COMPLEX-FORMATION BETWEEN EPHB2 AND SRC REQUIRES PHOSPHORYLATION OF TYROSINE-611 IN THE EPHB2 JUXTAMEMBRANE REGION

The cellular components of the neuronal signaling pathways of Eph rece ptor tyrosine kinases are only beginning to be elucidated. Here we sho w that in vivo tyrosine phosphorylation sites of the Eph receptors Eph A3, EphA4, and EphB2 in embryonic retina serve as binding sites for th e Src-homology 2 (SH2) domain of Src kinase, Furthermore, tyrosine-pho sphorylated EphB2 was detected in Src immunoprecipitates from transfec ted Cos cells, indicating that EphB2 and Src can physically associate. Interestingly, a form of Src with reduced electrophoretic mobility an d increased tyrosine phosphorylation was detected in Cos cells express ing tyrosine-phosphorylated EphB2, suggesting a functional interaction between EphB2 and Src. Yeast two-hybrid analysis in conjunction with site-directed mutagenesis demonstrated that phosphorylated tyrosine 61 1 in the juxtamembrane region of EphB2 is crucial for the interaction with the SH2 domain of Src, In contrast, binding of the carboxy-termin al SH2 domain of phospholipase C gamma was not abolished upon mutation of tyrosine 611 in EphB2, Phosphopeptide mapping of autophosphorylate d full-length EphB2, and wild-type and tyrosine to phenylalanine mutan ts of the EphB2 cytoplasmic domain fused to LexA, showed tyrosine 611 in the sequence motif YEDP as a major site of autophosphorylation in E phB2, Our mutational analysis also indicated that tyrosines 605 and 61 1 are important for EphB2 kinase activity. We propose Src kinase as a downstream effector that mediates the neuron's response to Eph recepto r activation.