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EphA receptors form a complex with caspase-8 to induce apoptotic cell death.

Lee H, Park S, Kang YS, Park S - Mol. Cells (2015)

Bottom Line: EphA4 also had a causative role in inducing apoptotic cell death with caspase-8, whereas EphA8 did not.Interestingly, we found that kinaseinactive EphA4 was well co-localized at the plasma membrane with catalytically inactive caspase-8, suggesting that an interaction between these mutant proteins was more stable.Therefore, we propose that Eph receptors physically associate with a transmembrane protein to form an apoptotic signaling complex and that this unidentified receptorlike protein acts as a biochemical linker between the Eph receptor and caspase-8.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science.

ABSTRACT
EphA7 has been implicated in the regulation of apoptotic cell death in neural epithelial cells. In this report, we provide evidence that EphA7 interacts with caspase-8 to induce apoptotic cell signaling. First, a pull-down assay using biotinylated ephrinA5-Fc showed that EphA7 coprecipitated with wild type caspase-8 or catalytically inactive caspase-8 mutant. Second, co-transfection of EphA7 with caspase-8 significantly increased the number of cleaved caspase-3 positive apoptotic cells under an experimental condition where transfection of EphA7 or caspase-8 alone did not affect cell viability or apoptosis. EphA4 also had a causative role in inducing apoptotic cell death with caspase-8, whereas EphA8 did not. Third, caspase-8 catalytic activity was essential for the apoptotic signaling cascade, whereas tyrosine kinase activity of the EphA4 receptor was not. Interestingly, we found that kinaseinactive EphA4 was well co-localized at the plasma membrane with catalytically inactive caspase-8, suggesting that an interaction between these mutant proteins was more stable. Finally, we observed that the extracellular region of the EphA7 receptor was critical for interacting with caspase-8, whereas the cytoplasmic region of EphA7 was not. Therefore, we propose that Eph receptors physically associate with a transmembrane protein to form an apoptotic signaling complex and that this unidentified receptorlike protein acts as a biochemical linker between the Eph receptor and caspase-8.

No MeSH data available.


Related in: MedlinePlus

Stable complex formation between EphA7 and inactive caspase-8. (A–C) The EphA7 expression vector was transiently transfected into HEK293 cells with varying amounts of the caspase-8 expression vector. The cells were treated with biotinylated ephrinA5-Fc for pull-down 16 h post-transfection. Protein complexes were subjected to Western blot analysis using anti-caspase-8 (A) or anti-EphA7 antibodies (B). Whole cell lysates were also analyzed by Western blot using anti-caspase-8 antibody (C). (D–F) Experiments were performed essentially as in (A–C), except that the inactive caspase-8 mutant was used instead of wild-type caspase-8.
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f2-molce-38-4-349: Stable complex formation between EphA7 and inactive caspase-8. (A–C) The EphA7 expression vector was transiently transfected into HEK293 cells with varying amounts of the caspase-8 expression vector. The cells were treated with biotinylated ephrinA5-Fc for pull-down 16 h post-transfection. Protein complexes were subjected to Western blot analysis using anti-caspase-8 (A) or anti-EphA7 antibodies (B). Whole cell lysates were also analyzed by Western blot using anti-caspase-8 antibody (C). (D–F) Experiments were performed essentially as in (A–C), except that the inactive caspase-8 mutant was used instead of wild-type caspase-8.

Mentions: As the transfection of 6 μg EphA7 mixed with 0.25 μg caspase-8 did not reduce EphA7 expression level, we further investigated whether EphA7 could cooperate with caspase-8 to promote apoptotic cell death under this transfection condition. As a control, cells transfected with vector alone barely displayed cleaved caspase-3 positive apoptotic cells (Figs. 2A and 2E, bar 1). Similarly, cells transfected with EphA7 or caspase-8 alone did not show a significant increase in the number of apoptotic cells (Figs. 2B, 2C, and 2E, bars 2 and 4). In contrast, co-transfection of EphA7 with caspase-8 resulted in an approximate 10-fold increase in the number of cleaved caspase-3 positive apoptotic cells (Figs. 2D and 2E, bar 3).


