NBA1/MERIT40 and BRE Interaction Is Required for the Integrity of Two Distinct Deubiquitinating Enzyme BRCC36-containing Complexes*

BRCC36-deubiquitinating enzyme (DUB) forms two different complexes through interactions with two different adaptor proteins Abraxas and ABRO1 in cells. Abraxas mainly localizes in the nucleus, mediating the interaction of BRCC36 with BRCA1. ABRO1 is mainly localized in the cytoplasm. Because it lacks the BRCA1-interacting motif, the ABRO1 complex does not interact with BRCA1. Both BRCC36-containing complexes contain common components including BRE and NBA1/MERIT40. Here, we found that the two complexes are assembled in a similar manner and NBA1 and BRE interaction is critical for maintaining the integrity of both of the complexes. Knockdown of NBA1 or BRE leads to decreased levels of components of the two BRCC36-containing complexes. We provided evidence that NBA1 interacts with BRE through a C-terminal conserved motif of the NBA1 protein and a C-terminal UEV domain of the BRE protein. Furthermore, the NBA1-BRE interaction is required for cellular resistance to ionizing irradiation and NBA1's role in recruiting BRCA1 to DNA damage sites. Together, these studies reveal critical interactions required for the formation and function of BRCC36-containing DUB complexes.

Modification of proteins by the covalent attachment of ubiquitin (Ub) 2 to the lysine (Lys) residue of a target protein is a key regulatory mechanism of many cellular processes including the DNA damage response (1,2). In response to ionizing irradiation, ubiquitination occurs at DNA damage sites in a central DNA damage kinases ATM/ATR-dependent manner (3). Ubiquitin Lys63 (K63) polyubiquitin chain linkages play important roles in the recruitment of repair factors in the DNA damage response (3). K63-linked polyubiquitin chains are assem-bled through a conserved heterodimer of an E2 complex composed of a catalytically active Ubc13 subunit and its inactive sequence homolog Mms2 that lacks the enzymatic active site (4,5). The formation of K63-polyubiquitins at DNA damage sites depends on two E3 ligases RNF8 and RNF168 (3,6). In addition, a HECT domain-containing E3 ligase HERC2 is also involved in facilitating the formation of K63-linked ubiquitin chains (7).
Deubiquitinating enzymes (DUBs) catalyze the removal of Ub from Ub-conjugated substrate proteins and it has become increasingly obvious that Ub deconjugation plays important roles in regulating Ub-dependent pathways (8,9). BRCC36 is a member of a small family of DUBs containing JAMM/MPNϩ domain proteins, which are Zn 2ϩ -binding metalloproteases (10 -12). It selectively cleaves K63-linked polyubiquitin (12)(13)(14), and is required for proper checkpoint regulation in response to DNA damage (13,(15)(16)(17). It plays an important role in recruiting BRCA1 to DNA damage sites, as BRCC36 deficiency leads to decreased BRCA1 accumulation at DNA damage sites (3).
BRCC36 is a component of the BRCA1 A complex (13,17,18). The BRCA1 A complex contains at least five different components, Abraxas, NBA1/MERIT40, BRE, Rap80, and BRCC36. It associates with BRCA1 through an interaction of Abraxas with the BRCA1 C-terminal BRCT domains (3). The BRCA1 A complex plays a critical role in the DNA damage-induced ubiquitin signaling that recruits BRCA1 to DNA damage sites. DNA damage-induced ATM/ATR phosphorylation on S139 of histone H2AX directly recruits MDC1 through MDC1's BRCT domains. The subsequent MDC1 phosphorylation-dependent recruitment of ubiquitin ligases RNF8/Ubc13 and RNF168/ UBC13 generates K63 Ub chains on the damaged chromatin that creates docking sites for the BRCA1 A complex for recruitment of BRCA1 to sites of damage (3). Down-regulation of each component of this complex compromises the recruitment of BRCA1 to DNA damage sites, leading to increased cell sensitivity to irradiation (IR) and inability of cells to arrest the cell cycle. In the BRCA1 A complex, Abraxas protein mediates the interaction of the complex with BRCA1 C-terminal BRCT domains via a phospho-SPXF motif. BRCC36 dimerizes with Abraxas forming an MPNϩ/MPN-domain-containing protein pair that is also observed in the lid of the 19S proteasome regulatory particle and the COP9 signalosome (16). The MPNϩ domain displays DUB activity while the MPN-domain lacks the critical residues for DUB activity. Rap80 contains two ubiquitin-interacting motif (UIM) domains that specifically bind to K63linked Ub chains, which is critical for BRCA1 recruitment to DNA damage sites (3). In addition, BRE contains two ubiquitin E2 variant (UEV) domains and the NBA1 protein contains a von Willebrand factor A (VWA) domain that is homologous to the VWA domain of the proteasome subunit Rpn10/S5A/PSMD4 (16). Thus, the BRCA1 A complex contains multiple ubiquitin binding domains and shares similarities with the lid complex of the 19S proteasome regulatory particle (16). However, it is not clear how the complex is assembled to facilitate its functions.
BRCC36 was also identified as a member of a cytoplasmic BRISC complex (BRCC36 isopeptidase complex) for cleavage of K63 Ub chains (14). It appears that the BRISC complex contains a paralog of Abraxas, ABRO1, as well as NBA1, BRE, and BRCC36. In the BRISC complex, the ABRO1 and BRCC36 interaction is minimally required for the K63-specific DUB activity of BRCC36 (19). Similar to the BRCA1 A complex, it is not clear how the multiple components are arranged in the complex to contribute to the integrity and activity of the complex.
