FIGURE 7.

Enhanced basal activities of PLCγ₂^R665W and PLCγ₂^L845F are specifically reduced by the Rac inhibitor EHT 1864. A, COS-7 cells were transfected with 500 ng/well empty vector (Control) or vector encoding wild-type PLCγ₂ (γ₂WT), PLCγ₂^R665W (γ₂R665W), PLCγ₂^L845F (γ₂L845F), or 30 ng/well of vector encoding PLCδ₁Δ44 (δ₁Δ44). Twenty four hours after transfection, the cells were incubated for 18 h with myo-[2-³H]inositol in the absence (Ø) or presence of 5 μm EHT 1864 or 5 μm of its inactive congener EHT 4063, followed by determination of inositol phosphate formation (left panel). The inset shows the structural formulas of EHT 1864, 5-(5-(7-(trifluoromethyl)quinolin-4-ylthio)pentyloxy)-2-(morpholino-methyl)-4H-pyran-4-one dihydrochloride and EHT 4063, 5-(5-(quinazolin-4-yloxy)pentyl-oxy)-2-((4-methylpiperazin-1-yl)methyl)-4H-pyran-4-one. In the right panel, COS-7 cells were transfected with 500 ng/well empty vector (Control) or vector encoding either wild-type PLCγ₂ (γ₂WT) or PLCγ₂^L845F (γ₂L845F). Twenty four hours after transfection, the cells were incubated for 18 h with myo-[2-³H]inositol in the absence or presence of EHT 1864 at the concentrations indicated at the abscissa. The IC₅₀ value of EHT 1864 for the inhibition of mutant PLCγ₂ activity obtained by non-linear curve fitting is shown above the graph in nanomolar. B, homogenates from cells functionally analyzed in A were subjected to SDS-PAGE and immunoblotting using an antibody reactive against the c-Myc epitope present on wild-type and mutant PLCγ₂ as well as on PLCδ₁Δ44.