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Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis.

Zong WX, Li C, Hatzivassiliou G, Lindsten T, Yu QC, Yuan J, Thompson CB - J. Cell Biol. (2003)

Bottom Line: In wild-type cells, this is associated with caspase 12 cleavage that is abolished in bax-/-bak-/- cells.In contrast, mitochondria-targeted Bak leads to enhanced caspase 7 and PARP cleavage in comparison with the ER-targeted Bak.These findings demonstrate that in addition to their functions at mitochondria, Bax and Bak also localize to the ER and function to initiate a parallel pathway of caspase activation and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Abramson Cancer Center, 421 Curie Blvd., BRB II/III, 445, Philadelphia, PA 19104-6160, USA. craig@mail.med.upenn.edu

ABSTRACT
Bax and Bak play a redundant but essential role in apoptosis initiated by the mitochondrial release of apoptogenic factors. In addition to their presence at the mitochondrial outer membrane, Bax and Bak can also localize to the ER. Agents that initiate ER stress responses can induce conformational changes and oligomerization of Bax on the ER as well as on mitochondria. In wild-type cells, this is associated with caspase 12 cleavage that is abolished in bax-/-bak-/- cells. In bax-/-bak-/- cells, introduction of Bak mutants selectively targeted to either mitochondria or the ER can induce apoptosis. However, ER-targeted, but not mitochondria-targeted, Bak leads to progressive depletion of ER Ca2+ and induces caspase 12 cleavage. In contrast, mitochondria-targeted Bak leads to enhanced caspase 7 and PARP cleavage in comparison with the ER-targeted Bak. These findings demonstrate that in addition to their functions at mitochondria, Bax and Bak also localize to the ER and function to initiate a parallel pathway of caspase activation and apoptosis.

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Caspase 12 functions downstream of the multidomain proapoptotic Bcl-2 family proteins. (A) ER stress–induced caspase 12 cleavage is dependent on Bax and Bak. Immortalized wild-type and bax−/−bak−/− MEFs and NIH3T3 cells were treated with brefeldin A (BFA; 10 μg/ml), thapsigargin (Thap; 2 μM), or tunicamycin (Tuni; 10 μg/ml) for 30 h. Caspase 12 level and processing were examined by immunoblotting of 20 μg of total cellular protein from samples as indicated. An ∼42-kD caspase 12 fragment is indicated by the arrow. Induction of CHOP expression is shown as an indicator of the ER stress response. A nonspecific band (NS) is shown as a loading control. (B) Caspase 12 kills bax−/−bak−/− cells. Wild-type and bax−/−bak−/− MEFs were cotransfected with pEGFP and constructs expressing caspase 3, caspase 9, caspase 12, and t-caspase 12. 24 h after transfection, cells were stained with DAPI, and cell death percentage was determined by the ratio of DAPI-positive to GFP-positive cells.
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fig3: Caspase 12 functions downstream of the multidomain proapoptotic Bcl-2 family proteins. (A) ER stress–induced caspase 12 cleavage is dependent on Bax and Bak. Immortalized wild-type and bax−/−bak−/− MEFs and NIH3T3 cells were treated with brefeldin A (BFA; 10 μg/ml), thapsigargin (Thap; 2 μM), or tunicamycin (Tuni; 10 μg/ml) for 30 h. Caspase 12 level and processing were examined by immunoblotting of 20 μg of total cellular protein from samples as indicated. An ∼42-kD caspase 12 fragment is indicated by the arrow. Induction of CHOP expression is shown as an indicator of the ER stress response. A nonspecific band (NS) is shown as a loading control. (B) Caspase 12 kills bax−/−bak−/− cells. Wild-type and bax−/−bak−/− MEFs were cotransfected with pEGFP and constructs expressing caspase 3, caspase 9, caspase 12, and t-caspase 12. 24 h after transfection, cells were stained with DAPI, and cell death percentage was determined by the ratio of DAPI-positive to GFP-positive cells.

Mentions: Caspase 12 is specifically localized to the ER and has been reported to be cleaved during ER stress–induced apoptosis (Nakagawa et al., 2000). To investigate whether ER stress–induced caspase 12 cleavage is dependent on Bax and Bak, wild-type, bax−/−bak−/−, and NIH3T3 cells were treated with brefeldin A, thapsigargin, and tunicamycin. No consistent differences in total caspase 12 expression were observed between wild-type MEFs, NIH3T3 cells, and bax−/−bak−/− MEFs. However, in both wild-type MEFs and NIH3T3 cells, an ∼47-kD fragment was revealed in untreated cells, indicating that there exists a basal processing of caspase 12 in these cells, which does not lead to apoptosis (Fig. 3Figure 3.


Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis.

