Nrdp1, through its C-terminal half, associates with BRUCE in vivo. (A) Cellular proteins associated with Nrdp1C. 293T cells were transiently transfected with empty vectors or the vectors encoding Nrdp1-FLAG or Nrdp1C-FLAG. The cells were labeled with [³⁵S] in the presence of 10 μM of MG132 for 3 h. Cell lysates were immunoprecipitated by using anti-FLAG resin and separated on a 4–12% gradient SDS–gel, and proteins on the gel were detected with a PhosphoImager. A re-immunoprecipitation experiment was performed after elution of the initial immunoprecipitate in the SDS sample buffer (right panel). Asterisk denotes a 30-kDa protein co-purified with Nrdp1C. (B) Analysis of the 500-kDa band by mass spectrometry. The proteins on the gel were detected by Coomassie staining, and then the 500-kDa band was sliced and subjected to mass spectrometry. (C) Detection of BRUCE co-immunoprecipitated with Nrdp1 or Nrdp1C by immunoblotting using anti-BRUCE antibodies. 293T cells were transfected as in (A). Asterisk denotes a nonspecific band.

Nrdp1 promotes BRUCE degradation by proteasomes. (A) Nrdp1 reduces cellular levels of BRUCE. 293T cells were either untransfected or transfected with FLAG-tagged Nrdp1 or its C-terminal half. BRUCE, c-IAP1, XIAP, actin, and Nrdp1 were detected by Western blotting. A nontagged Nrdp1, which could be detected by an antiserum against Nrdp1 in Western blot and was used to replace the FLAG-tagged Nrdp1 in some experiments, also reduced the levels of BRUCE. (B) The levels of BRUCE (red) were reduced in NIH/3T3 cells overexpressing the GFP–Nrdp1 fusion protein, as shown by immunostaining. More than 80% of GFP-positive cells showed almost complete disappearance of BRUCE. A representative cell that expresses GFP–Nrdp1 is indicated by an arrow. Staining with 4′-6-diamidino-2-phenylindole was used to visualize nuclei (blue). (C) Nrdp1 promotes BRUCE degradation by proteasomes. 293T cells were transfected with either control vectors or FLAG-tagged Nrdp1, labeled with ³⁵S-Met and ³⁵S-Cys for 12 h, and then chased for the indicated times with or without 10 μM MG132, 20 μM lactacystin, or 100 μM chloroquine. Numbers under the blot indicate relative amounts of BRUCE in the samples. The experiments were repeated at least three times. Each time, empty vectors were included as controls for Nrdp1. In some experiments, we also tried to use the C-terminal half of Nrdp1 as controls, and similar results were obtained.

Nrdp1 catalyzes BRUCE ubiquitination both in vitro and in vivo. (A) Nrdp1 in crude cell lysates promotes the formation of Ub conjugates, which were precipitated by anti-BRUCE antibodies. BRUCE on beads immunopurified from the 293T cells was incubated with the lysates of 293T cells transfected with no, wild-type (WT), or D56V mutant (Mut) Nrdp1. Ubiquitination of BRUCE was detected by immunoblotting using anti-Ub antibodies. (B) Re-immunoprecipitation of the ubiquitinated BRUCE. Following ubiquitination assay as in (A), the proteins were eluted from the beads with SDS sample buffer and re-immunoprecipitated with anti-BRUCE antibodies. BRUCE ubiquitination was visualized as in (A). (C) In vitro ubiquitination of BRUCE by Nrdp1 requires an exogenous E2, UbcH5c. BRUCE on beads was incubated in the ubiquitination buffer supplemented with no Nrdp1 or the immunopurified wild-type (WT) or D56V mutant (Mut) Nrdp1, the E1, and ¹²⁵I-labeled Ub in the presence or absence of UbcH5. After incubation, BRUCE was re-immunoprecipitated as in (B) and analyzed by Western blot or by PhosphoImager. (D) Nrdp1 enhances ubiquitination of BRUCE in vivo. 293T cells were transfected with empty vectors or vectors encoding Nrdp1-FLAG. BRUCE was immunoprecipitated from the lysates and separated on SDS–PAGE. BRUCE and ubiquitinated BRUCE were detected by Western blot using antibodies against BRUCE or Ub. Asterisk denotes a contaminating band from the antibody preparation.

Apoptotic stimuli induce proteasomal degradation of BRUCE. (A) Immunoblot analysis of the time course of BRUCE loss in 293T cells following treatment with etoposide (5 μM). (B) Lactacystin inhibits etoposide-induced degradation of BRUCE in 293T cells as analyzed by immunoblot. Lactacystin (20 μM) was added at 16 h after addition of etoposide, and was incubated with the cells for an additional 16 h. (C) Degradation of BRUCE is associated with apoptosis induced by etoposide or camptothecin (CPT). The cellular levels of IAPs and other apoptosis-related proteins were analyzed by Western blot following treatment either with the indicated concentrations of etoposide (for HeLa cells) or with 5 μg ml⁻¹ of camptothecin for 24 h or for indicated times (for MCF-7 and HT1080 cells).

Nrdp1 mediates the etoposide-induced loss of BRUCE. (A) Etoposide potentiates the Nrdp1-mediated BRUCE degradation. 293T cells were transfected with empty vectors or vectors encoding Nrdp1, incubated for 24 h, and then treated with the different concentrations of etoposide for 15 h. The levels of BRUCE, Nrdp1, c-IAP1, XIAP, and β-actin were detected by immunoblotting of cell lysates. (B) Nrdp1 siRNA reduces the decrease in BRUCE content induced by etoposide. 293T cells were transfected with BS/U6, BS/U6/GFP, or BS/U6/Nrdp1, and were split at 1 to 5 at 3 days after transfection. After 24 h, the cells were treated with or without etoposide (10 μM), and incubated for another 24 h. The levels of BRUCE, XIAP, and β-actin were determined by Western blot. The relative levels of Nrdp1 mRNA (normalized to those of GAPDH) were quantitated by real-time reverse transcriptase–PCR.