EphA receptors form a complex with caspase-8 to induce apoptotic cell death.

Lee H, Park S, Kang YS, Park S - Mol. Cells (2015)

Stable complex formation between EphA7 and inactive caspase-8. (A–C) The EphA7 expression vector was transiently transfected into HEK293 cells with varying amounts of the caspase-8 expression vector. The cells were treated with biotinylated ephrinA5-Fc for pull-down 16 h post-transfection. Protein complexes were subjected to Western blot analysis using anti-caspase-8 (A) or anti-EphA7 antibodies (B). Whole cell lysates were also analyzed by Western blot using anti-caspase-8 antibody (C). (D–F) Experiments were performed essentially as in (A–C), except that the inactive caspase-8 mutant was used instead of wild-type caspase-8.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4400310&req=5

f2-molce-38-4-349: Stable complex formation between EphA7 and inactive caspase-8. (A–C) The EphA7 expression vector was transiently transfected into HEK293 cells with varying amounts of the caspase-8 expression vector. The cells were treated with biotinylated ephrinA5-Fc for pull-down 16 h post-transfection. Protein complexes were subjected to Western blot analysis using anti-caspase-8 (A) or anti-EphA7 antibodies (B). Whole cell lysates were also analyzed by Western blot using anti-caspase-8 antibody (C). (D–F) Experiments were performed essentially as in (A–C), except that the inactive caspase-8 mutant was used instead of wild-type caspase-8.
Mentions: As the transfection of 6 μg EphA7 mixed with 0.25 μg caspase-8 did not reduce EphA7 expression level, we further investigated whether EphA7 could cooperate with caspase-8 to promote apoptotic cell death under this transfection condition. As a control, cells transfected with vector alone barely displayed cleaved caspase-3 positive apoptotic cells (Figs. 2A and 2E, bar 1). Similarly, cells transfected with EphA7 or caspase-8 alone did not show a significant increase in the number of apoptotic cells (Figs. 2B, 2C, and 2E, bars 2 and 4). In contrast, co-transfection of EphA7 with caspase-8 resulted in an approximate 10-fold increase in the number of cleaved caspase-3 positive apoptotic cells (Figs. 2D and 2E, bar 3).

Bottom Line: EphA4 also had a causative role in inducing apoptotic cell death with caspase-8, whereas EphA8 did not.Interestingly, we found that kinaseinactive EphA4 was well co-localized at the plasma membrane with catalytically inactive caspase-8, suggesting that an interaction between these mutant proteins was more stable.Therefore, we propose that Eph receptors physically associate with a transmembrane protein to form an apoptotic signaling complex and that this unidentified receptorlike protein acts as a biochemical linker between the Eph receptor and caspase-8.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science.

ABSTRACT
EphA7 has been implicated in the regulation of apoptotic cell death in neural epithelial cells. In this report, we provide evidence that EphA7 interacts with caspase-8 to induce apoptotic cell signaling. First, a pull-down assay using biotinylated ephrinA5-Fc showed that EphA7 coprecipitated with wild type caspase-8 or catalytically inactive caspase-8 mutant. Second, co-transfection of EphA7 with caspase-8 significantly increased the number of cleaved caspase-3 positive apoptotic cells under an experimental condition where transfection of EphA7 or caspase-8 alone did not affect cell viability or apoptosis. EphA4 also had a causative role in inducing apoptotic cell death with caspase-8, whereas EphA8 did not. Third, caspase-8 catalytic activity was essential for the apoptotic signaling cascade, whereas tyrosine kinase activity of the EphA4 receptor was not. Interestingly, we found that kinaseinactive EphA4 was well co-localized at the plasma membrane with catalytically inactive caspase-8, suggesting that an interaction between these mutant proteins was more stable. Finally, we observed that the extracellular region of the EphA7 receptor was critical for interacting with caspase-8, whereas the cytoplasmic region of EphA7 was not. Therefore, we propose that Eph receptors physically associate with a transmembrane protein to form an apoptotic signaling complex and that this unidentified receptorlike protein acts as a biochemical linker between the Eph receptor and caspase-8.

No MeSH data available.


Related in: MedlinePlus