Anti-EphA8 polyclonal antibody [Biotin]

We recently reported that the phosphotyrosine-binding (PTB) domain of Anks family proteins binds to EphA8, thereby positively regulating EphA8-mediated signaling pathways. In the current study, we identified a potential role for the SAM domains of Anks family proteins in EphA signaling. We found that SAM domains of Anks family proteins directly bind to ubiquitin, suggesting that Anks proteins regulate the degradation of ubiquitinated EphA receptors. Consistent with the role of Cbl ubiquitin ligases in the degradation of Eph receptors, our results revealed that the ubiquitin ligase c-Cbl induced the ubiquitination and degradation of EphA8 upon ligand binding. Ubiquitinated EphA8 also bound to the SAM domains of Odin, a member of the Anks family proteins. More importantly, the overexpression of wild-type Odin protected EphA8 and EphA2 from undergoing degradation following ligand stimulation and promoted EphA-mediated inhibition of cell migration. In contrast, a SAM domain deletion mutant of Odin strongly impaired the function of endogenous Odin, suggesting that the mutant functions in a dominant-negative manner. An analysis of Odin-deficient primary embryonic fibroblasts indicated that Odin levels play a critical role in regulating the stability of EphA2 in response to ligand stimulation. Taken together, our studies suggest that the SAM domains of Anks family proteins play a pivotal role in enhancing the stability of EphA receptors by modulating the ubiquitination process.

Endocytosis of Eph-ephrin complexes may be an important mechanism for converting cell-cell adhesion to a repulsive interaction. Here, we show that an endocytosis-defective EphA8 mutant forms a complex with EphAs and blocks their endocytosis in cultured cells. Further, we used bacterial artificial chromosome transgenic (Tg) mice to recapitulate the anterior > posterior gradient of EphA in the superior colliculus (SC). In mice expressing the endocytosis-defective EphA8 mutant, the nasal axons were aberrantly shifted to the anterior SC. In contrast, in Tg mice expressing wild-type EphA8, the nasal axons were shifted to the posterior SC, as predicted for the enhanced repellent effect of ephrinA reverse signalling. Importantly, Rac signalling was shown to be essential for EphA-ephrinA internalization and the subsequent nasal axonal repulsion in the SC. These results indicate that endocytosis of the Eph-ephrin complex is a key mechanism by which axonal repulsion is generated for proper guidance and topographic mapping. The EMBO Journal (2011) 30, 1593-1607. doi:10.1038/emboj.2011.44; Published online 22 February 2011 Subject Categories: signal transduction; neuroscience