- Email: [email protected]
BOK: Oddball of the BCL-2 Family
Spotlight
BOK: Oddball of the BCL-2 Family Elizabeth A. Brem1,@ and Anthony Letai2,*
unable to survive past the early pre-natal period [3]. This observation indicates that BOK is not able to completely compensate for loss of BAX and BAK. To help elucidate the potentially unique functions of BOK, Carpio et al. studied Bok-/- mice, which are phenotypically and developmentally normal [4]. It was previously shown that BOK can localize to the endoplasmic reticulum (ER) [5], so immortalized mouse embryonic fibroblasts (MEFs) from these Bok-/- animals were subjected to agents that target the ER stress pathway, namely thapsigargin and bortezomib. There was significantly less cell death in the Bok-/- cells as compared to their wild type counterparts, suggesting that BOK promotes apoptosis following ER stress [4]. However, BOK is not absolutely required for such death as there was still some dose-dependent decrease in viability in the Bok-/- MEFs.
BOK is a BCL-2 family member whose function has been difficult to elucidate. It has been recently demonstrated that BOK is regulated by the endoplasmic reticulum associated-degradation (ERAD) pathway, can induce mitochondrial outer membrane permeabilization (MOMP), and is not regulated by other members of the BCL-2 family. These findings demonstrate a novel mechanism for regulation of apoptosis, but it remains unclear in which specific contexts this mechanism may be most essential for Llambi et al. recently reported in Cell a series of experiments exploring how the cell death. The BCL-2 family of proteins regulates the permeabilization of the mitochondrial outer membrane, the point of commitment to apoptosis. The family contains both pro- and anti-apoptotic proteins. BAX and BAK are the key effectors that homo-oligomerize to form the actual mitochondrial pores. Without them, it has appeared that apoptosis in response to a wide number of insults cannot proceed.
pro-death function of BOK is regulated by ER stress. They identified cell lines with high levels of endogenous BOK (HCT116) and also created MEFs with dox-inducible BOK expression. In these models, it was demonstrated that BOK expression leads to increased cell death in the presence of agents that target the ERAD pathway. They showed that BOK can promote cell death in the absence of BAX and BAK and that the cell death induced by BOK is caspase-dependent. Unlike BAX and BAK, BOK does not appear to require the presence of activator BH3 peptides to induce MOMP. Also in contrast to BAX and BAK, the ability of BOK to induce apoptosis is not impaired by over-expression of the anti-apoptotic BCL-2 family members. The conclusion made is that BOK is constitutively active but not regulated by other members of the BCL-2 family. Rather, its protein levels are regulated by the ERAD pathway (Figure 1) [6].
More recent findings suggest that there may be circumstances where another protein, BOK, might perform a function similar to BAX and BAK. BOK was identified in 1997 in a rat cDNA screen for proteins that could bind MCL-1, a major anti-apoptotic member of the BCL-2 family [1]. BOK shares significant homology with BAX and BAK. Single knockout Bak or Bax deficient mice appear to have normal development [2]. However, Bak/Bax double knockout mice have multiple phe- There is some disagreement in the literanotypic abnormalities and are typically ture regarding whether resistance to
certain ER stressing agents is afforded by loss of BOK. Carpio et al. demonstrated relative resistance to thapsigargin in Bok-/- MEFs, but supplementary data from Llambi et al. and recent work by Fernandez-Marrero et al. do not [7]. It is not immediately clear where the discrepancies lie, but it may be due to strain or construct-specific effects, since each of the three Bok-/- mouse models was independently derived. The findings of Llambi et al. prompt additional questions. If BOK is indeed constitutively active, how is it that BOK can be readily detected in a variety of cell types [6,8]? There must be some mechanism other than protein stability alone modulating BOK function. Also, in how many cell types does this mechanism of BOK regulation function? To what extent is BOK required to mediate the cell death response induced in vivo by proteosome inhibitors? Bortezomib, the first FDA-approved proteasome inhibitor, was tested in a number of hematologic malignancies and solid tumors in phase I studies, but clinical benefit has been demonstrated on a large scale only in multiple myeloma and some nonHodgkin lymphomas. In the two multiple myeloma cell lines evaluated by Llambi et al., there was no significant BOK expression. Even within a tumor type, cell lines vary in the effect that silencing BOK has on survival [6]. Thus, data is so far lacking to demonstrate that stabilization of BOK is a mechanism that is significantly in play in vivo in response to proteasome inhibition. This work leads one to ask whether there might be ways to more specifically stabilize or increase expression of BOK; this may help induce MOMP in at least a subset of tumors that have lost BAX and BAK. Additionally, the lack of interaction of BOK with anti-apoptotic proteins makes it a particularly attractive candidate for gain-of-function interventions in cancers where anti-apoptotic BCL-2 family members are expressed at high levels.
