Inactivation of caspase-8 on mitochondria of Bcl-XL-expressing MCF7-Fas cells role for the bifunctional apoptosis regulator protein

Alexander H. Stegh, Bryan C. Barnhart, Jörg Volkland, Alicia Algeciras-Schimnich, Ning Ke, John C. Reed, Marcus E. Peter*

*Corresponding author for this work

Research output: Contribution to journalArticle

95 Citations (Scopus)

Abstract

Apoptosis induction through CD95 (APO-I/Fas) critically depends on generation of active caspase-8 at the death-inducing signaling complex (DISC). Depending on the cell type, active caspase-8 either directly activates caspase-3 (type I cells) or relies on mitochondrial signal amplification (type II cells). In MCF7-Fas cells that are deficient for pro-caspase-3, even high amounts of caspase-8 produced at the DISC cannot directly activate downstream effector caspases without mitochondrial help. Overexpression of Bcl-xL in these cells renders them resistant to CD95-mediated apoptosis. However, activation of caspase-8 in control (vector) and Bcl-xL transfectants of MCF7-Fas cells proceeds with similar kinetics, resulting in a complete processing of cellular caspase-8. Most of the cytosolic caspase-8 substrates are not cleaved in the Bcl-xL protected cells, raising the question of how Bcl-xL-expressing MCF7-Fas cells survive large amounts of potentially cytotoxic caspase-8. We now demonstrate that active caspase-8 is initially generated at the DISC of both MCF7-Fas-Vec and MCF7-Fas-Bcl-xL cells and that the early steps of CD95 signaling such as caspase-8-dependent cleavage of DISC bound c-FLIPL, caspase-8-dependent clustering, and internalization of CD95, as well as processing of procaspase-8 bound to mitochondria are very similar in both transfectants. However, events downstream of mitochondria, such as release of cytochrome c, only occur in the vector-transfected MCF7-Fas cells, and no in vivo caspase-8 activity can be detected in the Bcl-xL-expressing cells. Our data suggest that, in Bcl-xL-expressing MCF7-Fas cells, active caspase-8 is sequestered on the outer mitochondrial surface presumably by association with the protein "bifunctional apoptosis regulator" in a way that does not allow substrates to be cleaved, identifying a novel mechanism of regulation of apoptosis sensitivity by mitochondrial Bcl-xL.

Original languageEnglish (US)
Pages (from-to)4351-4360
Number of pages10
JournalJournal of Biological Chemistry
Volume277
Issue number6
DOIs
StatePublished - Feb 8 2002

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Mitochondria
Caspase 8
MCF-7 Cells
Apoptosis
Death Domain Receptor Signaling Adaptor Proteins
Proteins
Caspase 3
Effector Caspases
Substrates
Processing
Cytochromes c
Amplification
Cluster Analysis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Stegh, Alexander H. ; Barnhart, Bryan C. ; Volkland, Jörg ; Algeciras-Schimnich, Alicia ; Ke, Ning ; Reed, John C. ; Peter, Marcus E. / Inactivation of caspase-8 on mitochondria of Bcl-XL-expressing MCF7-Fas cells role for the bifunctional apoptosis regulator protein. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 6. pp. 4351-4360.
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abstract = "Apoptosis induction through CD95 (APO-I/Fas) critically depends on generation of active caspase-8 at the death-inducing signaling complex (DISC). Depending on the cell type, active caspase-8 either directly activates caspase-3 (type I cells) or relies on mitochondrial signal amplification (type II cells). In MCF7-Fas cells that are deficient for pro-caspase-3, even high amounts of caspase-8 produced at the DISC cannot directly activate downstream effector caspases without mitochondrial help. Overexpression of Bcl-xL in these cells renders them resistant to CD95-mediated apoptosis. However, activation of caspase-8 in control (vector) and Bcl-xL transfectants of MCF7-Fas cells proceeds with similar kinetics, resulting in a complete processing of cellular caspase-8. Most of the cytosolic caspase-8 substrates are not cleaved in the Bcl-xL protected cells, raising the question of how Bcl-xL-expressing MCF7-Fas cells survive large amounts of potentially cytotoxic caspase-8. We now demonstrate that active caspase-8 is initially generated at the DISC of both MCF7-Fas-Vec and MCF7-Fas-Bcl-xL cells and that the early steps of CD95 signaling such as caspase-8-dependent cleavage of DISC bound c-FLIPL, caspase-8-dependent clustering, and internalization of CD95, as well as processing of procaspase-8 bound to mitochondria are very similar in both transfectants. However, events downstream of mitochondria, such as release of cytochrome c, only occur in the vector-transfected MCF7-Fas cells, and no in vivo caspase-8 activity can be detected in the Bcl-xL-expressing cells. Our data suggest that, in Bcl-xL-expressing MCF7-Fas cells, active caspase-8 is sequestered on the outer mitochondrial surface presumably by association with the protein {"}bifunctional apoptosis regulator{"} in a way that does not allow substrates to be cleaved, identifying a novel mechanism of regulation of apoptosis sensitivity by mitochondrial Bcl-xL.",
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Inactivation of caspase-8 on mitochondria of Bcl-XL-expressing MCF7-Fas cells role for the bifunctional apoptosis regulator protein. / Stegh, Alexander H.; Barnhart, Bryan C.; Volkland, Jörg; Algeciras-Schimnich, Alicia; Ke, Ning; Reed, John C.; Peter, Marcus E.

