ARTICLE |
Correspondence to: Glenn D. Prestwich, Center for Cell Signaling, 421 Wakara Way, Suite 360, Salt Lake City, UT 84108. E-mail: gprestwich@pharm.utah.edu
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Summary |
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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 PtdInsPn quantitation is accomplished by extraction and separation of radiolabeled cellular PtdInsPns. Here we describe preparation of a covalent proteinPtdIns(3,4,5)P3 immunogen, characterization of binding selectivity of an anti-PtdIns(3,4,5)P3 IgM, and immunodetection of PtdIns(3,4,5)P3 in stimulated mammalian cells. This antibody has greater than three orders of magnitude selectivity for binding PtdIns(3,4,5)P3 relative to its precursor, phosphatidylinositol 4,5-bisphosphate (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 benchmarked 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)P3 and PtdIns(4,5)P2 in stimulated NIH 3T3 fibroblasts and human neutrophils were observed by immunofluorescence. In insulin- or PDGF-stimulated fibroblasts, PtdIns(3,4,5)P3 levels increased in the cytoplasm, peaking at 10 min. In contrast, increases in the PtdIns(4,5)P2 levels were detected in nuclei, corresponding to the production of new substrate following depletion by phosphoinositide (PI) 3-kinase. (J Histochem Cytochem 50:697708, 2002)
Key Words: phosphoinositide, anti-lipid IgM, immunofluorescence, growth factor stimulation, phosphoinositide 3-kinase, photoaffinity labeling, cell signaling, second messenger, neutrophil, fibroblast, nuclear lipids
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Introduction |
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PtdIns(3,4,5)P3 is a crucially important second messenger produced in response to cell stimulation, and a growing number of targets are being identified for this lipid signal (
Membrane-associated PtdIns(3,4,5)P3 can be visualized in individual cells using a GFP fusion of the PtdIns(3,4,5)P3-specific (
To quantify PtdIns(3,4,5)P3, the most common protocol requires cell stimulation in the presence of -[32P]-ATP, lipid extraction, separation by TLC, and quantification of the PtdIns(3,4,5)P3 produced by fluorography or scintillation counting. This approach is tedious and is limited to bulk analysis of millions of cells. Therefore, although one can detect PI 3-kinase in cells with isoform-selective anti-PI 3-kinase antibodies, there has not been a corresponding antibody to detect the lipid product itself. The availability of a reagent to permit direct determination of the cellular compartmentalization of PtdIns(3,4,5)P3 would provide an important new tool for studying PtdInsPn lipid signaling.
Antibodies against PtdIns(3,4,5)P3 would be indispensable for the study of signal transduction in cells and tissues. Unlike protein antigens, lipids are poorly recognized by antibodies. Therefore, the development of anti-lipid antibodies has progressed slowly. Antibodies against phospholipids have been produced by immunization with liposomes (
For phosphoinositide and inositol derivatives, several research teams have reported antibodies and their uses in ELISA and cell-based studies. The first monoclonal antibody (MAb) specific to PtdIns(4,5)P2 was obtained by immunization with liposomes containing native PtdIns(4,5)P2. This MAb inhibited oncogene-induced mitogenesis (
A different approach was required to convert PtdIns(3,4,5)P3, which appears to be a non-immunogenic hapten, to an immunogenic form. We selected an approach that was modeled on the successful preparation of anti-phosphatidylserine antibodies using an acyl-linked antigen (
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Materials and Methods |
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Synthesis of PtdIns(3,4,5)P3cBSA and PtdIns(3,4,5)P3 Conjugate
sn-1-O-succinoyl-sn-2-O-stearoyl-PtdIns(3,4,5)P3 (PtdIns (3,4,5) P3COOH) was synthesized according to modifications of protocols developed for the corresponding sn-1-aminohexanoyl derivatives (
Determination of Organic Phosphate
To aliquots (10100 µl) of PtdIns(3,4,5)P3cBSA or PtdIns (3,4,5)P3KLH conjugate solution was added 30 µl of 10% Mg(NO3)2, and the material was ashed by heating with a bunsen burner. The tube was allowed to cool and 3 ml of 0.5 N HCl was added, followed by 0.7 ml of the detection mixture (10% ascorbic acid:0.42% ammonium molybdate, 1:6 v/v). A phosphate standard was employed for calibration. The absorbance at 820 nm was measured after 30 min.