Trends in Cell Biology, June 2016, Vol. 26, No. 6
389
Canonical regulaon
subsequent work to identify those contexts where BOK-dependent death via stabilization of BOK protein is most important.
Non-canonical regulaon An-apoptoc signal
BH3-only
ERAD BOK
Proteosomal degradaon
BAX BAK MOMP
1
Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 2 Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA *Correspondence: [email protected]
MOMP
(A. Letai). @ Twitter: @DrLizBrem http://dx.doi.org/10.1016/j.tcb.2016.04.007 References 1. Hsu, S.Y. et al. (1997) Bok is a pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members. Proc. Natl. Acad. Sci. U.S.A. 94, 12401–12406 2. Ke, F.E. et al. (2013) Consequences of the combined loss of BOK and BAK or BOK and BAX. Cell Death Dis. 4, e650
Figure 1. BOK is a Pro-Apoptotic BCL-2 Family Member that Initiates Apoptosis Independent of BAX and BAK via a Non-Canonical Pathway. Until recently, it has been thought that BAX and BAK are essential for mitochondrial outer membrane permeabilization (MOMP) to occur. BH3-only proteins either directly or indirectly activate BAX and BAK by releasing them from anti-apoptotic BCL-2 family members, allowing BAX and BAK to oligodimerize at the mitochondrial outer membrane. An alternative mechanism may also be of importance in some cells where BOK can directly cause MOMP upon dysregulation of ER-associated degradation (ERAD) pathway components. BOK stability appears to be regulated by the ERAD pathway, rather than other BCL-2 family members, where it is targeted for degradation by the proteosome.
Previous to Llambi et al., there was a tidy circuit explaining BCL-2 family regulation of mitochondrial apoptosis. Effectors like BAX and BAK were activated by activator BH3 proteins to oligomerize and form pores. Monomeric effectors and
390
Trends in Cell Biology, June 2016, Vol. 26, No. 6
activators could be bound and inhibited by anti-apoptotic proteins. Now it appears that BOK breaks these simple rules with a novel ER stress regulation of protein stability. Now that we know that BOK can act in this unique way, it will be important for
3. Lindsten, T. et al. (2000) The combined functions of proapoptotic Bcl-2 family members Bak and Bax are essential for normal development of multiple tissues. Mol. Cell 6, 1389–1399 4. Carpio, M.A. et al. (2015) BCL-2 family member BOK promotes apoptosis in response to endoplasmic reticulum stress. Proc. Natl. Acad. Sci. U.S.A. 112, 7201–7206 5. Echeverry, N. et al. (2013) Intracellular localization of the BCL-2 family member BOK and functional implications. Cell Death Differ. 20, 785–799 6. Llambi, F. et al. (2016) BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation. Cell 165, 1–13 7. Fernandez-Marrero, Y. et al. (2016) Is BOK required for apoptosis induced by endoplasmic reticulum stress? Proc. Natl. Acad. Sci. U.S.A. 113, E492–E493 8. Ke, F.E. et al. (2012) BCL-2 family member BOK is widely expressed but its loss has only minimal impact in mice. Cell Death Differ. 19, 915–925
BOK: Oddball of the BCL-2 Family Elizabeth A. Brem1,@ and Anthony Letai2,*
unable to survive past the early pre-natal period [3]. This observation indicates that BOK is not able to completely compensate for loss of BAX and BAK. To help elucidate the potentially unique functions of BOK, Carpio et al. studied Bok-/- mice, which are phenotypically and developmentally normal [4]. It was previously shown that BOK can localize to the endoplasmic reticulum (ER) [5], so immortalized mouse embryonic fibroblasts (MEFs) from these Bok-/- animals were subjected to agents that target the ER stress pathway, namely thapsigargin and bortezomib. There was significantly less cell death in the Bok-/- cells as compared to their wild type counterparts, suggesting that BOK promotes apoptosis following ER stress [4]. However, BOK is not absolutely required for such death as there was still some dose-dependent decrease in viability in the Bok-/- MEFs.
