The Stability and Expression Level of Bok Are Governed by Binding to Inositol 1,4,5-Trisphosphate Receptors

Abstract

Bok is a member of the Bcl-2 protein family that governs the intrinsic apoptosis pathway, although the role that Bok plays in this pathway is unclear. We have shown previously in cultured cell lines that Bok interacts strongly with inositol 1,4,5-trisphosphate receptors (IP 3 Rs), suggesting that it may contribute to the structural integrity or stability of IP 3 R tetramers. Here we report that Bok is similarly IP 3 R-assocated in mouse tissues, that essentially all cellular Bok is IP 3 R bound, that it is the helical nature of the Bok BH4 domain, rather than specific amino acids, that mediates binding to IP 3 Rs, that Bok is dramatically stabilized by binding to IP 3 Rs, that unbound Bok is ubiquitinated and degraded by the proteasome, and that binding to IP 3 Rs limits the pro-apoptotic effect of overexpressed Bok. Agents that stimulate IP 3 R activity, apoptosis, phosphorylation, and endoplasmic reticulum stress did not trigger the dissociation of mature Bok from IP 3 Rs or Bok degradation, indicating that the role of proteasome-mediated Bok degradation is to destroy newly synthesized Bok that is not IP 3 R associated. The existence of this unexpected proteolytic mechanism that is geared toward restricting Bok to that which is bound to IP 3 Rs, implies that unbound Bok is deleterious to cell viability and helps explain the current uncertainty regarding the cellular role of Bok.Bok is a member of the Bcl-2 protein family that controls the intrinsic apoptosis pathway (1-3). Bok contains four Bcl-2 homology domains (BH1-4) 2 and shares greatest sequence homology with the pro-apoptotic proteins Bak and Bax (1-4). However, unlike Bak and Bax, which have clearly defined roles in mediating mitochondrial outer membrane permeabilization (5, 6), the cellular role of Bok is unclear. Key observations that pertain to our current understanding of the function of Bok are (i) that the atypical C-terminal transmembrane (TM) domain of Bok localizes it to membranes of the endoplasmic reticulum (ER) and Golgi (7), (ii) that Bok over-expression leads to apoptosis (7-10) if Bak or Bax are present (7), indicating that Bok lies upstream of Bak and Bax, (iii) that Bok Ϫ/Ϫ mice are phenotypically normal (4, 11, 12), while Bax Ϫ/Ϫ Bak Ϫ/Ϫ mice exhibit multiple severe defects (1), indicating that Bok cannot substitute for Bak and Bax, and (iv) that ER stress-induced apoptosis (13) can be suppressed in Bok Ϫ/Ϫ mouse cells in vitro and in vivo (12), although this result has not been seen by all groups (7). Overall, these data suggest that Bok plays a very different role from Bax and Bak, and that it may participate in the pathway between ER stress and apoptosis.Another intriguing facet of Bok cell biology is that it binds very strongly to inositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 Rs) (14), proteins that form tetrameric, IP 3 -, and Ca 2ϩ -gated Ca 2ϩ channels in ER membranes and play a key role in vertebrate cell signaling (15,16). Of the three mammalian IP 3 R types, Bok binding is strongest to IP 3 R1 and IP 3 R2 (14) and the...

The membrane activity of BOK involves formation of large, stable toroidal pores and is promoted by cBID

“…BOK has been shown to interact with IP3 receptors, protecting IP3Rs from caspase‐mediated degradation and BOK from proteasomal degradation . Degradation of BOK via the ubiquitin/proteasome was also shown by Llambi et al .…”

BOK displays cell death‐independent tumor suppressor activity in non‐small‐cell lung carcinoma

“…Consistent with this interpretation, BOK inhibited TGF-β2-induced cell migration, as assessed by Boyden chamber migration assay (Figure 5c). Furthermore, we detected TGFβ2-induced increase of ATF4 as well as a shorter form of BOK that lacks the N-terminus due to alternative translation initiation at Met 15 (13); ATF4, being required for cell invasion (24), was slightly diminished in TGFβ2-treated cell upon additional BOK overexpression and was accompanied by reduction of the shorter form of BOK (Figure 5e). …”

“…Importantly, we found that BOK levels were significantly decreased in tumors of lymph node positive patients and in less differentiated tumors compared to adjacent lung parenchyma (Figure 1b). This phenotype might be favoured by epigenetic mechanisms during tumor progression (Figure 3, 33) and/or by proteasome-dependent regulation of BOK levels (1, 13). Importantly, we found a poor correlation between BOK mRNA and protein levels.…”

“…Even though it is known that overexpressed BOK leads to the release of cytochrome-c from mitochondria leading to procaspase-9 activation, exact mechanisms of this pro-death function are controversially discussed as is its pro-apoptotic function under physiological conditions (1, 2, 5–10). Intriguingly, most of the cytoplasmic BOK seems to be localized at the endoplasmic reticulum (2, 11, 12), by a mechanism requiring its C-terminal tail-anchor (2), where it strongly interacts with IP3 receptors and is subject to posttranslational regulation through ubiquitination and proteasomal degradation (1, 13, 14). Significant amounts of BOK are also found within the nuclear compartment (2, 11) and non-apoptotic roles in regulation of proliferation (12), and even protective roles in response to specific stressors or in specialized cell types have been proposed (2, 15).…”

Potential Roles of Mitochondria-Associated ER Membranes (MAMs) in Traumatic Brain Injury

“…Recent evidence has indicated that Bok interacts strongly with IP3Rs and may contribute to the structural integrity or stability of IP3R tetramers (Schulman et al 2013). In return, Bok is dramatically stabilized by binding to IP3Rs and the proapoptotic effects of overexpressed Bok could be limited (Schulman et al 2016). PKB/Akt, which is a well-known prosurvival factor, exerts critical neuroprotective effects by phosphorylating downstream targets after TBI.…”