A permissive function of phosphoinositide 3-kinase in Ras activation mediated by inhibition of GTPase-activating proteins

The activation status of the guanosine triphosphate (GTP)-binding protein Ras is dictated by the relative intensities of two opposing reactions: the formation of active Ras-GTP complexes, promoted by guanine-nucleotide exchange factors (GEFs), and their conversion to inactive Ras-GDP as a result of the deactivating action of GTPase-activating proteins (GAPs). The relevance of phosphoinositide 3-kinase (PI 3-kinase) to these processes is still unclear. We have investigated the regulation of Ras activation by PI 3-kinase in the myelomonocytic U937 cell line. These cells exhibited basal levels of Ras-GTP, which were suppressed by two PI 3-kinase inhibitors and a dominant-negative PI 3-kinase. In addition, PI 3-kinase inhibition aborted Ras activation by all stimuli tested, including foetal calf serum (FCS) and phorbol 12-myristate 13-acetate (TPA). Significantly, TPA does not activate PI 3-kinase in U937 cells, indicating that PI 3-kinase has a permissive rather than an intermediary role in Ras activation. Investigation of the mechanism of PI 3-kinase action revealed that inhibition of PI 3-kinase does not affect nucleotide exchange on Ras but abrogates Ras-GTP accumulation through an increase in GAP activity. These findings establish blockage of GAP action as the mechanism underlying a permissive function of PI 3-kinase in Ras activation.