Ub Ligase Research Articles

BARD1 regulates BRCA1 apoptotic function by a mechanism involving nuclear retention

BRCA1 is involved in maintaining genomic integrity and, as a regulator of the G2/M checkpoint, contributes to DNA repair and cell survival. The overexpression of BRCA1 elicits diverse cellular responses including apoptosis due to the stimulation of specific signaling pathways. BRCA1 is normally regulated by protein turnover, but is stabilized by BARD1 which can recruit BRCA1 to the nucleus to form a ubiquitin E3 ligase complex involved in DNA repair or cell survival. Here, we identify BARD1 as a regulator of BRCA1-dependent apoptosis. Using transfected MCF-7 breast cancer cells, we found that BRCA1-induced apoptosis was independent of p53 and was stimulated by BRCA1 nuclear export. Conversely, BARD1 reduced BRCA1-dependent apoptosis by a mechanism involving nuclear sequestration. Regulation of apoptosis by BARD1 was reduced by BRCA1 cancer mutations that disrupt Ub ligase function. Transfection of BRCA1 N-terminal peptides that disrupted the cellular BRCA1–BARD1 interaction caused a loss of nuclear BRCA1 that correlated with increased apoptosis in single cell assays, but did not alter localization or expression of endogenous BARD1. Reducing BARD1 levels by siRNA caused a small increase in apoptosis. Our findings identify a novel apoptosis inhibitory function of BARD1 and suggest that nuclear retention of BRCA1–BARD1 complexes contributes to both DNA repair and cell survival.

The UbcH8 ubiquitin E2 enzyme is also the E2 enzyme for ISG15, an IFN-alpha/beta-induced ubiquitin-like protein.

Ubiquitin-(Ub) like proteins (Ubls) are conjugated to their targets by an enzymatic cascade involving an E1 activating enzyme, an E2 conjugating enzyme, and in some cases an E3 ligase. ISG15 is a Ubl that is conjugated to cellular proteins after IFN-alpha/beta stimulation. Although the E1 enzyme for ISG15 (Ube1L/E1(ISG15)) has been identified, the identities of the downstream components of the ISG15 conjugation cascade have remained elusive. Here we report the purification of an E2 enzyme for ISG15 and demonstrate that it is UbcH8, an E2 that also functions in Ub conjugation. In vitro assays with purified Ub E2 enzymes and in vivo RNA interference assays indicate that UbcH8 is a major E2 enzyme for ISG15 conjugation. These results indicate that the ISG15 conjugation pathway overlaps or converges with the Ub conjugation pathway at the level of a specific E2 enzyme. Furthermore, these results raise the possibility that the ISG15 conjugation pathway might use UbcH8-competent Ub ligases in vivo. As an initial test of this hypothesis, we have shown that a UbcH8-competent Ub ligase conjugates ISG15 to a specific target in vitro. These results challenge the concept that Ub and Ubl conjugation pathways are strictly parallel and nonoverlapping and have important implications for understanding the regulation and function of ISG15 conjugation in the IFN-alpha/beta response.

CHIP-Hsc70 Complex Ubiquitinates Phosphorylated Tau and Enhances Cell Survival

The microtubule-binding protein tau has been implicated in the neurofibrillary pathology of Alzheimer's disease. Within affected cells, ubiquitinated and hyperphosphorylated tau assembles into massive filamentous polymers. Eventually these tangle-bearing neurons die. The formation of neurofibrillary tangles closely parallels the progression and anatomic distribution of neuronal loss in Alzheimer's disease, suggesting that these lesions play a role in the disease pathogenesis. Mutations in the human tau gene cause autosomal dominant neurodegenerative disorders. These and other neurodegenerative conditions are also characterized by extensive neurofibrillary pathology. The mechanisms underlying tau-mediated neurotoxicity remain unclear; however, phosphorylated tau is a strong candidate for a toxic molecule, particularly those isoforms phosphorylated by the kinases glycogen synthase kinase 3beta and Cdk5. Here we show that Alzheimer tau binds to Hsc70, and its phosphorylation is a recognition requirement for the addition of ubiquitin (Ub) by the E3 Ub ligase CHIP (carboxyl terminus of the Hsc70-interacting protein) and the E2 conjugating enzyme UbcH5B. Other E3 Ub ligases including parkin and Cbl failed to ubiquitinate phosphorylated tau. CHIP could rescue phosphorylated tau-induced cell death, and therefore the CHIP-Hsc70 complex may provide a new therapeutic target for the tauopathies.

