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 participate 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 cytoplasmic domains and become tyrosine-phosphorylated when bound to each other. Tyrosine phosphorylation regulates EphB2 signaling and likely also ephrin-B1 signaling. Embryonic retina is a tissue that highly expresses both ephrin-B1 and EphB2. Although the expression patterns of EphB2 and ephrin-B1 in the retina are different, they partially overlap, and both proteins are substantially tyrosine-phosphorylated. To understand the role of ephrin-B1 phosphorylation, we have identified three tyrosines of ephrin-B1 as in vivo phosphorylation sites in transfected 293 cells stimulated with soluble EphB2 by using mass spectrometry and site-directed mutagenesis. These tyrosines are also physiologically phosphorylated in the embryonic retina, although the extent of phosphorylation at each site may differ. Furthermore, many of the tyrosines of EphB2 previously identified as phosphorylation sites in 293 cells (Kalo, M. S., and Pasquale, E. B. (1999) Biochemistry 38, 14396-14408) are also phosphorylated in retinal tissue. Our data underline the complexity of ephrin-Eph bidirectional signaling by implicating many tyrosine phosphorylation sites of the ligand-receptor complex.
Kalo
M S
MS
Burnham Institute, La Jolla, California 92037, USA.
Yu
H H
HH
Pasquale
E B
EB
D000595:Amino Acid Sequence; D001665:Binding Sites; D002465:Cell Movement; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D036387:Ephrin-B1; D036388:Ephrin-B2; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D012160:Retina / Q000196:embryology; D015398:Signal Transduction; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*
D036387:Ephrin-B1; D036388:Ephrin-B2; D008024:Ligands; D008565:Membrane Proteins; D014443:Tyrosine