EphA receptors form a complex with caspase-8 to induce apoptotic cell death.
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.
Affiliation: Department of Biological Science.
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
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).
No MeSH data available.