Pseudomonas exotoxin A-mediated apoptosis is Bak dependent and preceded by the degradation of Mcl-1 - PubMed (original) (raw)

Pseudomonas exotoxin A-mediated apoptosis is Bak dependent and preceded by the degradation of Mcl-1

Xing Du et al. Mol Cell Biol. 2010 Jul.

Abstract

Pseudomonas exotoxin A (PE) is a bacterial toxin that arrests protein synthesis and induces apoptosis. Here, we utilized mouse embryo fibroblasts (MEFs) deficient in Bak and Bax to determine the roles of these proteins in cell death induced by PE. PE induced a rapid and dose-dependent induction of apoptosis in wild-type (WT) and Bax knockout (Bax(-/-)) MEFs but failed in Bak knockout (Bak(-/-)) and Bax/Bak double-knockout (DKO) MEFs. Also a loss of mitochondrial membrane potential was observed in WT and Bax(-/-) MEFs, but not in Bak(-/-) or in DKO MEFs, indicating an effect of PE on mitochondrial permeability. PE-mediated inhibition of protein synthesis was identical in all 4 cell lines, indicating that differences in killing were due to steps after the ADP-ribosylation of EF2. Mcl-1, but not Bcl-x(L), was rapidly degraded after PE treatment, consistent with a role for Mcl-1 in the PE death pathway. Bak was associated with Mcl-1 and Bcl-x(L) in MEFs and uncoupled from suppressed complexes after PE treatment. Overexpression of Mcl-1 and Bcl-x(L) inhibited PE-induced MEF death. Our data suggest that Bak is the preferential mediator of PE-mediated apoptosis and that the rapid degradation of Mcl-1 unleashes Bak to activate apoptosis.

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Figures

FIG. 1.

Apoptosis of MEFs induced by PE. (A) WT MEFs were incubated with 1 to 50 ng/ml of PE for 24 h and subjected to apoptosis analysis by phosphatidylserine exposure (annexin V-PE) and 7-AAD uptake. (B) WT MEFs were incubated with 50 ng/ml of PE for 0 to 24 h and then subjected to apoptosis analysis as for panel A.

FIG. 2.

Bak is required for the apoptosis of MEFs induced by PE. (A) WT, Bax−/−, Bak−/−, and DKO MEFs were incubated with medium only or with 50 ng/ml of PE for 24 h, and the apoptosis was analyzed with annexin V-PE and 7-AAD. (B) Means and standard deviations (SD) of apoptotic cells (annexin V positive) from four independent assays. NT, no treatment (medium only). (C) MEFs were treated with different concentrations of PE for 8 h. The cells were then assayed for protein synthesis using a [3H]leucine incorporation assay. •, WT; ▪, Bak−/−; ▴, Bax−/−; ♦, DKO.

FIG. 3.

Bak is required to disrupt mitochondrial membrane potential. WT, Bax−/−, Bak−/−, and DKO MEFs were incubated with medium only or 50 ng/ml of PE for 24 h. The MEFs were stained with JC-1. Loss of ΔΨ_m_ is indicated by the decrease in red fluorescence.

FIG. 4.

Mcl-1 degradation is associated with apoptosis induced by PE. (A) WT MEFs were treated with 50 ng/ml of PE for up to 24 h, and the Mcl-1 expression level was detected by Western blotting. (B) WT, Bak−/−, Bax−/−, and DKO MEFs were treated with 50 ng/ml of PE for 20 h. Expression levels of Mcl-1, Bak, Bax, Bik, and Bcl-xL were detected by Western blotting. (C) WT MEFs were pretreated with 10 μM MG132 and then exposed to 50 ng/ml of PE for 20 h. The Mcl-1 expression level was detected by Western blotting. (D) The apoptosis of cells in panel C was analyzed with annexin V-PE and 7-AAD. Means and SD from triplicate wells are shown, representing data from three independent experiments.

FIG. 5.

Bak released from suppressor complexes after degradation of Mcl-1. (A) WT MEFs were treated with 50 ng/ml of PE for 20 h. Cell lysates were prepared with 1% CHAPS buffer and immunoprecipitated with anti-Mcl-1 antibody (Ab) (M) or anti-Bcl-xL antibody (B) combined anti-Mcl-1 and anti-Bcl-xL antibodies (M+B), or rabbit IgG (R). Bak, Mcl-1, and Bcl-xL in precipitated pellets were detected by Western blotting. At the bottom are shown endogenous Bak, Mcl-1, and Bcl-xL in whole-cell lysates (before immunoprecipitation). *, rabbit IgG light chain. (B) Cell lysates were prepared with 1% Triton X-100 buffer, and immunoprecipitation was done as for panel A.

FIG. 6.

Mcl-1 and Bcl-xL bound to Bak in MEFs, and overexpression protected MEFs from PE-induced cell death. (A) WT MEF lysates were prepared in CHAPS buffer. Rabbit IgG and antibodies against Bcl-2, Mcl-1, and Bcl-xL were used for immunoprecipitation. Immunoprecipitates equal to 3× WCL were loaded for Bak Western blots, and immunoprecipitates equal to 1× WCL were loaded for Mcl-1, Bcl-xL, and Bcl-2 Western blots. Serially diluted WCL was loaded as a quantity determination control. (B) EGFP-C1, EGFP-Ms-Mcl-1, and EGFP-Ms-Bcl-xL were transiently transfected into WT MEFs; 24 h later, 50 ng/ml PE was added and incubated for 20 h. The means and SD of apoptotic cells in GFP-positive populations from three independent assays are shown. (C) The cell lysates in panel B were prepared in 1% CHAPS buffer. Western blots were done with antibodies against Mcl-1, Bcl-xL, and GFP.

FIG. 7.

PE treatment did not release a significant amount of other Mcl-1-bound proapoptotic proteins. WT MEFs were treated with 50 ng/ml PE for 20 h. The cell lysates were prepared in 1% CHAPS buffer. Mcl-1 was immunoprecipitated from WCL with or without PE treatment, and Puma, Bax, Bim, and Mcl-1 were detected via Western blotting.

FIG. 8.

Bak knockout cannot protect MEFs from prolonged exposure to PE. (A) Bak−/− and DKO MEFs were treated with 50 to 1,000 ng/ml of PE for 24 h and analyzed by fluorescence-activated cell sorter (FACS) for apoptosis. (B) Bak−/− and DKO MEFs were treated with 50 ng/ml PE for up to 96 h and then subjected to apoptosis analysis. At the bottom are shown the means and SD of apoptotic cells from three independent assays.

FIG. 9.

The preferential role of Bak and Mcl-1 downregulation in apoptosis induced other protein synthesis inhibitors. (A) WT, Bak−/−, Bax−/−, and DKO MEFs were incubated with 50 ng/ml of ricin, 10 μg/ml of cycloheximide (CHX), or 50 nM staurosporine (STS) for 24 h and then analyzed by FACS for apoptosis. The means and SD of apoptotic cells from four independent assays are shown. NT, no treatment (medium only). (B) MEFs were treated with 50 ng/ml of PE, 50 ng/ml of ricin, 10 μg/ml of cycloheximide (CHX), or 50 nM staurosporine (STS) for 20 h. The expression level of Mcl-1 was detected by Western blotting.

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Pseudomonas exotoxin A-mediated apoptosis is Bak dependent and preceded by the degradation of Mcl-1 - PubMed (original) (raw)