Regulation of the p85/p110 Phosphatidylinositol 3′-Kinase: Stabilization and Inhibition of the p110α Catalytic Subunit by the p85 Regulatory Subunit

We propose a novel model for the regulation of the p85/p110␣ phosphatidylinositol 3-kinase.In insect cells, the p110␣ catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit.Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110␣ is reduced by 65 to 85% upon in vitro reconstitution with p85.Incubation of p110␣/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110␣.These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110␣ dimers by inducing a transition from an inhibited to a disinhibited state.In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15-to 20-fold by coexpression with p85.However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110␣ or by the growth of the HEK 293T cells at 30°C.These nonspecific interventions mimicked the effects of p85 on p110␣, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation.This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110.Furthermore, p85 protected p110 from thermal inactivation in vitro.Importantly, when we examined the effect of p85 on GST-p110␣ in mammalian cells at 30°C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110␣.Thus, we have experimentally distinguished two effects of p85 on p110␣: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity.Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110␣ is both stabilized and inhibited by dimerization with p85.