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Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol.

Breckenridge DG, Stojanovic M, Marcellus RC, Shore GC - J. Cell Biol. (2003)

Bottom Line: Cell.Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway.Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6.

ABSTRACT
Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

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Prolonged expression of p20 induces mitochondrial apoptosis. (A) Expression of p20 in KB cells. Cells were infected with Adp20 and cell lysates were collected and analyzed by immunoblotting at the times indicated post-infection. (B) KB and H1299 cells were infected with Adp20, and effector caspase (DEVDase) activity was measured at the indicated times post-infection by the ability of cell lysates to hydrolyze the fluorogenic caspase substrate DEVD-amc. Shown is a representative experiment. (C) KB cells were mock infected or infected with Adp20 for 35–40 h in the absence or presence of 50 μM zVAD-fmk, and equivalent amounts of post-mitochondrial supernatants were analyzed for the presence of cyt.c by SDS-PAGE and immunoblotting. The membrane was reprobed with anti-actin antibody to confirm equal loading. (D) Parental KB cells, or KB cells stably overexpressing BCL-2 or BCL-xL, were mock infected or infected with Adp20 in the absence or presence of 50 μM zVAD-fmk, and at 45 h post infection, cell death was assessed by trypan blue staining. Shown is mean ± SD of three independent experiments. (E) Wt, Bap31-, and Bap29,31- mouse ES cells were treated and analyzed as in D.
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fig2: Prolonged expression of p20 induces mitochondrial apoptosis. (A) Expression of p20 in KB cells. Cells were infected with Adp20 and cell lysates were collected and analyzed by immunoblotting at the times indicated post-infection. (B) KB and H1299 cells were infected with Adp20, and effector caspase (DEVDase) activity was measured at the indicated times post-infection by the ability of cell lysates to hydrolyze the fluorogenic caspase substrate DEVD-amc. Shown is a representative experiment. (C) KB cells were mock infected or infected with Adp20 for 35–40 h in the absence or presence of 50 μM zVAD-fmk, and equivalent amounts of post-mitochondrial supernatants were analyzed for the presence of cyt.c by SDS-PAGE and immunoblotting. The membrane was reprobed with anti-actin antibody to confirm equal loading. (D) Parental KB cells, or KB cells stably overexpressing BCL-2 or BCL-xL, were mock infected or infected with Adp20 in the absence or presence of 50 μM zVAD-fmk, and at 45 h post infection, cell death was assessed by trypan blue staining. Shown is mean ± SD of three independent experiments. (E) Wt, Bap31-, and Bap29,31- mouse ES cells were treated and analyzed as in D.

Mentions: Expression of p20 was observed by 10 h post-infection of KB cells with Adp20, and remained stable for over 50 h (Fig. 2 A). 30–40 h after infection, Adp20 induced the activation of caspases, measured by the hydrolysis of the caspase substrate DEVD-amc and by processing of procaspase-3, in many cell types including KB, H1299, HeLa, and Rat1 cells (Fig. 2 B; unpublished data). The mechanism of this caspase activation seemed to occur via the classical mitochondrial apoptosome stress pathway. For example, p20 expression resulted in the insertion of BAX into the OMM, homo-oligomerization of BAK, and release of cyt.c from mitochondria in the presence of the pan-caspase inhibitor, zVAD-fmk (Fig. 2 C; unpublished data). In contrast, p20-induced caspase activation was abrogated in APAF-1– cells (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200212059/DC1). Control adenovectors expressing either LacZ or the reverse tet transactivating protein (RTA) did not cause any of the aforementioned apoptotic changes (unpublished data). Inhibition of caspases using zVAD-fmk, or overexpression of BCL-2 or BCL-xL, blocked downstream morphological features of apoptosis including loss of plasma membrane integrity as assessed by trypan blue uptake (Fig. 2 D). In the absence of these inhibitors, cells showed typical signs of apoptosis, including nuclear condensation and fragmentation, membrane blebbing and cell surface exposure of phosphatidylserine (unpublished data).


Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol.

