially purified RING finger domains of BRCA1 and BARD1. A, GST or His6-tagged RING finger domains of BRCA1 and BARD1 were purified from bacteria, and each protein (5 µg) was resolved by SDS-PAGE followed by Coomassie Blue staining. B, the indicated amounts (µg) of the GST-fused RING finger domains of BRCA1 or BARD1 bound to glutathione-agarose beads and the His6-tagged RING finger domains of BRCA1 or BARD1 eluted in phosphate-buffered saline were mixed. The mixtures were subjected to UB ligation assay with Ez/UbcH5c.

BRCA1 and BARD1 Stabilize Each Other in Vivo-- There is a possibility that these two molecules are ubiquitination substrates of each other for targeted degradation. We therefore analyzed the steady-state levels of these proteins by Western blotting. Polyubiquitinated substrates are rapidly degraded by the 26 S proteasome and, correspondingly, the steady-state level of such substrates tends to drop detectably. However, the steady-state level of Myc-BRCA1-(1-772) was increased after co-transfection of HA-BARD1 in a dosage-dependent manner (Fig. 4A). Reciprocally, the steady-state level of HA-BARD1 was increased when it was cotransfected with Myc-BRCA1-(1-772) (Fig. 4B). These results indicate that BRCA1 and BARD1 stabilized each other while gaining the ability to ligate the polyubiquitin chain, suggesting that they are not substrates signaled by each other for degradation.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Stabilization of BRCA1 and BARD1 by their partner RING finger in vivo. A, cell lysates from 293T cells transfected with plasmids expressing Myc-BRCA1-(1-772) (lanes 1-4, 7.5 µg), and HA-BARD1 (lane 2, 0.2 µg; lane 3, 1.0 µg; lane 4, 7.5 µg) were subjected to anti-Myc or anti-HA immunoblotting. B, the steady-state level of Myc-BRCA1-(1-772) and HA-BARD1 was analyzed as described in A from the cells transfected with plasmids expressing Myc-BRCA1-(1-772) (lane 2, 0.5 µg; lane 3, 3.0 µg; lane 4, 14 µg) and HA-BARD1 (lanes 1-4, 1.0 µg) as indicated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The RING finger motif was thought to be a DNA binding site when BRCA1 was first identified as a tumor suppressor gene for familial breast and ovarian cancer, because it was related to the zinc finger, a known DNA binding motif (1). Moreover, the involvement of the BRCT domain in transcription suggests that BRCA1 is a transcription factor, and the RING finger takes part in DNA binding. However, despite tremendous effort, binding between the RING finger of BRCA1 and DNA has not been shown. Instead, the RING finger of BRCA1 has been shown to be important for a protein-protein interaction with another RING finger domain, which was identified and given the name BARD1 (5). A detailed biochemical analysis revealed, however, that the interaction between BRCA1 and BARD1 was mediated by regions outside the canonical RING finger motif (6), suggesting that the two RING fingers contained some additional function where the proximity of the two RINGs was important. In the present report, we provide evidence demonstrating that the RING heterodimer BRCA1-BARD1 functions as a Ub ligase. First, the BRCA1-BARD1 immunocomplex obtained from BRCA1- and BARD1-transfected cells contains a significant ability to promote polyubiquitination in vitro, and omission of either one of the two from the transfection eliminates this activity. Next, the bacterially purified RING finger domains of BRCA1 and BARD1 exhibit high Ub ligase activity. Again omission of either one of the two RING fingers eliminates this activity. Most importantly, in both experiments, a breast cancer-derived RING finger mutation of BRCA1, C61G, abolishes the ability to promote polyubiquitination. Although it is possible that the major reaction being monitored in the present study is autoubiquitination, BRCA1 and BARD1 are not substrates signaled by each other for degradation, because they stabilize each other in vivo. A common feature of RING finger ubiquitin ligases is autoubiquitination (Refs. 13-16 and 18). The importance of those autoubiquitinations in the function of RING type ubiquitin ligases remains to be determined.