AIRE functions as an E3 ubiquitin ligase.

Autoimmune regulator (AIRE) gene mutation is responsible for the development of autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy, an organ-specific autoimmune disease with monogenic autosomal recessive inheritance. AIRE is predominantly expressed in medullary epithelial cells of the thymus and is considered to play important roles in the establishment of self-tolerance. AIRE contains two plant homeodomain (PHD) domains, and the novel role of PHD as an E3 ubiquitin (Ub) ligase has just emerged. Here we show that the first PHD (PHD1) of AIRE mediates E3 ligase activity. The significance of this finding was underscored by the fact that disease-causing missense mutations in the PHD1 (C311Y and P326Q) abolished its E3 ligase activity. These results add a novel enzymatic function for AIRE and suggest an indispensable role of the Ub proteasome pathway in the establishment of self-tolerance, in which AIRE is involved.

A conserved catalytic residue in the ubiquitin-conjugating enzyme family.

Ubiquitin (Ub) regulates diverse functions in eukaryotes through its attachment to other proteins. The defining step in this protein modification pathway is the attack of a substrate lysine residue on Ub bound through its C-terminus to the active site cysteine residue of a Ub-conjugating enzyme (E2) or certain Ub ligases (E3s). So far, these E2 and E3 cysteine residues are the only enzyme groups known to participate in the catalysis of conjugation. Here we show that a strictly conserved E2 asparagine residue is critical for catalysis of E2- and E2/RING E3-dependent isopeptide bond formation, but dispensable for upstream and downstream reactions of Ub thiol ester formation. In contrast, the strictly conserved histidine and proline residues immediately upstream of the asparagine are dispensable for catalysis of isopeptide bond formation. We propose that the conserved asparagine side chain stabilizes the oxyanion intermediate formed during lysine attack. The E2 asparagine is the first non-covalent catalytic group to be proposed in any Ub conjugation factor.

The degradation of promyelocytic leukemia and Sp100 proteins by herpes simplex virus 1 is mediated by the ubiquitin-conjugating enzyme UbcH5a.

Infected cell protein 0 (ICP0) of herpes simplex virus 1 expresses two E3 ubiquitin (Ub) ligase activities mapping in the domains encoded by exons 2 and 3, respectively. Site 1 (exon 3) is responsible for the degradation of the E2 Ub-conjugating enzyme cdc34 whereas site 2 (exon 2) is associated with a ring finger and has been shown to mediate the degradation of promyelocytic leukemia (PML) and Sp100 proteins and the dispersal of nuclear domain 10 (ND10). In in vitro assays site 2 polyubiquitylates the E2 enzymes UbcH5a and UbcH6 but not other (e.g., UbcH7) enzymes. In this article, we show that ectopic expression of dominant negative UbcH5a carrying the substitution C85A delayed or blocked the degradation of PML and Sp100 and dispersal of ND10 whereas ectopic expression of wild-type UbcH5a or dominant negative UbcH6 and UbcH7 carrying the substitutions C131A and C86A, respectively, had no effect. These results link the degradation of PML and Sp100 and the dispersal of ND10 to the E3 activities of ICP0 associated with the UbcH5a E2 enzyme.

A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes.

Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).

Binding and recognition in the assembly of an active BRCA1/BARD1 ubiquitin-ligase complex.

BRCA1 is a breast and ovarian cancer tumor suppressor protein that associates with BARD1 to form a RINGRING heterodimer. The BRCA1BARD1 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 BRCA1BARD1 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 alpha-helix of the BRCA1 RING domain, a region disrupted by cancer-predisposing mutations. Unexpectedly, a second Ub-conjugating enzyme, UbcH7, also interacts with the BRCA1BARD1 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.

