The UBXN1 protein associates with autoubiquitinated forms of the BRCA1 tumor suppressor and inhibits its enzymatic function

Abstract

Although the BRCA1 tumor suppressor has been implicated in many cellular processes, the biochemical mechanisms by which it influences these diverse pathways are poorly understood. The only known enzymatic function of BRCA1 is the E3 ubiquitin ligase activity mediated by its highly conserved RING domain. In vivo, BRCA1 associates with the BARD1 polypeptide to form a heterodimeric BRCA1/BARD1 complex that catalyzes autoubiquitination of BRCA1 and trans ubiquitination of other protein substrates. In most cases, BRCA1-dependent ubiquitination generates polyubiquitin chains bearing an unconventional K6 linkage that does not appear to target proteins for proteasomal degradation. Since ubiquitin-dependent processes are usually mediated by cellular receptors with ubiquitin-binding motifs, we screened for proteins that specifically bind autoubiquitinated BRCA1. Here we report that the UBXN1 polypeptide, which contains a ubiquitin-associated (UBA) motif, recognizes autoubiquitinated BRCA1. This occurs through a bipartite interaction in which the UBA domain of UBXN1 binds K6-linked polyubiquitin chains conjugated to BRCA1 while the C-terminal sequences of UBXN1 bind the BRCA1/BARD1 heterodimer in a ubiquitin-independent fashion. Significantly, the E3 ligase activity of BRCA1/BARD1 is dramatically reduced in the presence of UBXN1, suggesting that UBXN1 regulates the enzymatic function of BRCA1 in a manner that is dependent on its ubiquitination status.

FIG. 1.

UBXN1 associates with BRCA1/BARD1 in vivo. (A) Map of human UBXN1, displaying the UBA and UBX domains. (B) 293 cells were transfected with vectors encoding Flag-tagged derivatives of cyclin A (lane 1), TAL1 (lane 2), UBXN1 (lane 3), and CtIP (lane 4) or with the empty vector (lane 5). Protein expression was confirmed by immunoblotting (IB) with Flag-specific antibodies. (C) Cell lysates were immunoprecipitated (IP) on Flag-specific agarose beads and fractionated by SDS-PAGE (lanes 1 to 5), along with an aliquot of untreated cell lysate (lane 6). Coimmunoprecipitation of endogenous BRCA1 and BARD1 polypeptides was determined by immunoblotting with the respective antisera (50). The electrophoretic mobilities of molecular size markers are indicated in kilodaltons.

FIG. 2.

UBXN can bind both BRCA1 and the BRCA1/BARD1 heterodimer independently of ubiquitin. Maltose-binding protein (MBP) (lane 2) and a polypeptide containing MBP fused to full-length UBXN1 (MBP-UBXN1) (lane 1) were expressed in E. coli, purified by affinity chromatography on amylose agarose, fractionated by SDS-PAGE, and detected by immunoblotting with an MBP-specific antibody (upper panel). In addition, 6H-HA-BR304, a polypeptide containing a hexahistidine tag, three tandem HA epitopes, and the N-terminal 304 residues of BRCA1, was expressed in E. coli either alone or together with full-length untagged BARD1. The 6H-HA-BR304 polypeptide (lane 4) or the 6H-HA-BR304/BARD1 heterodimer (lane 3), respectively, was then purified from E. coli lysates by nickel chromatography, fractionated by SDS-PAGE, and detected by immunoblotting with HA-specific antibodies (lower panel). To evaluate in vitro interactions between UBXN1 and BRCA1, the 6H-HA-BR304 polypeptide (lanes 6 and 8) and the 6H-HA-BR304/BARD1 heterodimer (lanes 5 and 7) were incubated with either MBP (lanes 7 and 8) or MBP-UBXN1 (lanes 5 and 6). Each reaction mixture was then loaded onto amylose-agarose beads, and the bound material was examined by immunoblotting with MBP-specific (upper panel) and HA-specific (lower panel) antibodies.

FIG. 3.

Ubiquitin-independent interaction with UBXN1 requires an N-terminal region of BRCA1 that includes the RING domain and its BARD1-binding sequences. (A) A panel of MBP-BRCA1 fusion proteins containing the indicated N-terminal BRCA1 residues was expressed in E. coli, purified by amylose chromatography, and analyzed by immunoblotting with an MBP-specific antibody. (B) To identify BRCA1 sequences required for interaction with UBXN1, each of the MBP-BRCA1 polypeptides was incubated with GST-UBXN1, a GST fusion protein containing full-length UBXN1. Each reaction mixture was then loaded onto glutathione-agarose beads, and the bound material was examined by immunoblotting with MBP-specific (upper panel) and GST-specific (lower panel) antibodies.

FIG. 4.

