EphB1 associates with Grb7 and regulates cell migration.
EphB1 is a member of the Eph family of receptor tyrosine kinases that play important roles in diverse biological processes including nervous system development, angiogenesis, and neural synapsis formation and maturation. Grb7 is an adaptor molecule implicated in the regulation of cell migration. Here we report identification of an interaction between Grb7 and the cytoplasmic domain of EphB1 by using Grb7 as a "bait" in a yeast two-hybrid screening. Co-immunoprecipitation was used to confirm the interaction of Grb7 with the cytoplasmic domain of EphB1 as well as the full-length receptor in intact cells. This interaction is mediated by the SH2 domain of Grb7 and requires tyrosine autophosphorylation of EphB1. Furthermore, Tyr-928 of EphB1 was identified as the primary binding site for Grb7. Stimulation of endogenous EphB1 in embryonal carcinoma P19 cells with its ligand ephrinB1 increased its association with Grb7, which is consistent with a role for the autophosphorylation of EphB1. We also found that EphB1 could phosphorylate Grb7 and mutation of either Tyr-928 or Tyr-594 to Phe decreased this activity. Finally, we show that EphB1 could stimulate fibroblast motility on extracellular matrix in a kinase-dependent manner, which also correlated with its association with Grb7. Consistent with this, co-expression of Grb7 with EphB1 further enhanced cell motility, whereas co-expression of the Grb7 SH2 domain abolished EphB1-stimulated cell migration. Together, our results identified a novel interaction between EphB1 with the adaptor molecule Grb7 and suggested that this interaction may play a role in the regulation of cell migration by EphB1.
Han
Dong Cho
DC
Department of Molecular Medicine, Cornell University, Ithaca, New York 14853, USA.
Shen
Tang-Long
TL
Miao
Hui
H
Wang
Bingcheng
B
Guan
Jun-Lin
JL
D000818:Animals; D016466:CHO Cells; D002465:Cell Movement / Q000502:physiology*; D006224:Cricetinae; D051382:GRB7 Adaptor Protein; D006801:Humans; D008024:Ligands; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D036225:Receptor, EphB1 / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000378:metabolism
C495955:GRB7 protein, human; D008024:Ligands; D011506:Proteins; D051382:GRB7 Adaptor Protein; D014443:Tyrosine; D036225:Receptor, EphB1