Production of the Monoclonal Antibody
Eight female mice (BALB/c) were immunized with 1 mg of PtdIns(3,4,5)P3cBSA conjugate in 1.0 ml of ammonium bicarbonate (pH 8.0) emulsified with an equal volume of Freund's complete adjuvant by multiple IP and SC injections. Booster injections were given at 2-week intervals with the same amounts of PtdIns(3,4,5)P3cBSA prepared with incomplete Freund's adjuvant. After the fourth boost the animals were bled and the antiserum titer was tested by ELISA. Three days after the final booster injection, positive-titer mice were sacrificed, and spleen cells were collected and fused with sp2/0 cells by the standard polyethylene glycol method. Two weeks after fusion the culture supernatants of the hybridomas were examined by ELISA. After cloning three times by limiting dilution, a hydridoma clone named RC6F8 was established. The cells were inoculated into incomplete Freund's adjuvant-primed mice and the resultant ascites fluid was used to prepare purified immunoglobulin according to the instructions of Immunopure IgM purification kit (Pierce).
Enzyme-linked Immunoabsorbent Assay
Ten microliters of 100 µg/ml PtdIns(3,4,5)P3 in 95% ethanol was added to microtiter wells and the ethanol was then evaporated in air, followed by high vacuum for 5 min. The plates were blocked with 5% non-fat milk in TBS (20 mM Tris, 0.15 M NaCl, pH 8.0) at RT for 1 hr. After washing three times with 5% nonfat milk in TBS, 50 µl of anti-PtdIns(3,4,5)P3 antibody RC6F8 in TBS was applied to the wells at RT for 1 h. Wells were washed three times with 5% non-fat milk, and then 50 µl of anti-mouse IgMperoxidase conjugate was added and incubated at RT for 1 hr. Wells were washed four times with TBS, 100 µl of TBM substrate solution (Sigma Chemical; St Louis, MO) was added, and absorbance at 370 nm was measured after 15 min.
Photoaffinity Labeling of RC6F8 Antibody
The purified RC6F8 IgM and a standard commercial mouse IgM (0.68 µM) were incubated with 20 µl of TBS containing 0.1 µCi (0.28 µM) of [3H]-BZDC-triester-PtdIns (3,4,5)P3 (
Measurement of PtdIns(3,4,5)P3 by HPLC
NIH 3T3 fibroblasts were grown to at least 60% confluency in 75-cm2 flasks, labeled, and phosphoinositide levels analyzed essentially as described (
Immunofluorescence (IF) Methodology
NIH 3T3 cells at logarithmic stage on coverslips were serum-starved overnight and stimulated with insulin (100 ng/ml) or PDGF (50 ng/ml). Reactions were stopped by washing the cells with cold TBS, and cells were processed for IF. Cells on glass coverslips were fixed with 2% formaldehyde and then permeabilized with 0.5% Triton X-100 in TBS. After blocking with 10% goat serum in TBS, either RC6F8 MAb ascites (1:50 dilution) or 10F8 (1:5000 dilution) was added and incubated at RT for 1 hr. After washing three times with blocking solution, FITC-labeled anti-mouse IgM (1:2000 dilution) was added and incubated at RT for 1 hr. After the cells were washed three times with deionized water, the fluorescence in the cells was observed using a confocal microscope (BioRad MRC 1024; Richmond, CA) (see below). Purified RC6F8 anti-PtdIns(3,4,5)P3 IgM and 2C11 anti-PtdIns(4,5)P2 IgM are now available from Echelon Research Laboratories.
Cell Stimulation
Neutrophils (500 µl of 5.5 x 106 cells/ml), freshly isolated by centrifugation (
Microscopy
Cells were examined using an inverted microscope (Nikon) and a BioRad laser scanning confocal microscope system (MRC 1024) with Laser Sharp acquisition software. Images of animal cells were collected using a x60 oil immersion objective. Post-acquisition enhancement of images was not performed except for gray-scale conversion to color using Laser Sharp software or Confocal Assistant software.
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Results |
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Preparation of Anti-PtdIns(3,4,5)P3 Antibodies
To increase the immunogenicity of PtdIns(3,4,5)P3 and to preserve the structural integrity of phosphoinositides, it is important to construct conjugates that do not modify the polar head group. Although several laboratories have tried to accomplish this by using lipids absorbed non-covalently to carriers (
A PtdIns(3,4,5)P3cationized BSA (cBSA) conjugate was synthesized, as illustrated in Fig 1. First, a diacylglycerylphosphoramidite was prepared bearing a benzyl ester of succinic acid as the sn-1-O-acyl group and stearate as the sn-2-O-acyl group. This phosphoramidite was coupled to the protected inositol head group and oxidized to the phosphate to yield the fully protected PtdIns(3,4,5)P3 analogue (
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Mice were immunized with the PtdIns(3,4,5)P3cBSA conjugate and serum was collected after four booster injections given every 2 weeks. Three days after the final booster injection, mice with high polyclonal anti-PtdIns(3,4,5)P3 antibody titers were sacrificed and the spleens were used to prepare hybridomas. Hybridomas were subcloned and selected for anti-PtdIns(3,4,5)P3 binding.
