Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry.

Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signaling lipid, phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P(3)). PtdIns(3,4,5)P(3) regulates major aspects of cellular behavior, and the use of both genetic and pharmacological intervention has revealed important isoform-specific roles for PI3Ks in health and disease. Despite this interest, current methods for measuring PtdIns(3,4,5)P(3) have major limitations, including insensitivity, reliance on radiolabeling, low throughput and an inability to resolve different fatty-acyl species. We introduce a methodology based on phosphate methylation coupled to high-performance liquid chromatography-mass spectrometry (HPLC-MS) to solve many of these problems and describe an integrated approach to quantify PtdIns(3,4,5)P(3) and related phosphoinositides (regio-isomers of PtdInsP and PtdInsP(2) are not resolved). This methodology can be used to quantify multiple fatty-acyl species of PtdIns(3,4,5)P(3) in unstimulated mouse and human cells (≥10(5)) or tissues (≥0.1 mg) and their increase upon appropriate stimulation.

Introduction

PI3Ks phosphorylate one or more of the three canonical phosphoinositides that are found in all eukaryotic cells, PtdIns, PtdIns4P and PtdIns(4,5)P2, to form PtdIns3P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3, respectively. These lipid products are now recognized as pivotal intracellular signals that act by dictating the localization and activity of several key regulators of cellular function1-2. The Class I PI3Ks, of which there are four isoforms in mammalian cells (α, β, δ and γ), can be activated by many groups of cell surface receptors to accelerate production of PtdIns(3,4,5)P3 in the inner leaflet of the plasma membrane. A diverse family of proteins can selectively bind PtdIns(3,4,5)P3 via their conserved PH domains, resulting in their activation and hence further translation and propagation of the original receptor signal.

Many cell processes are regulated by Class I PI3Ks, including growth, survival and movement. Remarkably, despite the fact they all make a common output signal, PtdIns(3,4,5)P3, the different Class I PI3K isoforms perform different roles in both physiology and pathology3. This, combined with the drugability of their active sites, has led to substantial investment in targeting the Class I PI3Ks in a variety of disease settings such as PI3Kγ and PI3Kδ in inflammation and PI3Kα in oncology.

The amphiphilic phosphoinositides, including PtdIns(3,4,5)P3, have been quantified by a number of methods. Broadly, these all show that the levels of PtdIns(3,4,5)P3 in un-stimulated cells are “very low” (typically undetectable) and rise five to fifty fold on stimulation, to reach concentrations that are, at most, 10% of cellular PtdIns(4,5)P2. These techniques have a variety of problems, including sensitivity, sample through-put, dynamic range and applicability across a range of sample formats4-6. Further, like all phospholipids, phosphoinositides contain a wide range of potential constituent fatty acid moieties and thus, in reality, comprise families of molecular species with a common headgroup. Classical chromatographic techniques and, more recently, mass-spectrometry-based lipidomics approaches have been developed that can resolve many families of fatty-acyl species, including those of the phosphoinositides4,7-12. PtdIns(3,4,5)P3 however, has proven very difficult to quantify using these approaches, primarily due to problems with recovery, stability and yield of the relevant ions7,9,13.

These issues have seriously hampered progress in understanding the Class I PI3K signaling pathway. In particular, the inability to quantify the pathway’s primary output signal is proving a major obstacle in developing an adequate, quantitative understanding of information flow through this pathway, including attempts to model the system. It has also hampered the development of biomarkers for reading out the impact of inhibitors of this pathway in vivo. This has lead to many workers using surrogate assays of pathway activity, such as phosphorylation levels of PKB, which are indirect and can be disconnected from PI3K signalling under some circumstances14. The inability to discriminate different fatty-acyl species of PtdIns(3,4,5)P3 has also prevented any serious attempt to elucidate their functional significance.

