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.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 Ub 1 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 G 1 /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...

BRCA1 is a breast and ovarian cancer tumor suppressor protein that associates with BARD1 to form a RING͞RING heterodimer. The BRCA1͞BARD1 RING complex functions as an ubiquitin (Ub) ligase with activity substantially greater than individual BRCA1 or BARD1 subunits. By using NMR spectroscopy and site-directed mutagenesis, we have mapped the binding site on the BRCA1͞BARD1 heterodimer for the Ub-conjugating enzyme UbcH5c. The results demonstrate that UbcH5c binds only to the BRCA1 RING domain and not the BARD1 RING. The binding interface is formed by the first and second Zn 2؉ -loops and central ␣-helix of the BRCA1 RING domain, a region disrupted by cancer-predisposing mutations. Unexpectedly, a second Ub-conjugating enzyme, UbcH7, also interacts with the BRCA1͞BARD1 complex with similar affinity, although it is not active in Ub-ligase activity assays. Thus, binding alone is not sufficient for BRCA1-dependent Ub-ligase activity.

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Clinicopathologic characteristics and prognosis of breast cancer patients associated with pregnancy and lactation were clarified by means of a case‐control study of matched non‐pregnant and non‐lactating patients with breast cancer. From 18 institutions in Japan, a total of 192 subjects with breast cancer diagnosed during pregnancy (72 cases) and lactation (120 cases) were collected between 1970 and 1988, accounting for 0.76% of all breast cancer patients. The duration of symptoms was longer and tumor size was larger in the study subjects. Although the disease‐free interval was longer than that in the control patients, the survival time was shorter. There was no characteristic difference in histologic type. Vascular invasion and lymph node metastasis were found more frequently in the subjects. The positive rates of estrogen receptor and progesterone receptor were lower in the subjects. The 5‐ and 10‐year survival rates of the study patients were 65% and 55%, respectively, and these survivals were significantly lower than those of the control (P < 0.001). The survival rates were poorer in the subjects, in accordance with stage and lymph node metastasis. The results suggest that most of the patients with breast cancer diagnosed during pregnancy and lactation are in a more advanced stage because of a delay in detection and diagnosis, and hence have unfavorable prognosis. Therefore, it is important to diagnose and treat early for improvement of prognosis in patients with breast cancer during pregnancy and lactation.

The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.The familial breast and ovarian cancer susceptibility gene product BRCA1 functions in multiple cellular processes that include DNA repair, transcriptional regulation, cell cycle control, and apoptosis (1-4). One possible biochemical function that could contribute to the cellular functions of BRCA1 is the ubiquitin (Ub) 1 ligase activity that arises when BRCA1 forms a RING heterodimer with BARD1 (5-10). Ub-protein isopeptide ligases (E3) catalyze the formation of poly-Ub chains on substrate proteins via isopeptide bonds that link the C-terminal Gly residue of one Ub molecule (activated in an ATP-dependent manner by the enzyme E1) to the ⑀NH 2 group of a Lys side chain in another Ub molecule (11). The most common poly-Ub chain is linked through Lys-48 of Ub and serves as a signal for rapid degradation of substrates by the proteasome-dependent proteolysis pathway (12). However, recent studies have revealed roles other than proteolysis for polyubiquitination (13). While Lys-48-and Lys-29-linked chains mediate proteasomedependent degradation (12, 14), Lys-63-linked chains are a signal for endocytosis, IB kinase activation, ribosome modification, and DNA repair (15-21). Therefore characterization of the poly-Ub chain linkage is important to predict biological function of Ub ligases.Several groups have characterized the type of poly-Ub linkages formed by the BRCA1-BARD1 heterodimer. It was reported that the poly-Ub chain built by the BRCA1-BARD1 ligase is linked through Ub Lys residues other than Lys-48, suggesting they may not serve as a degradation signal (6). Another group reported that BARD1 stimulates the formation of both Lys-48-and Lys-63-linked poly-Ub chains and that the BRCA1 autoubiquitylation by BARD1 mostly results in poly-Ub chains linked through Lys-63 (7)...

The breast and ovarian tumor suppressor BRCA1 constitutes a RING heterodimer E3 ligase with BARD1. BRCA1-associated protein 1 (BAP1) is a ubiquitin COOH-terminal hydrolase that was initially identified as a protein that bound to the RING finger domain of BRCA1. However, how BAP1 contributes to the E3 activity of BRCA1/BARD1 is unclear. Here, we report that BAP1 interacts with BARD1 to inhibit the E3 ligase activity of BRCA1/BARD1. Domains comprised by residues 182-365 of BAP1 interact with the RING finger domain of BARD1, and surface plasmon resonance spectroscopy (BIAcore) analyses showed that BAP1 interferes with the BRCA1/ BARD1 association. The perturbation resulted in inhibition of BRCA1 autoubiquitination and NPM1/B23 ubiquitination by BRCA1/BARD1. Although BAP1 was capable of deubiquitinating the polyubiquitin chains mediated by BRCA1/BARD1 in vitro, a catalytically inactive mutant of BAP1, C91S, still inhibited the ubiquitination in vitro and in vivo, implicating a second mechanism of action. Importantly, inhibition of BAP1 expression by short hairpin RNA resulted in hypersensitivity of the cells to ionizing irradiation and in retardation of S-phase progression. Together, these results suggest that BAP1 and BRCA1/BARD1 coordinately regulate ubiquitination during the DNA damage response and the cell cycle. [Cancer Res 2009;69(1):111-9]

, and BRCA1-BARD1 co-expression in cells causes NPM stabilization rather than degradation. This is consistent with the notion that this ligase catalyzes untraditional polyubiquitin chains. Given the many overlapped functions between NPM and BRCA1, we propose that NPM is a strong candidate as a substrate of the BRCA1-BARD1 ubiquitin ligase.

To elucidate the molecular mechanisms of mammary carcinogenesis and discover novel therapeutic targets for breast cancer, we previously carried out genome-wide expression profile analysis of