The Binding Properties and Biological Activities of Bcl-2 and Bax in Cells Exposed to Apoptotic Stimuli

Abstract

The oncogene product Bcl-2 protects cells from apoptosis whereas its homolog Bax functions to kill cells. Several binding partners of Bcl-2 and Bax have been isolated, but none of them has yet provided clues as to exactly how Bcl-2 and Bax work. According to one view, Bcl-2 and Bax interact with survival and death effector molecules, respectively, and neutralize each other through heterodimerization. Alternatively, Bcl-2 requires Bax for death protection, and additional proteins bind to the heterodimer to regulate its activity. Here we used a co-immunoprecipitation strategy to distinguish between these two possibilities. We show that the Bcl-2-Bax heterodimer is maintained, and no other protein associates stably in detectable amounts with Bcl-2, Bax, or the heterodimer in anti-Bcl-2 and anti-Bax immunoprecipitates from normal cells and cells exposed to apoptotic stimuli. Analysis of cells expressing various levels of Bcl-2 and Bax, however, revealed that the degree of protection against apoptosis does not correlate with the number of Bcl-2-Bax heterodimers but the amount of Bcl-2 that is free of Bax. In addition, the survival activity of Bcl-2 is unaffected when Bax expression is ablated by an antisense strategy. Our findings suggest that the Bcl-2-Bax heterodimer is a negative regulator of death protection, and that Bcl-2 requires neither Bax nor major, stable interactions with other cellular proteins to exert its survival function. We therefore propose that Bcl-2 acts as an enzyme (capturing substrates in a transient way), as a homodi-or multimer, or through the interaction with non-proteaceous targets (lipids, ions).

Bax activation and mitochondrial insertion during apoptosis

“…It was first thought to be involved in the ability of these proteins to form homoor hetero-oligomers. This assumption was demonstrated in the case of Bcl-2 and Bcl-xl binding of other BH3 proteins [16,17]. Indeed, the BH3 peptides which mimic Bax BH3 domain are able to interact with the hydrophobic groove present on the surface of these proteins and formed by the BH1, 2 and 3 domains.…”

Failure of Bcl-2 family members to interact with Apaf-1 in normal and apoptotic cells

“…Moreover, the failure of these molecules to form complexes was not due to the experimental conditions as anti-Bcl-2 effectively co-immunoprecipitated endogenous Bax, a known physiological partner of Bcl-2. 37,38 It was also highly unlikely that the putative Apaf-1/Bcl-2 complex was disrupted by the immunoprecipitation antibodies, because we used both polyclonal (data not shown) and monoclonal ( Figure 1) antibodies to Bcl-x L and Bcl-2. We also used two other detergents (digitonin, CHAPS) for cell lysis and immunoprecipitation and still did not find interactions between Apaf-1 and Bcl-2 or Apaf-1 and Bcl-x L (Figure 1e).…”

“…In some cases, the cells were treated for different time periods with apoptotic agents such as the proteasomal inhibitor MG132 to determine whether interactions between Flag-Apaf-1 and Bcl-2 or Flag-Apaf-1 and Bcl-x L are only No interaction between Apaf-1 and Bcl-2 family members S Conus et al formed when the latter two proteins act as death suppressors. 37 Cell lysates prepared in the presence of the detergent Nonidet P-40 (NP-40) were subjected to anti-Flag, anti-Bcl-2 or anti-Bcl-x L immunoprecipitations, and the immunoprecipitates were Western-blotted for the detection of co-precipitating proteins. As shown in Figures 1 and 3 The lack of interaction between Flag-Apaf-1 and Bcl-2 or Bcl-x L was not due to the Flag-tag, as the same results were obtained by transfecting non-tagged Apaf-1 and using an anti-Apaf-1 specific antibody (Figure 1d).…”

“…It has however been reported that detergents can either promote or disrupt interactions between Bcl-2 family members. 36,37 We therefore needed to investigate the interaction between Apaf-1 and Bcl-2 family members in an experimental system that does not require detergent extraction. We chose an immunofluorescence analysis on fixed, permeabilized rat embryo fibroblasts overexpressing the proteins and show that three Bcl-2 family members Bcl-2, Bcl-x L , or Bax cannot attract cytoplasmic Apaf-1 to membranes in normal and apoptotic cells.…”

Sponge Bcl-2 homologous protein (BHP2-GC) confers distinct stress resistance to human HEK-293 cells

“…18 It is assumed that the ratio of pro-survival versus pro-apoptotic dimers is crucial for the resistance of cells to apoptosis. 19 While heterodimerization is not required for the pro-survival function 20 such a process is thought to be essential for the pro-apoptotic activity, especially via the BH3 region. 21 Members of the Bcl-2 family that comprise only domains BH1 and BH2 belong to the pro-survival proteins, e.g.…”