NBA1 is a novel protein that contains a VWA domain that is similar to the Rpn10 subunit of the lid of the 19S regulatory subunit (16). NBA1 deficiency leads to decreased levels of Abraxas, BRE, and BRCC36 (16,17,20). In this study, we found that NBA1 and BRE interaction plays an important role in maintaining the integrity of both of the BRCC36-containing complexes. In the BRCA1 A complex, the NBA1 and BRE interaction is required for recruiting BRCA1 to sites of DNA damage and maintaining a proper DNA damage response.
Cell Lines and Cell Culture-U2OS cells were grown in the McCoy's 5A medium supplemented with 10% FBS, 100 g/ml penicillin/streptomycin. 293T cells were grown in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% FBS, 100 g/ml penicillin/streptomycin. NBA1-deficient and BRE-deficient stable cell lines were generated by infecting U2OS cells with retrovirus of MSCV vectors (22) containing shRNAs against NBA1 or BRE sequences with selection of puromycin (0.8 g/ml) for 1 week. For NBA1-or BRE-deficient cells rescued with expression of wild type or mutants of NBA1 or BRE, U2OS cells were first infected with retroviruses expressing wild type or mutants of NBA1 or BRE. Stable cell lines expressing these proteins were then infected with retroviruses containing the appropriate shRNAs that target the 3Ј-UTR sequence of the corresponding genes. To prepare doxycyclin inducible shNBA1 stable cell lines, U2OS cells were infected with lentiviruses expressing shRNAs against the NBA1 gene expressed under a tetracycline-inducible promoter and selection was performed with puromycin (0.8 g/ml).
Immunofluorescence-Cells grown on coverslips were fixed with 3.6% formaldehyde for 15 min, permeablized with 0.5% Triton X-100 solution and incubated with primary antibodies for 2 h followed by appropriate Alexa 488-conjugated (green; Invitrogen) and Cy3-conjugated (red; Amersham Biosciences) secondary antibodies. For immunostaining of cells expressing GFP-NLS-NBA1 or NBA1 mutants, cells were first treated with 0.5% Triton X-100 in PBS prior to fixation to extract soluble NBA1 or its mutant proteins. All images were obtained with a Nikon TE2000 inverted microscope with a Photometrics Cool-Snap HQ camera.
Cell Lysis and Immunoprecipitation-As previously described (16), cells were lysed in NETN buffer (50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5% Nonidet P-40] with protease inhibitors and protein phosphatase inhibitors, 1 mM NaF, and 1 mM Na 3 VO 4 . Immunoprecipitations (IP) were carried out in the same buffer with appropriate antibodies and protein A/G-Sepharose beads (Santa Cruz Biotechnology) overnight at 4°C. FLAG IP was carried out using Flag (M2) beads (Sigma).
Cellular Fractionation-U2OS cells, untreated or treated with 10 Gy IR, were cultured at 37°C for 2 h, trypsinized, and collected. Total cell extracts (TCE) were prepared by resuspending cells directly in Laemmli buffer (3% SDS, 5% 2-mercaptoethanol, 10% glycerol, 50 mM Tris) followed by sonication. For fractionation experiments, cells were resuspended in buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 1.5 mM MgCl 2 , 0.34 M sucrose, 10% glycerol, 5 mM NaF, 1 mM Na 3 VO 4 , 1 mM dithiothreitol (DTT), and protease cocktails) containing 0.1% Triton X-100, and incubated on ice for 5 min for permeabilization. Cells were then centrifuged at 4000 rpm for 5 min at 4°C, and supernatants were collected for preparation of cytoplasmic proteins, while pellets were further processed for nuclear proteins. The supernatants were further centrifuged at 16,000 rpm for 15 min at 4°C to remove cell debris and insoluble aggregates and the supernatants (S2 fraction; cytoplasmic proteins) were collected. The pellets were first washed with ice-cold buffer A, then resuspended in buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, a protease inhibitor mixture) and incubated for 30 min on ice. After centrifugation at 4500 rpm for 5 min at 4°C, the supernatants (S3 fraction, soluble nucleoplasmic protein) were collected. The final chromatin pellet (P3) was washed with buffer B and resuspended in Laemmli buffer and sonicated for 15 s before analysis.
Colony Formation Assay-U2OS stable cell lines were seeded at low density and irradiated with 4 Gy ionizing irradiation using a 137 Cs source. The cells were then cultured at 37°C for 14 days to allow colonies to form. Colonies were stained with 2% methylene blue/50% ethanol. Colonies containing 50 or more cells were counted. Statistical data were analyzed by the t test.