BOK is a BCL-2 family member whose function has been difficult to elucidate. It has been recently demonstrated that BOK is regulated by the endoplasmic reticulum associated-degradation (ERAD) pathway, can induce mitochondrial outer membrane permeabilization (MOMP), and is not regulated by other members of the BCL-2 family. These findings demonstrate a novel mechanism for regulation of apoptosis, but it remains unclear in which specific contexts this mechanism may be most essential for Llambi et al. recently reported in Cell a series of experiments exploring how the cell death. The BCL-2 family of proteins regulates the permeabilization of the mitochondrial outer membrane, the point of commitment to apoptosis. The family contains both pro- and anti-apoptotic proteins. BAX and BAK are the key effectors that homo-oligomerize to form the actual mitochondrial pores. Without them, it has appeared that apoptosis in response to a wide number of insults cannot proceed.
pro-death function of BOK is regulated by ER stress. They identified cell lines with high levels of endogenous BOK (HCT116) and also created MEFs with dox-inducible BOK expression. In these models, it was demonstrated that BOK expression leads to increased cell death in the presence of agents that target the ERAD pathway. They showed that BOK can promote cell death in the absence of BAX and BAK and that the cell death induced by BOK is caspase-dependent. Unlike BAX and BAK, BOK does not appear to require the presence of activator BH3 peptides to induce MOMP. Also in contrast to BAX and BAK, the ability of BOK to induce apoptosis is not impaired by over-expression of the anti-apoptotic BCL-2 family members. The conclusion made is that BOK is constitutively active but not regulated by other members of the BCL-2 family. Rather, its protein levels are regulated by the ERAD pathway (Figure 1) [6].
More recent findings suggest that there may be circumstances where another protein, BOK, might perform a function similar to BAX and BAK. BOK was identified in 1997 in a rat cDNA screen for proteins that could bind MCL-1, a major anti-apoptotic member of the BCL-2 family [1]. BOK shares significant homology with BAX and BAK. Single knockout Bak or Bax deficient mice appear to have normal development [2]. However, Bak/Bax double knockout mice have multiple phe- There is some disagreement in the literanotypic abnormalities and are typically ture regarding whether resistance to
certain ER stressing agents is afforded by loss of BOK. Carpio et al. demonstrated relative resistance to thapsigargin in Bok-/- MEFs, but supplementary data from Llambi et al. and recent work by Fernandez-Marrero et al. do not [7]. It is not immediately clear where the discrepancies lie, but it may be due to strain or construct-specific effects, since each of the three Bok-/- mouse models was independently derived. The findings of Llambi et al. prompt additional questions. If BOK is indeed constitutively active, how is it that BOK can be readily detected in a variety of cell types [6,8]? There must be some mechanism other than protein stability alone modulating BOK function. Also, in how many cell types does this mechanism of BOK regulation function? To what extent is BOK required to mediate the cell death response induced in vivo by proteosome inhibitors? Bortezomib, the first FDA-approved proteasome inhibitor, was tested in a number of hematologic malignancies and solid tumors in phase I studies, but clinical benefit has been demonstrated on a large scale only in multiple myeloma and some nonHodgkin lymphomas. In the two multiple myeloma cell lines evaluated by Llambi et al., there was no significant BOK expression. Even within a tumor type, cell lines vary in the effect that silencing BOK has on survival [6]. Thus, data is so far lacking to demonstrate that stabilization of BOK is a mechanism that is significantly in play in vivo in response to proteasome inhibition. This work leads one to ask whether there might be ways to more specifically stabilize or increase expression of BOK; this may help induce MOMP in at least a subset of tumors that have lost BAX and BAK. Additionally, the lack of interaction of BOK with anti-apoptotic proteins makes it a particularly attractive candidate for gain-of-function interventions in cancers where anti-apoptotic BCL-2 family members are expressed at high levels.
