Activation of phosphoinositide 3'-hydroxykinase (P13K) is required for mitogenic signal transduction by several growth factors and oncogenes. P13K is a heterodimer consisting of a p85 regulatory subunit and a p110 catalytic subunit. In the current study, we report the cloning and characterization of the p110 alpha catalytic subunit of human P13K. This clone is highly homologous (> 99% amino acid identity) to bovine brain p110 alpha, but contains 10 amino acid differences from the human p110 alpha sequence previously reported. Comparison of this sequence with known Ser/Thr kinases and p110 homologs highlighted several conserved residues within the putative kinase domain. Mutational analysis of these residues (Asp915, (Asp933 + Phe934)) yielded P13K mutants with virtually complete loss of phosphoinositide phosphorylating activity. Expression of the wild-type p110 alpha protein in CHO cells is sufficient to activate the serum response element derived from the promoter of c-fos, an immediate early gene product. In contrast, the catalytically impaired p110 alpha mutants as well as the p85 alpha subunit of P13K were inactive in the fos assay. These studies suggest that the mitogenic signal transduction pathway mediated by P13K is dependent upon the enzymatic activity of the p110 alpha subunit of P13K.

Phosphorylation of phosphoinositides by phosphoinositide 3'-hydroxykinase (PI3K) occurs at a lipid/water interface. We have determined that highly purified recombinant human P13K binds tightly to vesicle interfaces composed primarily of phosphatidylinositol (PI) or 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM). The rate of desorption of PI3K from the vesicle interface is slow and does not significantly affect the observed product formation kinetics. Observations which demonstrate that PI3K is tightly bound to the vesicle lipid/water interface include the following: (1) product formation plateaus rapidly, even in the presence of active enzyme and excess substrate; (2) total product formation is proportional to the amount of PI3K; (3) initial product formation rates are unaffected by bulk lipid concentration but are dependent on the interfacial substrate concentration; and (4) PI3K partitions with lipid vesicles in sedimentation gradients. This enzymatic profile has been referred to as catalysis in the "scooting" mode (Berg et al., 1991). A kinetic analysis of PI3K catalysis in the scooting mode is presented. The interfacial Km,app for PI was determined to be approximately 6.0 mol % in PI/DMPM vesicles. The ratio of specificity constants (kcat/Km) for PI, phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-diphosphate (PIP2) utilization was determined to be near unity. These results provide a rigorous enzymological framework for the kinetic analysis of PI3K inhibitors.