TY - JOUR
T1 - Regulation of phosphoinositide-3-kinase by G protein βγ subunits in a rat osteosarcoma cell line
AU - Morris, A. J.
AU - Rudge, S. A.
AU - Mahlum, C. E.
AU - Jenco, J. M.
PY - 1995/9
Y1 - 1995/9
N2 - Rat osteosarcoma 17/2.8 cells (Ros 17/2.8 cells) were labeled with [32P]PO42-, 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 βγ 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 βγ subunits (16-fold activation with 16 μM βγ subunits). Half-maximal effects of the βγ subunits were observed at a concentration of 0.6 μM, and activation was blocked by preincubation of the βγ subunits with an excess of recombinant G(iα2). βγ Subunits did not activate the p85α/p110β form of phosphoinositide-3-kinase purified from sf9 cells after expression with the use of baculovirus vectors.
AB - Rat osteosarcoma 17/2.8 cells (Ros 17/2.8 cells) were labeled with [32P]PO42-, 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 βγ 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 βγ subunits (16-fold activation with 16 μM βγ subunits). Half-maximal effects of the βγ subunits were observed at a concentration of 0.6 μM, and activation was blocked by preincubation of the βγ subunits with an excess of recombinant G(iα2). βγ Subunits did not activate the p85α/p110β form of phosphoinositide-3-kinase purified from sf9 cells after expression with the use of baculovirus vectors.
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M3 - Article
C2 - 7565635
AN - SCOPUS:0029155815
SN - 0026-895X
VL - 48
SP - 532
EP - 539
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 3
ER -