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

Abstract

Signal transduction pathways are tightly controlled by positive and negative regulators. We have previously identified Odin (also known as ankyrin repeat and sterile alpha motif domain-containing 1A; gene symbol ANKS1A) as a negative regulator of growth factor signaling; however, the mechanisms through which Odin regulates these pathways remain to be elucidated. To determine how Odin negatively regulates growth factor signaling, we undertook a proteomic approach to systematically identify proteins that interact with Odin using the SILAC strategy. In this study, we identified 18 molecules that were specifically associated in a protein complex with Odin. Our study established that the complete family of 14-3-3 proteins occur in a protein complex with Odin, which is also supported by earlier reports that identified a few members of the 14-3-3 family as Odin interactors. Among the novel protein interactors of Odin were CD2-associated protein, SH3 domain kinase binding protein 1 and DAB2 interacting protein. We confirmed 8 of the eighteen interactions identified in the Odin protein complex by co-immunoprecipitation experiments. Finally, a literature-based network analysis revealed that Odin interacting partners are involved in various cellular processes, some of which are key molecules in regulating receptor endocytosis.

Figure 1

Tyrosine phosphorylation of Odin is induced by EGFR signaling. (A) Wild type (WT) but not kinase dead (KD) EGFR, induces tyrosine phosphorylation. Tyrosine phosphorylation status of HEK 293T cells expressing proteins the indicated constructs was assessed by Western blotting with anti-phosphotyrosine antibodies (4G10). The level of expression of WT and KD EGFR was determined by using anti-EGFR antibodies (bottom panel). (B) Odin is tyrosine phosphorylated in WT but not KD EGFR-expressing cells. Odin was immunoprecipitated from HEK 293T cells expressing the EGFR constructs as indicated in the figure. Tyrosine phosphorylation of Odin was assessed by Western blotting with anti-phosphotyrosine antibodies (4G10) and the total amount of Odin was detected by reprobing the membrane with anti-FLAG antibodies.

Figure 2

A schematic illustration of the SILAC methodology to identify proteins in a protein complex with Odin. Cells grown in light medium were transfected with human EGFR and empty vector as control while cells grown in heavy medium were transfected with human EGFR and FLAG-tagged Odin. Cell lysates were subjected to immunoprecipitation using anti-FLAG antibodies. After washing, the immunoprecipitates were mixed, and the bound proteins were eluted by FLAG peptides. The proteins were resolved by SDS-PAGE. The gel was stained and the protein bands excised, digested with trypsin, and analyzed by LC-MS/MS. The absence of light MS ion peak indicates a true protein interactor of Odin.

Figure 3

Odin expression is confirmed by heavy SILAC label. MS spectra of two representative peptides from Odin are shown in panels A and C. Panels B and D show the corresponding MS/MS spectra along with the peptide sequence. * indicates ¹³C₆-Lys or ¹³C₆-Arg.

Figure 4

Two serine phosphorylation sites identified in Odin. MS spectra of two phosphopeptides from Odin are shown in panels A and C. Panels B and D show the corresponding MS/MS spectra along with the peptide sequence. pS refers to phosphoserine. * indicates ¹³C₆-Lys or ¹³C₆-Arg.

Figure 5

Proteins identified by the SILAC strategy. MS (panel A) and MS/MS (panel B) spectra of a representative peptide, SVDFDSLTVR, from CD2 associated protein are shown. MS (panel C) and MS/MS (panel D) spectra of a representative peptide, AIIIFVPVPQLK, from ribosomal protein S7 are shown. * indicates ¹³C₆-Lys or ¹³C₆-Arg.

Figure 6

A schematic illustration of interactome network of Odin. Protein-protein interactions indicated by black lines were those reported in the literature. Novel protein-protein interactions indicated by red lines indicate the proteins identified as components of the Odin protein complex in this study.

Figure 7

Validation of protein-protein interactions by co-immunoprecipitation experiments. The Odin protein complex was harvested by immunoprecipitation with anti-FLAG antibodies. The cell lysates and corresponding immunoprecipitates were resolved by SDS-PAGE and probed with antibodies against talin 2 (A), SH3KBP1 (B), CD2AP (C), ARHGAP10 (D), mortalin (E), 14-3-3ε (F), ζ (G) and γ (H).