Expression of siRNAs for BRUCE promotes apoptosis or enhances sensitivity to TNFα-induced apoptosis in diverse cell types. (A) BRUCE siRNA reduces the cellular levels of BRUCE in HeLa cells. HeLa cells were cotransfected with BS/U6 (control) or BS/U6/BRUCE (siRNA-BRUCE) and the vectors encoding GFP (pEGFP, Clontech), and were split at 1 to 4 at 3 days after transfection. After 16 h, the cells were fixed with methanol and immunostained with mouse anti-BRUCE antibodies/anti-mouse IgG–Cy3 conjugates to visualize BRUCE (red) under a confocal microscope. Visualization of nuclei (blue) was achieved by co-staining the cells with 4′-6-diamidino-2-phenylindole. The siRNA vectors and GFP vectors were cotransfected at a ratio of 10:1 to ensure that the GFP-positive cells also received the siRNA vectors. More than 90% GFP-expressing cells showed reduced levels of BRUCE. A representative GFP-positive cell with reduced levels of BRUCE is indicated by an arrow following transfection with BS/U6/BRUCE. (B) Expression of siRNA for BRUCE causes apoptosis in HeLa cells. HeLa cells were transfected and processed as in (A), but were finally stained with 2 μM Hoechst 33342 (without fixing with methanol) to visualize their nuclei. In some experiments, the control BS/U6 was replaced by BS/U6/GFP, but similar results were obtained. Apoptotic cells with condensed chromatin (upper panels) were counted under a microscope using UV lights. All the cells (lower panels) were photographed under visible lights. The levels of apoptosis were expressed as a percentile of the number of apoptotic cells, relative to total cell number. Transfection efficiency with both treatments was about 27%. (C) Expression of siRNA-BRUCE promotes caspase 3 activation in HeLa cells. HeLa cells were transfected and split as in (A). At 10 h after splitting, the cells were either untreated or treated with 20 ng ml⁻¹ TNFα for 6 h. The levels of β-actin and active caspase 3 were analyzed by immunoblotting. (D) Expression of siRNA-BRUCE potentiates caspase 3 activation induced by TNFα in 293T cells. 293T cells were transfected and split as in (A). At 16 h after splitting, the cells were either untreated or treated with 20 ng ml⁻¹ TNFα for 6 h. The levels of BRUCE, β-actin, and active caspase 3 were analyzed by immunoblotting. (E) The cell death induced by BRUCE RNAi is resistant to overexpression of Bcl-x_L. MCF-7/Fas and MCF-7/Fas/Bcl-x_L cells were transfected and processed as in (A). Percentage of cell death (round) was determined for the transfected cells, which express GFP. To monitor whether the transfected Bcl-x_L functions properly, both cell lines were treated with 20 ng ml⁻¹ TNFα in the presence of 1 μM cycloheximide (CHX) for 5 h. (F) The cell death induced by BRUCE RNAi is independent of caspase activation in MCF-7/Fas cells. MCF-7/Fas cells were transfected, split, treated with TNFα/CHX, and analyzed as in (E). In addition, the cells were untreated or treated with 20 μM of z-VAD-fmk at 2 days after transfection, and 24 h later, fresh z-VAD-fmk was added following splitting.

Overexpression of Nrdp1 sensitizes cells to caspase 3 activation and apoptosis. (A) Overexpression of Nrdp1 inhibits growth of NIH/3T3 cells. NIH/3T3 cells were transfected with empty pcDNA3.1(+) vector (Invitrogen) or with the vector encoding wild-type (WT), mutant, or truncated Nrdp1. At 3 days after transfection, the cells were subcultured, and the same number of cells for each transfection was seeded. The transfection efficiency was about 15% in all the cases, as monitored in parallel using a GFP-expressing vector. Then, the cells were selected for 8 days by 800 μg ml⁻¹ geneticin (Gibco) and were photographed. The percentage of colonies relative to cells transfected with the empty vector (768±295 colonies plate⁻¹) is presented under each photograph. (B) Overexpression of Nrdp1 sensitizes cells to apoptosis induced by TNFα. (Left panel) HeLa cells were cotransfected with GFP and either empty vectors or the vectors encoding Nrdp1 for 24 h, and then were treated with 10 ng ml⁻¹ TNFα and 1 μM of cycloheximide for 3 h. The cells were finally stained with 2 μM Hoechst 33342 to visualize their nuclei. Apoptotic cells with condensed chromatin in GFP-positive cells were counted under a microscope using UV lights. (Right panel) 293T cells were transfected with either empty vectors or the vectors encoding wild-type or mutant (D56V) Nrdp1 for 16 h, and then were treated with 20 ng ml⁻¹ TNFα for 6 h. The cells were finally stained with 2 μM Hoechst 33342 to visualize their nuclei. Apoptotic cells with condensed chromatin were counted under a microscope using UV lights. (C) Nrdp1 overexpression promotes caspase 3 activation in response to TNFα. 293T cells were transfected as in (B), and then were treated with 20 ng ml⁻¹ TNFα for 6 h. (Upper panel) Caspase activity was assayed by using DEVD-7-amino-4-methylcoumarin as a substrate. The caspase activity in untreated cells (23.9 pmol min⁻¹ mg⁻¹ protein) was assigned as 100. (Lower panels) Levels of active caspase 3, BRUCE, and Nrdp1 were detected by immunoblotting. β-Actin is a control for loading. The band marked with an asterisk is probably ubiquitinated Nrdp1.