Ligand Binding Selectivity
The monoclonal anti-PtdIns(3,4,5)P3 antibody named RC6F8 (IgM class) was obtained from mouse ascites fluid. Three different methods were employed to test the specificity of the antibody. First, using ELISA, the antibody RC6F8 specifically recognized PtdIns (3,4,5)P3 (Fig 2). The RC6F8 IgM had very low affinity for PtdIns(4,5)P2, for the two monophosphates PtdIns(3)P and PtdIns(4)P, for the nonphosphorylated PtdIns, and for other phospholipids. In these assays, lipids were nonspecifically adsorbed to the plastic microtiter plate wells. The dipalmitoyl (di-C16)PtdIns (3,4,5)P3 was essentially water-soluble at the low coating concentrations employed, and adhered poorly compared to the essentially insoluble mono- and bisphosphates di-C16PtdIns(4,5)P2, di-C16PtdIns(3)P, di-C16PtdIns(4)P, and di-C16PtdIns. Therefore, even though the same amount of each lipid was coated in the wells, the amount of the PtdIns(3,4,5)P3 to be tested in the wells was less than other lipids after the washing steps in the ELISA. This screen therefore underestimates both the affinity and the selectivity of the MAb for PtdIns(3,4,5)P3.
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Second, we employed ELISA to confirm the antibody affinity and selectivity for PtdIns(3,4,5)P3. Using a competitive ELISA, the RC6F8 IgM was incubated with soluble lipids until equilibrium was reached. For this experiment, we employed dioctanoyl (di-C8) analogues of each of the PtdInsPns. The proportion of antibody that remained unbound at equilibrium was then measured by an indirect ELISA using PtdIns(3,4,5)P3KLH immobilized as the capture antigen in the wells. The results showed that di-C16PtdIns (3,4,5)P3 had greater than 100-fold discrimination for di-C8PtdIns(3,4,5)P3 relative to the other PtdInsPns tested (Fig 3). This result confirms the importance of all three phosphates correctly positioned on the inositol head group in the antibodyantigen recognition. The 3- and 4-monophosphates and the 3,5-, 3,4-, and 4,5-bisphosphates showed significantly reduced affinity for this antibody. Moreover, the soluble head group [Ins(1,3,4,5)P4] showed minimal displacement of the RC6F8PtdIns(3,4,5)P3 interaction.
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Third, we demonstrated ligand selectivity by photolabeling the MAb with three [3H]-BZDC-triester-modified photoaffinity labels (
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Sensitivity of the Antibody
We examined the affinity of the RC6F8 MAb for PtdIns(3,4,5)P3. The ELISA can detect 6 pmol/liter PtdIns(3,4,5)P3 (data not shown). Because it has been estimated that the intracellular concentration of PtdIns(3,4,5)P3 can achieve values of up to 200 µM in neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine (fMLP) (
Quantification of PtdIns(3,4,5)P3 in NIH 3T3 Cells
Because growth factors are known to stimulate PI 3-kinase activity, we first measured PtdIns(3,4,5)P3 levels in [3H]-myo-inositol-loaded NIH 3T3 fibroblasts at several time points after PDGF stimulation. We observed that PtdIns(3,4,5)P3 levels increased rapidly. Fig 5 shows significant increases at 3 min, 15 min, and 60 min, with levels over tenfold above the stimulation base line. This finding is intriguing and served as an impetus to examine the localization and levels of PDGF-stimulated PtdIns(3,4,5)P3 accumulation in NIH 3T3 cells using the RC6F8 MAb.
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Localization of PtdIns(3,4,5)P3 in NIH 3T3 Cells
To locate PtdIns(3,4,5)P3 in cells, several experiments were performed with NIH 3T3 fibroblasts. Cells were stimulated with either insulin or PDGF and both PtdIns(3,4,5)P3 and PtdIns(4,5)P2 were detected by IF using specific MAbs, FITC-labeled goat anti-mouse IgM secondary antibody, and laser scanning confocal fluorescence microscopy (Fig 6 and Fig 7). In both insulin- and PDGF-stimulated cells, an increase in PtdIns(3,4,5)P3 was detected within 1 min of stimulation (not shown) and was sustained up to 120 min after stimulation. Preabsorption of the RC6F8 MAb with PtdIns(3,4,5)P3 before using the MAb to detect cellular PtdIns(3,4,5)P3 ablated detection, while pre-adsorption with PtdIns(4,5)P2 did not reduce PtdIns (3,4,5)P3 detection. When cellular PI 3-kinase activity was inhibited with LY294002 before growth factor stimulation, no increase in PtdIns(3,4,5)P3 was observed (data not shown).
