Identifying transient protein-protein interactions in EphB2 signaling by blue native PAGE and mass spectrometry

Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues, and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands lead to activation of signal transduction pathways and formation of many transient protein–protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well understood. We used blue native PAGE and MS to analyze the transient protein–protein interactions that resulted from the stimulation of EphB2 receptors by their ephrinB1‐Fc ligands. We analyzed the phosphotyrosine‐containing protein complexes immunoprecipitated from the cell lysates of both unstimulated (−) and ephrinB1‐Fc‐stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein–protein interactions upon ephrinB1‐Fc stimulation. These data led us to investigate the roles of proteins such as FAK, WAVEs and Nischarin in EphB2 signaling.