Expression Plasmids and shRNA Plasmids-Retroviral expression constructs for HA-FLAG-NBA1, GFP-BRE, or GFP-NBA1 were made using MSCV vectors carrying HA-FLAG or GFP tag at the N terminus as described (16). GFP-NLS-NBA1 wild type or mutants were generated by cloning the corresponding cDNA fragments into a MSCV vector containing GFP tag and a SV40 nuclear localization signal (NLS). Deletion mutants of NBA1 or BRE were generated by cloning the corresponding cDNA fragments into the above retroviral vectors. All site mutants of NBA1 or BRE were generated with the Quick-ChangeII site-directed mutagenesis kit (Stratagene, CA), according to the manufacturer's protocol. The shRNAs against NBA1 and BRE were cloned into a MSCV vector under a mir30 sequence context as described (22). The shRNAs against NBA1 were shNBA1 # 1 CCTTCAATCTGGAAGGACT (targeting to the 3Ј-UTR of NBA1 mRNA) and shNBA1#2 CTCAAT-GTCTCCCAGAAGA. The shRNAs against BRE were shBRE#1 CACAAATTATGGTGCAGTA (targeting to the 3Ј-UTR of BRE mRNA) and shBRE#2 GCTACAAACTGTTT-GAGGA. The shNBA1#1 sequence was also cloned into a lentiviral construct with shRNA expression regulated by a doxcyclin-inducible promoter.

Two Distinct BRCC36-deubiquitinating Complexes Are
Formed in a Similar Manner through Abraxas and Its Paralog ABRO1 Protein-From tandem affinity purification (TAP) and mass spectrometry analysis of NBA1-associated proteins, we found that NBA1 associates with a protein that is a paralog of the Abraxas protein, termed ABRO1 (16). Previously, we have described that ABRO1 is 39% identical to Abraxas at the N-terminal region of the protein (21). Both ABRO1 and Abraxas contain an MPN domain at the N terminus and a coiled-coil domain at the central region of the protein. While Abraxas contains the phospho-SPXF motif at its C terminus that associates with the BRCT domains of the BRCA1 protein, ABRO1 does not contain this motif (Fig. 1A). We tested the association of NBA1 with endogenous ABRO1 using antibodies against ABRO1 protein for immunoprecipitations. Indeed, we found that ABRO1 associates with the endogenous NBA1. In addition, ABRO1 also associates with BRE and BRCC36 of the BRCA1 A complex (Fig. 1B). However, the BRCA1 A complex component, Rap80, failed to interact with ABRO1 ( Fig. 1B). In addition, as ABRO1 lacks the phospho-SPXF motif that binds to the BRCT domain of BRCA1, it does not interact with BRCA1 as expected (Fig. 1B). To investigate whether ABRO1 binds to NBA1, BRE, and BRCC36 in a similar manner as Abraxas protein, we made deletion mutants of ABRO1 and tested their binding to different components of the BRCA1 A complex. We found that, similar to Abraxas (16,18), the N terminus ABR domain of ABRO1 binds to NBA1 and BRE, and that the coiled-coil domain is required for binding to BRCC36 (Fig. 1C). Interestingly, Abraxas does not interact with ABRO1 ( Fig. 1B). Thus, the Abraxas complex (BRCA1 A complex) and the ABRO1 complex (BRISC complex) represent two distinct complexes consisting of the common components NBA1, BRE, and BRCC36. To further understand the nature of these two complexes, we examined the localization of these two complexes using cell fractionation. We found that while Abraxas/ Rap80 are mostly localized in the nuclear chromatin fraction, ABRO1 is mainly localized in the cytoplasm (Fig. 1D). NBA1, BRE, and BRCC36 are mostly localized in the cytoplasm, similar to ABRO1, thus suggesting that most of the NBA1, BRE, and BRCC36 protein in the cells are present in the ABRO1 complex.
We also examined the localization of ABRO1 using immunofluoresence with ABRO1 antibodies or a GFP-tagged ABRO1 protein. We found that ABRO1 mainly localized in the cytoplasm with some localization in the nucleus as well ( Fig. 1E and supplemental Fig. S1). The localization of ABRO1 did not appear to change in response to IR (Fig. 1E).
NBA1 Is Required for the Integrity of the Abraxas/BRCA1 A Complex and ABRO1 Complex-Previously, we and others have found that NBA1 is required for cells to maintain proper levels of Abraxas, BRE, and BRCC36 (16,17,23). We found that NBA1 is also required for maintaining the levels of ABRO1 protein (Fig. 2). In the NBA1 shRNA-treated U2OS cells, the levels of ABRO1 protein were decreased, similar to Abraxas, BRE and BRCC36 ( Fig. 2A). When we expressed an NBA1 cDNA lacking the shRNA target site in U2OS cells treated with NBA1 shRNA#1, the protein levels of Abraxas, BRE and BRCC36, as well as the levels of ABRO1, were rescued (Fig. 2B). We then established a stable U2OS cell line that allowed us to inducibly knock down NBA1 using an inducible expression construct of shRNA#1 against NBA1 gene. Upon knockdown of NBA1 by expression of shRNA against NBA1 (from day 2-3), the protein levels of Abraxas, ABRO1, BRE or BRCC36 were quickly decreased, correlating to decreased levels of NBA1. However, the levels of Rap80 took a much longer time to decrease (more than 7 days) (Fig. 2C). We also observed that NBA1 is required for maintaining levels of component proteins in the BRCA1 A complex and the ABRO1 complex in human normal fibroblast BJ cells and human mammary epithelial MCF10A cells (supplemental Fig. S2). The decrease of protein levels was not due to the decrease of mRNA levels for genes encoding these proteins based on RT-PCR analysis (supplemental Fig. S3). Upon treatment with a proteasome inhibitor MG132, protein levels of Abraxas, ABRO1, or Rap80 were restored to some extent in stable cell lines with NBA1 knockdown or inducible NBA1 knockdown, suggesting that the decreased protein level is at least partly due to the proteasomal degradation (supplemental Fig. S4). We also attempted to measure protein stability following cycloheximide (CHX) treatment in cells. All of the component proteins were quite stable when NBA1 was present, however, levels of these proteins greatly decreased when NBA1 was absent (supplemental Fig.  S5).
