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...

We have identified two highly conserved RING finger proteins, ROC1 and ROC2, that are homologous to APC11, a subunit of the anaphase-promoting complex. ROC1 and ROC2 commonly interact with all cullins while APC11 specifically interacts with APC2, a cullin-related APC subunit. YeastROC1 encodes an essential gene whose reduced expression resulted in multiple, elongated buds and accumulation of Sic1p and Cln2p. ROC1 and APC11 immunocomplexes can catalyze isopeptide ligations to form polyubiquitin chains in an E1- and E2-dependent manner. ROC1 mutations completely abolished their ligase activity without noticeable changes in associated proteins. Ubiquitination of phosphorylated I kappa B alpha can be catalyzed by the ROC1 immunocomplex in vitro. Hence, combinations of ROC/APC11 and cullin proteins proteins potentially constitute a wide variety of ubiquitin ligases.

Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Here, we report that human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. As with SKP1-CUL1, the DDB1-CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically expressed DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevented UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. We suggest that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4A(DDB1), in response to UV irradiation.

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.

The expression of the replication-dependent histone mRNAs is tightly regulated during the cell cycle. As cells progress from G 1 to S phase, histone mRNA levels increase 35-fold, and they decrease again during G 2 phase. Replication-dependent histone mRNAs are the only metazoan mRNAs that lack polyadenylated tails, ending instead in a conserved stem-loop. Much of the cell cycle regulation is posttranscriptional and is mediated by the 3 stem-loop. A 31-kDa stem-loop binding protein (SLBP) binds the 3 end of histone mRNA. The SLBP is necessary for pre-mRNA processing and accompanies the histone mRNA to the cytoplasm, where it is a component of the histone messenger RNP. We used synchronous CHO cells selected by mitotic shakeoff and HeLa cells synchronized at the G 1 /S or the M/G 1 boundary to study the regulation of SLBP during the cell cycle. In each system the amount of SLBP is regulated during the cell cycle, increasing 10-to 20-fold in the late G 1 and then decreasing in the S/G 2 border. SLBP mRNA levels are constant during the cell cycle. SLBP is regulated at the level of translation as cells progress from G 1 to S phase, and the protein is rapidly degraded as they progress into G 2 . Regulation of SLBP may account for the posttranscriptional component of the cell cycle regulation of histone mRNA.The replication-dependent histone mRNAs are tightly regulated during the cell cycle, increasing 35-fold as cells progress from G 1 to S phase (18). There is only a three-to five-fold increase in the rate of transcription of the histone genes (7,19), indicating that much of the regulation is posttranscriptional. The posttranscriptional component of cell cycle regulation is mediated by the 3Ј end of the histone mRNA (32, 35), which is a highly conserved stem-loop (34). The only processing step necessary for formation of the mature histone mRNA is an endonucleolytic cleavage to form the 3Ј end (14). Cleavage is directed by two cis-acting elements, the stem-loop which binds to the hairpin binding factor (37, 38), and a purine-rich sequence about 10 nucleotides (nt) 3Ј of the cleavage site that binds the 5Ј end of U7 snRNA (3,39,55). Hairpin binding factor is composed of a single 31-kDa protein, the stem-loop binding protein (SLBP) (10, 60) or hairpin binding protein (33), which is required for processing in vivo (44) and which remains with the mature mRNA as a component of the cytoplasmic messenger RNP (mRNP) (9, 17). At least one additional factor, a heat-labile factor, is required for processing, but this factor has not been well defined biochemically (15).Two posttranscriptional regulatory steps contribute to the cell cycle regulation of histone mRNA concentrations. Processing is regulated as cells progress from G 1 to S phase, and the half-life of histone mRNA is reduced to about 10 min at the end of S phase (18), resulting in the destruction of histone mRNA prior to mitosis. We (60) and others (33) recently cloned the cDNA for the SLBP from several species using the yeast three-hybrid system (50). Here we show tha...

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]

Synoviolin, also called HRD1, is an E3 ubiquitin ligase and is implicated in endoplasmic reticulum -associated degradation. In mammals, Synoviolin plays crucial roles in various physiological and pathological processes, including embryogenesis and the pathogenesis of arthropathy. However, little is known about the molecular mechanisms of Synoviolin in these actions. To clarify these issues, we analyzed the profile of protein expression in synoviolinnull cells. Here, we report that Synoviolin targets tumor suppressor gene p53 for ubiquitination. Synoviolin sequestrated and metabolized p53 in the cytoplasm and negatively regulated its cellular level and biological functions, including transcription, cell cycle regulation and apoptosis. Furthermore, these p53 regulatory functions of Synoviolin were irrelevant to other E3 ubiquitin ligases for p53, such as MDM2, Pirh2 and Cop1, which form autoregulatory feedback loops. Our results provide novel insights into p53 signaling mediated by Synoviolin.