The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo

We have tested the binding specificities of the pleckstrin homology (PH) do mains of protein kinase B (PKB) and GRP1 (general receptor for phosphoinosi tides-l), expressed as green fluorescent protein (GFP) fusion proteins [PH( PKB)GFP and PH(GRP1)GFP respectively] in HEK 293 cells and Swiss 3T3 cells, using confocal microscopy. Stimulation of HEK 293 cells with insulin cause d a small, but sustained, increase in PtdIns(3,4,5)P-3 levels, detected usi ng a radioligand displacement assay, which was mirrored by the translocatio n of PH(PKB)GFP and PH(GRP1)GFP from the cytosol to the plasma membrane of live, transfected cells. Similar results were obtained using Swiss 3T3 cell s stimulated with platelet-derived growth factor (PDGF) and expressing eith er PH(PKB)GFP or PH(GRP1)GFP. Biochemical analyses confirmed the accumulati on of both PtdIns(3,4,5)P-3 and PtdIns(3,4)P-2 in response to PDGF, but onl y the latter was present at increased levels in Swiss 3T3 cells 30 min afte r an oxidative stress (1 mM H2O2). Concomitantly, only PH(PKB)GFP, and not PH(GRP1)GFP, was localized at plasma membranes after 30 min of treatment wi th H2O2. The fusion proteins appear accurately to report the spatial and te mporal distribution of PtdIns(3,4,5)P-3 and Ptdins(3,4)P-2 in intact cells. These results establish the lipid selectivity of these PH domains in vivo, and further emphasize the overlapping, but distinct, second messenger role s of PtdIns(3,4,5)P-3 and PtdIns(3,4)P-2.