D-3 phosphoinositide metabolism in cells treated with platelet-derived growth factor

Despite extensive analysis of phosphoinositide 3-hydroxykinases (PI 3-kinases) at the molecular level, comparatively little is known about the mechanisms by which products of these enzymes exert their expected second-messenger functions. This study examines the metabolism of D-3 phosphoinositides in mouse Ph-N2 fibroblasts lacking the platelet-derived growth factor (PDGF) α-receptor. Treatment of these cultures with BB PDGF, but not AA PDGF, resulted in transient activation of PI 3-kinase activity measured in vitro. Treatment of myo-[³H]inositol-labelled Ph-N2 cells with BB PDGF resulted in the rapid induction of PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ and, to a smaller extent, PtdIns3P. The appearance of PtdIns(3,4,5)P₃ preceded that of PtdIns(3,4)P₂ and PtdIns3P after the addition of PDGF, suggesting that PtdIns(4,5)P₂ is the preferred substrate of the agonist-stimulated PI 3-kinase in intact cells. Treatment of both resting and PDGF-stimulated cells with the fungal metabolite wortmannin resulted in pronounced, selective effects on the levels of all D-3 phosphoinositides. Kinetic studies with this PI 3-kinase inhibitor revealed the presence of at least two independent routes for the biosynthesis of D-3 phosphoinositides in PDGF-treated cells.