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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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ER stress induces Bax and Bak conformational changes and oligomerization at the ER. (A) ER stresses induce the conformational changes of Bax and Bak. HeLa, MCF7, and 293T cells were treated with thapsigargin (Thap; 2 μM) or tunicamycin (Tuni; 5 μg/ml) for 36 h. Cells were fixed in 0.25% paraformaldehyde in PBS for 5 min. Cells were incubated with a control antibody (mouse IgG1) and conformation-sensitive antibodies against Bax or Bak, followed by incubation with FITC-conjugated secondary antibody. (B) ER stress induces Bax oligomerization at the ER. Wild-type MEFs were treated with brefeldin A (BFA; 10 μg/ml), Thap (2 μM), or Tuni (10 μg/ml) for 24 h. Cells were resuspended in hypotonic buffer A and disrupted. 5 mM BMH cross-linking reagent was added to cross-link the oligomerized proteins. Cells were subjected to subcellular fractionation to obtain the HM and LM fractions. 20 μg of total protein was separated on a 4–12% gradient NuPAGE gel. A polyclonal anti-Bax antibody was used to detect Bax. COX IV and calnexin are shown as indicators of the purity of the fractionation and as loading controls.
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fig2: ER stress induces Bax and Bak conformational changes and oligomerization at the ER. (A) ER stresses induce the conformational changes of Bax and Bak. HeLa, MCF7, and 293T cells were treated with thapsigargin (Thap; 2 μM) or tunicamycin (Tuni; 5 μg/ml) for 36 h. Cells were fixed in 0.25% paraformaldehyde in PBS for 5 min. Cells were incubated with a control antibody (mouse IgG1) and conformation-sensitive antibodies against Bax or Bak, followed by incubation with FITC-conjugated secondary antibody. (B) ER stress induces Bax oligomerization at the ER. Wild-type MEFs were treated with brefeldin A (BFA; 10 μg/ml), Thap (2 μM), or Tuni (10 μg/ml) for 24 h. Cells were resuspended in hypotonic buffer A and disrupted. 5 mM BMH cross-linking reagent was added to cross-link the oligomerized proteins. Cells were subjected to subcellular fractionation to obtain the HM and LM fractions. 20 μg of total protein was separated on a 4–12% gradient NuPAGE gel. A polyclonal anti-Bax antibody was used to detect Bax. COX IV and calnexin are shown as indicators of the purity of the fractionation and as loading controls.

Mentions: It has been established that during apoptosis, Bax and Bak undergo conformational change. This process seems to be required for the proapoptotic activity of Bax and Bak (Hsu et al., 1997; Gross et al., 1998; Desagher et al., 1999; Griffiths et al., 1999; Wei et al., 2000; Makin et al., 2001). To determine whether these proapoptotic proteins also undergo conformational changes during ER stress–induced apoptosis, HeLa, MCF7, and 293T cells were treated with thapsigargin and tunicamycin. These drugs trigger ER-mediated apoptosis by perturbing the normal function of the ER. Thapsigargin inhibits the ER Ca2+ uptake from the cytosol. Tunicamycin is an inhibitor of protein N-glycosylation. Antibodies against Bax and Bak that recognize their proapoptotic-competent forms were used. These antibodies were previously used to demonstrate the activation of Bax and Bak in response to other apoptotic stimuli (Griffiths et al., 1999; Mandic et al., 2001). In all three cell lines tested, ER stress treatment resulted in an increase of the active forms of Bax and Bak, indicating that Bax and Bak undergo conformational changes from the inactive forms to the active forms upon ER stress treatment (Fig. 2Figure 2.


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)

ER stress induces Bax and Bak conformational changes and oligomerization at the ER. (A) ER stresses induce the conformational changes of Bax and Bak. HeLa, MCF7, and 293T cells were treated with thapsigargin (Thap; 2 μM) or tunicamycin (Tuni; 5 μg/ml) for 36 h. Cells were fixed in 0.25% paraformaldehyde in PBS for 5 min. Cells were incubated with a control antibody (mouse IgG1) and conformation-sensitive antibodies against Bax or Bak, followed by incubation with FITC-conjugated secondary antibody. (B) ER stress induces Bax oligomerization at the ER. Wild-type MEFs were treated with brefeldin A (BFA; 10 μg/ml), Thap (2 μM), or Tuni (10 μg/ml) for 24 h. Cells were resuspended in hypotonic buffer A and disrupted. 5 mM BMH cross-linking reagent was added to cross-link the oligomerized proteins. Cells were subjected to subcellular fractionation to obtain the HM and LM fractions. 20 μg of total protein was separated on a 4–12% gradient NuPAGE gel. A polyclonal anti-Bax antibody was used to detect Bax. COX IV and calnexin are shown as indicators of the purity of the fractionation and as loading controls.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172724&req=5

fig2: ER stress induces Bax and Bak conformational changes and oligomerization at the ER. (A) ER stresses induce the conformational changes of Bax and Bak. HeLa, MCF7, and 293T cells were treated with thapsigargin (Thap; 2 μM) or tunicamycin (Tuni; 5 μg/ml) for 36 h. Cells were fixed in 0.25% paraformaldehyde in PBS for 5 min. Cells were incubated with a control antibody (mouse IgG1) and conformation-sensitive antibodies against Bax or Bak, followed by incubation with FITC-conjugated secondary antibody. (B) ER stress induces Bax oligomerization at the ER. Wild-type MEFs were treated with brefeldin A (BFA; 10 μg/ml), Thap (2 μM), or Tuni (10 μg/ml) for 24 h. Cells were resuspended in hypotonic buffer A and disrupted. 5 mM BMH cross-linking reagent was added to cross-link the oligomerized proteins. Cells were subjected to subcellular fractionation to obtain the HM and LM fractions. 20 μg of total protein was separated on a 4–12% gradient NuPAGE gel. A polyclonal anti-Bax antibody was used to detect Bax. COX IV and calnexin are shown as indicators of the purity of the fractionation and as loading controls.
Mentions: It has been established that during apoptosis, Bax and Bak undergo conformational change. This process seems to be required for the proapoptotic activity of Bax and Bak (Hsu et al., 1997; Gross et al., 1998; Desagher et al., 1999; Griffiths et al., 1999; Wei et al., 2000; Makin et al., 2001). To determine whether these proapoptotic proteins also undergo conformational changes during ER stress–induced apoptosis, HeLa, MCF7, and 293T cells were treated with thapsigargin and tunicamycin. These drugs trigger ER-mediated apoptosis by perturbing the normal function of the ER. Thapsigargin inhibits the ER Ca2+ uptake from the cytosol. Tunicamycin is an inhibitor of protein N-glycosylation. Antibodies against Bax and Bak that recognize their proapoptotic-competent forms were used. These antibodies were previously used to demonstrate the activation of Bax and Bak in response to other apoptotic stimuli (Griffiths et al., 1999; Mandic et al., 2001). In all three cell lines tested, ER stress treatment resulted in an increase of the active forms of Bax and Bak, indicating that Bax and Bak undergo conformational changes from the inactive forms to the active forms upon ER stress treatment (Fig. 2Figure 2.

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
Related in: MedlinePlus