The SAM Domains of Anks Family Proteins Are Critically Involved in Modulating the Degradation of EphA Receptors

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.Activation of Eph receptor tyrosine kinases (RTKs) by ephrin ligands stimulates intracellular signaling pathways that regulate diverse cell behaviors such as axon guidance, cell adhesion, and cell migration (1). Activated Eph receptors also initiate negative signaling events that counteract or alter positive signals, thereby modulating biological outcomes. Negative signaling events associated with Eph RTKs include metalloprotease-mediated cleavage of ephrins and trans endocytosis of Eph-ephrin complexes (9, 15, 24). These negative regulatory mechanisms may be important in the repulsive mechanism responsible for retraction of cellular processes. Some studies suggest that c-Cbl, a RING finger E3 ligase, participates in activated Eph receptor signal termination. Ligand stimulation induces the tyrosine phosphorylation of c-Cbl and facilitates the degradation of Eph receptors (19, 23). More recent studies have shown that the E3 ligase activity of c-Cbl is activated through tyrosine phosphorylation by Src family kinases and that c-Cbl is recruited to activated Eph receptors and induces the ubiquitination and degradation of the receptors (6, 14). These studies point to an important role for Cbl family ubiquitin (Ub) ligases in mediating the ubiquitination of activated Eph RTKs and in fine-tuning Eph receptor signaling pathways.Emerging evidence points to a critical role for Eph receptors in human diseases such as diabetes and cancer (2, 13, 17). For example, EphA2 overexpression has been found in many types of malignant tumors. Overexpression of EphA2 in nontransformed epithelial cells enhances tumorigenic and metastatic potential, whereas downregulation of EphA2 expression suppresses tumor growth and metastasis (4). In addition, either soluble ephrin-A ligand or a monoclonal antibody that activates and degrades EphA2 has been shown to inhibit the growth of human tumor xenografts in nude mice (5, 12). More recent evidence reveals that EphA2 cooperates with Erb2 (also known as Neu) to promote tumor progression in mice (3). These findings strongly suggest that EphA2 and possibly other Eph receptors function in tumor progression in the context of either specific oncogenes or tumor suppressors. In this respect, understanding the negative regulation of Eph receptors, such as their degradation, may have important implications in the design of effective antitumor therapeutics.Recently, we showed that Anks family proteins act as key scaffolding molecules in EphA8-mediated signaling pathways (20). Anks family proteins contain six ankyrin repeats at their N terminus, two SAM domains, and a phosphotyrosine-binding (PTB) domain at their C terminus (22). Odin and AβPP intracellular domain-associated protein 1b (AIDA-1b) belong to this protein family. Several isoforms of AIDA-1b have been described, and the regions encoding the PTB domain and the two SAM domains are very well conserved among all isoforms (7). Interestingly, AIDA-1 has been implicated in reducing AβPP processing through the inhibition of γ-secretase activity (7) and in increasing the global protein biosynthetic capacity in response to long-term neuronal stimulation through the regulation of nucleolar assembly (10). Functions attributed to Odin have been limited to its negative role in platelet-derived growth factor (PDGF)-mediated cell proliferation (16). In contrast to AIDA-1 proteins, Odin appears to be abundantly and ubiquitously expressed in many different mammalian cell lines, and its expression is restricted to the mouse embryonic brain rather than the adult brain (20). We recently reported that the PTB domains of Anks family proteins are crucial for the association of these proteins with the juxtamembrane (JM) domain of EphA8; however, an as-yet-unidentified motif in Anks family proteins also contributes to stable complex formation between these two proteins (20).While the SAM domains of Anks family proteins are highly conserved among all isoforms, the function of this domain is not well understood. In the current study, we identified a potential role for SAM domains in EphA signaling. We showed that while the ubiquitin ligase c-Cbl mediates the ubiquitination and degradation of EphA8 upon ligand binding, the SAM domains of Anks family proteins associate with ubiquitinated EphA8 receptor and are critically involved in inhibiting the degradation of EphA2 and EphA8 receptors. These results suggest that the fine-tuning of EphA RTK signaling is regulated by a delicate balance between the activity of c-Cbl E3 ligase and Anks family proteins.