Trends in Cell Biology, June 2016, Vol. 26, No. 6
389
Canonical regulaon
subsequent work to identify those contexts where BOK-dependent death via stabilization of BOK protein is most important.
Non-canonical regulaon An-apoptoc signal
BH3-only
ERAD BOK
Proteosomal degradaon
BAX BAK MOMP
1
Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 2 Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA *Correspondence: [email protected]
MOMP
(A. Letai). @ Twitter: @DrLizBrem http://dx.doi.org/10.1016/j.tcb.2016.04.007 References 1. Hsu, S.Y. et al. (1997) Bok is a pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members. Proc. Natl. Acad. Sci. U.S.A. 94, 12401–12406 2. Ke, F.E. et al. (2013) Consequences of the combined loss of BOK and BAK or BOK and BAX. Cell Death Dis. 4, e650
Figure 1. BOK is a Pro-Apoptotic BCL-2 Family Member that Initiates Apoptosis Independent of BAX and BAK via a Non-Canonical Pathway. Until recently, it has been thought that BAX and BAK are essential for mitochondrial outer membrane permeabilization (MOMP) to occur. BH3-only proteins either directly or indirectly activate BAX and BAK by releasing them from anti-apoptotic BCL-2 family members, allowing BAX and BAK to oligodimerize at the mitochondrial outer membrane. An alternative mechanism may also be of importance in some cells where BOK can directly cause MOMP upon dysregulation of ER-associated degradation (ERAD) pathway components. BOK stability appears to be regulated by the ERAD pathway, rather than other BCL-2 family members, where it is targeted for degradation by the proteosome.
Previous to Llambi et al., there was a tidy circuit explaining BCL-2 family regulation of mitochondrial apoptosis. Effectors like BAX and BAK were activated by activator BH3 proteins to oligomerize and form pores. Monomeric effectors and
390
Trends in Cell Biology, June 2016, Vol. 26, No. 6
activators could be bound and inhibited by anti-apoptotic proteins. Now it appears that BOK breaks these simple rules with a novel ER stress regulation of protein stability. Now that we know that BOK can act in this unique way, it will be important for
3. Lindsten, T. et al. (2000) The combined functions of proapoptotic Bcl-2 family members Bak and Bax are essential for normal development of multiple tissues. Mol. Cell 6, 1389–1399 4. Carpio, M.A. et al. (2015) BCL-2 family member BOK promotes apoptosis in response to endoplasmic reticulum stress. Proc. Natl. Acad. Sci. U.S.A. 112, 7201–7206 5. Echeverry, N. et al. (2013) Intracellular localization of the BCL-2 family member BOK and functional implications. Cell Death Differ. 20, 785–799 6. Llambi, F. et al. (2016) BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation. Cell 165, 1–13 7. Fernandez-Marrero, Y. et al. (2016) Is BOK required for apoptosis induced by endoplasmic reticulum stress? Proc. Natl. Acad. Sci. U.S.A. 113, E492–E493 8. Ke, F.E. et al. (2012) BCL-2 family member BOK is widely expressed but its loss has only minimal impact in mice. Cell Death Differ. 19, 915–925