Figure 13. Unlike the PLAID PLCγ₂ deletion mutant PLC_γ2_Δ20–22, neither APLAID and ibrutinib resistance PLCγ₂ mutant S707Y nor the ibrutinib resistance PLCγ₂ mutant R665W are activated by cool temperatures.

(A) left panel, COS-7 cells were transfected with empty vector (Control) or 500 ng/well of vector encoding either wild-type PLCγ₂ (WT) or PLCγ₂R665W (R665W), or 100 ng/well of vector encoding either PLCγ₂S707Y (S707Y) or PLCγ₂Δ20-22 (Δ20-22). Twenty-four hours after transfection, the cells were incubated for 18 h with myo-[2-³H]inositol at the indicated temperatures and inositol phosphate formation was then determined. The levels of inositol phosphate formation in control cells (Control) and cells expressing wild-type PLCγ₂ (WT), PLCγ₂R665W (R665W), or PLCγ₂S707Y (S707Y) are shown in expanded scale on the center panel. Right panel, determination of the 10-degree temperature coefficients, Q₁₀. The data shown in panel A on the cool temperature responses of cells expressing wild-type PLCγ₂ (WT), PLCγ₂Δ20–22 (Δ20–22), PLCγ₂R665W (R665W), and PLCγ₂S707Y (S707Y) was taken to determine the Q₁₀ values of these responses as detailed in Experimental Procedures. The individual temperatures T_i were plotted against, with the interpolated maximum activity of PLCγ₂Δ20-22 at 32° C chosen as the reference activity A_ref and reference temperature, T_ref, respectively. The data of the linear components was analyzed by non-linear least square curve fitting to a polynominal first order (straight line) equation. The slope of the curve of PLCγ₂Δ20–22 between 27° C and 31° C was not significantly different by global curve fitting from the slope obtained for wild-type PLCγ₂ (P = 0.9356 and P = 0.2121, respectively). The slopes of the curves of PLCγ₂S707Y and PLCγ₂R665W were not significantly different by global curve fitting from each other but different from the slope obtained for PLCγ₂Δ20–22 between 27° C and 31° C and wild-type PLCγ₂ (P = 0.9356 and P = 0.2121, respectively). (B) Unlike PLCγ₂Δ20–22, where a decrease in the incubation temperature resulted in a progressive loss of the Rac2^G12V stimulatory activity, the mutant S707Y is also stimulated by Rac2^G12V at low temperatures. COS-7 cells were transfected with 50 ng/well of vector encoding either PLCγ₂S707Y (S707Y) or PLCγ₂Δ20–22 (Δ20–22) and 25 ng/well of empty vector or vector encoding Rac2^G12V. Twenty four hours after transfection, the cells were incubated for 18 h with myo-[2-³H]inositol at the indicated temperatures and inositol phosphate formation was then determined.