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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

The EphA7 cytoplasmic region is not essential for apoptotic activation of caspase-8. (A) Schematic diagrams showing EphA7 deletion mutants used for expression in HEK293 cells. (B) Each of these deletion mutants was co-transfected with caspase-8 into HEK293 cells. Whole cell lysates were directly subjected to Western blot analysis using anti-EphA7 or anti-caspase-8 antibody 20 h post-transfection. (C) HEK293 cells were co-transfected with the indicated constructs. The cells were subjected to a cell surface binding assay using ephrinA5-Fc 16 h post-transfection, as described in Fig. 6. (D) The data in (C) were quantified. Data represent means ± standard errors. *P < 0.001; paired Student’s t-test. (E) HEK293 cells were co-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. Data represent means ± standard errors. *P < 0.001; paired Student’s t-test. (F) Transfected cells were precipitated with biotinylated ephrinA5-Fc, as described in Figs. 1 and 2. Cell lysates were subjected to Western blot analysis using anti-EphA7 or anti-caspase-8 antibody.
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f6-molce-38-4-349: The EphA7 cytoplasmic region is not essential for apoptotic activation of caspase-8. (A) Schematic diagrams showing EphA7 deletion mutants used for expression in HEK293 cells. (B) Each of these deletion mutants was co-transfected with caspase-8 into HEK293 cells. Whole cell lysates were directly subjected to Western blot analysis using anti-EphA7 or anti-caspase-8 antibody 20 h post-transfection. (C) HEK293 cells were co-transfected with the indicated constructs. The cells were subjected to a cell surface binding assay using ephrinA5-Fc 16 h post-transfection, as described in Fig. 6. (D) The data in (C) were quantified. Data represent means ± standard errors. *P < 0.001; paired Student’s t-test. (E) HEK293 cells were co-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. Data represent means ± standard errors. *P < 0.001; paired Student’s t-test. (F) Transfected cells were precipitated with biotinylated ephrinA5-Fc, as described in Figs. 1 and 2. Cell lysates were subjected to Western blot analysis using anti-EphA7 or anti-caspase-8 antibody.

Mentions: We further investigated whether a specific domain in the EphA7 cytoplasmic region is crucial for interacting with caspase-8. Thus, we constructed two different deletion mutants, such as EphA7-D1 and -D2 (Fig. 6A). The EphA7-D1 mutant lacked an entire cytoplasmic region except for the first 10 aa of the juxtamembrane region. In contrast, the EphA7–D2 mutant had an intact juxtamembrane region but no tyrosine kinase domain or COOH-terminal region. Expression of these deletion mutants was highly detected in 293 cells (Fig. 6B). In addition, a cell surface binding assay using ephrinA5-Fc revealed that the cell surface localization of the deletion mutants was indistinguishable from that of wild-type EphA7 (Figs. 6C and 6D). Strikingly, these two EphA7 deletion mutants were equally effective in triggering apoptotic cell death, similar to wild-type EphA7 when they were co-expressed with caspase-8 (Fig. 6E, bars 3 and 4). More importantly, it was observed that the level of caspase-8 co-precipitated with the EphA7 deletion mutants was significantly elevated to a similar degree as observed in wild-type EphA7 (Fig. 6F). Taken together, these findings suggest that the extracellular region of EphA receptors is critical for interacting with caspase-8. We propose that Eph receptors physically interact with a transmembrane protein, which is not yet identified, but acts as a biochemical linker between the Eph receptor and caspase-8.


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

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

The EphA7 cytoplasmic region is not essential for apoptotic activation of caspase-8. (A) Schematic diagrams showing EphA7 deletion mutants used for expression in HEK293 cells. (B) Each of these deletion mutants was co-transfected with caspase-8 into HEK293 cells. Whole cell lysates were directly subjected to Western blot analysis using anti-EphA7 or anti-caspase-8 antibody 20 h post-transfection. (C) HEK293 cells were co-transfected with the indicated constructs. The cells were subjected to a cell surface binding assay using ephrinA5-Fc 16 h post-transfection, as described in Fig. 6. (D) The data in (C) were quantified. Data represent means ± standard errors. *P < 0.001; paired Student’s t-test. (E) HEK293 cells were co-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. Data represent means ± standard errors. *P < 0.001; paired Student’s t-test. (F) Transfected cells were precipitated with biotinylated ephrinA5-Fc, as described in Figs. 1 and 2. Cell lysates were subjected to Western blot analysis using anti-EphA7 or anti-caspase-8 antibody.
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f6-molce-38-4-349: The EphA7 cytoplasmic region is not essential for apoptotic activation of caspase-8. (A) Schematic diagrams showing EphA7 deletion mutants used for expression in HEK293 cells. (B) Each of these deletion mutants was co-transfected with caspase-8 into HEK293 cells. Whole cell lysates were directly subjected to Western blot analysis using anti-EphA7 or anti-caspase-8 antibody 20 h post-transfection. (C) HEK293 cells were co-transfected with the indicated constructs. The cells were subjected to a cell surface binding assay using ephrinA5-Fc 16 h post-transfection, as described in Fig. 6. (D) The data in (C) were quantified. Data represent means ± standard errors. *P < 0.001; paired Student’s t-test. (E) HEK293 cells were co-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. Data represent means ± standard errors. *P < 0.001; paired Student’s t-test. (F) Transfected cells were precipitated with biotinylated ephrinA5-Fc, as described in Figs. 1 and 2. Cell lysates were subjected to Western blot analysis using anti-EphA7 or anti-caspase-8 antibody.
Mentions: We further investigated whether a specific domain in the EphA7 cytoplasmic region is crucial for interacting with caspase-8. Thus, we constructed two different deletion mutants, such as EphA7-D1 and -D2 (Fig. 6A). The EphA7-D1 mutant lacked an entire cytoplasmic region except for the first 10 aa of the juxtamembrane region. In contrast, the EphA7–D2 mutant had an intact juxtamembrane region but no tyrosine kinase domain or COOH-terminal region. Expression of these deletion mutants was highly detected in 293 cells (Fig. 6B). In addition, a cell surface binding assay using ephrinA5-Fc revealed that the cell surface localization of the deletion mutants was indistinguishable from that of wild-type EphA7 (Figs. 6C and 6D). Strikingly, these two EphA7 deletion mutants were equally effective in triggering apoptotic cell death, similar to wild-type EphA7 when they were co-expressed with caspase-8 (Fig. 6E, bars 3 and 4). More importantly, it was observed that the level of caspase-8 co-precipitated with the EphA7 deletion mutants was significantly elevated to a similar degree as observed in wild-type EphA7 (Fig. 6F). Taken together, these findings suggest that the extracellular region of EphA receptors is critical for interacting with caspase-8. We propose that Eph receptors physically interact with a transmembrane protein, which is not yet identified, but acts as a biochemical linker between the Eph receptor and caspase-8.

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