Skp2 Regulates Myc Protein Stability and Activity

Myc is an oncoprotein transcription factor that plays a prominent role in cancer. Like many transcription factors, Myc is an unstable protein that is destroyed by ubiquitin (Ub)-mediated proteolysis. Here, we report that the oncoprotein and Ub ligase Skp2 regulates Myc ubiquitylation and stability. Because of the growing number of Ub ligases that function as transcriptional coactivators, we speculated that Skp2 might also regulate Myc's transcriptional activity. Consistent with this model, we also show that Skp2 is a transcriptional coactivator for Myc, recognizing an essential element within the Myc activation domain and activating Myc target genes. These data suggest that Skp2 functions to connect Myc activity and destruction, and reveal an unexpected oncoprotein connection that may play an important role in controlling cell growth in normal and cancer cells.

Ubiquitin ligases and the immune response.

Ubiquitin (Ub)-protein conjugation represents a novel means of posttranscriptional modification in a proteolysis-dependent or -independent manner. E3 Ub ligases play a key role in governing the cascade of Ub transfer reactions by recognizing and catalyzing Ub conjugation to specific protein substrates. The E3s, which can be generally classified into HECT-type and RING-type families, are involved in the regulation of many aspects of the immune system, including the development, activation, and differentiation of lymphocytes, T cell-tolerance induction, antigen presentation, immune evasion, and virus budding. E3-promoted ubiquitination affects a wide array of biological processes, such as receptor downmodulation, signal transduction, protein processing or translocation, protein-protein interaction, and gene transcription, in addition to proteasome-mediated degradation. Deficiency or mutation of some of the E3s like Cbl, Cbl-b, or Itch, causes abnormal immune responses such as autoimmunity, malignancy, and inflammation. This review discusses our current understanding of E3 Ub ligases in both innate and adaptive immunity. Such knowledge may facilitate the development of novel therapeutic approaches for immunological diseases.

Development of a high throughput time-resolved fluorescence resonance energy transfer assay for TRAF6 ubiquitin polymerization.

Interleukin 1 receptor activation innervates a cascade of signal transduction events that ultimately lead to the activation of inflammatory and immune response genes. TRAF6 is a Ub ligase (E3) involved in this pathway, and inhibition of this critical enzyme may provide a means for treating inflammatory and immune diseases. A TR-FRET assay has been developed and evaluated for HTS for TRAF6 inhibitors. Bio-Ub and Eu-Ub were polymerized in the presence of Ub activating enzyme E1, conjugating enzyme E2, and TRAF6. Following a 2-h incubation, the reaction was stopped with a buffer containing 10 m M EDTA and the fluorescence donor SA-APC. Fluorescence energy transfer from Eu to APC was measured as a ratio of fluorescence intensity at 655 nm to that at 615 nm (excitation at 340 nm). This homogeneous assay has been optimized and validated in a 384-well format. A window of five- to eightfold and Z' factor of 0.6-0.8 suggests that this assay can be applied to screen for inhibitors of the polyubiquitination activity of TRAF6.

Enhancement of BRCA1 E3 Ubiquitin Ligase Activity through Direct Interaction with the BARD1 Protein

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 Lys(48)- and Lys(63)-linked poly-Ub chains. However, the enhancement of BRCA1 autoubiquitylation by BARD1 mostly resulted in poly-Ub chains linked through Lys(63), 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.

Paramyxoviruses SV5 and HPIV2 assemble STAT protein ubiquitin ligase complexes from cellular components.

Signal transducer and activator of transcription (STAT) proteins are normally long-lived, but infection with certain Paramyxoviruses results in efficient loss of IFN-responsive STAT1 or STAT2. Expression of a virus-encoded protein called "V" is sufficient to mediate the destruction of STAT proteins. STAT degradation is blocked by proteasome inhibitors, strongly implicating the ubiquitin (Ub)-proteasome targeting system. We demonstrate that cellular expression of V proteins from simian virus 5 (SV5) and type II human parainfluenza virus (HPIV2) induces polyubiquitylation of STAT1 and STAT2 targets. In vitro, the V proteins catalyze Ub transfer in an ATP-dependent process that requires both Ub-activating (E1) and Ub-conjugating (E2) activities. Furthermore, SV5 and HPIV2 V-interacting protein partners were isolated by affinity purification from human cells and reveal a complex of associated cellular proteins. This complex includes both STAT1 and STAT2, and the damaged DNA binding protein, DDB1. In addition, a protein related to a family of cellular Ub ligase complex subunits, cullin 4A (Cul4A), associated with the V proteins. The roles of both DDB1 and Cul4A in STAT1 degradation by SV5 infection were analyzed using small interfering RNAs. These findings demonstrate the assembly of a V-dependent degradation complex that includes STAT1, STAT2, DDB1, and Cul4A. In agreement with prior nomenclature on SCF-type cellular E3 enzymes, we refer to this complex as VDC.

