NBA1/MERIT40 and BRE interaction is required for the integrity of two distinct deubiquitinating enzyme BRCC36-containing complexes

Abstract

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.

FIGURE 1.

NBA1 is a component of both the Abraxas/BRCA1 A and ABRO1 complexes. A, diagram for the domain structure of Abraxas and ABRO1. MPN, an MPN (Mpr1, Pad1 N-terminal) domain; CC, a coiled-coil domain; 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.

FIGURE 2.

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 μg/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.

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 HA- and 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.

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 (Δ299–329) 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 (Δ315–329), 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 μg of purified NBA1 WT or PR mt incubated with 1 μg of purified recombinant Gst-BRE. D, Coomassie staining of the purified proteins in the SDS-PAGE gel.

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.

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.

FIGURE 7.

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 Δ315–329) 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 HA- and Flag-tagged wild type (WT), or PR mutant (PR mt), a deletion mutant (Δ315–329), 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* = 0.0014; P** = 0.0026.