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. 1999 Oct;19(10):6673-81.
doi: 10.1128/MCB.19.10.6673.

"V体育官网" The BH3 domain of Bcl-x(S) is required for inhibition of the antiapoptotic function of Bcl-x(L)

B S Chang  1 , A Kelekar, M H Harris, J E Harlan, S W Fesik, C B Thompson
Affiliations

The BH3 domain of Bcl-x(S) is required for inhibition of the antiapoptotic function of Bcl-x(L)

B S Chang et al. Mol Cell Biol. 1999 Oct.

Abstract

bcl-x is a member of the bcl-2 family of genes. The major protein product, Bcl-x(L), is a 233-amino-acid protein which has antiapoptotic properties. In contrast, one of the alternatively spliced transcripts of the bcl-x gene codes for the protein Bcl-x(S), which lacks 63 amino acids present in Bcl-x(L) and has proapoptotic activity VSports手机版. Unlike other proapoptotic Bcl-2 family members, such as Bax and Bak, Bcl-x(S) does not seem to induce cell death in the absence of an additional death signal. However, Bcl-x(S) does interfere with the ability of Bcl-x(L) to antagonize Bax-induced death in transiently transfected 293 cells. Mutational analysis of Bcl-x(S) was conducted to identify the domains necessary to mediate its proapoptotic phenotype. Deletion mutants of Bcl-x(S) which still contained an intact BH3 domain retained the ability to inhibit survival through antagonism of Bcl-x(L). Bcl-x(S) was able to form heterodimers with Bcl-x(L) in mammalian cells, and its ability to inhibit survival correlated with the ability to heterodimerize with Bcl-x(L). Deletion mutants of Bax and Bcl-2, which lacked BH1 and BH2 domains but contained a BH3 domain, were able to antagonize the survival effect conferred by Bcl-x(L). The results suggest that BH3 domains from both pro- and antiapoptotic Bcl-2 family members, while lacking an intrinsic ability to promote programmed cell death, can be potent inhibitors of Bcl-x(L) survival function. .

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Figures

FIG. 1
FIG. 1
Bcl-xS can prevent Bcl-xL rescue of Bax-induced cell death. (A) 293 cells, plated in six-well dishes, were transiently transfected with the indicated combinations of constructs, each at 1 μg per transfection. The amount of total DNA per transfection was maintained at 3 μg. Cells were harvested 24 h posttransfection, and the subdiploid percentage of the subpopulation of cells expressing the marker for transfection, EGFP, was measured. Means and standard deviations of at least three independent experiments are shown. (B) Transfections were conducted as for panel A, but cells were harvested at 15 h posttransfection. Cells were lysed, and proteins were separated by SDS-PAGE. Proteins were blotted with a polyclonal anti-Bcl-x antibody and a polyclonal anti-Bax antibody.
FIG. 2
FIG. 2
Alignment of Bcl-xS with deletion mutants of Bcl-xS. (Top) Schematic of Bcl-xS with alpha-helical domains (based on homology to Bcl-xL) highlighted above. TM, transmembrane domain at the C-terminal end of Bcl-xS. (Bottom) Deletion mutants used in this study. Thin lines represent gaps of sequence missing from the deletion constructs. The asterisk identifies the Leu 90-to-Ala point mutation. All constructs, including Bcl-xS, have N-terminal FLAG epitope tags.
FIG. 3
FIG. 3
The BH3 domain is necessary for the proapoptotic phenotype of Bcl-xS. (A) Bax, Bcl-xL, Bcl-xS, and mutants of Bcl-xS were transfected in equal quantities (1 μg each) as indicated. Cells were harvested at 24 h posttransfection, and the subdiploid percentage of the EGFP-positive population was measured. Means and standard deviations of three independent experiments are shown. (B) Transfections were conducted as for panel A. Cells were harvested at 24 h posttransfection, and intracellular staining with an anti-FLAG monoclonal antibody (M2) was performed. Fluorescence was measured by flow cytometry. Results are representative of three independent experiments.
FIG. 4
FIG. 4
Heterodimerization with Bcl-xL correlates with the presence of the BH3 domain in Bcl-xS. Cells were transfected with equal amounts of Bcl-xL and Bcl-xS or Bcl-xS deletion mutants. All Bcl-xS constructs had an N-terminal FLAG epitope. Cells were harvested and lysed after 15 h. (A) Western blot analysis of the whole-cell lysates visualized with an anti-Bcl-x antibody (S-18). Note that the deletion mutants of Bcl-xS lack the S-18 epitope. (B) Western blot analysis of whole-cell lysates with an anti-FLAG antibody. (C) Whole-cell lysates were immunoprecipitated with anti-FLAG, and immunoprecipitates were blotted with an anti-Bcl-x antibody (S-18). (D) Western blot analysis with M2 (anti-FLAG) of immunoprecipitates following immunoprecipitation with M2. Note that the immunoglobulin light chain (∗) comigrates with Bcl-xS. WB, Western blot; IP, immunoprecipitation.
FIG. 5
FIG. 5
The α2 helices of Bcl-x, Bax, and Bcl-2 all antagonize Bcl-xL. (A) Cells in six-well dishes were transfected with the indicated constructs, all at 1 μg per transfection. Total DNA concentration was maintained at 3 μg per transfection. The EGFP-positive, subdiploid percentage was measured 24 h after transfection. Means and standard deviations of three independent experiments are shown. (B) Cells were transfected with 1 μg of each indicated construct per transfection. Two micrograms of control vector was used in each transfection. (C) α2 constructs of Bcl-xS, Bcl-2, and Bax. Bold lines represent regions of cDNAs retained in deletion constructs. (D) Yeast cells expressing either Bax, Bax α2, Bcl-xS, or Bcl-xS α2 under the control of a galactose-inducible promoter were grown in the presence of glucose or galactose.
FIG. 6
FIG. 6
The α2 helix of Bax heterodimerizes with Bcl-xL to a greater degree than wild-type Bax. (A) Cells plated in 10-cm dishes were transfected with a total of 20 μg of constructs per transfection (10 μg of each construct per transfection). Both Bax and Bax α2 had an N-terminal HA epitope tag. WB, Western blot. There is a nonspecific band in all lanes that migrated close to HA-Bax. (B) Lysates were immunoprecipitated (IP) with an anti-HA antibody. Immunoprecipitates were separated by SDS-PAGE and blotted with anti-Bcl-x and anti-HA antibodies. The top panel shows the amount of Bcl-x which coimmunoprecipitates with Bax and Bax α2. The bottom panel is an anti-HA Western blot. The light chain migrated near HA-Bax.
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