NBA1 Binds to Components of the Abraxas/BRCA1 A Complex or ABRO1 Complex through Its C Terminus PXXR Motif-Because NBA1 plays a critical role in maintaining the levels of components of the Abraxas/BRCA1 A complex and the ABRO1 complex, thus maintaining the integrity of these complexes, we decided to examine how NBA1 interacts with the other components in the complex. We generated a series of mutants of NBA1 and determined their interactions with other components of the complex (Fig. 3). We have previously found that NBA1 contains a VWA domain spanning 94 -298 amino acids of the protein (16). In addition, its N terminus contains multiple serine residues that were reported phosphorylated in largescale proteomic analysis for phosphorylation sites on the proteins (24 -28). Its C-terminal 310 -314 amino acids have been suggested to be important for its localization to DNA damage sites (23). Thus, we generated deletion mutants for the N ter-minus and C terminus of the NBA1 protein, as well as point mutants in the VWA-domain for amino acids that are conserved either among various VWA domains or among NBA1 proteins from different species (16). Exogenously expressed HA-Flag-tagged NBA1 appeared as two bands in Western blot analysis (Fig. 3A). The slower migrating form of NBA1 disappeared when the lysates were treated with a protein phosphatase indicating a phosphorylation modification (supplemental Fig. S6). We found that while the N-terminal deletion mutants and VWA point mutants of NBA1 appeared to interact with Rap80, BRE, BRCC36, Abraxas, and ABRO1, similarly as the wild type NBA1, the C-terminal deletion mutants, ⌬299 -329, and ⌬306 -329 were defective in their ability to associate with components of the Abraxas/BRCA1 A complex and the ABRO1 complex (Fig. 3A, lanes 10 and 14). We then further analyzed the sequence of the C terminus of the NBA1 protein and generated NBA1 ⌬315-329 and ⌬323-329 mutants as controls. We noticed that a PXXR motif exists at the C terminus of NBA1 pSPxF, a phosphoserine motif that binds to the BRCT domains of BRCA1. B, NBA1 is present in both Abraxas and ABRO1 associated protein complexes. Immunoprecipitation of endogenous Abraxas or ABRO1 was performed with lysates of 293 T cells using appropriate antibodies. Immunoprecipitates were then separated by SDS-PAGE and Western blot analyses were carried out using antibodies against Abraxas, ABRO1, NBA1, BRE, BRCC36, Rap80, and BRCA1. C, ABRO1 interacts with NBA1, BRE, and BRCC36 in a manner similar to Abraxas. The left panel is a schematic representation of ABRO1 wild type and deletion mutants. 293T cells were transfected with DNA constructs expressing GFP-tagged wild-type ABRO1 or various deletion mutants of ABRO1. The cell lysates were immunoprecipitated with GFP antibodies and separated by SDS-PAGE. Immunoblot analyses were carried out using antibodies against NBA1, BRCC36, BRE, and GFP. D, ABRO1 complex localizes differently to the Abraxas complex in cells. U2OS cells were treated with or without 10 Gy ionizing irradiation (IR), incubated for 2 h at 37°C. Cellular fractionations were performed as described under "Experimental Procedures." Different fractions from cells treated with 10 Gy IR or untreated (CTL) were loaded in equal amount, separated by SDS-PAGE, transferred to nitrocellulose membrane, and blotted with the indicated antibodies. (TCE, total protein; S2, cytoplasmic protein fraction; S3, nuclear soluble protein fraction; P3, chromatin-bound protein fraction). E, ABRO1 protein does not form IR-induced foci. U2OS cells were untreated or treated with 10 Gy IR, cultured at 37°C for 2 h, fixed with 3.6% formaldehyde, and immunofluorescence was carried out with antibodies against ABRO1 and BRCA1 and appropriate secondary antibodies.
protein with the two amino acids proline (Pro-302) and arginine (Arg-305) completely conserved in a line-up of the NBA1 sequences from different species (Fig. 3B). Mutation of these two amino acids to alanine (PR mutant) revealed that the PXXR motif is required for the binding of NBA1 with BRE, BRCC36, Rap80, Abraxas, and ABRO1 (Fig. 3C). To further analyze the requirement of the PXXR motif for these interactions, we also generated the single mutants, P302A and R305A, for comparison. While the mutation of P302 did not appear to greatly affect the binding of NBA1 to the other components, the R305A mutant did, although to a lesser extent compared with the PR mutant of NBA1 (Fig. 3C). These results indicate that although both proline 302 and arginine 305 are important for these interactions, arginine 305 plays a more critical role in the binding. We also generated and examined a R305K mutant of NBA1 in which the arginine 305 residue is replaced with a similarly positively charged lysine residue. Although the R305K mutant was defective in binding to BRCC36, BRE, and ABRO1, it appeared to bind to Rap80 and Abraxas quite similarly to the wild-type NBA1 protein (Fig. 3D).
