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. 1997 Nov 11;94(23):12401-6.
doi: 10.1073/pnas.94.23.12401.

Bok is a pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members

S Y Hsu  1 , A Kaipia, E McGee, M Lomeli, A J Hsueh
Affiliations

Bok is a pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members

S Y Hsu et al. Proc Natl Acad Sci U S A. .

Abstract

In the intracellular death program, hetero- and homodimerization of different anti- and pro-apoptotic Bcl-2-related proteins are critical in the determination of cell fate. From a rat ovarian fusion cDNA library, we isolated a new pro-apoptotic Bcl-2 gene, Bcl-2-related ovarian killer (Bok). Bok had conserved Bcl-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region present in other Bcl-2 proteins, but lacked the BH4 domain found only in anti-apoptotic Bcl-2 proteins. In the yeast two-hybrid system, Bok interacted strongly with some (Mcl-1, BHRF1, and Bfl-1) but not other (Bcl-2, Bcl-xL, and Bcl-w) anti-apoptotic members. This finding is in direct contrast to the ability of other pro-apoptotic members (Bax, Bak, and Bik) to interact with all of the anti-apoptotic proteins. In addition, negligible interaction was found between Bok and different pro-apoptotic members. In mammalian cells, overexpression of Bok induced apoptosis that was blocked by the baculoviral-derived cysteine protease inhibitor P35. Cell killing induced by Bok was also suppressed following coexpression with Mcl-1 and BHRF1 but not with Bcl-2, further indicating that Bok heterodimerized only with selective anti-apoptotic Bcl-2 proteins. Northern blot analysis indicated that Bok was highly expressed in the ovary, testis and uterus. In situ hybridization analysis localized Bok mRNA in granulosa cells, the cell type that underwent apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic Bcl-2 protein with restricted tissue distribution and heterodimerization properties could facilitate elucidation of apoptosis mechanisms in reproductive tissues undergoing hormone-regulated cyclic cell turnover.

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Figures

Figure 1
Figure 1
Sequence comparison between Bok and different Bcl-2 family members. (A) Sequence alignment of Bok with several members of the Bcl-2 family proteins. The ORF for Bok predicts a protein of 213 amino acids in length. Asterisks indicate two potential phosphorylation sites. Shaded residues are identical in at least three of the six Bcl-2 proteins shown. Consensus sequences for these proteins are listed on top of the sequence alignment. The conserved BH domains (–4) and the C-terminal transmembrane region are indicated. The GenBank accession number for Bok is AF027954. r: rat, m: mouse, h: human. (B) Comparison of BH3 domain sequences in different pro-apoptotic Bcl-2 proteins. (C) Phylogenetic relatedness of different Bcl-2 family members. Full-length polypeptides for all Bcl-2 members except Mcl-1 (amino acids 121–330 only) are used for comparison.
Figure 2
Figure 2
Bok only interacts with selective anti-apoptotic Bcl-2 proteins in the yeast two-hybrid system. (A) Upper: yeast cells were grown in the selective media containing 5 mM 3-aminotriazole and without tryptophan, leucine, and histidine. Prominent growth of yeast colonies expressing Bok fused to the GAL4 activation domain together with Mcl-1, BHRF1, or Bfl-1 fused to the GAL4 binding domain could be seen. Minimal growth of yeast colonies was found in cells that express the same Bok expressing vector together with Bcl-2, Bcl-xL, Bcl-w, BAD, Bax, Bak, or Bik fused to the GAL4 binding domain. In addition, prominent growth of colonies expressing Bcl-xL and different pro-apoptotic Bcl-2 proteins indicated that the lack of growth in yeast cells expressing Bok and different pro-apoptotic family members was not due to suppression of cell growth by these pro-apoptotic proteins. Lower: growth of yeast colonies transformed with the same vector pairs maintained in a nonselective media. (B) Summary of protein-protein interactions between pairs of pro-apoptotic proteins (Bok, Bak, Bik, and Bax) and different anti-apoptotic Bcl-2 members. The positive signs indicate prominent (++) or moderate (+) yeast cell growth whereas the negative signs (−) indicate the absence of reporter gene expression.
Figure 3
Figure 3
Overexpression of Bok promotes apoptosis in mammalian cells. (A) Morphology of CHO cells transfected with an expression vector encoding Bok. Normal cell morphology was found in cells transiently transfected with the empty pcDNA3 expression vector (2.1 μg DNA per 35-mm dish) or the vector containing Bok cDNA in reverse orientation (Bok Rev). Cells were also transfected with the Bok expression vector without or with an equal amount of the P35-expressing construct. Arrowheads indicate apoptotic cells whereas darkly stained cells represent transfected cells. (B) Internucleosomal DNA fragmentation induced by Bok and partial inhibition by the baculoviral protease inhibitor P35. CHO cells were treated as described in A. At 18 h after transfection, cellular DNA was extracted for analysis of DNA fragmentation using a 3′end labeling method. Quantitative estimation of low molecular weight DNA is shown at the bottom of the figure (mean ± SEM, n = 3). (C) Quantitative analysis of cell killing by Bok and the inhibitory effects of P35. The number of β-gal-expressing cells (mean ± SEM, n = 3) was determined at 36 h after transfection. Data from cells transfected with three independent clones (1, 2, and 3) encoding Bok are presented as a percentage of viable cells in the control group. CHO cells were transfected with a total of 2.1 μg plasmid DNA including 2.0 μg of pcDNA3 expression constructs and 0.1 μg of the pCMV-β-gal reporter. In cells transfected with two different pcDNA3 expression plasmids, 1.0 μg each was used. Similar results were obtained in three separate experiments.
Figure 4
Figure 4
Suppression of Bok-induced apoptosis by selective anti-apoptotic Bcl-2 members in CHO cells. Cell killing by Bok and the antagonistic effects of Mcl-1 and BHRF1 were analyzed. Cell transfection and estimation of apoptosis were as described in the Fig. 3 legend. Coexpression of Bcl-2 was ineffective in suppressing Bok-induced apoptosis.
Figure 5
Figure 5
Expression of Bok mRNA transcripts in rat tissues. (A) For Northern blot analysis, poly(A)+-selected RNA from different tissues of rats at 27 days of age or from isolated granulosa cells of estrogen-treated rats was hybridized with a 32P-labeled Bok cRNA probe. After washing, the blots were exposed to x-ray films at −70°C for five days. Subsequent hybridization with a glyceraldehyde-3-phosphate dehydrogenase cRNA probe was performed to estimate nucleic acid loading (8 h exposure). Specific Bok transcripts are indicated by an arrow. (B) In situ hybridization analysis of Bok expression in the ovary. Ovaries from immature equine chorionic gonadotropin-treated rats were probed with the antisense Bok cRNA. Upper, Left: hybridization signals in representative primary (1°) and secondary (2°) but not primordial (Pmd) follicles (magnification, ×100). Upper, Right (magnification, ×200) and Lower, Left (magnification, ×100): positive signals in granulosa cells of preantral follicles and an antral follicle, respectively. Lower, Right: no signal was found in a section hybridized with the sense Bok probe. O, oocyte; G, granulosa cells; T, theca cells; A, antrum.
Figure 6
Figure 6
Conservation of Bok in diverse vertebrate species. Southern blot analysis of genomic DNA from different vertebrate species was performed. DNA was digested with the EcoRI enzyme and probed with a Bok cDNA probe. Following hybridization at 68°C, the membrane was washed under high stringency conditions (1% SDS/0.1 × SSC at 65°C) before exposure.
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