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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

EphA4 autokinase activity is dispensable for caspase8-dependent apoptotic signaling. (A–G) HEK293 cells were transiently transfected with the indicated constructs. The cells were subjected to immunocytochemical staining using anti-cleaved caspase3 antibody 16 h post-transfection, as described in Fig. 3. (H) Data represent means ± standard errors. * P < 0.001; single-factor analysis of variance.
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f4-molce-38-4-349: EphA4 autokinase activity is dispensable for caspase8-dependent apoptotic signaling. (A–G) HEK293 cells were transiently transfected with the indicated constructs. The cells were subjected to immunocytochemical staining using anti-cleaved caspase3 antibody 16 h post-transfection, as described in Fig. 3. (H) Data represent means ± standard errors. * P < 0.001; single-factor analysis of variance.

Mentions: Our results demonstrate that EphA4 or EphA7 physically interact with caspase-8 to induce the apoptotic signaling cascade. To further test whether caspase-8 catalytic activity is essential for triggering the apoptotic signaling cascade, the catalytically inactive caspase-8 mutant (C362A) was co-transfected into 293 cells with EphA4 or EphA7. Similar to the result for caspase-8 transfection alone, we did not observe a significant increase in apoptotic cell death in the transfected cells (Figs. 4D, 4G, and 4H, bars 4 and 7). We also tested whether EphA4 tyrosine kinase activity is essential for triggering apoptotic cell death with caspase-8. For this purpose, the tyrosine at EphA4 aa 653, an ATP binding residue, was replaced by phenylalanine. Strikingly, the tyrosine kinase inactive EphA4 mutant was as effective as wild-type EphA4 in enhancing apoptotic cell death with caspase-8 (Figs. 4F and 4H, bar 6). This finding suggests that Eph receptor tyrosine kinase activity is dispensable for its specific interaction with caspase-8 to trigger the apoptotic signaling cascade.


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

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

EphA4 autokinase activity is dispensable for caspase8-dependent apoptotic signaling. (A–G) HEK293 cells were transiently transfected with the indicated constructs. The cells were subjected to immunocytochemical staining using anti-cleaved caspase3 antibody 16 h post-transfection, as described in Fig. 3. (H) Data represent means ± standard errors. * P < 0.001; single-factor analysis of variance.
© Copyright Policy
Related In: Results  -  Collection

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

f4-molce-38-4-349: EphA4 autokinase activity is dispensable for caspase8-dependent apoptotic signaling. (A–G) HEK293 cells were transiently transfected with the indicated constructs. The cells were subjected to immunocytochemical staining using anti-cleaved caspase3 antibody 16 h post-transfection, as described in Fig. 3. (H) Data represent means ± standard errors. * P < 0.001; single-factor analysis of variance.
Mentions: Our results demonstrate that EphA4 or EphA7 physically interact with caspase-8 to induce the apoptotic signaling cascade. To further test whether caspase-8 catalytic activity is essential for triggering the apoptotic signaling cascade, the catalytically inactive caspase-8 mutant (C362A) was co-transfected into 293 cells with EphA4 or EphA7. Similar to the result for caspase-8 transfection alone, we did not observe a significant increase in apoptotic cell death in the transfected cells (Figs. 4D, 4G, and 4H, bars 4 and 7). We also tested whether EphA4 tyrosine kinase activity is essential for triggering apoptotic cell death with caspase-8. For this purpose, the tyrosine at EphA4 aa 653, an ATP binding residue, was replaced by phenylalanine. Strikingly, the tyrosine kinase inactive EphA4 mutant was as effective as wild-type EphA4 in enhancing apoptotic cell death with caspase-8 (Figs. 4F and 4H, bar 6). This finding suggests that Eph receptor tyrosine kinase activity is dispensable for its specific interaction with caspase-8 to trigger the apoptotic signaling cascade.

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