FIGURE 3.

Lipid kinase activities of phospholipid vesicle-associated class I_B PI3Ks. A, an experimental model of phospholipid vesicles recruiting equal amounts of PI3Kγ variants. To obtain equal recruitment of PI3Kγ variants, we designed phospholipid vesicles where p110γ and p87–p110γ were recruited via interaction with anionic phosphatidylserine (PS) and p101–p110γ via interaction with Gβ₁γ₂. B and C, lipid kinase activities of PI3Kγ variants equally associated with phospholipid vesicles in the presence and absence of 100 nm Gβ₁γ₂ were examined as described previously (35). To achieve equal association of PI3Kγ variants, phospholipid vesicles were prepared and incubated with 32 nm concentrations of enzyme as detailed under “Experimental Procedures.” The upper panels show representative immunoblots of sedimented phospholipid vesicles probed with specific anti-p110γ antiserum (top rows) and autoradiographs demonstrating formation of ³²P-labeled PtdIns(3,4,5)P₃ under identical experimental conditions (bottom rows). The histograms represent statistical evaluations (mean values ± S.E.) of four (A) or three (B) independent experiments. p101–p110γ displays higher basal and Gβ₁γ₂-induced lipid kinase activities as compared with p110γ and p87–p110γ, whereas the association of each PI3Kγ variants with phospholipid vesicles was comparable.