BRCA1-BARD1 may constitute a novel type of RING containing Ub ligase that forms a heterodimeric RING finger complex. However, the stoichiometry of RING finger molecules in complexes of known RING Ub ligases has not yet been elucidated. It remains to be determined whether dimerization, either as homo- or heterodimers, between two RING fingers represents a general mechanism for activating Ub ligase activity. Supporting this possibility, Myc-ROC1 can be detected in HA-ROC1 immunocomplexes precipitated from cells transfected with Myc-ROC1 and HA-ROC1.²

During the S phase of the cell cycle, the steady-state levels of BRCA1 reach a maximum, and BARD1 colocalizes with BRCA1 into nuclear dots (23). Consequently it is likely that an in vivo Ub ligase activity of BRCA1-BARD1 would reach a maximum in S phase. Whether the formation of the heterodimer is a regulated event possibly resulting in the stabilization of BRCA1 and in vivo Ub ligase activity remains to be determined. To further understand the biological function of the BRCA1-BARD1 Ub ligase, determining the substrates presumably targeted for degradation is critical. Nuclear proteins that are degraded or down-regulated in S phase may be candidate substrates for the BRCA1-BARD1 Ub ligase. The high Ub ligase activity of BRCA1-BARD1 presented here should make identifying those candidates easier.

The fact that the two RING finger domains of BRCA1 and BARD1 are necessary for one biochemical function indicates that the oncogenic potential or the phenotypes caused by mutations in the domains of those two molecules should be the same. Indeed, germline mutations in the BARD1 gene in primary breast and ovarian cancers have been reported (24). BARD1 is potentially as important a gene as BRCA1 in tumor suppression of breast and ovarian cancers. By correlating each mutation of the two molecules and BRCA1-BARD1 in vitro Ub ligase activity, it may now be possible to predict the oncogenic potential for breast and ovarian cancer in families with germline missense point mutations in the RING finger domains of BRCA1 or BARD1. As such, the finding that the BRCA1-BARD1 heterodimer complex acts as a Ub ligase should provide a new insight to further clinical research into the roles of BRCA1 and BARD1 in tumor suppression.

	ACKNOWLEDGEMENTS

We thank Dr. Yue Xiong and Christopher W. Jenkins for helpful discussions and critical reading of the manuscripts, Dr. W-H. Lee for kindly providing BRCA1 cDNA. We also thank Takako Kuwahara for secretarial assistance.

	FOOTNOTES

^* This study was supported by a grant-in-aid for general scientific research from the Ministry of Education, Science, Sports, and Culture of Japan, and by a grant-in-aid from the Tokyo Biochemical Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-44-977-8111; Fax: 81-44-976-5964; E-mail: to@marianna-u.ac.jp.

Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.C000881200

² T. Ohta, unpublished results.

	ABBREVIATIONS

The abbreviations used are: Ub, ubiquitin; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; SCF, SKP1-CUL1 (CDC53)- F-box protein complex; APC, anaphase-promoting complex; PAGE, polyacrylamide gel electrophoresis, GST, glutathione S-transferase; HA, hemagglutinin; DTT, dithiothreitol; IPTG, isopropyl-1-thio--D-galactopyranoside.

	REFERENCES

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				E. Reinstein and A. Ciechanover Narrative review: protein degradation and human diseases: the ubiquitin connection. Ann Intern Med, November 7, 2006; 145(9): 676 - 684. [Abstract] [Full Text] [PDF]

				S-M Karppinen, R B Barkardottir, K Backenhorn, T Sydenham, K Syrjakoski, J Schleutker, T Ikonen, K Pylkas, K Rapakko, H Erkko, et al. Nordic collaborative study of the BARD1 Cys557Ser allele in 3956 patients with cancer: enrichment in familial BRCA1/BRCA2 mutation-negative breast cancer but not in other malignancies J. Med. Genet., November 1, 2006; 43(11): 856 - 862. [Abstract] [Full Text] [PDF]

				C. Chen, A. K. Seth, and A. E. Aplin Genetic and Expression Aberrations of E3 Ubiquitin Ligases in Human Breast Cancer Mol. Cancer Res., October 1, 2006; 4(10): 695 - 707. [Abstract] [Full Text] [PDF]

				R. Tsunematsu, M. Nishiyama, S. Kotoshiba, T. Saiga, T. Kamura, and K. I. Nakayama Fbxw8 is essential for cul1-cul7 complex formation and for placental development. Mol. Cell. Biol., August 1, 2006; 26(16): 6157 - 6169. [Abstract] [Full Text] [PDF]

				Z. Li, R. Cao, M. Wang, M. P. Myers, Y. Zhang, and R.-M. Xu Structure of a Bmi-1-Ring1B Polycomb Group Ubiquitin Ligase Complex J. Biol. Chem., July 21, 2006; 281(29): 20643 - 20649. [Abstract] [Full Text] [PDF]