Pairs of dipeptides synergistically activate the binding of substrate by ubiquitin ligase through dissociation of its autoinhibitory domain.

Protein degradation by the ubiquitin (Ub) system controls the concentrations of many regulatory proteins. The degradation signals (degrons) of these proteins are recognized by the system's Ub ligases (complexes of E2 and E3 enzymes). Two substrate-binding sites of UBR1, the E3 of the N-end rule pathway in the yeast Saccharomyces cerevisiae, recognize basic (type 1) and bulky hydrophobic (type 2) N-terminal residues of proteins or short peptides. A third substrate-binding site of UBR1 targets CUP9, a transcriptional repressor of the peptide transporter PTR2, through an internal (non-N-terminal) degron of CUP9. Previous work demonstrated that dipeptides with destabilizing N-terminal residues allosterically activate UBR1, leading to accelerated in vivo degradation of CUP9 and the induction of PTR2 expression. Through this positive feedback, S. cerevisiae can sense the presence of extracellular peptides and react by accelerating their uptake. Here, we show that dipeptides with destabilizing N-terminal residues cause dissociation of the C-terminal autoinhibitory domain of UBR1 from its N-terminal region that contains all three substrate-binding sites. This dissociation, which allows the interaction between UBR1 and CUP9, is strongly increased only if both type 1- and type 2-binding sites of UBR1 are occupied by dipeptides. An aspect of autoinhibition characteristic of yeast UBR1 also was observed with mammalian (mouse) UBR1. The discovery of autoinhibition in Ub ligases of the UBR family indicates that this regulatory mechanism may also control the activity of other Ub ligases.

Characterization of the novel E3 ubiquitin ligase encoded in exon 3 of herpes simplex virus-1-infected cell protein 0

Infected cell protein 0 (ICP0) of herpes simplex virus-1 is a 775-aa residue multifunctional protein that acts as a promiscuous transactivator linked to the degradation of several proteins. ICP0 is the only protein known which encodes two physically separated E3 ubiquitin (Ub) ligase domains, one, designated herpes virus Ub ligase 1 (HUL-1) located in a domain encoded in exon 3 and one designated herpes virus Ub ligase 2 (HUL-2) associated with the really interesting new gene (RING) finger domain encoded by exon 2. We report the following: (i) ICP0 residues 543-680 are sufficient for HUL-1 E3 activity and necessary determinants are encoded between residues 616 and 680. (ii) In substrate independent in vitro ubiquitylation reactions, a chimeric protein containing the HUL-1 domain promotes the ubiquitylation of itself and the ubiquitin conjugating enzyme (E2) cdc34 and interacts with cdc34. (iii) The mechanism of HUL-1 E3 function does not involve formation of a thioester between the HUL-1 domain and Ub. (iv) Residues 621-625 are essential for in vitro HUL-1 E3 activity and interaction between the HUL-1 domain and cdc34, suggesting that this interaction is required for HUL-1 E3 function.

Interaction between parkin and alpha‐synuclein

Mutations in the parkin gene have been identified in autosomal recessive Parkinson's disease (ARPD) throughout the world. Several groups have shown that parkin acts as an ubiquitin (Ub) ligase in in vivo and in vitro studies and identified several molecules to be ubiquitinated by parkin. Two rare mutations, Ala53Thr and Ala30Pro, in the α‐synuclein (αS) gene have been identified in autosomal dominant PD cases. αS is a major constituent of Lewy bodies the pathological hallmark of PD. Therefore, αvS metabolism is likely to play a central role in the pathogenesis of PD. We investigated the possible interaction between the two familial PD‐linked gene products, parkin and αS. We first demonstrated that parkin epitopes can be found within Lewy bodies by immunohistochemistry and immuno‐EM. We also detected parkin biochemically in affinity purified Lewy bodies. Furthermore, we identified a protein complex in normal human brain that includes parkin as an E3 Ub ligase, UbcH7 as its associated E2 protein and a novel 22 kDa glycosylated isoform of αS (αSp22) as its substrate. In an in vitro ubiquitination assay, αSp22 was further modified by normal but not mutant parkin into high Mr polyubiquitinated species. We observed accumulation of nonubiquitinated αSp22 in ARPD brain homogenates. Importantly enzymatic digestion of αSp22 abolish the binding of recombinant parkin with deglycosylated αS. We conclude that αSp22 is a substrate for parkin's Ub ligase activity in normal brain, and that loss of function in parkin‐deficient ARPD causes pathological αSp22 accumulation. These findings demonstrate a critical functional relationship between the two PD‐linked gene products.

