FIGURE 11.

PLCγ₂ ibrutinib resistance mutations R665W and L845F synergize to enhance basal enzyme activity and to sensitize the enzyme to stimulation by activated Vav1 and Rac2. A, COS-7 cells were transfected either with 500 ng/well empty vector (Co., control) or increasing amounts (15, 50, 150, and 500 ng/well) of vector encoding either PLCγ₂ (WT), PLCγ₂^R665W (R665W), PLCγ₂^L845F (L845F), or the compound mutant PLCγ₂^R665W/L845F (R665W/L845F). B, COS-7 cells were transfected with 15 ng/well either empty vector (Co., Control) or vector encoding either PLCγ₂ (WT), PLCγ₂^R665W (R665W), PLCγ₂^L845F (L845F), or the compound mutant PLCγ₂^R665W/L845F (R665W/L845F), together with 25 ng/well empty vector or vector encoding Rac2 or Rac2^G12V. C, left panel, COS-7 cells were transfected as indicated with 15 ng/well vector encoding either PLCγ₂^R665W (R665W), PLCγ₂^L845F (L845F), or PLCγ₂^R665W/L845F (R665W/L845F) and increasing amounts of vector encoding Rac2^G12V. Right panel, COS-7 cells were transfected with 50 ng/well vector encoding either PLCγ₂^R665W (R665W), PLCγ₂^L845F (L845F), or PLCγ₂^R665W/L845F (R665W/L845F) and increasing amounts of vector encoding Vav1ΔN. The ED₅₀ values of vector encoding Rac2^G12V or Vav1ΔN for the stimulation of mutant PLCγ₂ activity obtained by non-linear curve fitting are shown above the graphs in nanograms/well. In all panels, the cells were incubated 24 h after transfection for 20 h with myo-[2-³H]inositol, and inositol phosphate formation was determined. D, upper panel, 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 or antibody reactive against β-actin. Lower panel, homogenates from cells functionally analyzed in B were subjected to SDS-PAGE and immunoblotting using an antiserum reactive against Rac2.