Kinetic and structural analyses reveal residues in phosphoinositide 3-kinase α that are critical for catalysis and substrate recognition

Abstract

Phosphoinositide 3-kinases (PI3Ks) are ubiquitous lipid kinases that activate signaling cascades controlling cell survival, proliferation, protein synthesis, and vesicle trafficking. PI3Ks have dual kinase specificity: a lipid kinase activity that phosphorylates the 3-hydroxyl of phosphoinositides and a protein-kinase activity that includes autophosphorylation. Despite the wealth of biochemical and structural information on PI3K, little is known about the identity and roles of individual active-site residues in catalysis. To close this gap, we explored the roles of residues of the catalytic domain and the regulatory subunit of human PI3K in lipid and protein phosphorylation. Using site-directed mutagenesis, kinetic assays, and quantitative mass spectrometry, we precisely mapped key residues involved in substrate recognition and catalysis by PI3K. Our results revealed that Lys-776, located in the P-loop of PI3K, is essential for the recognition of lipid and ATP substrates and also plays an important role in PI3K autophosphorylation. Replacement of the residues His-936 and His-917 in the activation and catalytic loops, respectively, with alanine dramatically changed PI3K kinetics. Although H936A inactivated the lipid kinase activity without affecting autophosphorylation, H917A abolished both the lipid and protein kinase activities of PI3K. On the basis of these kinetic and structural analyses, we propose possible mechanistic roles of these critical residues in PI3K catalysis.