Mutation of EphB4 juxtamembrane tyrosine residues or tyrosine kinase inhibition abolishes the cell growth effect of transduced EphB4.

(A) Wild-type EphB4 (EphB4wt), mutants EphB4 (EphB4m590 and EphB4m596), and pcDNA3 (vector) were stably transfected in 32D cells and selected for G418-resistance for 3 weeks. Cell proliferation assay was performed in the presence of 0.1 ng/mL IL-3. Data represent the mean ± SD of 3 experiments (each in duplicate). (B) Schematic representation of mutations inEphB4 intracellular domain. (C). EphB4-expressing and control 32D (pcDNA3) cells were incubated in 0.1 ng/mL IL-3 with 50 μM PD98059 (protein kinase C inhibitor) or 100 μM genistein (tyrosine kinase inhibitor) for 48 hours. Data represent the mean ± SD for 3 experiments (each in duplicate). (D) Apoptosis analysis. EphB4-expressing and control 32D (pcDNA3) cells were incubated in 0.1 ng/mL IL-3 for 3 days. Cells were stained with Annexin V-FITC antibodies and analyzed by FACS.