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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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BAP31, but not BAP29, is cleaved during Fas-mediated apoptosis. (A) Schematic representation of human BAP31, the p20 caspase cleavage product, and BAP29 in the ER membrane. Both BAP31 and BAP29 contain three transmembrane domains, a cytosolic tail containing a coiled coil domain (boxed region), and terminate with a canonical KKXX ER retrieval sequence. The caspase-8 recognition sites in BAP31 are shown. (B) KB cells were untreated or stimulated with 500 ng/ml anti-Fas activating antibody (CH11) and 10 μg/ml cycloheximide (CHX) for 7 h, and cell lysates were analyzed by SDS-PAGE and immunoblotting with anti-BAP31 (left) or anti-BAP29 (right) pAbs. The positions of BAP31, its p27 and p20 cleavage products, and BAP29 are indicated. (C) Adenoviral-expressed p20-HA (Adp20) localizes to the ER. H1299 cells were infected with Adp20 for 20 h, then fixed and double stained with anti-HA and anti-calreticulin antibodies or anti-HA and anti-TOM20 antibodies.
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fig1: BAP31, but not BAP29, is cleaved during Fas-mediated apoptosis. (A) Schematic representation of human BAP31, the p20 caspase cleavage product, and BAP29 in the ER membrane. Both BAP31 and BAP29 contain three transmembrane domains, a cytosolic tail containing a coiled coil domain (boxed region), and terminate with a canonical KKXX ER retrieval sequence. The caspase-8 recognition sites in BAP31 are shown. (B) KB cells were untreated or stimulated with 500 ng/ml anti-Fas activating antibody (CH11) and 10 μg/ml cycloheximide (CHX) for 7 h, and cell lysates were analyzed by SDS-PAGE and immunoblotting with anti-BAP31 (left) or anti-BAP29 (right) pAbs. The positions of BAP31, its p27 and p20 cleavage products, and BAP29 are indicated. (C) Adenoviral-expressed p20-HA (Adp20) localizes to the ER. H1299 cells were infected with Adp20 for 20 h, then fixed and double stained with anti-HA and anti-calreticulin antibodies or anti-HA and anti-TOM20 antibodies.

Mentions: BAP31 and its cellular homologue and heterodimerizing partner, BAP29 (Adachi et al., 1996), are structurally conserved proteins sharing identical topology in the ER membrane and 47% sequence identity in man. Both proteins initiate with the NH2 terminus facing the lumen, followed by three transmembrane regions and a cytosolic tail containing a long coiled coil domain ending in a canonical KKXX ER retrieval sequence (Fig. 1 A). Human BAP31 contains two identical caspase cleavage sites (AAVD.G) at D164 and D238 that are preferentially cleaved by caspase-8 (Ng et al., 1997; Wang et al., 2003). Fig. 1 B shows that in human KB cells stimulated with agonistic anti-Fas antibody BAP31 was cleaved generating the p27 and p20 membrane-embedded fragments. Only the former cleavage site is conserved in mouse Bap31, suggesting that cleavage at D164 is critical. The two caspase cleavage sequences are not conserved in BAP29, which remained structurally intact during apoptosis (Fig. 1 B).


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)

BAP31, but not BAP29, is cleaved during Fas-mediated apoptosis. (A) Schematic representation of human BAP31, the p20 caspase cleavage product, and BAP29 in the ER membrane. Both BAP31 and BAP29 contain three transmembrane domains, a cytosolic tail containing a coiled coil domain (boxed region), and terminate with a canonical KKXX ER retrieval sequence. The caspase-8 recognition sites in BAP31 are shown. (B) KB cells were untreated or stimulated with 500 ng/ml anti-Fas activating antibody (CH11) and 10 μg/ml cycloheximide (CHX) for 7 h, and cell lysates were analyzed by SDS-PAGE and immunoblotting with anti-BAP31 (left) or anti-BAP29 (right) pAbs. The positions of BAP31, its p27 and p20 cleavage products, and BAP29 are indicated. (C) Adenoviral-expressed p20-HA (Adp20) localizes to the ER. H1299 cells were infected with Adp20 for 20 h, then fixed and double stained with anti-HA and anti-calreticulin antibodies or anti-HA and anti-TOM20 antibodies.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172754&req=5

fig1: BAP31, but not BAP29, is cleaved during Fas-mediated apoptosis. (A) Schematic representation of human BAP31, the p20 caspase cleavage product, and BAP29 in the ER membrane. Both BAP31 and BAP29 contain three transmembrane domains, a cytosolic tail containing a coiled coil domain (boxed region), and terminate with a canonical KKXX ER retrieval sequence. The caspase-8 recognition sites in BAP31 are shown. (B) KB cells were untreated or stimulated with 500 ng/ml anti-Fas activating antibody (CH11) and 10 μg/ml cycloheximide (CHX) for 7 h, and cell lysates were analyzed by SDS-PAGE and immunoblotting with anti-BAP31 (left) or anti-BAP29 (right) pAbs. The positions of BAP31, its p27 and p20 cleavage products, and BAP29 are indicated. (C) Adenoviral-expressed p20-HA (Adp20) localizes to the ER. H1299 cells were infected with Adp20 for 20 h, then fixed and double stained with anti-HA and anti-calreticulin antibodies or anti-HA and anti-TOM20 antibodies.
Mentions: BAP31 and its cellular homologue and heterodimerizing partner, BAP29 (Adachi et al., 1996), are structurally conserved proteins sharing identical topology in the ER membrane and 47% sequence identity in man. Both proteins initiate with the NH2 terminus facing the lumen, followed by three transmembrane regions and a cytosolic tail containing a long coiled coil domain ending in a canonical KKXX ER retrieval sequence (Fig. 1 A). Human BAP31 contains two identical caspase cleavage sites (AAVD.G) at D164 and D238 that are preferentially cleaved by caspase-8 (Ng et al., 1997; Wang et al., 2003). Fig. 1 B shows that in human KB cells stimulated with agonistic anti-Fas antibody BAP31 was cleaved generating the p27 and p20 membrane-embedded fragments. Only the former cleavage site is conserved in mouse Bap31, suggesting that cleavage at D164 is critical. The two caspase cleavage sequences are not conserved in BAP29, which remained structurally intact during apoptosis (Fig. 1 B).

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