Localization of agonist-sensitive PtdIns(3,4,5)P3 reveals a nuclear pool that is insensitive to PTEN expression

Phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5) P 3 ] is a lipid second messenger, produced by Type I phosphoinositide 3-kinases (PI 3-kinases), which mediates intracellular responses to many growth factors. Although PI 3-kinases are implicated in events at both the plasma membrane and intracellular sites, including the nucleus, direct evidence for the occurrence of PtdIns(3,4,5) P 3 at non-plasma membrane locations is limited. We made use of the pleckstrin homology (PH) domain of general receptor for phosphoinositides (Grp1) to detect PtdIns(3,4,5) P 3 in an on-section labeling approach by quantitative immunogold electron microscopy. Swiss 3T3 cells contained low levels of PtdIns(3,4,5) P 3 that increased up to 15-fold upon stimulation with platelet-derived growth factor (PDGF). The signal was sensitive to PI 3-kinase inhibitors and present mainly at plasma membranes, including lamellipodia, and in a surprisingly large pool within the nuclear matrix. Comparatively little labeling was observed in endomembranes. A similar distribution of PtdIns(3,4,5) P 3 was observed in U87MG cells, which lack the PtdIns(3,4,5) P 3 phosphatase, PTEN. Re-expression of PTEN into U87MG cells ablated plasma membrane PtdIns(3,4,5) P 3 , but not the nuclear pool of this lipid even when PTEN was targeted to nuclei. These data have important implications for the versatility of PI 3-kinase signaling and for the proposed functions of PTEN in the nucleus.