Regulation of phosphoinositide-3-kinase by G protein βγ subunits in a rat osteosarcoma cell line

A. J. Morris, S. A. Rudge, C. E. Mahlum, J. M. Jenco

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)532-539
Number of pages8
JournalMolecular Pharmacology
Volume48
Issue number3
StatePublished - Sep 1995

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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