The UBA domain is not required for ubiquitin-independent binding between UBXN1 and BRCA1. (A) A panel of MBP-UBXN1 fusion proteins containing the indicated segments of UBXN1 was expressed in E. coli and purified by amylose chromatography. (B) The MBP-UBXN1 fusion proteins were incubated with a full-length BRCA1/BARD1 heterodimer purified from baculovirus-infected Sf9 cells (53). Each reaction mixture was then loaded onto amylose-agarose beads, and the bound material was fractionated by SDS-PAGE (lanes 1 to 7), together with an aliquot of the input BRCA1/BARD1 heterodimer (lane 8). Immunoblotting was performed with MBP-specific (upper panel) and BRCA1-specific (lower panel) antibodies.

FIG. 5.

Preparation of free K6-linked polyubiquitin chains and BRCA1 polypeptides conjugated with K6-linked polyubiquitin. Three parallel in vitro ubiquitination reactions were conducted using the purified full-length BRCA1/BARD1 heterodimer and the UbcH5c ubiquitin conjugating enzyme, as described previously (53). The reaction mixtures were supplied with either wild-type ubiquitin (Ub-wt) (lanes 1 and 2) or a mutant ubiquitin (Ub-R6) in which lysine residue 6 is substituted with arginine (lane 3). In addition, a control reaction was performed in the absence of ATP (lane 2). The reaction products were then analyzed by immunoblotting with a ubiquitin-specific (A) or a BRCA1-specific (B) antibody. The electrophoretic mobilities of free K6-linked polyubiquitin (K6-Ub_n), free ubiquitin monomers (Ub), K6-linked polyubiquitinated BRCA1 (BRCA1-Ub_n), and unmodified BRCA1 (BRCA1) are indicated. Aliquots of the three reaction mixtures (lanes 1 to 3) were also subjected to affinity chromatography on Ni-NTA agarose beads, and the resulting nickel-unbound (lanes 4 to 6) and nickel-bound (lanes 7 to 9) fractions were analyzed by immunoblotting. The nickel-unbound fraction of the reaction mixture supplied with the wild type (lane 4) was subsequently used as the source of free K6-linked polyubiquitin and free ubiquitin monomers for the experiment with results shown in Fig. 6, while the nickel-bound fraction (lane 7) was used as the source of both polyubiquitinated and unmodified BRCA1/BARD1 heterodimers for the experiment with results shown in Fig. 7.

FIG. 6.

The UBA domain of UBXN1 binds K6-linked polyubiquitin chains. The nickel-unbound fraction of a BRCA1-driven ubiquitination reaction mixture (Fig. 5, lane 4) was incubated with MBP (lanes 8 and 12) or the indicated MBP-UBXN1 fusion proteins (lanes 2 to 7 and 9 to 11). The MBP-UBXN1 fusion proteins contain full-length wild-type UBXN1 (residues 1 to 297) (lanes 2 and 9), the indicated segments of UBXN1 (lanes 3 to 7), or full-length mutant UBXN1 bearing the M13T (lane 10) or R219A (lane 11) amino acid substitution. Each reaction mixture was then loaded onto amylose-agarose beads, and the bound material was separated by SDS-PAGE (lanes 2 to 12), together with aliquots of the input nickel-unbound fraction (lanes 1 and 13). The presence of the MBP fusion proteins was confirmed by immunoblotting with MBP-specific antibodies (upper panel), while the associations with K6-linked polyubiquitin and free monoubiquitin were evaluated by immunoblotting with Ub-specific antibodies (lower panel).

FIG. 7.

UBXN1 preferentially binds autoubiquitinated BRCA1/BARD1 heterodimers. The nickel-bound fraction of a BRCA1-driven ubiquitination reaction mixture (Fig. 5, lane 7) was incubated with MBP (lane 1), the MBP-UBXN1 fusion protein (lane 2), an MPB fusion protein containing the known ubiquitin-binding protein RAD23A (lane 3), or NEMO (lane 4). Each reaction mixture was then loaded onto amylose-agarose beads, and the bound material was fractionated by SDS-PAGE, together with an aliquot of the input nickel-bound fraction (lane 5). The presence of the MBP fusion proteins was confirmed by immunoblotting with MBP-specific antibodies (A), while the association with polyubiquitinated and unmodified BRCA1/BARD1 heterodimers was evaluated by immunoblotting with BRCA1-specific antibodies (B).

FIG. 8.

UBXN1 inhibits the enzymatic activity of the BRCA1/BARD1 heterodimer. In vitro ubiquitination reactions were conducted in the presence (lanes 2 to 5) or absence (lane 1) of UbcH5c and either the full-length wild-type BRCA1/BARD1 heterodimer (B/B-wt) (lanes 1 to 4) or an enzymatically defective heterodimer containing the I26A mutation of BRCA1 (B/B-I26A) (lane 5). The indicated reaction mixtures were supplemented with either purified maltose-binding protein (MBP) (lane 4) or an MBP fusion protein containing full-length UBXN1 (MBP-UBXN1) (lane 3). Aliquots of each reaction mixture were then evaluated by immunoblotting with ubiquitin-specific (A), BRCA1-specific (B), and MBP-specific (C) antibodies. The electrophoretic mobilities of K6-linked polyubiquitin (K6-Ub_n), free ubiquitin monomers (Ub), K6-linked polyubiquitinated BRCA1 (BRCA1-Ub_n), and unmodified BRCA1 (BRCA1) are indicated.

