Intracellular localization of the BCL-2 family member BOK and functional implications

Abstract

The pro-apoptotic BCL-2 family member BOK is widely expressed and resembles the multi-BH domain proteins BAX and BAK based on its amino acid sequence. The genomic region encoding BOK was reported to be frequently deleted in human cancer and it has therefore been hypothesized that BOK functions as a tumor suppressor. However, little is known about the molecular functions of BOK. We show that enforced expression of BOK activates the intrinsic (mitochondrial) apoptotic pathway in BAX/ BAK-proficient cells but fails to kill cells lacking both BAX and BAK or sensitize them to cytotoxic insults. Interestingly, major portions of endogenous BOK are localized to and partially inserted into the membranes of the Golgi apparatus as well as the endoplasmic reticulum (ER) and associated membranes. The C-terminal transmembrane domain of BOK thereby constitutes a 'tail-anchor' specific for targeting to the Golgi and ER. Overexpression of full-length BOK causes early fragmentation of ER and Golgi compartments. A role for BOK on the Golgi apparatus and the ER is supported by an abnormal response of Bok-deficient cells to the Golgi/ER stressor brefeldin A. Based on these results, we propose that major functions of BOK are exerted at the Golgi and ER membranes and that BOK induces apoptosis in a manner dependent on BAX and BAK.

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

“…A previous study reported no difference in annexin V positivity between WT and independently derived Bok −/− MEFs treated with TG, and even an increased sensitivity of Bok −/− cells to BFA exposure (4). In contrast, here we observe apoptotic resistance in SV40-transformed Bok −/− MEFs exposed to six different ER stress agents, including TG and BFA, at various time points and over a broad dose range, as analyzed by XTT, caspase-3/7, clonogenic, and annexin V assays.…”

“…Nevertheless, Bok −/− mice show no overt defects in these tissues either (3,14,15). Thus, we explored whether BOK could subserve a specific apoptotic function independent from BAX and BAK at the ER, its site of predominant intracellular localization (4).…”

“…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)(3)(4)(5)(6)(7)(8)(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)(10)(11).…”

Bag6 complex contains a minimal tail-anchor–targeting module and a mock BAG domain

“…Overexpression of Bag6 in HeLa cells exposed to ricin, an apoptosis inducer, leads to an increase in endogenous Bcl-2 protein levels, whereas Bag6 knockdown causes downregulation of Bcl-2 proteins (50). Several proteins that belong to the Bcl-2 family, including Bcl-2, MCL1, BAX, and BOK, are tail-anchored and reside both at the ER and the mitochondria (51)(52)(53). The Bag6 complex then would play an important role in regulating the localization and turnover of these Bcl-2 proteins, and this role could be disrupted by Bag6 cleavage.…”

Bok is a genuine multi-BH-domain protein that triggers apoptosis in the absence of Bax and Bak and augments drug response

“…Because these experiments were performed in MCF7 cells, which are proficient in Bax and Bak (Neise et al, 2008), Bok might function as a BH3-only protein, for example, like Puma (Jabbour et al, 2008), and induce cytochrome c release indirectly by blocking anti-apoptotic proteins or by directly activating Bax and Bak, which in turn mediate cytochrome c release. This is supported by experiments showing that Bok alone is insufficient for apoptosis induction in Bax −/− /Bak −/− MEFs and that Bok depends on Bax and Bak to mediate release of cytochrome c (Echeverry et al, 2013). However, it should be considered that the inability of endogenous Bok to mediate apoptosis in the absence of Bax and Bak might be due to its low or even absent expression level in MEF Bax −/−…”