Differential Regulation of Phosphoinositide 3-Kinase Adapter Subunit Variants by Insulin in Human Skeletal Muscle

The role of phosphoinositide 3-kinase (PI 3-kinase) in insulin signaling was evaluated in human skeletal muscle. Insulin stimulated both antiphosphotyrosine-precipitable PI 3-kinase activity and 3-O-methylglucose transport in isolated skeletal muscle (both ≈2–3-fold). Insulin stimulation of 3-O-methylglucose transport was inhibited by the PI 3-kinase inhibitor LY294002 (IC50 = 2.5 μm). The PI 3-kinase adapter subunits were purified from muscle lysates using phosphopeptide beads based on the Tyr-751 region of the platelet-derived growth factor receptor. Immunoblotting of the material adsorbed onto the phosphopeptide beads revealed the presence of p85α, p85β, p55PIK/p55γ, and p50 adapter subunit isoforms. In addition, p85α-NSH2 antibodies recognized four adapter subunit variants of 54, 53, 48, and 46 kDa, the latter corresponding to the p50 splice variant. Serial immunoprecipitations demonstrated that these four proteins were associated with a large proportion of the total PI 3-kinase activity immunoprecipitated by p85α-NSH2 domain antibodies. Antibodies to p85β, p55PIK/p55γ, and the p50 adapter subunit also immunoprecipitated PI 3-kinase activity from human muscle lysates. A large proportion of the total cellular pool of the 53-kDa variant, p50, and p55PIK was present in antiphosphotyrosine immunoprecipitates from unstimulated muscle, whereas these immunoprecipitates contained only a very small proportion of the cellular pool of p85α, p85β, and the 48-kDa variant. Insulin greatly increased the levels of the 48-kDa variant in antiphosphotyrosine immunoprecipitates and caused smaller -fold increases in the levels of p85α, p85β, and the 53-kDa variant. The levels of p50 and p55PIK were not significantly changed. These properties indicate mechanisms by which specificity is achieved in the PI 3-kinase signaling system.