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 Pas 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 Pas 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 Pas 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 Pas activation, Investigation of the mechanism of PI 3 kinase action revealed that inhibition of PI 3 kinase does not affect nucleotide exchange on Pas 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 Pas activation. (C) 2000 Elsevier Science Ltd. All rights reserved.