Receptor tyrosine kinases (RTKs) are a large family of transmembrane receptors that contain a tyrosine kinase domain in their cytoplasmic domains. Oligomerization of RTKs induces transphosphorylation of their cytoplasmic domains, which leads to activation of their tyrosine kinase activity. Phosphorylated tyrosine residues on downstream molecules serve as docking sites for other cytoplasmic signaling molecules containing phosphotyrosine binding domains such as SH2 and PTB domains to form multimeric protein complexes.
The activation of RTKs induced by ligand binding is tightly controlled by ‘positive’ and ‘negative’ regulatory mechanisms. One such negative mechanism is removal of activated RTKs from the cell surface by the process of endocytosis in which the Cbl family of E3 ubiquitin ligases and other adapter proteins play a key role. Cbl contains an SH2 domain that binds to phosphotyrosine residues on activated RTKs and a RING finger domain that is responsible for ubiquitination of the RTKs. Cbl also serves as a scaffold protein to recruit other adapter proteins such as CD2-associated protein (CD2AP) and SH3 domain kinase binding protein 1 (SH3KBP1) in close proximity of RTKs. Endophilins, pre-associated with SH3KBP1, regulate clathrin-coated vesicles that can induce negative curvature and invagination of the plasma membrane during the early step of RTK internalization. This mechanism has been observed during internalization of several RTKs, including EGFR, PDGFR, c-Met and c-Kit [1
]. Internalized RTKs are sorted into multivesicular bodies and targeted for endosomal degradation. Overall, the negative regulatory pathways of RTKs that attenuate and terminate signals have not been studied well.
Using a mass spectrometry-based proteomic approach, we previously identified a number of signaling molecules undergoing tyrosine phosphorylation in the EGFR signaling pathway, including Odin [4
]. Odin is a ubiquitously expressed 130 kDa cytosolic protein that contains six ankyrin repeat domains at its N-terminus, two sterile alpha motifs (SAM) and a phosphotyrosine binding domain (PTB) at its C-terminus. Our group has previously shown that overexpression of Odin causes inhibition of c-Fos promoter activity and that microinjection of Odin cDNA into NIH3T3 cells inhibits PDGF-induced mitogenesis [5
]. We have also examined the effects of loss of expression of Odin by generating Odin-deficient mice. 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
]. However, the molecular mechanisms by which Odin affects cellular proliferation/growth mediated by RTK signaling are still poorly characterized.
Stable isotope labeling by amino acids in cell culture (SILAC) has been previously used to identify specific protein-protein interactions [10
]. The advantages of the SILAC method are that it not only uses mass spectrometry for comprehensive identification of protein complexes but can also allows differentiation of true interacting proteins from proteins that bind non-specifically. To better understand the function of Odin, we employed a SILAC-based proteomic approach to characterize the interactome of Odin in activated EGFR signaling. Our quantitative mass spectrometric analysis identified 18 out of 193 proteins as true interacting partners of Odin. Further, Western blotting results confirmed eight protein-protein interactions. By searching Human Protein Reference Database (HPRD) [13
] for known interactions among 18 novel protein interactors of Odin, we constructed an interaction network of the Odin protein complex, in which several Odin interacting partners also bind to each other. This literature-based functional analysis also revealed that proteins involved in endocytosis are enriched in the Odin protein complex suggesting that Odin may play a role in endocytosis of EGFR as a negative regulator.