The C Terminus PXXR Motif of NBA1 Is Required to Maintain the Protein Levels of the Abraxas/BRCA1 A Complex and ABRO1 Complex Components-To investigate how NBA1 is involved in maintaining the integrity of the Abraxas/BRCA1 A complex and the ABRO1 complex, we established stable NBA1-knockdown cells expressing shRNA-resistant cDNA encoding various NBA1 mutants. We found that mutant ⌬299 -329 which abolished binding to the other components of the complexes also failed to rescue the levels of the component proteins (Fig. 4A, lane 13). On the contrary, the NBA1 mutants that retained the ability to bind rescued the levels of BRE, BRCC36, Rap80, Abraxas, and ABRO1 (Fig. 4A). Furthermore, the C terminus PXXR motif appeared to be required for NBA1's role in maintaining protein levels of the members of the Abraxas/ BRCA1 A and ABRO1 complexes. While expression of wildtype NBA1 or a mutant that retained binding ability (⌬315-329) could rescue the protein levels of various components in the NBA1 shRNA-treated cells, expression of a NBA1 PR mutant could not (Fig. 4B).
Previously, it was shown that NBA1 directly interacts with BRE (23) and yeast two-hybrid data also suggested that NBA1 and BRE interact directly (29). Therefore, we tested whether the PXXR motif is required for NBA1 binding to BRE. We used purified His-tagged NBA1 wild type or the PR mutant, incubated with purified Gst-BRE protein in an in vitro binding assay. We found that mutation of the PXXR motif decreased binding of NBA1 to BRE (Fig. 4, C and D).
BRE Is Also Required for the Integrity of the BRCA1 A Complex and ABRO1 Complex-Because NBA1 interacts with BRE and is required for the integrity of the Abraxas/BRCA1 A and

. NBA1 is required to maintain protein levels of Abraxas, ABRO1, BRE, and BRCC36 in the Abraxas/BRCA1 A and the ABRO1 complexes.
A, protein levels of Abraxas, ABRO1, BRE, and BRCC36 were decreased in the NBA1-shRNA-treated cells. Retroviral expression vector containing two different shRNA hairpins against NBA1, as well as a control retroviral shRNA construct against the firefly gene (FF), were introduced into U2OS cells and selected for stable cell lines with knockdown of the NBA1 protein. Cell lysates were then analyzed by Western blot analysis using indicated antibodies. B, protein levels of Abraxas, ABRO1, BRE, and BRCC36 were restored in the NBA1-shRNA-treated cells complemented with HA-Flag-tagged NBA1 expressed from an NBA1 cDNA lacking the shRNA targeting site. U2OS cells were infected with retrovirus expressing HA-Flag-NBA1 and shNBA1#1 sequentially as described under "Experimental Procedures." Cell lysates were analyzed by Western blots using the indicated antibodies. * represents a nonspecific protein band recognized by the antibodies. C, protein levels of components of the Abraxas/BRCA1 A and ABRO1 complexes were decreased in cells in which NBA1 knockdown was induced. U2OS cells were infected with a lentiviral construct carrying shRNA#1 hairpin against NBA1 regulated by a doxycycline inducible promoter. Cells were cultured in the absence of doxycycline until induction. For induction, 2 g/ml doxycycline was added to the medium. Time points were taken at different days after induction of NBA1 shRNA expression. Cell lysates were analyzed by Western blot using indicated antibodies.
the ABRO1 complexes, we examined whether BRE is required for the formation of the complexes. We found that similar to NBA1, knocking down BRE led to decreased levels of Abraxas, ABRO1, NBA1, BRCC36, and Rap80 (Fig. 5A). When we expressed a BRE cDNA lacking the shRNA target site in BRE shRNA-treated cells, the protein levels of various components were rescued (Fig. 5B). BRE contains two ubiquitin-conjugating enzyme variant (UEV) domains that lack a cysteine residue necessary for the formation of a thioester-ubiquitin intermediate, the function of which in the BRCA1 A complex has not been fully investigated (16). We generated a series of both N-terminal and C-terminal deletion mutants of BRE and examined the interaction of these mutants with NBA1 and other components of the BRCA1 A complex and ABRO1. We found that while the C-terminal region including the second UEV domain seemed responsible for the interaction with NBA1, a much larger region in BRE including both the N-terminal UEV domain and the C-terminal UEV domain appeared critical for binding to Abaxas, ABRO1, Rap80, and BRCC36 (Fig. 5C). When we mutated a stretch of three conserved residues in the second UEV domain of BRE (Tyr 349 -Ser 350 -Pro 351 ) to alanine (supplemental Fig. S7), it abolished the binding to NBA1 (Fig. 5D). This mutant (YSP mt) was also slightly compromised in its binding to Abraxas, Rap80, BRCC36, and ABRO1 (Fig. 5D).
