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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

Physical association between EphA7 and caspase-8. HEK293 cells were transiently transfected with the indicated constructs, and the cells were treated with biotinylated ephrinA5-Fc on ice for 1 h 24 h post-transfection. Cell lysates were further incubated with streptavidin-agarose beads prior to pull-down (PD). Protein complexes were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Western blot analysis using anti-caspase-8 (A) or EphA7 antibodies (B). Whole cell lysates (WCL) were also analyzed by Western blot using anti-caspase-8 antibody (C). (D-G) HEK293 cells were transiently transfected as above and the cell lysates were precipitated with anti-caspase-8 antibody 16 h post-transfection. Protein complexes were resolved by SDS-PAGE and subjected to Western blot analysis using the indicated antibodies.
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f1-molce-38-4-349: Physical association between EphA7 and caspase-8. HEK293 cells were transiently transfected with the indicated constructs, and the cells were treated with biotinylated ephrinA5-Fc on ice for 1 h 24 h post-transfection. Cell lysates were further incubated with streptavidin-agarose beads prior to pull-down (PD). Protein complexes were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Western blot analysis using anti-caspase-8 (A) or EphA7 antibodies (B). Whole cell lysates (WCL) were also analyzed by Western blot using anti-caspase-8 antibody (C). (D-G) HEK293 cells were transiently transfected as above and the cell lysates were precipitated with anti-caspase-8 antibody 16 h post-transfection. Protein complexes were resolved by SDS-PAGE and subjected to Western blot analysis using the indicated antibodies.

Mentions: The EphA7-mediated signaling pathway has been implicated in triggering apoptotic cell death during early brain development (Depaepe et al., 2005; Kim et al., 2013; Park et al., 2013). To explore the potential mechanism underlying the apoptotic signaling pathway downstream of EphA7, we investigated whether EphA7 can form a complex with caspase-8 in intact cells. For this purpose, 293 cells were transiently transfected with the EphA7 expression vector. Endogenous expression of EphA7 was not detectable in vector-transfected cells whereas ectopic expression of EphA7 was abundantly induced by transient transfection of the expression vector (data not shown; Fig. 1B). In contrast, endogenous caspase-8 expression was highly detectable, even in the absence of additional transfection (Fig. 1C). When the EphA7 receptor complexes were precipitated with biotinylated ephrinA5-Fc, caspase-8 specifically co-precipitated in the EphA7-transfected cells but not in the vector-transfected cells (Fig. 1A). In addition, co-immunoprecipitation using anti-caspse-8 antibody revealed that EphA7 was also associated with caspase-8 in EphA7-transfected cells (Figs. 1D and 1E, lane 1). To further confirm specific complex formation between EphA7 and caspase-8, EphA7 and caspase-8 were transfected into 293 cells. However, EphA7 level decreased markedly in the co-transfected cells, likely due to the high level of caspse-8 expression (Figs. 1F and 1G, lane 2). Although the EphA7 expression level was low in the co-transfected cells, it was evident that EphA7 was co-precipitated with caspase-8 (Fig. 1D, lane 2).


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

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

Physical association between EphA7 and caspase-8. HEK293 cells were transiently transfected with the indicated constructs, and the cells were treated with biotinylated ephrinA5-Fc on ice for 1 h 24 h post-transfection. Cell lysates were further incubated with streptavidin-agarose beads prior to pull-down (PD). Protein complexes were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Western blot analysis using anti-caspase-8 (A) or EphA7 antibodies (B). Whole cell lysates (WCL) were also analyzed by Western blot using anti-caspase-8 antibody (C). (D-G) HEK293 cells were transiently transfected as above and the cell lysates were precipitated with anti-caspase-8 antibody 16 h post-transfection. Protein complexes were resolved by SDS-PAGE and subjected to Western blot analysis using the indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

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

f1-molce-38-4-349: Physical association between EphA7 and caspase-8. HEK293 cells were transiently transfected with the indicated constructs, and the cells were treated with biotinylated ephrinA5-Fc on ice for 1 h 24 h post-transfection. Cell lysates were further incubated with streptavidin-agarose beads prior to pull-down (PD). Protein complexes were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Western blot analysis using anti-caspase-8 (A) or EphA7 antibodies (B). Whole cell lysates (WCL) were also analyzed by Western blot using anti-caspase-8 antibody (C). (D-G) HEK293 cells were transiently transfected as above and the cell lysates were precipitated with anti-caspase-8 antibody 16 h post-transfection. Protein complexes were resolved by SDS-PAGE and subjected to Western blot analysis using the indicated antibodies.
Mentions: The EphA7-mediated signaling pathway has been implicated in triggering apoptotic cell death during early brain development (Depaepe et al., 2005; Kim et al., 2013; Park et al., 2013). To explore the potential mechanism underlying the apoptotic signaling pathway downstream of EphA7, we investigated whether EphA7 can form a complex with caspase-8 in intact cells. For this purpose, 293 cells were transiently transfected with the EphA7 expression vector. Endogenous expression of EphA7 was not detectable in vector-transfected cells whereas ectopic expression of EphA7 was abundantly induced by transient transfection of the expression vector (data not shown; Fig. 1B). In contrast, endogenous caspase-8 expression was highly detectable, even in the absence of additional transfection (Fig. 1C). When the EphA7 receptor complexes were precipitated with biotinylated ephrinA5-Fc, caspase-8 specifically co-precipitated in the EphA7-transfected cells but not in the vector-transfected cells (Fig. 1A). In addition, co-immunoprecipitation using anti-caspse-8 antibody revealed that EphA7 was also associated with caspase-8 in EphA7-transfected cells (Figs. 1D and 1E, lane 1). To further confirm specific complex formation between EphA7 and caspase-8, EphA7 and caspase-8 were transfected into 293 cells. However, EphA7 level decreased markedly in the co-transfected cells, likely due to the high level of caspse-8 expression (Figs. 1F and 1G, lane 2). Although the EphA7 expression level was low in the co-transfected cells, it was evident that EphA7 was co-precipitated with caspase-8 (Fig. 1D, lane 2).

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