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...

NSM is safe with a low complication rate. No significant difference was observed in cumulative local recurrence rate, cumulative distant disease recurrence rate, and overall survival between patients who underwent NSM or conventional total mastectomy, confirming that NSM was surgically and oncologically safe.

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Ebp2 plays an essential role in biogenesis of 60S ribosomal subunits. We determined the genetic interactions between EBP2 and RPL36A/B, which encodes ribosomal protein L36a/b. RPL36A/B was a multicopy suppressor to ebp2 mutants, and the suppression was not common to defects in ribosome biogenesis resulting from other mutations of assembly factors. Disruption of RPL36A or RPL36B caused synthetic enhancement of the growth defect of the ebp2-14 allele at high temperatures. Disruption of RPL36B led to a more severe growth defect than that of RPL36A due to imbalances in the expression levels of the duplicated genes. Primer-extension analysis revealed that L36a/b is required for the processing of 27SA2, 27SA3, and 27SBL pre-rRNAs. Two-hybrid analysis indicated that Ebp2 interacts with ribosomal proteins L36a/b, L34a/b, and L8, which in mature ribosomes are located adjacent to each other in close proximity to the 3' end of 5.8S rRNA. These results suggest that Ebp2 functions cooperatively with ribosomal proteins L36, L34, and L8 in biogenesis of the 60S ribosomal subunit.

Reduced ribosome biogenesis in response to environmental conditions is a key feature of cell adaptation to stress. For example, ribosomal genes are transcriptionally repressed when cells are exposed to tunicamycin, a protein glycosylation inhibitor that induces endoplasmic reticulum stress and blocks vesicular trafficking in the secretory pathway. Here, we describe a novel regulatory model, in which tunicamycin-mediated stress induces the accumulation of long-chain sphingoid bases and subsequent activation of Pkh1/2 signaling, which leads to decreased expression of ribosomal protein genes via the downstream effectors Pkc1 and Sch9. Target of rapamycin complex 1 (TORC1), an upstream activator of Sch9, is also required. This pathway links ribosome biogenesis to alterations in membrane lipid composition under tunicamycin-induced stress conditions. Our results suggest that sphingolipid/Pkh1/2-TORC1/Sch9 signaling is an important determinant for adaptation to tunicamycin-induced stress.