Autoregulation of Class II Alpha PI3K Activity by Its Lipid-Binding PX-C2 Domain Module

Class II phosphoinositide 3-kinases (PI3K-C2) are large multidomain enzymes that control cellular functions ranging from membrane dynamics to cell signaling via synthesis of 3′-phosphorylated phosphoinositides. Activity of the alpha isoform (PI3K-C2α) is associated with endocytosis, angiogenesis, and glucose metabolism. How PI3K-C2α activity is controlled at sites of endocytosis remains largely enigmatic. Here we show that the lipid-binding PX-C2 module unique to class II PI3Ks autoinhibits kinase activity in solution but is essential for full enzymatic activity at PtdIns(4,5)P 2 -rich membranes. Using HDX-MS, we show that the PX-C2 module folds back onto the kinase domain, inhibiting its basal activity. Destabilization of this intramolecular contact increases PI3K-C2α activity in vitro and in cells, leading to accumulation of its lipid product, increased recruitment of the endocytic effector SNX9, and facilitated endocytosis. Our studies uncover a regulatory mechanism in which coincident binding of phosphoinositide substrate and cofactor selectively activate PI3K-C2α at sites of endocytosis. Using recombinantly expressed protein, Wang et al. discovered that the lipid kinase activity of PI3K-C2α is inhibited by its C-terminal lipid-binding domains. Disruption of the inhibitory interface by mutation or lipid engagement of the C terminus with PtdIns(4,5)P 2 stimulates kinase activity, while cellular hyperactivation of PI3K-C2α enhances PtdIns(3,4)P 2 production and endocytosis.