A monoclonal antibody to visualize Ptdlns(3,4,5)P3 in cells

Phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P-3] is a second messenger produced in response to agonist stimulation. Traditionally, visualization of phosphoinositide polyphosphates (PtdInsP(n)) in living cells is accomplished using chimeric green fluorescent protein (GFP)-pleckstrin homology (PH) domain proteins, while PtdInsP(n), quantitation is accomplished by extraction and separation of radiolabeled cellular PtdInsP(n)s Here we describe preparation of a covalent protein-PtdIns(3,4,5)P3 immunogen, characterization of binding selectivity of an anti-PtdIns(3,4,5)P-3 IgM, and immunodetection of PtdIns(3,4,5)P-3 in stimulated mammalian cells. This antibody has greater than three orders of magnitude selectivity for binding PtdIns(3,4,5)P-3 relative to its precursor, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2), and is therefore optimal for studies of cell function. The immunodetection in plate let-derived growth factor (PDGF)-stimulated NIH 3T3 cells was benchmarked against HPLC analysis of [H-3]-myo-inositol-labeled cellular PtdInSP(n)s. In addition, the changes in subcellular amounts and localizations of both PtdInS(3,4,5)P-3 and PtdIns(4,5)P-2 in stimulated NIH 3T3 fibroblasts and human neutrophils were observed by immunofluorescence. In insulin- or PDGF-stimulated fibroblasts, PtdIns(3,4,5)P-3 levels increased in the cytoplasm, peaking at 10 min. In contrast, increases in the PtdIns(4,5)P-2 levels were detected in nuclei, corresponding to the production of new substrate following depletion by phosphoinositide (PI) 3-kinase.