A monoclonal antibody to visualize PtdIns(3,4,5)P(3) 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)P(3) 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 platelet-derived growth factor (PDGF)-stimulated NIH 3T3 cells was benchmarked against HPLC analysis of [3H]-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.
Chen
Riyan
R
Center for Cell Signaling, Salt Lake City, Utah 84108, USA.
Kang
Veronica H
VH
Chen
Jian
J
Shope
Joseph C
JC
Torabinejad
Javad
J
DeWald
Daryll B
DB
Prestwich
Glenn D
GD
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000378:metabolism*; D000918:Antibody Specificity; D001201:Ascites / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D018375:Neutrophil Activation; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis* / Q000276:immunology; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007328:Insulin; D008024:Ligands; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010982:Platelet-Derived Growth Factor; C060974:phosphatidylinositol 3,4,5-triphosphate