We now present an integrated HPLC-MS based methodology that allows absolute and relative measurements of the concentration of PtdIns(3,4,5)P3 and its different fatty-acyl species in small numbers of cells (≥ 105), either in suspension or adhered to tissue culture plates, and small samples of tissue.

Results

Establishing a quantitative assay for PtdIns(3,4,5)P3

As part of our synthetic studies making phosphoinositides we observed that precursor compounds whose phosphates were protected with benzyl groups, in contrast to their de-protected forms, gave good ESI mass spectra. We therefore reasoned that chemically protecting the acidic phosphate groups of PtdIns(3,4,5)P3 in biological extracts might offer a route to solving the major stability and sensitivity issues associated with its quantification by MS.

It was clear that the key to success would be the use of a mild and efficient method for the protection reaction. We chose to use trimethylsilyl diazomethane (TMS-diazomethane), because it provides a relatively fast and clean method for the esterification of protonated phosphate groups at room temperature15. Further, the solvent mixture into which PtdIns(3,4,5)P3 is extracted from cells most efficiently (a mixture of chloroform, methanol and H2O with traces of HCl16-17) is an ideal environment for TMS-diazomethane reactions. Using TMS-diazomethane we were able to achieve rapid and complete methylation of the phosphate groups in PtdIns(3,4,5)P3 with some mono-methylation of free hydroxyl groups in the inositol ring but no modification of unsaturated fatty acyl chains (see below). The methylated PtdIns(3,4,5)P3 species were more efficiently transferred into the mass spectrometer, with a reduced number of ionic species, compared to their underivatised counterparts, both simplifying and effectively sensitizing the MS analysis.

We chromatographed derivatised lipid extracts through an in-line C4 column prior to infusion into a triple quadrapole mass spectrometer in the positive ion mode. This step concentrated the methylated PtdIns(3,4,5)P3 species but co-elution with more abundant molecules necessitated a selective, second phase of mass fragmentation analysis (usually termed multiple reaction monitoring or MRM). Molecular ions selected in the first quadrapole were fragmented in the second quadrapole using collision parameters optimized for cleavage between the phosphoinositide headgroup and the glycerol unit, yielding a charged fragment corresponding to the diacylglycerol (DAG) and the neutral loss of a fragment corresponding to the methylated inositol phosphate headgroup (a neutral loss of 598amu for the major methylated species of PtdInsP3; Supplementary Fig. 1). Since the mass of the charged DAG fragments can be determined and the products of further fragmentation can also be analyzed, then, in the context of previous work, it is possible to assign the likely structure of the DAGs and the parent lipids with some confidence. Further, whilst the focus of the study was on the measurement of PtdIns(3,4,5)P3, the same principle of ‘measurement through neutral loss’ could be applied to the measurement of other lipids that fragmented similarly, for example measuring the DAG produced via loss of 490amu for the methylated headgroup of PtdInsP2 or 213amu for the methylated headgroup of PtdSer, providing useful parallel data.

We first analyzed and quantified phosphoinositides in human neutrophils. Control or fMLP-stimulated human neutrophils were quenched with chloroform, methanol and HCl to create a single-phase extract into which non-biological C17:0/C16:0-PtdIns(3,4,5)P3 was spiked as an internal standard (ISD). The samples were extracted, methylated and infused into the mass spectrometer. We initially performed neutral loss scans on parent ions whose masses were in a range consistent with either potential esterified PtdInsP2 or PtdIns(3,4,5)P3 species, with the detector set to measure daughter ions (putative DAGs) that were smaller by either 490 or 598amu (see Supplementary Table 1 for a list of relevant masses and associated structures). This revealed two relatively abundant species of PtdInsP2 (m/z 1095 and 1117) in both control and stimulated cells (Fig. 1a,b) and two abundant species of PtdIns(3,4,5)P3 (m/z 1203 and 1225) that were more concentrated in the stimulated cells (Fig. 1c,d). The daughter ions from neutrophil PtdInsP2 with a parental m/z of 1117, and neutrophil PtdIns(3,4,5)P3 with a parental m/z of 1225, created fragments identical to those derived from C18:0/C20:4-PtdIns(4,5)P2 or –PtdIns(3,4,5)P3 synthetic standards (Supplementary Figs. 2 and 3), strongly suggesting the biological phosphoinositides were both C18:0/C20:4, stearoyl/arachidonoyl species. The masses and further fragmentation of the additional PtdInsP2 (m/z 1095) and PtdIns(3,4,5)P3 (m/z 1203) species that we identified suggest they both contained C18:0/C18:1 (stearoyl/oleoyl) fatty acids (data not shown). We also detected other, lower abundance species but did not analyze them further.