NBA1 and BRE Interaction Is Required for NBA1 Localization to DNA Damage Sites-We next examined whether the interaction of NBA1 with BRE is required for NBA1 accumulation to DNA damage sites. The NBA1 PR mutant and R305A mutant that decreased the binding to BRE were fused with GFP-tag and stably expressed in U2OS cells. GFP-tagged wild type NBA1 was used as a control in the experiment. We found that mutation in the PXXR motif of the NBA1 protein compromised NBA1's ability to form IR induced foci (IRIF) (supplemental Fig. S8). To exclude the possibility that mutation of the PXXR motif affected NBA1's localization to the nucleus, we forced expression of NBA1 or its mutants in the nucleus by expressing GFP-tagged proteins with a SV40 nuclear localization signal (NLS). We found that similarly, the GFP and NLS tagged PR mutant of NBA1 failed to localize to DNA damage sites (Fig. 6A). In addition, when we expressed a GFP-tagged FIGURE 3. NBA1 binds to components of the Abraxas complex or ABRO1 through a C terminus PXXR motif. A, NBA1 C-terminal 299 to 306 aa is required for binding. 293T cells were transiently transfected with expression constructs carrying HA-and Flag-tagged NBA1 wild-type (WT) or various mutants as indicated. Immunoprecipitations were carried out with anti-FLAG antibodies from total cell lysates. Immunoprecipitates were then analyzed by Western blot with antibodies to Rap80, BRE, BRCC36, ABRO1, Abraxas, or HA. B, identification of a conserved PXXR motif in the alignment of the NBA1 C-terminal region sequence of different species. C and D, PXXR motif is required for the binding. 293T cells were transiently transfected with expression constructs carrying HAand Flag-tagged NBA1 WT or various mutants that lack the intact PXXR motif. Immunoprecipitates from anti-FLAG-immunoprecipitated proteins (IP) were separated by SDS-PAGE and analyzed by Western blot using indicated antibodies.
BRE mutant (BRE YSP mt) that failed to bind NBA1, we found that the accumulation of this mutant protein to DNA damage sites also significantly decreased in response to IR (Fig. 6B and  supplemental Fig. S9).
NBA1 Interaction with BRE Is Required for a Proper DNA Damage Response-NBA1 is required for optimum recruitment of BRCA1 to DNA damage sites. NBA1 deficiency leads to decreased IRIF formation of BRCA1, although the BRCA1 protein level was not affected in the NBA1-deficient cells (supplemental Fig. S10). Similarly, knockdown of BRE leads to decreased BRCA1 IR induced foci (IRIF) formation while not affecting the total BRCA1 protein levels in cells (Refs. 16 -18, 23, and supplemental Fig. S10). Thus, we examined whether the NBA1 interaction with BRE through the PXXR motif is required for NBA1 function in recruiting BRCA1. We found that in NBA1-deficient cells stably expressing HA-Flag-tagged wild type NBA1 or a NBA1 deletion mutant (⌬315-329) that was intact for binding to BRE, BRCA1 IRIF formation was rescued, however in NBA1-deficient cells expressing a tagged PXXR mutant of NBA1 (PR mt), rescue of IRIF formation of BRCA1 failed (Fig. 7, A and B). Furthermore, NBA1-shRNAtreated cells displayed increased sensitivity to irradiation. While HA-and Flag-tagged wild type NBA1 or a NBA1 mutant (⌬315-329) capable of binding to BRE rescued the sensitivity of cells to IR, cells complemented with the PR mutant of NBA1 remained sensitive to IR (Fig. 7C).

DISCUSSION
It appears that BRCC36 forms two different complexes through interactions with two different adaptor proteins, FIGURE 4. NBA1-BRE interaction is required for the integrity of the BRCA1 A complex and ABRO1 complex. A, deletion of the C-terminal tail of NBA1 (⌬299 -329) failed to rescue the decreased protein levels of components of the Abraxas complex and ABRO1 in the NBA1-deficient cells. U2OS cells were infected with retrovirus expressing HA-Flag NBA1 and shNBA1#1 sequentially as described under "Experimental Procedures." Cell lysates were analyzed by Western blots using indicated antibodies. * represents a nonspecific protein band recognized by the antibodies. B, NBA1 C-terminal PXXR motif is required for maintaining proper protein levels of BRE, Abraxas, ABRO1, BRCC36. U2OS cells were infected with viruses containing vector (MSCV), wild-type NBA1 (WT), NBA1 PR mutant (PR mt), or a deletion mutant (⌬315-329), followed by infection with an shRNA hairpin against BA1 (shNBA1#1). Cell lysates were analyzed by Western blot using indicated antibodies. * represents a nonspecific protein band recognized by the antibodies. C, NBA1 PR mutant lacking the PXXR motif failed to interact with BRE in vitro. His-tagged NBA1 wild-type (WT) or mutant in the PXXR motif (PR mt) and GST-tagged BRE were expressed and purified from bacteria. A pull-down assay was carried out with His-tagged beads of 1 g of purified NBA1 WT or PR mt incubated with 1 g of purified recombinant Gst-BRE. D, Coomassie staining of the purified proteins in the SDS-PAGE gel.

NBA1-BRE Interaction Stabilizes BRCC36-containing Complexes
namely Abraxas and ABRO1 (supplemental Fig. S12). Abraxas primarily localizes in the nucleus, mediating the interaction of BRCC36 with BRCA1. The Abraxas/BRCA1 A complex contains at least Abraxas, Rap80, BRE, NBA1, and BRCC36 proteins. ABRO1 is mainly localized in the cytoplasm. Because it lacks the BRCA1 interacting motif, ABRO1 does not interact with BRCA1. The ABRO1/BRISC complex contains at least ABRO1, BRE, NBA1, and BRCC36. While Rap80 localizes in the nucleus and is only present in the Abraxas/BRCA1 A complex, BRCC36, BRE, and NBA1 are expressed both in the nucleus and cytoplasm and are present in both complexes. From cell fractionation analysis, we noticed that a major portion of the BRCC36, BRE, and NBA1 proteins were localized in the cytoplasm, displaying an expression pattern similar to the ABRO1 protein. Thus, the ABRO1/BRISC complex is a dominant BRCC36-containing DUB complex in the cytoplasm.
