Figure 6.
Involvement of tyrosine kinase(s) in Gβγ induction of Rac-GEF activity of Ras-GRF1. (A) Tyrosine-phosphorylation of Ras-GRF1 on Gβγ expression. Proteins within anti-FLAG antibody M2 (+) or control antibody (−) immunoprecipitates from 293 cells transfected with pCMV5-FLAG-Ras-GRF alone or pCMV5-FLAG-Ras-GRF and expression plasmids for Gβγ were stained with an antiphosphotyrosine antibody. Where indicated, cells were treated with herbimycin A (1 μg/ml) or PP2 (30 μM) for 24 h. A representative result of three independent experiments is shown. (B) Effect of tyrosine kinase inhibitors on Rac-GEF activity of Ras-GRF1. The 293 cells were transfected with a control vector (○), pCMV5-FLAG-Ras-GRF alone (▵), or pCMV5-FLAG-Ras-GRF with pCMV5-β1 and pCMV5-γ2 (▴, □, ■). During serum starvation, cells were treated with herbimycin A (1 μg/ml, □), PP2 (30 μM, ■), or control solvent (○, ▵, ▴). Cell lysates were prepared after serum starvation. Anti-FLAG M2 immunoprecipitates were subjected to [3H]GDP binding assays. Data are shown as described in Fig. 1A.