Figure 1

Analysis of phosphoinositides in control and fMLP-stimulated human neutrophils. (a-d) Neutral loss scans of a derivatised phosphoinositide extract, from un-stimulated control (a, c) or fMLP-stimulated (b, d) neutrophils, ISD = internal standard. The two most abundant species of endogenous PtdIns(3,4,5)P3 and PtdInsP2 are labeled with full masses and corresponding fatty acid species of DAG unit. (e) Overlay of m/z chromatograms for parent ions with masses similar to derivatised C18:0/C20:4-Ptdins(3,4,5)P3 from extracts from 1 ×105 human neutrophils, highlighting elution at 10.75 min (ions which increase with fMLP-stimulation in a wortmannin-sensitive manner). (f) Overlay of MRM chromatograms (m/z 1,225 → m/z 627 + 598) of samples from e. Data in a-d were collected using a Quattro Ultima mass spectrometer and data in e-f were collected using a QTRAP4000 mass spectrometer. Each are representative traces from several independent experiments. A full list of relevant structures and associated masses are shown in Supplementary Table 1; sn-1/sn-2 assignment is based on biological precedent and further fragmentation of DAG species.

The selectivity of the neutral loss transition is evident from comparing traces quantifying all ions derived from the neutrophil extract with an m/z of 1225 (Fig. 1e) to traces quantifying only those ions with an m/z of 1225 that underwent an m/z 1225 to m/z 627 + 598 transition (Fig. 1f). The clarity with which fMLP increased the levels of the species quantified and the extent to which wortmannin, a selective PI3K inhibitor, prevented this increase provided biological confirmation that the assignment of ions to parental PtdIns(3,4,5)P3 species was correct.

We refined our approach into a reliable and quantitative assay for targeted PtdIns(3,4,5)P3 species. We found that synthetic ISD or C18:0/C20:4-PtdIns(3,4,5)P3 that had been spiked into primary neutrophil extracts, esterified and processed for MS analysis was stable in mixtures of methanol/water (80%/20%) for ≥ 23h at room temperature (Fig. 2a,b) and could be detected in un-stimulated extracts when as little as 0.25ng had been added with excellent signal to noise (Fig. 2c). Further, both biological and synthetic PtdIns(3,4,5)P3 species were routinely methylated to the same relative extent (Supplementary Fig. 4 and Supplementary Table 2) and were detected with linear efficiency and sensitivity over at least a 0.5-20ng range (Fig. 2d and Supplementary Fig. 5). If increasing amounts of synthetic C18:0/C20:4 PtdIns(4,5)P2 were added to neutrophil extracts, it had no effect on the quantification of a parallel, internal spike of C18:0/C20:4-PtdIns(3,4,5)P3 (Supplementary Fig. 6), showing the fidelity of the assay. If 10 ng ISD was spiked into primary extracts from different numbers of neutrophils then there was a reduction in both its, and the endogenous PtdInsP2 and PtdIns(3,4,5)P3’s, recovery with increasing cell number (Supplementary Fig. 7). However, when the amount of the endogenous C18:0/C20:4-PtdInsP2 or -PtdIns(3,4,5)P3 detected in these samples was corrected for the recovery of the internal ISD, a linear relationship between endogenous PtdInsP2 or PtdIns(3,4,5)P3 and cell number was observed (Fig. 2e, parallel data for C18:0/C20:4-PtdInsP2 are shown in Supplementary Fig. 7).