In this study, we found that these two complexes were assembled in a similar manner. Through analysis of the deletion mutants of ABRO1, it appears that ABRO1 interacts with NBA1, BRE, or BRCC36 in a similar manner as Abraxas does in the BRCA1 A complex (18). The two BRCC36-containing complexes share common components BRE and NBA1. Furthermore, we found that the NBA1 and BRE interaction is required for maintaining the integrity of both of the complexes. Knocking down NBA1 leads to decreased protein levels of BRE, BRCC36, as well as Abraxas and ABRO1. Similarly, BRE knockdown also decreased the levels of NBA1, BRCC36, Abraxas, and ABRO1. NBA1 and BRE directly interact with each other. Many protein dimers require association to maintain each others stability, such as BRCA1/BARD1 dimer (30,31) or ATR/Atrip complex (32). Because the disruption of NBA1-BRE interaction leads to decreased protein levels of all the complex components, it suggests that the integrity of the complex is required for the protein stability of each of the components and NBA1-BRE interaction plays a critical role in maintaining the integrity of the complex. Decreased protein levels of complex components are likely due to protein degradation in a proteasome-de- FIGURE 5. BRE is required to maintain protein levels of Abraxas and ABRO1 complex components. A, protein levels were decreased in BRE shRNA-treated cells. U2OS cells were infected with retrovirus containing the shRNAs against BRE (shBRE#1 and shBRE#2), as well as retrovirus containing a control hairpin (FF). Cell lysates from indicated stable cell lines were analyzed with antibodies against different proteins in the Abraxas and ABRO1 complexes. * represents a nonspecific protein band recognized by the antibodies. B, expression of GFP-tagged BRE restored expression levels of components of the Abraxas complex and ABRO1 in BRE-deficient cells. U2OS cells were infected with viruses containing vector (MSCV) or GFP-tagged BRE protein, followed by infection with an shRNA hairpin against NBA1 (shNBA1#1). Cell lysates from indicated cells lines were analyzed with proper antibodies. * represents a nonspecific protein band recognized by the antibodies. C, defining the region on BRE that is required for binding to NBA1. 293T cells were transiently transfected with GFP-tagged BRE wild type (WT) or deletion mutants as indicated in the right panel. Anti-GFP-immunoprecipitated proteins (IP) from total cell lysates were separated by SDS-PAGE and analyzed by Western blot with indicated antibodies. D, amino acids YSP in the UEV2 domain of BRE are required for the binding with NBA1. 293T cells were transiently transfected with GFP-tagged wild type BRE (WT) or the YSP to AAA mutant of BRE (YSP mt). Anti-GFP-immunoprecipitated proteins (IP) from total cell lysates were separated by SDS-PAGE and analyzed by Western blot with indicated antibodies. pendent manner (supplemental Fig. S4). Further analysis of proteolytic pathways may shed additional light as to the formation and stability of these complexes.
NBA1 plays a critical role in maintaining the integrity of the BRCC36-containing complexes through its C-terminal interaction with BRE. Mutations that disrupt NBA1 interaction with BRE compromised the formation of both the Abraxas/BRCA1 A complex and the ABRO1/BRISC complex. Furthermore, these mutants are defective in NBA1 role in the DNA damage response and the recruitment of BRCA1 to sites of DNA damage. The interaction of NBA1 and BRE requires a PXXR motif in the C terminus of the NBA1 protein. Mutation of both the proline and arginine residues in the PXXR motif of NBA1 disrupted the direct binding of NBA1 to BRE in vitro. Mutation of the arginine residue in the PXXR motif to alanine (R305A) largely abolished the binding of NBA1 to ABRO1, BRCC36, and BRE while maintaining some binding to Rap80 and Abraxas. Similarly, a mutation of the arginine to lysine (R305K) substantially decreased the binding of NBA1 to the ABRO1, BRE, and BRCC36, yet had minimal effect on the binding of NBA1 to Rap80 and Abraxas. Because a major portion of the NBA1, BRE, and BRCC36 proteins were present in the cytoplasm with FIGURE 6. NBA1 PXXR motif is required for its localization to DNA damage sites. A, GFP-tagged NBA1 PR mutant lacking the PXXR motif failed to form IRIF. U2OS cells were infected with retrovirus containing a SV40 NLS and GFP-tagged NBA1 wild-type (WT), PR mutant or R305A mutant. Cells were treated with 10 Gy IR, incubated at 37°C for 2 h. Cells were then treated with 0.5% Triton X-100 at room temperature for 5 min for extraction of soluble proteins, then fixed with 3.6% formaldehyde. Immunofluorescence was carried out with antibodies against BRCA1, GFP, and appropriate secondary antibodies. B, GFP-tagged BRE mutant that failed to bind NBA1 is defective in forming IRIF. U2OS cells infected with retroviruses expressing GFP-tagged BRE wild-type (WT) or mutant (YSP mt) were treated with 10 Gy IR and cultured for 2 h at 37°C. Cells were then treated, fixed, and stained as described above.
