The pivotal role of
phosphatidylinositol 3-kinase (PI 3-kinase) in signal transduction has been well established in recent years. Receptor-regulated forms of
PI 3-kinase are thought to phosphorylate
phosphatidylinositol 4,5-bisphosphate (
PtdIns(4,5)P2) at the 3-position of the
inositol ring to give the putative
lipid second messenger,
phosphatidylinositol 3,4,5-trisphosphate (
PtdIns(3,4, 5)P3). Cellular levels of
PtdIns(3,4,5)P3 are currently measured by time-consuming procedures involving radiolabeling with high levels of 32PO4, extraction, and multiple chromatography steps. To avoid these lengthy and hazardous procedures, many laboratories prefer to assay
PI 3-kinase activity in
cell extracts and/or appropriate immunoprecipitates. Such approaches are not readily applied to measurements of
PtdIns(3,4,5)P3 in extracts of animal tissues. Moreover, they can be misleading since the association of PI 3-kinases in molecular complexes is not necessarily correlated with the
enzyme's activity state. Direct measurements of
PtdIns(3,4,5)P3 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)P3 metabolism. We now report a simple, reproducible
isotope dilution assay which detects
PtdIns(3,4,5)P3 at subpicomole sensitivity, suitable for measurements of both basal and stimulated levels of
PtdIns(3,4,5)P3 obtained from samples containing approximately 1 mg of cellular
protein. Total
lipid extracts, containing
PtdIns(3,4,5)P3, are first subjected to alkaline hydrolysis which results in the release of the polar head group
Ins(1,3,4,5)P4. The latter is measured by its ability to displace [32P]
Ins(1,3,4,5)P4 from a highly specific
binding protein present in cerebellar membrane preparations. We show that this assay solely detects
PtdIns(3,4,5)P3 and does not suffer from interference by other compounds generated after alkaline hydrolysis of total cellular
lipids. Measurements on a wide range of cells, including rat-1 fibroblasts, 1321N1
astrocytoma cells, HEK 293 cells, and rat adipocytes, show
wortmannin-sensitive increased levels of
PtdIns(3,4,5)P3 upon stimulation with appropriate agonists. The enhanced utility of this procedure is further demonstrated by measurements of
PtdIns(3,4,5)P3 levels in tissue derived from whole animals. Specifically, we show that stimulation with
insulin increases
PtdIns(3,4,5)P3 levels in rat skeletal muscle in vivo with a time course which parallels the activation of
protein kinase B in the same samples.