FIGURE 6.

Enhanced Rac2- and Rac2^G12V-stimulated activity of PLCγ₂^R665W as well as enhanced basal activity of PLCγ₂^R665W and PLCγ₂^L845F are prevented by a point mutation of PLCγ₂, F897Q, mediating resistance of the enzyme to stimulation by activated Rac2. A, COS-7 cells were transfected with 500 ng/well empty vector (Co., Control) or vector encoding either wild-type PLCγ₂ (WT), PLCγ₂^F897Q (F897Q), PLCγ₂^R665W (R665W), or PLCγ₂ R665W/F897Q (R665W|F897Q), together with 25 ng/well empty vector or vector encoding Rac2 or Rac2^G12V (left panel). In the right panel, COS-7 cells were transfected with 500 ng/well (from left to right) empty vector (Co.) or vector encoding either wild-type PLCγ₂, PLCγ₂^F897Q, PLCγ₂^R665W, PLCγ₂ R665W/F897Q, PLCγ₂^L845F, or PLCγ₂ L845F/F897Q. Note that the vectors encoding mutant PLCγ₂ were used at the same maximal amount (500 ng/well) to observe the stimulation by wild-type Rac2 (left panel) and enhanced basal activity of the PLCγ₂ mutants (right panel).Twenty four hours after transfection, the cells were incubated for 20 h with myo-[2-³H] inositol, and inositol phosphate formation was determined. B, homogenates from cells functionally analyzed in A were subjected to SDS-PAGE and immunoblotting using an antibody reactive against the c-Myc epitope.