Cloning and mutagenesis of the p110α subunit of human phosphoinositide 3′-hydroxykinase

Abstract

Activation of phosphoinositide 3′-hydroxykinase (PI3K) is required for mitogenic signal transduction by several growth factors and oncogenes. PI3K 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 p110α catalytic subunit of human PI3K. This clone is highly homologous (>99% amino acid identity) to bovine brain p110α, but contains 10 amino acid differences from the human p110α 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 PI3K mutants with virtually complete loss of phosphoinositide phosphorylating activity. Expression of the wild-type p110α 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 p110α mutants as well as the p85α subunit of PI3K were inactive in the fos assay. These studies suggest that the mitogenic signal transduction pathway mediated by PI3K is dependent upon the enzymatic activity of the p110α subunit of PI3K.