BCL-2 family member BOK promotes apoptosis in response to endoplasmic reticulum stress

B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) is a BCL-2 family protein with high homology to the multidomain proapoptotic proteins BAX and BAK, yet Bok^−/− and even Bax^−/−Bok^−/− and Bak^−/−Bok^−/− mice were reported to have no overt phenotype or apoptotic defects in response to a host of classical stress stimuli. These surprising findings were interpreted to reflect functional compensation among the BAX, BAK, and BOK proteins. However, BOK cannot compensate for the severe apoptotic defects of Bax^−/−Bak^−/− mice despite its widespread expression. Here, we independently developed Bok^−/− mice and found that Bok^−/− cells are selectively defective in their response to endoplasmic reticulum (ER) stress stimuli, consistent with the predominant subcellular localization of BOK at the ER. Whereas Bok^−/− mouse embryonic fibroblasts exposed to thapsigargin, A23187, brefeldin A, DTT, geldanamycin, or bortezomib manifested reduced activation of the mitochondrial apoptotic pathway, the death response to other stimuli such as etoposide, staurosporine, or UV remained fully intact. Multiple organs in Bok^−/− mice exhibited resistance to thapsigargin-induced apoptosis in vivo. Although the ER stress agents activated the unfolded protein response, both ATF4 and CHOP activation were diminished in Bok^−/− cells and mice. Importantly, BAX and BAK were unable to compensate for the defective apoptotic response to ER stress observed in SV40-transformed and primary Bok^−/− cells, and in vivo. These findings support a selective and distinguishing role for BOK in regulating the apoptotic response to ER stress, revealing—to our knowledge—the first bona fide apoptotic defect linked to Bok deletion.BAX and BAK are essential B-cell lymphoma-2 (BCL-2) family proteins that porate the mitochondrial outer membrane in response to a broad range of apoptotic stimuli (1). BCL-2 ovarian killer (BOK) exhibits ∼70–80% sequence homology to BAX and BAK, sharing BH1-3 domains and a carboxy-terminal transmembrane domain (2). Like BAX and BAK, BOK is widely expressed and induces cell death upon transient overexpression (2–9). In the context of BOK overexpression, BOK-induced cell death manifests classical apoptotic features, including the release of cytochrome c, activation of caspase-3, and nuclear and DNA fragmentation (6, 9–11). In addition, overexpression of antiapoptotic MCL-1, BFL-1/A1, and BHRF-1 inhibit BOK-mediated cell death (2, 7, 12, 13). Based on these similarities, it has been suggested that BOK acts in a redundant fashion to BAX and BAK and may be responsible for the apparently normal development of numerous organs in Bax^−/−Bak^−/− mice (14).Despite these initial observations, the physiologic role of BOK in responding to cell stress has remained enigmatic. Indeed, the development and characterization of Bok-deficient mice revealed little to no phenotype (3). Moreover, leukocyte subsets from Bok^−/− mice showed no alteration in their death response to classical proapoptotic agents, such as etoposide, dexamethasone, Fas ligand, ABT-737, or cytokine deprivation (3). Even Bok^−/−Bax^−/− and Bok^−/−Bak^−/− doubly deficient mice are mostly normal, and cells derived from these mice are equally susceptible to death stimuli (15), in stark contrast to the severe apoptotic blockade of Bax^−/−Bak^−/− cells. Also intriguing, a functional role for Bok as a tumor suppressor was suggested by its genetic location in 1 of the 20 most deleted regions in all human cancers (16).To investigate a role for BOK in the apoptotic pathway, we generated Bok^−/− mice and cells. Based on the recent localization of BOK at the endoplasmic reticulum (ER) (4), we focused our inquiry on ER stress pathways, including the unfolded protein response (UPR). We find that Bok^−/− cells manifest reduced activation of the classical mitochondrial apoptotic pathway in response to a battery of ER stress stimuli. Moreover, the apoptotic deficiency is selective, with no observed differences in response to other inducers of apoptosis. From a mechanistic standpoint, we find that BOK requires downstream BAX/BAK activation to induce mitochondrial apoptosis, as BOK expression can rescue the defective response of Bok^−/− cells, but not Bax^−/−Bak^−/− cells. These findings support a selective role for BOK in regulating the apoptotic response to ER stress, a functional activity that corresponds to its recent subcellular localization at the ER.