ACTIVATION OF PHOSPHOINOSITIDE 3-KINASE BY INTERACTION WITH RAS AND BY POINT MUTATION

We have reported previously that Ras interacts with the catalytic subu nit of phosphoinositide 3-kinase (PI 3-kinase) in a GTP-dependent mann er, The affinity of the interaction of Ras . GTP with p85 alpha/p110 a lpha is shown here to be similar to 150 nM. The site of interaction on the p110 alpha and beta isoforms of PI 3-kinase lies between amino ac id residues 133 and 314. A point mutation in this region, K227E, block s the GTP-dependent interaction of PI 3-kinase p110 alpha 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 Ras binding site on the catalytic activity of PI 3-kinase. Using an in vitro reconstit ution assay, it is shown that the interaction of Ras . GTP, but not Ra s . GDP, with PI 3-kinase leads to an increase in its enzymatic activi ty, This stimulation is synergistic with the effect of tyrosine phosph opeptide binding to p85, particularly at suboptimal peptide concentrat ions. These data show that PI 3-kinase is regulated by a number of mec hanisms, and that Ras contributes to the activation of this lipid kina se synergistically with tyrosine kinases.