Rat
osteosarcoma 17/2.8 cells (Ros 17/2.8 cells) were labeled with [32P]PO4(2-), and their levels of
inositol lipids were determined after stimulation with
thrombin.
Thrombin stimulated a
pertussis toxin-sensitive rapid accumulation of phosphatidylinositol-3,4,5-trisphosphate [
PtdIns(3,4,5)P3] with lesser increases in levels of phosphatidylinositol-3,4-bisphosphate [
PtdIns(3,4)P2] and phosphatidylinositol-3-phosphate [
PtdIns3P] that were slower in onset. Ros 17/2.8 cell homogenates contained
phosphatase activities that hydrolyzed
PtdIns(3,4,5)P3 to
PtdIns(3,4)P2 and
PtdIns3P. Phosphoinositide-3-kinase activity was determined in Ros 17/2.8 cell homogenates using exogenously provided
PtdIns(4,5)P2. Guanosine-5'-3-O-(thio)triphosphate caused an approximately 3-fold increase in phosphoinositide-3-kinase activity in a manner that was blocked by high concentrations of guanosine-5'-2-O-(thio)diphosphate. Purified bovine brain
G protein beta gamma subunits also increased phosphoinositide-3-kinase activity modestly in Ros 17/2.8 cell homogenates. Ros 17/2.8 cell homogenates contained
phosphatase activities that sequentially dephosphorylated
PtdIns(3,4,5)P3 to
PtdIns(3,4)P2 and
PtdIns3P. Two peaks of phosphoinositide-3-kinase activity were resolved by
anion exchange chromatography of a Ros 17/2.8 cell cytosolic extract. The later elution of these was selectively activated by beta gamma subunits (16-fold activation with 16 microM beta gamma subunits). Half-maximal effects of the beta gamma subunits were observed at a concentration of 0.6 microM, and activation was blocked by preincubation of the beta gamma subunits with an excess of recombinant Gi alpha 2. beta gamma Subunits did not activate the p85 alpha/p110 beta form of phosphoinositide-3-kinase purified from sf9 cells after expression with the use of baculovirus vectors.