FIG. 9.

UBXN1 inhibits BRCA1 autoubiquitination in vivo. 293 cells were transfected with a Flag-tagged segment of BRCA1 (ΔBRCA1, amino acids 1 to 771) with (lanes 5 and 6) or without (lanes 3 and 4) the I26A mutation. All cultures were also cotransfected with exogenous BARD1 and ubiquitin, and the indicated cultures were transfected with a vector encoding UBXN1 (lanes 2, 4, and 6) or the corresponding empty vector (lanes 1, 3, and 5). The cells were lysed 48 h posttransfection, and chromatin extracts were examined by immunoblotting with FLAG-specific antibodies (A) and UBXN1-specific antiserum (B). As shown, the autoubiquitinated ΔBRCA1 conjugates detected with Flag-specific antibodies (lane 3) were eliminated upon overexpression of UBXN1 (lane 4). wt, wild type.

FIG. 10.

The ubiquitin-binding activity of UBXN1 is required for inhibition of BRCA1 enzymatic function. In vitro ubiquitination reactions were conducted in the presence (lanes 2 to 7) or absence (lane 1) of UbcH5c and either the full-length wild-type BRCA1/BARD1 heterodimer (B/B-wt) (lanes 1 to 6) or an enzymatically defective heterodimer containing the I26A mutation of BRCA1 (B/B-I26A) (lane 7). The indicated reaction mixtures were also supplemented with either purified maltose-binding protein (MBP) (lane 6) or MBP fusion proteins containing full-length UBXN1 (MBP-UBXN1) (lanes 3 to 5). The MBP-UBXN1 fusions harbored the wild-type UBXN1 sequence (lane 3) or contained a missense mutation in either the UBA (M13T) (lane 4) or the UBX (R219A) (lane 5) domain. Aliquots of each reaction mixture were then immunoblotted with ubiquitin-specific (A), BRCA1-specific (B), and MBP-specific (C) antibodies. The electrophoretic mobilities of K6-linked polyubiquitin (K6-Ub_n), free ubiquitin monomers (Ub), K6-linked polyubiquitinated BRCA1 (BRCA1-Ub_n), and unmodified BRCA1 (BRCA1) are indicated.

FIG. 11.

The ubiquitin-binding activity of UBXN1 is required for in vivo inhibition of BRCA1 enzymatic function. The indicated cultures of 293 cells were cotransfected with BARD1 and a Flag-tagged derivative of ΔBRCA1 (lanes 3 to 10). All cultures were cotransfected with exogenous ubiquitin, and the indicated cultures were transfected with wild-type (lanes 5 and 6), M13T mutant (lanes 7 and 8), or R219A mutant (lanes 9 and 10) UBXN1. The indicated cultures were subjected to 12 Gy of ionizing radiation (IR) at 1 h prior to harvest. Cells were lysed 48 h posttransfection, and chromatin extracts were examined by immunoblotting with FLAG-specific antibodies (upper panel), UBXN1-specific antiserum (middle panel), and γH2AX phosphospecific antiserum (lower panel). As shown, the autoubiquitinated ΔBRCA1 conjugates detected with Flag-specific antibodies (lanes 3 and 4) were eliminated upon overexpression of wild-type UBXN1 (lane 5 and 6) and R219A mutant UBXN1 (lanes 9 and 10) but not M13T mutant UBXN1 (lanes 7 and 8).

FIG. 12.

UBXN1 overexpression does not affect homology-directed repair of double-strand DNA breaks. The effects of UBXN1 overexpression were evaluated with TOSA4 cells, a subclone of 293T cells that contains a single integrated copy of the DR-GFP recombination substrate (8). (A) TOSA4 cells were cotransfected with the ISceI endonuclease (lanes 2, 4, 6, and 8) and wild-type (lanes 3 and 4), M13T mutant (lanes 5 and 6), or R219A mutant (lanes 7 and 8) UBXN1. At 48 h posttransfection, the levels of endogenous (lanes 1 and 2) and overexpressed (lanes 3 to 8) UBXN1 were assessed by immunoblotting. (B) To evaluate homology-directed repair of ISceI-induced double-strand breaks in DR-GFP, the number of GFP-positive TOSA4 cells was quantitated at 48 h posttransfection by flow cytometry. The error bars indicate standard deviations. As expected, ISceI expression induced homology-directed repair of DR-GFP (compare lanes 1 and 2). However, repair levels were not altered upon overexpression of wild-type (lane 4) or mutant (lanes 6 and 8) UBXN1.