				J.-R. J. Yeh, R. Ju, C. M. Brdlik, W. Zhang, Y. Zhang, M. E. Matyskiela, J. D. Shotwell, and C. M. Crews Targeted gene disruption of methionine aminopeptidase 2 results in an embryonic gastrulation defect and endothelial cell growth arrest PNAS, July 5, 2006; 103(27): 10379 - 10384. [Abstract] [Full Text] [PDF]

				X. Yu, S. Fu, M. Lai, R. Baer, and J. Chen BRCA1 ubiquitinates its phosphorylation-dependent binding partner CtIP. Genes & Dev., July 1, 2006; 20(13): 1721 - 1726. [Abstract] [Full Text] [PDF]

				M. C Fleisch, C. A Maxwell, and M.-H. Barcellos-Hoff The pleiotropic roles of transforming growth factor beta in homeostasis and carcinogenesis of endocrine organs. Endocr. Relat. Cancer, June 1, 2006; 13(2): 379 - 400. [Abstract] [Full Text] [PDF]

				B. P. Schlegel, F. M. Jodelka, and R. Nunez BRCA1 Promotes Induction of ssDNA by Ionizing Radiation. Cancer Res., May 15, 2006; 66(10): 5181 - 5189. [Abstract] [Full Text] [PDF]

				J Gronwald, T Huzarski, B Byrski, K Medrek, J Menkiszak, A N Monteiro, P Sun, J Lubinski, and S A Narod Cancer risks in first degree relatives of BRCA1 mutation carriers: effects of mutation and proband disease status J. Med. Genet., May 1, 2006; 43(5): 424 - 428. [Abstract] [Full Text] [PDF]

				S. Sankaran, L. M. Starita, A. M. Simons, and J. D. Parvin Identification of Domains of BRCA1 Critical for the Ubiquitin-Dependent Inhibition of Centrosome Function. Cancer Res., April 15, 2006; 66(8): 4100 - 4107. [Abstract] [Full Text] [PDF]

				A. A. Horwitz, S. Sankaran, and J. D. Parvin Direct Stimulation of Transcription Initiation by BRCA1 Requires Both Its Amino and Carboxyl Termini J. Biol. Chem., March 31, 2006; 281(13): 8317 - 8320. [Abstract] [Full Text] [PDF]

				J. R. Morris, L. Pangon, C. Boutell, T. Katagiri, N. H. Keep, and E. Solomon Genetic analysis of BRCA1 ubiquitin ligase activity and its relationship to breast cancer susceptibility Hum. Mol. Genet., February 15, 2006; 15(4): 599 - 606. [Abstract] [Full Text] [PDF]

				A. M. Simons, A. A. Horwitz, L. M. Starita, K. Griffin, R. S. Williams, J.N. M. Glover, and J. D. Parvin BRCA1 DNA-Binding Activity Is Stimulated by BARD1 Cancer Res., February 15, 2006; 66(4): 2012 - 2018. [Abstract] [Full Text] [PDF]

				J. Silke, T. Kratina, D. Chu, P. G. Ekert, C. L. Day, M. Pakusch, D. C. S. Huang, and D. L. Vaux Determination of cell survival by RING-mediated regulation of inhibitor of apoptosis (IAP) protein abundance PNAS, November 8, 2005; 102(45): 16182 - 16187. [Abstract] [Full Text] [PDF]

				S. Sankaran, L. M. Starita, A. C. Groen, M. J. Ko, and J. D. Parvin Centrosomal Microtubule Nucleation Activity Is Inhibited by BRCA1-Dependent Ubiquitination Mol. Cell. Biol., October 1, 2005; 25(19): 8656 - 8668. [Abstract] [Full Text] [PDF]

				E M Rosen, S Fan, and C Isaacs BRCA1 in hormonal carcinogenesis: basic and clinical research Endocr. Relat. Cancer, September 1, 2005; 12(3): 533 - 548. [Abstract] [Full Text] [PDF]

				M K Sauer and I L Andrulis Identification and characterization of missense alterations in the BRCA1 associated RING domain (BARD1) gene in breast and ovarian cancer J. Med. Genet., August 1, 2005; 42(8): 633 - 638. [Abstract] [Full Text] [PDF]