The kinase-null EphB6 receptor undergoes transphosphorylation in a complex with EphB1.
Uniquely for the Eph family of receptor tyrosine kinases, the EphB6 receptor is catalytically inactive due to the alteration of several critical residues in its kinase domain. This has cast doubt upon its ability to participate in cytoplasmic signaling events. We show here that despite its lack of kinase activity, EphB6 undergoes inducible tyrosine phosphorylation upon stimulation with the Eph-B receptor subfamily ligand ephrin-B1. We also demonstrate, for the first time, evidence of cross-talk between Eph receptors. Overexpression of a catalytically active member of the Eph-B subfamily, EphB1, resulted in increased EphB6 phosphorylation. EphB1-induced EphB6 phosphorylation was ligand-dependent and required the functional catalytic activity of EphB1. EphB1 not only transphosphorylated EphB6, but together they also formed a stable hetero-complex. In addition, we identify the proto-oncogene c-Cbl as an EphB6-binding protein. Although EphB6-Cbl association appeared to be constitutive, Cbl required a functional phosphotyrosine binding domain in order to bind the receptor, whereas its RING finger motif ubiquitin-transfer domain was not necessary. Our findings demonstrate that EphB6 is an actively signaling receptor that undergoes transphosphorylation upon ligand binding and that can initiate specific cytoplasmic signaling events.
Freywald
Andrew
A
Immunology and Allergy, Department of Paediatrics, Infection, Immunity, Injury and Repair Program, Research Institute, The Hospital for Sick Children and the University of Toronto, Toronto M5G 1X8, Canada.
Sharfe
Nigel
N
Roifman
Chaim M
CM
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D017931:DNA Primers; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D000090063:Proto-Oncogene Mas; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D036224:Receptor, EphB4; D036261:Receptor, EphB6; D036081:Receptors, Eph Family; D014443:Tyrosine / Q000378:metabolism
D017931:DNA Primers; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D036261:Receptor, EphB6; D014443:Tyrosine; D020794:Receptor Protein-Tyrosine Kinases; D036224:Receptor, EphB4; D036081:Receptors, Eph Family