FIGURE 5.

C1 domain is required for PMT activity. A, NFAT- and SRE-luciferase reporter assays in 293T cells expressing either C-PMT wild type (WT) or C-PMT ΔC1(4H). 293T cells were co-transfected with the plasmids for C-PMTs and either NFAT- or SRE-luciferase. The luciferase activity in the cells was determined 48 h after the transfection as described under “Experimental Procedures.” B, stimulation of phospholipase C activity by C-PMT. Swiss3T3 cells, which had been labeled with myo-[³H]inositol for 48 h, were treated with the HVJ envelope vector containing 1.25 or 12.5 μg of C-PMT (residues 569–1285), or C-PMT ΔC1(4H) (residues 671–1285), and intracellular [³H]inositol phosphates, which are the products of the enzymatic action of phospholipase C, were measured as described under “Experimental Procedures.” Each data point represents the mean of at least triplicate measurements; the error bar shows S.D. Representative results from three independent experiments are shown. C, SDS-PAGE of purified recombinant C-PMT and C-PMT ΔC1(4H). One microgram of each recombinant protein was applied to a lane and visualized by Coomassie Brilliant Blue staining.