A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition

Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells. This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins. Here, we show that expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface. MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity. In vitro, addition of a MIR2–glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion; this reaction is abolished by mutation of the Zn-coordinating residues of the PHD domain. Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice.

NEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)–like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3 − /− mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57Kip2. These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. β-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3 −/− cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.

Ubiquitin-Dependent 26S Proteasomal Pathway: A Role in the Degradation of Native Human Liver CYP3A4 Expressed in Saccharomyces Cerevisiae?

Cytochrome P450, CYP3A4, is the dominant human liver endoplasmic reticulum (ER) hemoprotein enzyme, responsible for the metabolism of over 60% of clinically relevant drugs. We have previously shown that mechanism-based suicide inactivation of CYP3A4 and its rat liver ER orthologs, CYPs 3A, via heme-modification of their protein moieties, results in their ubiquitin (Ub)-dependent 26S proteasomal degradation (Korsmeyer et al. (1999) Arch. Biochem. Biophys. 365, 31; Wang et al. (1999) Arch. Biochem. Biophys. 365, 45). This is not surprising given that the heme-modified CYP3A proteins are structurally damaged. To determine whether the turnover of the native enzyme similarly recruited this pathway, we heterologously expressed this protein in wild-type Saccharomyces cerevisiae and mutant strains (hrd1Δ, hrd2-1, and hrd3Δ) previously shown to be deficient in the Ub-dependent 26S proteasomal degradation of the polytopic ER protein 3-hydroxy-3-methylglutaryl-CoA reductase (isoform Hmg2p), the rate-limiting enzyme in sterol biosynthesis, as well as in strains deficient in ER-associated Ub-conjugating enzymes, Ubc6p and/or Ubc7p (Hampton et al. (1996) Mol. Biol. Cell 7, 2029; Hampton and Bhakta (1997) Proc. Natl. Acad. Sci. USA 94, 12,944). Our findings reveal that in common with the degradation of Hmg2p, that of native CYP3A4 also requires Hrd2p (a subunit of the 19S cap complex of the 26S proteasome) and Ubc7p, and to a much lesser extent Hrd3p, a component of the ER-associated Ub-ligase complex. In contrast to Hmg2p-degradation, that of native CYP3A4 does not appear to absolutely require Hrd1p, another component of the ER-associated Ub–ligase complex. Furthermore, studies in a S. cerevisiae pep4Δ strain proven to be deficient in the vacuolar degradation of carboxypeptidase Y indicated that CYP3A4 degradation is also largely independent of vacuolar (lysosomal) proteolytic function. The degradation of two other native ER proteins, Sec61p and Sec63p, normal components of the ER translocon, were also examined in parallel and found to be stabilized to some extent in HRD2- and UBC7-deficient strains. Together these findings attest to the remarkable mechanistic diversity in the normal degradation of ER proteins.

Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity.

BRCA1 is a breast and ovarian cancer-specific tumor suppressor that seems to be involved in transcription and DNA repair. Here we report that BRCA1 exhibits a bona fide ubiquitin (Ub) protein ligase (E3) activity, and that cancer-predisposing mutations within the BRCA1 RING domain abolish its Ub ligase activity. Furthermore, these mutants are unable to reverse gamma-radiation hypersensitivity of BRCA1-null human breast cancer cells, HCC1937. Additionally, these mutations within the BRCA1 RING domain are not capable of restoring a G(2) + M checkpoint in HCC1937 cells. These results establish a link between Ub protein ligase activity and gamma-radiation protection function of BRCA1, and provide an explanation for why mutations within the BRCA1 RING domain predispose to cancer. Furthermore, we propose that the analysis of the Ub ligase activity of RING-domain mutations identified in patients may constitute an assay to predict predisposition to cancer.