Cloning, expression, purification and characterization of the human class Ia phosphoinositide 3-kinase isoforms.

3524 The Class I Phosphoinositide 3 –Kinases (PI3Ks) are lipid kinases that phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositides. Although closely related, experimental evidence suggests that the four Class I PI3Ks may be functionally distinct. In order to identify their unique kinetic parameters, the three human Class Ia PI3K (α, β, δ) p110 catalytic domains were cloned and co-expressed with the p85α regulatory domain in Sf9 cells. None of the p110 subunits were successfully expressed in the absence of p85α. Successful expression and purification of each p85α/p110 protein required using an excess of the p110 vector over the p85 vector during co-infection of Sf9 cells. Proteins were purified as the p85α/p110 complex by nickel affinity chromatography through an N-terminal His-tag on the p110 subunit using an imidazole gradient. The identity of each purified isoform was confirmed by mass spectral analysis and immunoblotting. The kinetics of the three p85α/p110 proteins, the Class Ib p110 γ catalytic domain, and bovine PI3K were investigated using phosphatidylinositol 4,5-bisphosphate (PIP2) as the substrate in a PIP2/Phosphatidylserine (PS) liposome. All five enzymes exhibited reaction velocities that were dependent on the surface concentration of PIP2. The maximal activity for each human isoform was obtained at PIP2 concentrations ranging from 2.5 mol % PIP2 for p110γ to 10 mol % PIP2 for p85α/p110α. The specific activity of p85α/p110α was three to five times higher than the other human isoforms. These kinetic differences may contribute to the unique roles of these isoforms in cells.