Model to illustrate EGF-induced interaction between Akt and PLC-γ1. Before EGF stimulation, both PLC-γ1 and Akt are located in the cytosol. The conformation of inactive PLC-γ1 blocks the interaction between PLC-γ1 SH3 domain and the proline-rich motifs of Akt (left). After EGF stimulation, PLC-γ1 translocated to the plasma membrane, and its Y783 was phosphorylated by EGFR, which exposes its SH3 domain for interaction. Simultaneously, Akt was activated by EGFR through PI3K and translocated to the plasma membrane. At the plasma membrane, the PLC-γ1 SH3 domain and Akt proline-rich motifs bind to each other (right).

Regulation of EGF-induced PLC-γ1 and Akt association by PLC-γ1 Y771 and Y783. COS-7 cells were transfected with YFP-tagged wild-type PLC-γ1 or mutant PLC-γ1 Y771F, Y783F, and Y/Y. The cells were either not treated or treated with 100 ng/ml EGF for 30 min. The cell lysates were then immunoprecipitated with anti-Akt antibody. The resulting immunoprecipitates were subjected to immunoblotting with a polyclonal anti-GFP antibody. The PLC-γ1 blot served as the loading control, and the GFP blot of total lysate served as the control to show the level of expression.

Colocalization of PLC-γ1 and Akt in COS-7 cells. (A) COS-7 cells were either nontreated or treated with 100 ng/ml EGF for indicated times. The cells were then fixed and stained with anti-PLC-γ1 and anti-Akt antibodies. Colocalization (yellow) of Akt (green) and PLC-γ1 (red) is indicated by arrow. Bar, 20 μM. (B) Serum-starved COS-7 cells were either nontreated or treated with 100 ng/ml EGF for 5 or 30 min. The total cell homogenates were subcellularly fractionated into PM, EN, and Cyt. Ten percent protein of each fraction was loaded to 8% SDS-PAGE and immunoblotted with antibodies to Akt, PLC-γ1, EEA1, and EGFR.

Effect of PLC-γ1 S1248 phosphorylation on EGF-induced cell motility. (A) COS-7 cells were transfected with either YFP-tagged wild-type PLC-γ1 or mutant PLC-γ1 S1248A. Confluent monolayers of serum-starved cells were not treated or treated with 100 ng/ml EGF as indicated. Cell motility was examined by wound healing assay as described in Materials and Methods. In the merged image, red shows the cells at 0 h, and green shows the cells at 8 h. All of cells migrated from the left to right as indicated. (B) Quantitation of the data from A. The data were quantitated as described in Materials and Methods.

Both P424 and P467 of Akt are required for the association of PLC-γ1 and Akt. (A) COS-7 cells were transfected with GFP-Akt or GFP-tagged Akt mutants P424A, P467A, and P/P. The cells were either not treated or treated with 100 ng/ml EGF for 30 min. After treatment, the cell lysates were immunoprecipitated with anti-PLC-γ1 antibody, and the resulting immunoprecipitates were immunoblotted with a polyclonal anti-GFP antibody. The PLC-γ1 blot served as the loading control, and the GFP blot of total lysate served as the control of expression. (B) COS-7 cells were transfected with GFP-Akt-P/P. The cells were then fixed and stained with anti-Akt antibody. The localization of Akt was indicated by arrow. Bar, 20 μM.

Effects of PLC-γ1–Akt interaction and PLC-γ1 activity on EGF-induced cell motility. (A) COS-7 cells were transfected with wild-type GFP-Akt or mutant Akt P/P. Confluent monolayers of serum-starved cells were not treated or treated with 10 μM U73122 and/or 100 ng/ml EGF as indicated. Cell motility was examined by wound healing assay as described in Materials and Methods. In the merged image, red shows the cells at 0 h, and the green shows the cells at 8 h. All of cells migrated from the left to right as indicated. (B) Quantitation of the data from A. The data were quantitated as described in Materials and Methods.

