Affiliation:
1. Center for Cell Signaling, Salt Lake City, Utah
2. Echelon Research Laboratories, Salt Lake City, Utah
3. Department of Medicinal Chemistry, The University of Utah, Salt Lake City, Utah
4. Department of Biology, Utah State University, Logan, Utah
Abstract
Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] is a second messenger produced in response to agonist stimulation. Traditionally, visualization of phosphoinositide polyphosphates (PtdInsPn) in living cells is accomplished using chimeric green fluorescent protein (GFP)-pleckstrin homology (PH) domain proteins, while PtdInsPnquantitation is accomplished by extraction and separation of radiolabeled cellular PtdInsPns. Here we describe preparation of a covalent protein-PtdIns(3,4,5)P3immunogen, characterization of binding selectivity of an anti-PtdIns(3,4,5)P3IgM, and immunodetection of PtdIns(3,4,5)P3in stimulated mammalian cells. This antibody has greater than three orders of magnitude selectivity for binding PtdIns(3,4,5)P3relative to its precursor, phosphatidylinositol 4,5-bis-phosphate (PtdIns(4,5)P2), and is therefore optimal for studies of cell function. The immunodetection in platelet-derived growth factor (PDGF)-stimulated NIH 3T3 cells was bench-marked against HPLC analysis of [3H]-myo-inositol-labeled cellular PtdInsPns. In addition, the changes in subcellular amounts and localizations of both PtdIns(3,4,5)P3and PtdIns(4,5)P2in stimulated NIH 3T3 fibroblasts and human neutrophils were observed by immunofluorescence. In insulin- or PDGF-stimulated fibroblasts, PtdIns(3,4,5)P3levels increased in the cytoplasm, peaking at 10 min. In contrast, increases in the PtdIns(4,5)P2levels were detected in nuclei, corresponding to the production of new substrate following depletion by phosphoinositide (PI) 3-kinase.