Zong WX, Li C, Hatzivassiliou G, Lindsten T, Yu QC, Yuan J, Thompson CB - J. Cell Biol. (2003)

Caspase 12 functions downstream of the multidomain proapoptotic Bcl-2 family proteins. (A) ER stress–induced caspase 12 cleavage is dependent on Bax and Bak. Immortalized wild-type and bax−/−bak−/− MEFs and NIH3T3 cells were treated with brefeldin A (BFA; 10 μg/ml), thapsigargin (Thap; 2 μM), or tunicamycin (Tuni; 10 μg/ml) for 30 h. Caspase 12 level and processing were examined by immunoblotting of 20 μg of total cellular protein from samples as indicated. An ∼42-kD caspase 12 fragment is indicated by the arrow. Induction of CHOP expression is shown as an indicator of the ER stress response. A nonspecific band (NS) is shown as a loading control. (B) Caspase 12 kills bax−/−bak−/− cells. Wild-type and bax−/−bak−/− MEFs were cotransfected with pEGFP and constructs expressing caspase 3, caspase 9, caspase 12, and t-caspase 12. 24 h after transfection, cells were stained with DAPI, and cell death percentage was determined by the ratio of DAPI-positive to GFP-positive cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172724&req=5

fig3: Caspase 12 functions downstream of the multidomain proapoptotic Bcl-2 family proteins. (A) ER stress–induced caspase 12 cleavage is dependent on Bax and Bak. Immortalized wild-type and bax−/−bak−/− MEFs and NIH3T3 cells were treated with brefeldin A (BFA; 10 μg/ml), thapsigargin (Thap; 2 μM), or tunicamycin (Tuni; 10 μg/ml) for 30 h. Caspase 12 level and processing were examined by immunoblotting of 20 μg of total cellular protein from samples as indicated. An ∼42-kD caspase 12 fragment is indicated by the arrow. Induction of CHOP expression is shown as an indicator of the ER stress response. A nonspecific band (NS) is shown as a loading control. (B) Caspase 12 kills bax−/−bak−/− cells. Wild-type and bax−/−bak−/− MEFs were cotransfected with pEGFP and constructs expressing caspase 3, caspase 9, caspase 12, and t-caspase 12. 24 h after transfection, cells were stained with DAPI, and cell death percentage was determined by the ratio of DAPI-positive to GFP-positive cells.
Mentions: Caspase 12 is specifically localized to the ER and has been reported to be cleaved during ER stress–induced apoptosis (Nakagawa et al., 2000). To investigate whether ER stress–induced caspase 12 cleavage is dependent on Bax and Bak, wild-type, bax−/−bak−/−, and NIH3T3 cells were treated with brefeldin A, thapsigargin, and tunicamycin. No consistent differences in total caspase 12 expression were observed between wild-type MEFs, NIH3T3 cells, and bax−/−bak−/− MEFs. However, in both wild-type MEFs and NIH3T3 cells, an ∼47-kD fragment was revealed in untreated cells, indicating that there exists a basal processing of caspase 12 in these cells, which does not lead to apoptosis (Fig. 3Figure 3.

Bottom Line: In wild-type cells, this is associated with caspase 12 cleavage that is abolished in bax-/-bak-/- cells.In contrast, mitochondria-targeted Bak leads to enhanced caspase 7 and PARP cleavage in comparison with the ER-targeted Bak.These findings demonstrate that in addition to their functions at mitochondria, Bax and Bak also localize to the ER and function to initiate a parallel pathway of caspase activation and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Abramson Cancer Center, 421 Curie Blvd., BRB II/III, 445, Philadelphia, PA 19104-6160, USA. craig@mail.med.upenn.edu

ABSTRACT
Bax and Bak play a redundant but essential role in apoptosis initiated by the mitochondrial release of apoptogenic factors. In addition to their presence at the mitochondrial outer membrane, Bax and Bak can also localize to the ER. Agents that initiate ER stress responses can induce conformational changes and oligomerization of Bax on the ER as well as on mitochondria. In wild-type cells, this is associated with caspase 12 cleavage that is abolished in bax-/-bak-/- cells. In bax-/-bak-/- cells, introduction of Bak mutants selectively targeted to either mitochondria or the ER can induce apoptosis. However, ER-targeted, but not mitochondria-targeted, Bak leads to progressive depletion of ER Ca2+ and induces caspase 12 cleavage. In contrast, mitochondria-targeted Bak leads to enhanced caspase 7 and PARP cleavage in comparison with the ER-targeted Bak. These findings demonstrate that in addition to their functions at mitochondria, Bax and Bak also localize to the ER and function to initiate a parallel pathway of caspase activation and apoptosis.

Show MeSH