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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

Apoptotic activation of caspase-8 is highly dependent on Eph receptor subtype. (A–F) HEK293 cells were transiently transfected with the indicated constructs as described in Fig. 3. Note that EphA8 does not promote apoptotic cell death even in the presence of caspase-8. (G) Cleaved caspase-3 stained apoptotic cells were counted as described in Fig. 3E but are presented as a percentage of total GFP+ cells. Caspase-8 was subcloned into a bicistronic pIRES-GFP vector. Data represent ± standard errors. *P < 0.001; single-factor analysis of variance.
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f3-molce-38-4-349: Apoptotic activation of caspase-8 is highly dependent on Eph receptor subtype. (A–F) HEK293 cells were transiently transfected with the indicated constructs as described in Fig. 3. Note that EphA8 does not promote apoptotic cell death even in the presence of caspase-8. (G) Cleaved caspase-3 stained apoptotic cells were counted as described in Fig. 3E but are presented as a percentage of total GFP+ cells. Caspase-8 was subcloned into a bicistronic pIRES-GFP vector. Data represent ± standard errors. *P < 0.001; single-factor analysis of variance.

Mentions: We further assessed whether other EphA receptors also synergistically cooperated with caspase-8 to enhance apoptotic cell death in 293 cells. As a positive control, co-transfection of TNFR-1 with caspase-8 was sufficient to induce massive apoptotic cell death in 293 cells (Figs. 3A, 3B, and 3G, bar 7). Interestingly, co-expression of EphA4 with caspase-8 also resulted in increasing apoptotic cell death to a similar degree as observed in EphA7/caspase-8 transfected cells (Figs. 3C–3E, and 3G, bars 2–5). However, EphA8 was not effective for inducing apoptotic cell death with caspase-8 (Figs. 3F and 3G, bar 6). EphA2 cooperated with caspase-8 to enhance apoptotic cell death to a similar degree as observed with EphA7 or EphA4 (data not shown). Taken together, our findings indicate that the Eph receptor/caspase-8 complex triggers the apoptotic signaling cascade and that this apoptotic protein complex is highly dependent on the type of Eph receptor.


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

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

Apoptotic activation of caspase-8 is highly dependent on Eph receptor subtype. (A–F) HEK293 cells were transiently transfected with the indicated constructs as described in Fig. 3. Note that EphA8 does not promote apoptotic cell death even in the presence of caspase-8. (G) Cleaved caspase-3 stained apoptotic cells were counted as described in Fig. 3E but are presented as a percentage of total GFP+ cells. Caspase-8 was subcloned into a bicistronic pIRES-GFP vector. Data represent ± 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

f3-molce-38-4-349: Apoptotic activation of caspase-8 is highly dependent on Eph receptor subtype. (A–F) HEK293 cells were transiently transfected with the indicated constructs as described in Fig. 3. Note that EphA8 does not promote apoptotic cell death even in the presence of caspase-8. (G) Cleaved caspase-3 stained apoptotic cells were counted as described in Fig. 3E but are presented as a percentage of total GFP+ cells. Caspase-8 was subcloned into a bicistronic pIRES-GFP vector. Data represent ± standard errors. *P < 0.001; single-factor analysis of variance.
Mentions: We further assessed whether other EphA receptors also synergistically cooperated with caspase-8 to enhance apoptotic cell death in 293 cells. As a positive control, co-transfection of TNFR-1 with caspase-8 was sufficient to induce massive apoptotic cell death in 293 cells (Figs. 3A, 3B, and 3G, bar 7). Interestingly, co-expression of EphA4 with caspase-8 also resulted in increasing apoptotic cell death to a similar degree as observed in EphA7/caspase-8 transfected cells (Figs. 3C–3E, and 3G, bars 2–5). However, EphA8 was not effective for inducing apoptotic cell death with caspase-8 (Figs. 3F and 3G, bar 6). EphA2 cooperated with caspase-8 to enhance apoptotic cell death to a similar degree as observed with EphA7 or EphA4 (data not shown). Taken together, our findings indicate that the Eph receptor/caspase-8 complex triggers the apoptotic signaling cascade and that this apoptotic protein complex is highly dependent on the type of Eph receptor.

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