FIGURE 5.

Interaction of 3BP2 with PLC-γ2 and Vav1. A, shown is a schematic diagram of biotinylated synthesized 3BP2 peptides used in this experiment. Phosphorylated or non-phosphorylated 3BP2 peptides Ser¹⁶⁶–Ser¹⁸², Glu¹⁷⁷–Lys¹⁹³, and Gly⁴³⁸–Ser⁴⁵³ are shown. Positions of phosphorylation sites are indicated with asterisks. Tyr¹⁷⁴, Tyr¹⁸³, or Tyr⁴⁴⁶ was phosphorylated in each phosphorylated 3BP2 peptide, respectively. These tyrosine residues were not phosphorylated in non-phosphorylated 3BP2 peptides. B, affinity purification by peptide binding experiments is shown. Daudi cell lysates were reacted with either phosphorylated or non-phosphorylated biotinylated synthesized 3BP2 peptides prebound to streptavidin beads. Interactions of these peptides with PLC-γ2 and Vav1 were analyzed by SDS-PAGE and immunoblotting with anti-PLC-γ2 and anti-Vav1 antibodies. C, interaction of 3BP2 peptides with GST-SH2 domain fusion proteins is shown. Bacterial GST, GST fusion proteins of PLC-γ2-SH2 (N+C), or Vav1-SH2 was reacted with each 3BP2 peptide prebound to streptavidin beads. Interactions of these peptides with SH2 domains were analyzed by SDS-PAGE and immunoblotting with anti-GST mAb. The last two lanes show the positive controls of GST and GST-SH2 fusion proteins of PLC-γ2 (N+C) and Vav1. D, shown is a co-immunoprecipitation experiment. Ramos-T cells were transiently transfected with HA-Myc-3BP2 wild type (WT) and HA-myc-3BP2-Y183F (Y183F). After 24 h cells were unstimulated (−) or stimulated with anti-IgM mAb for 3 min (+). Anti-HA immunoprecipitates (IP) were separated by SDS-PAGE and analyzed by immunoblotting with anti-phospho-PLC -γ2 (upper panel) and anti-HA antibodies (bottom panel). Molecular size markers are indicated at the left in kilodaltons. The results are representative of three independent experiments.