Bax degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression

Abstract

Previously we reported that proteasome inhibitors were able to overcome Bcl-2-mediated protection from apoptosis. Here we show that inhibition of the proteasome activity in Bcl-2-overexpressing cells accumulates the proapoptotic Bax protein to mitochondria/cytoplasm, where it interacts to Bcl-2 protein. This event was followed by release of mitochondrial cytochrome c into the cytosol and activation of caspase-mediated apoptosis. In contrast, proteasome inhibition did not induce any apparent changes in Bcl-2 protein levels. In addition, treatment with a proteasome inhibitor increased levels of ubiquitinated forms of Bax protein, without any effects on Bax mRNA expression. We also established a cell-free Bax degradation assay in which an in vitro-translated, (35)S-labeled Bax protein can be degraded by a tumor cell protein extract, inhibitable by addition of a proteasome inhibitor or depletion of the proteasome or ATP. The Bax degradation activity can be reconstituted in the proteasome-depleted supernatant by addition of a purified 20S proteasome or proteasome-enriched fraction. Finally, by using tissue samples of human prostate adenocarcinoma, we demonstrated that increased levels of Bax degradation correlated well with decreased levels of Bax protein and increased Gleason scores of prostate cancer. Our studies strongly suggest that ubiquitin/proteasome-mediated Bax degradation is a novel survival mechanism in human cancer cells and that selective targeting of this pathway should provide a unique approach for treatment of human cancers, especially those overexpressing Bcl-2.

Figure 1

Proteasome inhibitor LLnV induces Bax accumulation, cytochrome c release, and PARP cleavage in Bcl-2-overexpressing Jurkat T cells. (A–C) Jurkat T cells overexpressing Bcl-2 (0 h) were treated with 50 μM LLnV for up to 8 h, followed by preparation of cytosol and mitochondrial fractions. Both fractions were immunoblotted first by an antibody to cytochrome c (Cyto C, 17 kDa; A and C) and then reblotted by anticytochrome oxidase subunit II (COX, 26 kDa; B). Note: 20 μg protein from the cytosol, and 40 μg protein from the mitochondrial, preparation was used in each lane. (D–G) Whole-cell extracts (70 μg per lane) of the above treated cells were immunoblotted with specific antibodies to PARP (D), Bax (clone N-20; E), Bcl-2 (F), or actin (G). Molecular masses of PARP, the PARP cleavage fragment (p85), Bax, Bcl-2, and actin are 113, 85, 21, 26, and 40 kDa, respectively. Positions of protein markers are indicated at right. (H) Bcl-2-expressing Jurkat cells (Control) were treated with 50 μM LLnV for 8 h, followed by reverse transcription–PCR (RT-PCR). For the first-strand cDNA synthesis, 0.2 (lanes 1 and 4), 0.6 (lanes 2 and 5), and 1.8 μg (lanes 3 and 6) of the total RNA were used. The positions of Bax (538 bp) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA (983 bp) are indicated. Lane M is DNA molecular weight marker.

Figure 2

Proteasome inhibition accumulates Bax to mitochondria/cytoplasm and increases interaction between Bax and Bcl-2 proteins. Jurkat cells (0 h; A and B) or Jurkat cells overexpressing Bcl-2 (0 h; C and D) were treated by either 10 μM lactacystin (A–C), 50 μM LLnV (D), or 50 μM LLM (A and B) for the indicated h. (A and B) Western blotting with antibodies to PARP and Bax were performed as described in Fig. 1. (C) Immunohistochemistry (see Materials and Methods). Localization of Bax protein (green) and nuclei (red) are shown. (D) Bax immunoprecipitates (IP), prepared by an agarose-conjugated N-20 Bax antibody, were immunoblotted (IB) first with the 6A7 Bax antibody (Upper) and then reblotted with a Bcl-2 antibody (Lower).

Figure 3

Bax degradation depends on ubiquitination, proteasome, and ATP. (A) Jurkat cells (0 h) were treated with either 50 μM LLnV or 10 μM lactacystin, followed by preparation of Bax immunoprecipates (with clone 6A7), which were immunoblotted with an ubiquitin antibody. Positions of putative ubiquitinated Bax proteins (p47 and p55) are indicated at left. The nature of the ≈30-kDa and ≈84-kDa bands remains unclear. (B) The ³⁵S-labeled Bax (Upper) or Bcl-2 (Lower) protein (1 μl) were incubated at 37°C for 2 h with either buffer Z only (lane 1) or 100 μg protein extract of MCF-7 cells grown exponentially (lane 2) or pretreated for 8 h with 50 μM LLnV (lane 3). (C) The ³⁵S-labeled Bax protein (1 μl) was incubated with either buffer Z only (lane 1) or 100 μg MCF-7 cell lysate at 37°C for 4 h, in the presence of inhibitors N-carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (LLL), LLnV, N-acetyl-L-leucyl-L-leucyl-norleucinal (LLnL), and LLM (100 μM; lanes 3–8) or an equal volume of DMSO (lane 2) in buffer Z. A weak band of ≈16 kDa (indicated by an arrow) is probably an intermediate product of proteasome-mediated Bax degradation. (D) The proteasome in MCF-7 whole-cell lysate (W lys, lane 1) was precipitated by using either ultraspin (ULS) or a proteasome subunit α6 antibody (IP). Both supernatant (lanes 2 and 4) and pellet (lanes 3 and 5) fractions were examined by Western blot assay using antibody to the proteasome α6 subunit (33 kDa). (E) Bax degradation assay was performed as in B, with addition of buffer Z (lane 1) or 100 μg protein from MCF-7 whole-cell lysate (lane 2), ultraspun supernatant (lane 3) or plus the pellet (lane 4), immunodepleted supernatant (lane 5), or plus a purified 20S proteasome (2 μg; lane 6), or the purified proteasome alone (2 μg; lane 7). (F) Bax degradation assay was performed as in B, in the absence (lane 3) or presence of 10 mM ATP (lane 2) or 10 mM ATP-γ-S (lane 4).

Figure 4

Correlation of decreased Bax protein levels and increased Bax degradation in advanced human prostate cancer specimens (marked by increased Gleason scores). (A–C) Whole-tissue extracts (100 μg/lane) were immunoblotted with specific antibodies to proliferating cell nuclear antigen (PCNA, 36 kDa; A), Bax (N-20; B) or actin (C). (D and E) Bax or Bcl-2 degradation activity was assayed by incubating an ³⁵S-labeled Bax or Bcl-2 protein (1 μl) with either buffer Z only (lane 1) or 200 μg protein extract prepared from either prostate adenocarcinomas with different grades (lanes 3–8) or a benign prostate hyperplasia (as a control; lane 2) at 37°C for 4 h in buffer Z. (F) Whole tissue extracts (200 μg) were immunoprecipated (IP) with 6A7 Bax antibody, followed by immunoblot (IB) with an ubiquitin antibody. Position of the putative ubiquitinated Bax, p55, is indicated. n/s indicates a possible nonspecific band.