Phosphorylation of tyrosine residues in the kinase domain and juxtamembrane region regulates the biological and catalytic activities of eph receptors

Members of the Eph family of receptor tyrosine kinases exhibit a striking d egree of amino acid homology, particularly notable in the kinase and membra ne-proximal regions. A mutagenesis approach was taken to address the functi ons of specific conserved tyrosine residues within these catalytic and juxt amembrane domains. Ligand stimulation of wild-type EphB2 in neuronal NG108- 15 cells resulted in an upregulation of catalytic activity and an increase in cellular tyrosine phosphorylation, accompanied by a retraction of neurit ic processes. Tyrosine-to-phenylalanine substitutions within the conserved juxtamembrane motif abolished these responses. The mechanistic basis for th ese observations was examined using the highly related EphA4 receptor in a continuous coupled kinase assay. Tandem mass spectrometry experiments confi rmed autophosphorylation of the two juxtamembrane tyrosine residues and als o identified a tyrosine within the kinase domain activation segment as a ph osphorylation site. Kinetic analysis revealed a decreased affinity for pept ide substrate upon substitution of activation segment or juxtamembrane tyro sines. Together, our data suggest that the catalytic and therefore biologic al activities of Eph receptors are controlled by a two-component inhibitory mechanism, which is released by phosphorylation of the juxtamembrane and a ctivation segment tyrosine residues.