Fig. 4.
Active H-Ras recruits p110γ to cell membranes. (A) Subcellular distribution of fluorescently labeled p110γ in living cells. HEK cells were transfected with plasmids encoding H-Ras (H-RasG12V or H-RasS17N) and YFP-tagged p110γ as indicated above. The localization of YFP-labeled p110γ subunits in representative, starved (18 h) HEK cells (cLSM slices of 0.8 μm) out of 3 independent experiments is shown. p110γ translocated to the plasma membrane in the presence of H-RasG12V. (Scale bar: 10 μm.) (B) Colocalization of fluorescently labeled p110γ and immunostained H-Ras. HEK cells were transfected with plasmids encoding H-Ras (H-RasG12V or H-RasS17N) and YFP-tagged p110γ as indicated above. The localization of YFP-labeled p110γ (yellow) and immunostained H-Ras (red) is shown in representative fixed HEK cells from 3 independent experiments (cLSM slices of 0.8 μm). p110γ colocalized at the plasma membrane with H-RasG12V. (Scale bar: 10 μm.) (C) Membrane recruitment of GFP-Grp1PH in living cells. HEK cells were transfected with plasmids encoding Gβγ, PI3Kγ (p87/p110γ or p87/p110γCAAX), GFP-Grp1PH, and NF1 as indicated above. Shown are representative, starved (18 h) HEK cells (cLSM slices of 0.8 μm) from 4 independent experiments. Unlike p87/p110γ activity, p87/p110γCAAX activity was unchanged in the presence of NF1. (Scale bar: 10 μm.) The right panels present the quantification of the membrane translocation of GFP-Grp1PH in the corresponding experiments. The data represent the mean ± SD of 4 independent experiments analyzing 24 cells.