Breckenridge DG, Stojanovic M, Marcellus RC, Shore GC - J. Cell Biol. (2003)

Prolonged expression of p20 induces mitochondrial apoptosis. (A) Expression of p20 in KB cells. Cells were infected with Adp20 and cell lysates were collected and analyzed by immunoblotting at the times indicated post-infection. (B) KB and H1299 cells were infected with Adp20, and effector caspase (DEVDase) activity was measured at the indicated times post-infection by the ability of cell lysates to hydrolyze the fluorogenic caspase substrate DEVD-amc. Shown is a representative experiment. (C) KB cells were mock infected or infected with Adp20 for 35–40 h in the absence or presence of 50 μM zVAD-fmk, and equivalent amounts of post-mitochondrial supernatants were analyzed for the presence of cyt.c by SDS-PAGE and immunoblotting. The membrane was reprobed with anti-actin antibody to confirm equal loading. (D) Parental KB cells, or KB cells stably overexpressing BCL-2 or BCL-xL, were mock infected or infected with Adp20 in the absence or presence of 50 μM zVAD-fmk, and at 45 h post infection, cell death was assessed by trypan blue staining. Shown is mean ± SD of three independent experiments. (E) Wt, Bap31-, and Bap29,31- mouse ES cells were treated and analyzed as in D.
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fig2: Prolonged expression of p20 induces mitochondrial apoptosis. (A) Expression of p20 in KB cells. Cells were infected with Adp20 and cell lysates were collected and analyzed by immunoblotting at the times indicated post-infection. (B) KB and H1299 cells were infected with Adp20, and effector caspase (DEVDase) activity was measured at the indicated times post-infection by the ability of cell lysates to hydrolyze the fluorogenic caspase substrate DEVD-amc. Shown is a representative experiment. (C) KB cells were mock infected or infected with Adp20 for 35–40 h in the absence or presence of 50 μM zVAD-fmk, and equivalent amounts of post-mitochondrial supernatants were analyzed for the presence of cyt.c by SDS-PAGE and immunoblotting. The membrane was reprobed with anti-actin antibody to confirm equal loading. (D) Parental KB cells, or KB cells stably overexpressing BCL-2 or BCL-xL, were mock infected or infected with Adp20 in the absence or presence of 50 μM zVAD-fmk, and at 45 h post infection, cell death was assessed by trypan blue staining. Shown is mean ± SD of three independent experiments. (E) Wt, Bap31-, and Bap29,31- mouse ES cells were treated and analyzed as in D.
Mentions: Expression of p20 was observed by 10 h post-infection of KB cells with Adp20, and remained stable for over 50 h (Fig. 2 A). 30–40 h after infection, Adp20 induced the activation of caspases, measured by the hydrolysis of the caspase substrate DEVD-amc and by processing of procaspase-3, in many cell types including KB, H1299, HeLa, and Rat1 cells (Fig. 2 B; unpublished data). The mechanism of this caspase activation seemed to occur via the classical mitochondrial apoptosome stress pathway. For example, p20 expression resulted in the insertion of BAX into the OMM, homo-oligomerization of BAK, and release of cyt.c from mitochondria in the presence of the pan-caspase inhibitor, zVAD-fmk (Fig. 2 C; unpublished data). In contrast, p20-induced caspase activation was abrogated in APAF-1– cells (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200212059/DC1). Control adenovectors expressing either LacZ or the reverse tet transactivating protein (RTA) did not cause any of the aforementioned apoptotic changes (unpublished data). Inhibition of caspases using zVAD-fmk, or overexpression of BCL-2 or BCL-xL, blocked downstream morphological features of apoptosis including loss of plasma membrane integrity as assessed by trypan blue uptake (Fig. 2 D). In the absence of these inhibitors, cells showed typical signs of apoptosis, including nuclear condensation and fragmentation, membrane blebbing and cell surface exposure of phosphatidylserine (unpublished data).

Bottom Line: Cell.Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway.Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6.

ABSTRACT
Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

Show MeSH
Related in: MedlinePlus