In: Journal of Biological Chemistry, Vol. 277, No. 6, 08.02.2002, p. 4351-4360.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Inactivation of caspase-8 on mitochondria of Bcl-XL-expressing MCF7-Fas cells role for the bifunctional apoptosis regulator protein

AU - Stegh, Alexander H.

AU - Barnhart, Bryan C.

AU - Volkland, Jörg

AU - Algeciras-Schimnich, Alicia

AU - Ke, Ning

AU - Reed, John C.

AU - Peter, Marcus E.

PY - 2002/2/8

Y1 - 2002/2/8

N2 - Apoptosis induction through CD95 (APO-I/Fas) critically depends on generation of active caspase-8 at the death-inducing signaling complex (DISC). Depending on the cell type, active caspase-8 either directly activates caspase-3 (type I cells) or relies on mitochondrial signal amplification (type II cells). In MCF7-Fas cells that are deficient for pro-caspase-3, even high amounts of caspase-8 produced at the DISC cannot directly activate downstream effector caspases without mitochondrial help. Overexpression of Bcl-xL in these cells renders them resistant to CD95-mediated apoptosis. However, activation of caspase-8 in control (vector) and Bcl-xL transfectants of MCF7-Fas cells proceeds with similar kinetics, resulting in a complete processing of cellular caspase-8. Most of the cytosolic caspase-8 substrates are not cleaved in the Bcl-xL protected cells, raising the question of how Bcl-xL-expressing MCF7-Fas cells survive large amounts of potentially cytotoxic caspase-8. We now demonstrate that active caspase-8 is initially generated at the DISC of both MCF7-Fas-Vec and MCF7-Fas-Bcl-xL cells and that the early steps of CD95 signaling such as caspase-8-dependent cleavage of DISC bound c-FLIPL, caspase-8-dependent clustering, and internalization of CD95, as well as processing of procaspase-8 bound to mitochondria are very similar in both transfectants. However, events downstream of mitochondria, such as release of cytochrome c, only occur in the vector-transfected MCF7-Fas cells, and no in vivo caspase-8 activity can be detected in the Bcl-xL-expressing cells. Our data suggest that, in Bcl-xL-expressing MCF7-Fas cells, active caspase-8 is sequestered on the outer mitochondrial surface presumably by association with the protein "bifunctional apoptosis regulator" in a way that does not allow substrates to be cleaved, identifying a novel mechanism of regulation of apoptosis sensitivity by mitochondrial Bcl-xL.

AB - Apoptosis induction through CD95 (APO-I/Fas) critically depends on generation of active caspase-8 at the death-inducing signaling complex (DISC). Depending on the cell type, active caspase-8 either directly activates caspase-3 (type I cells) or relies on mitochondrial signal amplification (type II cells). In MCF7-Fas cells that are deficient for pro-caspase-3, even high amounts of caspase-8 produced at the DISC cannot directly activate downstream effector caspases without mitochondrial help. Overexpression of Bcl-xL in these cells renders them resistant to CD95-mediated apoptosis. However, activation of caspase-8 in control (vector) and Bcl-xL transfectants of MCF7-Fas cells proceeds with similar kinetics, resulting in a complete processing of cellular caspase-8. Most of the cytosolic caspase-8 substrates are not cleaved in the Bcl-xL protected cells, raising the question of how Bcl-xL-expressing MCF7-Fas cells survive large amounts of potentially cytotoxic caspase-8. We now demonstrate that active caspase-8 is initially generated at the DISC of both MCF7-Fas-Vec and MCF7-Fas-Bcl-xL cells and that the early steps of CD95 signaling such as caspase-8-dependent cleavage of DISC bound c-FLIPL, caspase-8-dependent clustering, and internalization of CD95, as well as processing of procaspase-8 bound to mitochondria are very similar in both transfectants. However, events downstream of mitochondria, such as release of cytochrome c, only occur in the vector-transfected MCF7-Fas cells, and no in vivo caspase-8 activity can be detected in the Bcl-xL-expressing cells. Our data suggest that, in Bcl-xL-expressing MCF7-Fas cells, active caspase-8 is sequestered on the outer mitochondrial surface presumably by association with the protein "bifunctional apoptosis regulator" in a way that does not allow substrates to be cleaved, identifying a novel mechanism of regulation of apoptosis sensitivity by mitochondrial Bcl-xL.

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