AKT mediates EGF-induced serine phosphorylation of PLC-γ1. (A) COS-7 cells were treated with 100 ng/ml EGF for indicated times with or without 100 nM wortmannin. The total cell lysates were immunoblotted with antibodies to Akt, pAkt, PLC-γ1, and pS1248 of PLC-γ1. (B) COS-7 cells without transfection were treated with 100 ng/ml EGF or EGF and 100 nM wortmannin or U73122. COS-7 cells transfected with GFP-Akt or GFP-tagged mutant Akt P/P were either not treated or treated with EGF. Then, the cell lysates were immunoblotted with anti-pS1248 antibody. Immunoblotting with anti-tubulin antibody was used as loading control and immunoblotting with anti-GFP antibody was used to show the expression level of transfected Akt. (C) Quantification of the data from three independent experiments as described in B. The PLC-γ1 S1248 phosphorylation levels were expressed as the percentage of the level after EGF stimulation. (D) COS-7 cells were treated with 100 ng/ml EGF or EGF with PKA inhibitors H89/KT5270 or with both wortmannin and H89/KT5270. Then, the cell lysates were immunoblotted with anti-pS1248 antibody. (E) Quantification of the data from three independent experiments as described in D. The PLC-γ1 S1248 phosphorylation levels were expressed as the percentage of the level after EGF stimulation.

The EGF-induced association of PLC-γ1 and Akt. (A) COS-7 cells were either not treated or treated with 100 ng/ml EGF for 30 min. The cell lysates were immunoprecipitated with either anti-Akt or anti-PLC-γ1 antibodies and then blotted with anti-PLC-γ1 and anti-Akt antibodies. In controls, the primary antibodies were replaced by normal rabbit IgG and mouse IgG. (B) Serum-starved COS-7 cells were stimulated with 100 ng/ml EGF for 5, 15, 30, 60, or 120 min. Alternatively, serum-starved COS-7 cells were pretreated by 10 μM U73122 for 10 min and then treated with 100 ng/ml EGF for 15 min. The cell lysates were then immunoprecipitated with anti-Akt antibodies, followed by blotting with anti-PLC-γ1 antibody. (C) Serum-starved COS-7 cells were either nontreated or treated with 100 ng/ml EGF for 5 or 30 min. The total cell homogenates were subcellularly fractionated into Mem and Cyt. The proteins of each fraction were immunoprecipitated with antibody to Akt, and the immunoprecipitates were immunoblotted with anti-PLC-γ1 and anti-Akt antibodies. (D) Serum-starved COS-7 cells were either nontreated or treated with 100 ng/ml EGF for 15 min, and the cell lysates were then immunoprecipitated with anti-PLC-γ1 antibodies followed by goat anti-mouse IgG conjugated with agarose. After washing three times, GST-Akt was added and incubated for 30 min. The final precipitates were washed and blotted with GST antibody.

SH3 domain of PLC-γ1 is required for the association. (A) The association of Akt with various GST fusion PLC-γ1 domains by GST pull-down. COS-7 cells were not treated, treated with 100 ng/ml EGF for 30 min, or pretreated with 100 nM wortmannin for 30 min followed by 100 ng/ml EGF for 30 min. The cell lysates were incubated with various GST fusion PLC-γ1 proteins bound to glutathione-agarose beads. Bound proteins were analyzed by immunoblotting with anti-Akt antibody. Immunoblotting with anti-GST antibody was used as loading controls. (B) COS-7 cells were transfected with wild type, ΔPH-PLC-γ1, and ΔSH3-PLC-γ1. The cells were either not treated or treated with 100 ng/ml EGF for 30 min. The cell lysates were subjected to immunoprecipitation with monoclonal anti-Akt antibody, and the resulting immunoprecipitates were subjected to immunoblotting with a polyclonal anti-GFP antibody, which reacts to both GFP and YFP. (C) In vitro binding assay. GST and GST-PLC-γ1SH3 beads were incubated with purified Akt for 30 min. After washing three times, the final precipitates were blotted with antibodies to GST and Akt.