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Serum-starved fibroblasts were stimulated with insulin for 0, 5, or 30 min and PtdIns(3,4,5)P3 and PtdIns(4,5)P2 distributions were visualized by IF (Fig 6). Consistent with previous studies (
To compare PtdIns(3,4,5)P3 production and localization with another PtdInsPn molecule acting as a second messenger in NIH 3T3 cells, PtdIns(4,5)P2 was detected by IF using the anti-PtdIns(4,5)P2 MAb 10F8 (
To confirm the specificity of the 10F8 MAb for cellular lipids, we tested whether the antibody reacted with any proteins present in NIH 3T3 cell extracts. For this determination, a lysate of NIH 3T3 cells (1 x 106 cells) was subjected to SDS-PAGE, and then the proteins were electrotransferred to the PVDF membrane and subjected to Western blotting analysis with the antibody. Results showed that the antibody did not react with any proteins present in NIH 3T3 cell lysate (data not shown).
Stimulated Production of PtdIns(3,4,5)P3 and PtdIns(4,5)P2 by Human Neutrophils
It had been shown by metabolic labeling and lipid extraction that PtdIns(3,4,5)P3 levels increase rapidly in neutrophils on stimulation with fMLP (
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Discussion |
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The distribution of PtdInsPns, their relocalization and association with target proteins upon cell activation, and their metabolism in cells is still not clearly delineated. Answering these questions unequivocally is necessary to obtain a coherent view of phosphoinositide signaling pathways. Here we utilized an anti-PtdIns(3,4,5)P3 MAb as a detection tool and showed approximate cellular level changes and demonstrated distribution of PtdIns(3,4,5)P3 in intact cells. Previously, PtdInsPn level changes have been alluded to, indirectly, as PI 3-kinase activity, or measured by laborious metabolic labeling followed by lipid extraction and HPLC. The PtdIns(3,4,5)P3 MAb enabled us to easily measure and visualize changes in PtdIns (3,4,5)P3 after cell stimulation in vivo. The specificity of the antibody can be further exploited to quantitate PI 3-kinase activity and PtdIns(3,4,5)P3 levels in cells, and can be used to specifically disturb the cellular activity by introduction into living cells.
The results described here illustrate two major aspects of the utility of this anti-PtdIns(3,4,5)P3 antibody. First, it is clear that the plasma membrane is not the only site at which PtdIns(3,4,5)P3 is produced in fibroblasts and neutrophils. The RC6F8 antibody identifies cytoplasmic pools of PtdIns(3,4,5)P3 that are associated with membrane ruffling (
PtdIns(3,4,5)P3 was also detected in the nucleus of stimulated cells. Interestingly, PtdIns(3,4,5)P3-binding proteins have been identified in the nuclei of the neuronal cells (
The difference in duration of PtdIns(3,4,5)P3 levels and PI 3-kinase activity on stimulation poses interesting speculations. Previous studies showed that insulin stimulation resulted in a slow rise and sustained PI 3-kinase activity in 3T3-L1 cells, whereas PDGF resulted in rapid and transient PI 3-kinase activity (
The change in PtdIns(3,4,5)P3 and PtdIns(4,5)P2 levels in neutrophils suggests the immediate precursor of PtdIns(3,4,5)P3 to be PtdIns(4,5)P2. However, in NIH 3T3 cells the primary precursor of PtdIns(3,4,5)P3 may not be PtdIns(4,5)P2, because the accumulation of PtdIns(3,4,5)P3 does not correspond directly to a decrease in PtdIns(4,5)P2. It has been suggested that, in NIH 3T3 cells, PtdIns(3,4,5)P3 formation can occur via the PtdIns(3)P to PtdIns(3,4)P2 to PtdIns(3,4,5)P3 pathway (
In summary, the generation of a PtdIns(3,4,5)P3-specific MAb has enabled us to address fundamental issues in phosphoinositide signaling. This tool should now facilitate discovery of potential drug candidates and development of diagnostic tools for cancer and other pathologies (
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Acknowledgments |
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NIH (Grant NS29632), the American Cancer Society (Grant RPG-00-126-01-TBE), and the Utah Centers of Excellence Program provided financial support for the antigen preparation and antibody development.
We thank Echelon Research Laboratories for providing dipalmitoyl and dioctanoyl PIPn derivatives and Dr D.W. Neklason for assistance with manuscript preparation. Drs L. Feng, and J. Peng provided radioligands and assistance with IgM photoaffinity labeling. Special thanks to A. Weyrich (U Utah) for assistance with neutrophil preparations. Mr C. Jones and Ms K. Manabe (Utah State) assisted with HPLC and microscopy. Anti-PtdIns(4,5)P2 IgMs 2C11 and 10F8 were initially obtained from Dr G. Schiavo (Imperial Cancer Research Fund, London). In more recent studies, the 2C11 and RC6F8 antibodies were obtained from Echelon.
Received for publication January 2, 2002; accepted January 9, 2002.
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