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 degree of amino acid homology, particularly notable in the kinase and membrane-proximal regions. A mutagenesis approach was taken to address the functions of specific conserved tyrosine residues within these catalytic and juxtamembrane 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 neuritic processes. Tyrosine-to-phenylalanine substitutions within the conserved juxtamembrane motif abolished these responses. The mechanistic basis for these observations was examined using the highly related EphA4 receptor in a continuous coupled kinase assay. Tandem mass spectrometry experiments confirmed autophosphorylation of the two juxtamembrane tyrosine residues and also identified a tyrosine within the kinase domain activation segment as a phosphorylation site. Kinetic analysis revealed a decreased affinity for peptide substrate upon substitution of activation segment or juxtamembrane tyrosines. Together, our data suggest that the catalytic and therefore biological activities of Eph receptors are controlled by a two-component inhibitory mechanism, which is released by phosphorylation of the juxtamembrane and activation segment tyrosine residues.
Binns
K L
KL
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Taylor
P P
PP
Sicheri
F
F
Pawson
T
T
Holland
S J
SJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D036387:Ephrin-B1; D007700:Kinetics; D008565:Membrane Proteins / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D016501:Neurites / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010649:Phenylalanine; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D036183:Receptor, EphB2; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism
D036387:Ephrin-B1; D008565:Membrane Proteins; D014443:Tyrosine; D010649:Phenylalanine; D020794:Receptor Protein-Tyrosine Kinases; D036183:Receptor, EphB2