In vivo tyrosine phosphorylation sites of activated ephrin-B1 and EphB2 from neural tissue

EphB2 is a receptor tyrosine kinase of the Eph family and ephrin-B1 is one of its transmembrane ligands. In the embryo, EphB2 and ephrin-B1 participat e in neuronal axon guidance, neural crest cell migration, the formation of blood vessels, and the development of facial structures and the inner ear. Interestingly, EphB2 and ephrin-B1 can both signal through their cytoplasmi c domains and become tyrosine-phosphorylated when bound to each other. Tyro sine phosphorylation regulates EphB2 signaling and likely also ephrin-B1 si gnaling. Embryonic retina is a tissue that highly expresses both ephrin-B1 and EphB2. Although the expression patterns of EphB2 and ephrin-B1 in the r etina are different, they partially overlap, and both proteins are substant ially tyrosine-phosphorylated. To understand the role of ephrin-B1 phosphor ylation, we have identified three tyrosines of ephrin-B1 as in vivo phospho rylation sites in transfected 293 cells stimulated with soluble EphB2 by us ing mass spectrometry and site-directed mutagenesis. These tyrosines are al so physiologically phosphorylated in the embryonic retina, although the ext ent of phosphorylation at each site may differ. Furthermore, many of the ty rosines of EphB2 previously identified as phosphorylation sites in 293 cell s (Kalo, M. S., and Pasquale, E. B. (1999) Biochemistry 38,14396-14408) are also phosphorylated in retinal tissue. Our data underline the complexity o f ephrin-Eph bidirectional signaling by implicating many tyrosine phosphory lation sites of the ligand-receptor complex.