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

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Abstract
Phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P3] 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)P3 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)P3 in an on-section labeling approach by quantitative immunogold electron microscopy. Swiss 3T3 cells contained low levels of PtdIns(3,4,5)P3 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)P3 was observed in U87MG cells, which lack the PtdIns(3,4,5)P3 phosphatase, PTEN. Re-expression of PTEN into U87MG cells ablated plasma membrane PtdIns(3,4,5)P3, 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.