Figure 5.

Single-molecule and bulk studies of the effect of lipid composition on the kinase activities of membrane-bound PI3Kα and PKCα. (A–C) Representative data from a new, to our knowledge, single-molecule TIRF assay of PI3K lipid kinase activity at 21.5°C ± 0.5°C on standard PE/PS/DAG/PIP₂ supported bilayers, where the fluorescent high-affinity PIP₃-sensor GRP1 PH protein was used to tag and detect each molecule of product PIP₃ lipid (Materials and Methods). (A) Raw TIRF images show accumulation of the PIP₃ sensor on the supported bilayer as the reaction proceeds in the absence or presence of a PI3K activator (pY₂ peptide). (B) Increase in the number of total PIP₃ product molecules with time as the PI3K lipid kinase reaction proceeds on supported bilayers of different lipid compositions (Table 2). Again, the negative control lacking the pY₂ peptide activator shows minimal activity. (C) Specific lipid kinase activity per PI3Kα molecule for each bilayer composition, determined from the ratio of total lipid kinase activity to the density of bound kinase on the membrane surface (Fig. 4C). (D) Relative specific kinase activity of PKCα for each bilayer composition, determined from the ratio of total bulk PKC kinase activity (Materials and Methods) to the density of bound kinase on the membrane surface (Fig. 4A). Single-molecule TIRF conditions as detailed in the Fig. 4 legend.