ABRO1, it seemed that mutation of the arginine residue in the PXXR motif mainly disrupt the ABRO1 complex and affect the Abraxas complex to a much lesser extent. Although it is possible that the arginine residue (R305) is modified to promote its interaction with BRE, it is more likely that both the proline residue (P302) and the arginine residue (R305) play a structure role in the interaction between NBA1 and BRE. Furthermore, it is likely that the structural interactions between NBA1 and BRE in two different complexes are not completely the same, and are possibly influenced by the presence of the Rap80 protein in the BRCA1 A complex. Rap80 is only present in the A complex in the nucleus. Interestingly, although knocking down NBA1 or BRE led to decreased levels of all the component proteins in the complexes, the levels of Rap80 were decreased in a much slower manner than the rest of the component proteins.
BRE contains two UEV domains (16), and appears to interact with NBA1 through the second UEV domain. Recently it was suggested that the first UEV domain of BRE is required for an interaction with Abraxas in the BRCA1 A complex (33).
Through deletion analyses of BRE, we confirmed that the N terminus of BRE is required for interactions with Abraxas or Rap80. In fact, a much larger region of BRE including both UEV domains appeared to be critical for binding to Abraxas and Rap80. The second UEV domain at the C terminus is required for BRE interaction with NBA1 and appears to interact with the PXXR motif present in the C terminus of the NBA1 protein.
The crystal structure from a UEV domain of TSG101, a protein that functions in both HIV-1 budding and the vacuoloar protein sorting pathway, indicates that the UEV domain contacts the Ile 44 surface and an adjacent loop of ubiquitin through a highly solvated interface (34). The TSG101 UEV domain also binds a PTAP peptide motif independently of its binding to ubiquitin (34,35). It is likely that the second UEV domain of BRE also binds the PXXR motif of NBA1 independently to its binding to ubiquitin. Alternatively, its binding to the PXXR motif might be required for the binding of ubiquitin or vice versa. Definitive evidence for these interactions awaits higher resolution structural analysis. . NBA1-BRE interaction is required for BRCA1 localization to DNA damage sites and cellular resistance to IR. A and B, NBA1-PR mutant lacking the PXXR motif failed to rescue the recruitment of BRCA1 to DNA damage sites in NBA1-deficient cells. U2OS cells carrying expression constructs of an empty control vector (MSCV) or NBA1 cDNA lacking an shRNA targeted site of wild-type or mutant (PR mt and ⌬315-329) were treated with either a control hairpin targeting a firefly gene sequence (FF) or shRNA#1 to NBA1. The protein levels of endogenous NBA1 and exogenously expressed HA-Flag-tagged NBA1 wild type or mutants are shown in Fig. 4B. The corresponding stable cell lines cells were treated with 10 Gy IR. After 2 h of incubation, the cells were fixed for immunofluorescence with appropriate antibodies. Cells were counted three times independently. A total of about 800 cells were analyzed, and cells with 10 or more foci were counted as positive. Representative images from the staining of corresponding cells are shown in (B). C, NBA1-PR mutant lacking the PXXR motif failed to rescue the increased IR sensitivity of the NBA1-deficient cells. NBA1-deficient cells expressing either shRNA-resistant NBA1 cDNA containing HAand Flag-tagged wild type (WT), or PR mutant (PR mt), a deletion mutant (⌬315-329), or a control shRNA hairpin (FF) were generated for the analyses of cellular resistance to IR using a colony-forming assay. A control cell line expressing empty vector and a control shRNA hairpin (MSCV/FF) or an NBA1 shRNA (MSCV/ shNBA1) were used as controls. A colony-forming assay was performed as described under "Experimental Procedures." Colonies were counted and normalized as a percentage of colonies formed at 0 Gy. Error bars indicate S.D. The p values indicated are: P* ϭ 0.0014; P** ϭ 0.0026.
The Abraxas/BRCA1 A complex plays a critical role in the recruitment of BRCA1 to DNA damage sites, and in DNA damage repair and cell cycle checkpoint control. Previously we have found that the BRCA1 A complex possesses multiple ubiquitin binding motifs and shares similarities with the lid complex of the 19S proteasome regulatory particle. Although the significance of the similarities is not completely understood, it is likely that the complex is formed to facilitate the DUB activity of BRCC36. Indeed, recently it was shown that the presence of Abraxas, BRE, NBA1, and Rap80 is required for BRCC36 DUB activity in the BRCA1 A complex (33,36). For ABRO1/BRISC complex, ABRO1 dimerization with BRCC36 is required for BRCC36 to display a minimal DUB activity (19). However, the BRCC36 DUB activity doesn't appear to be required for its interaction with components of the complexes because a DUB mutant of BRCC36 interacts with various component proteins similarly as the wild-type BRCC36 (supplemental Fig. S11). Our studies showed that the interaction of NBA1 with BRE and the integrity of this complex is essential for the function of the BRCA1 A complex to recruit BRCA1 to DNA damage sites and for cellular resistance to IR. The ABRO1/BRISC complex appears to associate with the COP9 signalosome (14). Recently, it was also suggested that BRISC deficiency enhances formation of the BRCA1-Rap80 interaction and increased BRCA1 levels at the DNA damage sites (33,36). However, the exact role of the ABRO1/BRISC complex needs further investigation to be understood. Two recent genome wide association studies (GWAS) implicated that common genetic variants in NBA1 may predispose women to serous ovarian or hormone negative breast cancer (37,38). Because NBA1 is not only involved in the BRCA1 A complex but also in the ABRO1/BRISC complex, it awaits further characterization as to whether ABRO1/BRISC complex plays a role in the development of tumors.