A NOVEL, RAPID, AND HIGHLY SENSITIVE MASS ASSAY FOR PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE (PTDINS(3,4,5)P-3) AND ITS APPLICATION TO MEASURE INSULIN-STIMULATED PTDINS(3,4,5)P-3 PRODUCTION IN RAT SKELETAL-MUSCLE IN-VIVO

The pivotal role of phosphatidylinositol S-kinase (PI 3-kinase) in sig nal transduction has been well established in recent years. Receptor-r egulated forms of PI 3-kinase are thought to phosphorylate phosphatidy linositol 4,5-bisphosphate (PtdIns(4,5)P-2) at the 3-position of the i nositol ring to give the putative lipid second messenger, phosphatidyl inositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3). Cellular levels of Pt dIns(5,4,5)P-3 are currently measured by time-consuming procedures inv olving radiolabeling with high levels of (PO4)-P-32, extraction, and m ultiple chromatography steps. To avoid these lengthy and hazardous pro cedures, many laboratories prefer to assay PI 3-kinase activity in cel l extracts and/or appropriate immunoprecipitates. Such approaches are not readily applied to measurements of PtdIns(3,4,5)P-3 in extracts of animal tissues. Moreover, they can be misleading since the associatio n of PI 3-kinases in molecular complexes is not necessarily correlated with the enzyme's activity state, Direct measurements of PtdIns(3,4,5 )P-3 would also be desirable since its concentration may be subject to additional control mechanisms such as activation or inhibition of the phosphatases responsible for PtdIns(3,4,5)P-3 metabolism. We now repo rt a simple, reproducible isotope dilution assay which detects PtdIns( 3,4,5)P-3 at subpicomole sensitivity, suitable for measurements of bot h basal and stimulated levels of PtdIns(3,4,5)P-3 obtained from sample s containing approximately 1 mg of cellular protein. Total lipid extra cts, containing PtdIns(3,4,5)P-3, are first subjected to alkaline hydr olysis which results ill the release of the polar head group Ins(1,3,4 ,5)P-4. The latter is measured by its ability to displace [P-32]Ins(1, 3,4,5)P-4 from a highly specific binding protein present in cerebellar membrane preparations. We show that this assay solely detects PtdIns( 3,4,5)P-3 and does not suffer from interference by other compounds gen erated after alkaline hydrolysis of total cellular lipids, Measurement s on a wide range of cells, including rat-1 fibroblasts, 1321N1 astroc ytoma cells, HEK 293 cells, and rat adipocytes, show wortmannin-sensit ive increased levels of PtdIns(3,4,5)P-3 upon stimulation with appropr iate agonists. The enhanced utility of this procedure is further demon strated by measurements of PtdIns(3,4,5)P-3 levels in tissue derived f rom whole animals. Specifically, we show that stimulation with insulin increases PtdIns(3,4,5)P-3 levels in rat skeletal muscle in vivo with a time course which parallels the activation of protein kinase B in t he same samples.