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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

Co-localization of the kinase-inactive EphA4 mutant with the catalytically inactive caspase-8 mutant on the cell surface. (A–C) HEK293 cells were transfected with both kinase-inactive EphA4 mutant and wild-type caspase-8 expression vectors. The cells were treated with preclustered ephrinA5-Fc on ice for 1 h at 22 h post-transfection. EphrinA5-Fc (1 μg) was incubated for clustering with goat anti-human IgG antibody for 30 min on ice. The cells were washed, fixed, and subjected to immunocytochemical stain-using goat anti-human IgG antibody (conjugated with FITC) and anti-caspase-8 antibody (pre-incubated with rhodamine-conjugated secondary antibody). (D–F) Experiments were performed essentially as in (A–C), except that the inactive caspase-8 mutant was transfected instead of wild-type caspase-8.
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f5-molce-38-4-349: Co-localization of the kinase-inactive EphA4 mutant with the catalytically inactive caspase-8 mutant on the cell surface. (A–C) HEK293 cells were transfected with both kinase-inactive EphA4 mutant and wild-type caspase-8 expression vectors. The cells were treated with preclustered ephrinA5-Fc on ice for 1 h at 22 h post-transfection. EphrinA5-Fc (1 μg) was incubated for clustering with goat anti-human IgG antibody for 30 min on ice. The cells were washed, fixed, and subjected to immunocytochemical stain-using goat anti-human IgG antibody (conjugated with FITC) and anti-caspase-8 antibody (pre-incubated with rhodamine-conjugated secondary antibody). (D–F) Experiments were performed essentially as in (A–C), except that the inactive caspase-8 mutant was transfected instead of wild-type caspase-8.

Mentions: Although the kinase-inactive EphA4 mutant showed a similar result to that of wild type EphA4 for triggering apoptotic signaling, it was not efficiently internalized into the cells, and it more efficiently co-precipitated with the catalytically inactive caspase-8 mutant (data not shown). This result suggests that kinase-inactive EphA4 more stably complexes with catalytically inactive caspase-8 in cells. To confirm this possibility, we performed immunofluorescence staining using anti-caspase8 antibody and ephrinA5-Fc, which is bound to EphA4 and is visualized with Fc-specific secondary antibodies. As a result, the kinase-inactive EphA4 mutant was primarily localized to the cell surface, whereas wild-type caspase-8 was found in the cytoplasm (Figs. 5A–5C). The kinase-inactive EphA4 mutant was barely co-localized with wild-type caspase-8 on the cell surface, possibly due to their transient interaction on the plasma membrane. In contrast, when kinase-inactive EphA4 was co-expressed with catalytically inactive caspase-8, the two proteins co-localized well on the plasma membrane, suggesting that the interaction between the two proteins was prolonged due to the inactive mutation (Figs. 5D–5F). Taken together, our results indicate that the EphA4 or EphA7 receptor transiently associates with caspase-8 to trigger apoptotic cell death and that Eph receptor tyrosine kinase activity is not essential for this apoptotic process.


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

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

Co-localization of the kinase-inactive EphA4 mutant with the catalytically inactive caspase-8 mutant on the cell surface. (A–C) HEK293 cells were transfected with both kinase-inactive EphA4 mutant and wild-type caspase-8 expression vectors. The cells were treated with preclustered ephrinA5-Fc on ice for 1 h at 22 h post-transfection. EphrinA5-Fc (1 μg) was incubated for clustering with goat anti-human IgG antibody for 30 min on ice. The cells were washed, fixed, and subjected to immunocytochemical stain-using goat anti-human IgG antibody (conjugated with FITC) and anti-caspase-8 antibody (pre-incubated with rhodamine-conjugated secondary antibody). (D–F) Experiments were performed essentially as in (A–C), except that the inactive caspase-8 mutant was transfected instead of wild-type caspase-8.
© Copyright Policy
Related In: Results  -  Collection

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

f5-molce-38-4-349: Co-localization of the kinase-inactive EphA4 mutant with the catalytically inactive caspase-8 mutant on the cell surface. (A–C) HEK293 cells were transfected with both kinase-inactive EphA4 mutant and wild-type caspase-8 expression vectors. The cells were treated with preclustered ephrinA5-Fc on ice for 1 h at 22 h post-transfection. EphrinA5-Fc (1 μg) was incubated for clustering with goat anti-human IgG antibody for 30 min on ice. The cells were washed, fixed, and subjected to immunocytochemical stain-using goat anti-human IgG antibody (conjugated with FITC) and anti-caspase-8 antibody (pre-incubated with rhodamine-conjugated secondary antibody). (D–F) Experiments were performed essentially as in (A–C), except that the inactive caspase-8 mutant was transfected instead of wild-type caspase-8.
Mentions: Although the kinase-inactive EphA4 mutant showed a similar result to that of wild type EphA4 for triggering apoptotic signaling, it was not efficiently internalized into the cells, and it more efficiently co-precipitated with the catalytically inactive caspase-8 mutant (data not shown). This result suggests that kinase-inactive EphA4 more stably complexes with catalytically inactive caspase-8 in cells. To confirm this possibility, we performed immunofluorescence staining using anti-caspase8 antibody and ephrinA5-Fc, which is bound to EphA4 and is visualized with Fc-specific secondary antibodies. As a result, the kinase-inactive EphA4 mutant was primarily localized to the cell surface, whereas wild-type caspase-8 was found in the cytoplasm (Figs. 5A–5C). The kinase-inactive EphA4 mutant was barely co-localized with wild-type caspase-8 on the cell surface, possibly due to their transient interaction on the plasma membrane. In contrast, when kinase-inactive EphA4 was co-expressed with catalytically inactive caspase-8, the two proteins co-localized well on the plasma membrane, suggesting that the interaction between the two proteins was prolonged due to the inactive mutation (Figs. 5D–5F). Taken together, our results indicate that the EphA4 or EphA7 receptor transiently associates with caspase-8 to trigger apoptotic cell death and that Eph receptor tyrosine kinase activity is not essential for this apoptotic process.

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