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

Abstract

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.

RINL, Guanine Nucleotide Exchange Factor Rab5-Subfamily, Is Involved in the EphA8-Degradation Pathway with Odin

“…Since odin has been shown to protect EphA8 from degradation [18], we supposed that RINL might be involved in this degradation process. For this purpose, HeLa cells were co-transfected with myc-RINL and EphA8-FLAG.…”

Tandem SAM Domain Structure of Human Caskin1: A Presynaptic, Self-Assembling Scaffold for CASK

“…All currently characterized SAM polymers, including SHANK3 polymers, form helices containing six SAM domains per helical turn. Besides Caskin1 and Caskin2, many proteins contain more than one SAM domain in tandem, including AIDA-1 (Jordan et al, 2007), ANKS1 (Kim et al, 2010), the liprin protein family (Serra-Pagès et al, 1998; Spangler and Hoogenraad, 2007), SARM1 (Belinda et al, 2008), and kazrinE (Nachat et al, 2009). The first natural tandem SAM domain structure of AIDA-1 was recently solved by NMR which shows its two SAM domains each adopt characteristic five α-helix bundles and associate intra-molecularly through an EH-ML interface to give soluble, monomeric tandem SAMs covalently connected by a disordered linker (Kurabi et al, 2009).…”

The interactome of a PTB domain-containing adapter protein, Odin, revealed by SILAC

“…Although, Odin-deficient mice do not display any obvious phenotype, mouse embryonic fibroblasts (MEFs) generated from these mice exhibit a hyperproliferative phenotype compared to wild-type-derived MEFs [7] implicating Odin as a negative regulator in growth factor signaling. More recently, Odin was identified as a downstream scaffold protein in EphA receptor signaling and suggested to play a pivotal role in EphA receptor signaling [8, 9]. However, the molecular mechanisms by which Odin affects cellular proliferation/growth mediated by RTK signaling are still poorly characterized.…”