Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation

Abstract

We have reported previously that Ras interacts with the catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase) in a GTP-dependent manner. The affinity of the interaction of Ras·GTP with p85α/p110α is shown here to be ~ 150 nM. The site of interaction on the p110α and β isoforms of PI 3-kinase lies between amino acid residues 133 and 314. A point mutation in this region, K227E, blocks the GTP-dependent interaction of PI 3-kinase p110α with Ras in vitro and the ability of Ras to activate PI 3-kinase in intact cells. In addition, this mutation elevates the basal activity of PI 3-kinase in intact cells, suggesting a direct influence of the Pas binding site on the catalytic activity of PI 3-kinase. Using an in vitro reconstitution assay, it is shown that the interaction of Ras·GTP, but not Ras·GDP, with PI 3-kinase leads to an increase in its enzymatic activity. This stimulation is synergistic with the effect of tyrosine phosphopeptide binding to p85, particularly at suboptimal peptide concentrations. These data show that PI 3-kinase is regulated by a number of mechanisms, and that Ras contributes to the activation of this lipid kinase synergistically with tyrosine kinases.