Fig. 5.

Interaction between PLC-γ1 and Rac1 is critical for EGF-induced Rac1 activation. The activation of Rac1 was determined by pulldown with GST-fusion PAK Rac1-binding domain as described in Materials and Methods. Briefly, the cell lysates were incubated with GST-PAK bound to glutathione-agarose beads. Bound proteins were analyzed by immunoblotting with anti-Rac1 antibody. Immunoblotting with anti-GST antibody was used as loading control. A, EGF-induced activation of Rac1 in BT20 and Cos-7 cells. BT20 and Cos7 cells were stimulated with EGF for the indicated times, and the activation of Rac1 was determined as above. B, The effects of the PLC-γ1 SH3 domain on EGF-induced activation of Rac1. Cos7 cells were transfected with YFP-tagged wt PLC-γ1 or mutant PLC-γ1ΔSH3. 293T cells were transfected with wt EGFR and YFP-tagged wt PLC-γ1 or mutant PLC-γ1ΔSH3. The cells were either not treated or treated with EGF (100 ng/ml) for 30 min. The Rac1 activation was determined by GST-PAK pulldown. C, The effects of Rac1 ¹⁰⁶PNTP¹⁰⁹ motif on EGF-induced activation of Rac1 in Cos7 cells. Cells were transfected with GFP-tagged wt Rac1 or mutant Rac1PP/AA. The cells were either not treated or treated with EGF (100 ng/ml) for 30 min. The Rac1 activation was determined by GST-PAK pulldown. D, The effects of PLC-γ1 SH3 domain on EGF-induced activation of Rac1 in 293T cells. Cells were transfected with wild-type EGFR and YFP-tagged wt PLC-γ1 or mutant PLC-γ1ΔSH3. The cells were either not treated or treated with EGF (100 ng/ml) for 30 min. The Rac1 activation was determined by GST-PAK pulldown. E, The effects of Rac1 ¹⁰⁶PNTP¹⁰⁹ motif on EGF-induced activation of Rac1 in 293T cells. Cells were transfected with wild-type EGFR and GFP-tagged wt Rac1 or mutant Rac1PP/AA. The cells were either not treated or treated with EGF (100 ng/ml) for 30 min. The Rac1 activation was determined by GST-PAK pulldown.