Figure 2

Validation of the robustness, signal-to-noise and linearity of the assay. a) C17:0/C16:0-PtdIns(3,4,5)P3 internal standard (ISD) spiked into un-stimulated human neutrophil extract; (b) C18:0/C20:4-PtdIns(3,4,5)P3 standard spiked into un-stimulated human neutrophil extract; (c) 0.25 ng C18:0/C20:4-PtdIns(3,4,5)P3 standard spiked into un-stimulated neutrophil extract; (d) linear response to increasing amounts of C18:0/C20:4-PtdIns(3,4,5)P3 added to extracts of un-stimulated neutrophils (2.25 × 106); (e) linear relationship between cell number (fMLP-stimulated neutrophils) and estimated endogenous C18:0/C20:4-PtdIns(3,4,5)P3 by using the ISD to correct for recovery. The term ‘response ratio’ in (b,d,e) is the integrated ion current response to the defined phosphoinositide divided by that to the ISD. (f) MRM chromatograms for 1μg each of synthetic C18:0/C20:4-PtdIns(4,5)P2 (i), synthetic C18:0/C20:4-PtdIns(3,4,5)P3 (ii) and ISD (C17:0/C16:0-PtdIns(3,4,5)P3) (iii) in water, demonstrating equal recovery/detection.

These results all indicate that through use of an internal, non-biological standard, C17:0/C16:0-PtdIns(3,4,5)P3 (ISD), to correct for recovery through the extraction, methylation and mass spectrometry phases, this is a very sensitive and robust method to measure the relative levels of individual PtdIns(3,4,5)P3 species in cells. Correction to the recovery of the ISD also proved to be a good normalization factor for other lipids measured in parallel, typically up to twenty three other lipids, including five different molecular species of PtdIns(3,4,5)P3 and PtdInsP2 from a single extract. We also noted that the ionizations of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in HPLC eluate were very similar, hence their ion currents were good indicators of their relative amounts (Fig. 2f). A completely confident measure of the absolute amounts of a particular molecular species however, requires the synthesis of the equivalent molecule to both calibrate the method and confirm the fragmentation analysis. This was only done for C18:0/C20:4-PtdIns(3,4,5)P3 and C18:0/C20:4-PtdIns(4,5)P2 in the current study.

Quantifying PtdIns(3,4,5)P3 species in fMLP-stimulated human neutrophils

We used this approach to measure changes in PtdInsP2 and PtdIns(3,4,5)P3 molecular species in human neutrophils stimulated with fMLP, a setting where much previous work with other methods18-21 has given a clear bench mark. We found that fMLP stimulated a small, transient and parallel reduction in the levels of both C18:0/C20:4- and C18:0/C18:1-PtdInsP2 (Fig. 3a), that is known to result from activation of PLCβ22, and a parallel increase in C18:0/C20:4- and C18:0/C18:1-PtdIns(3,4,5)P3, that is known to result from activation of PI3Kγ23-24 (Fig. 3b). The scale and kinetics of these responses were entirely in-keeping with earlier, quantitative data for total PtdIns(3,4,5)P3 measured by competitive binding assay18, ‘fat-blot’ assay20 and metabolic labeling with either [3H]-myo-inositol or [32P]-Pi19,21. Further, our analysis accurately defines the basal as well as the stimulated levels of PtdIns(3,4,5)P3 in 105 cells and, through calibration with synthetic C18:0/C20:4-PtdIns(3,4,5)P3, absolute molar amounts of this species.

Figure 3

fMLP-stimulated changes in C18:0/C20:4 and C18:0/C18:1 PtdInsP2 and PtdIns(3,4,5)P3 species in human neutrophils. (a-b) The levels of the C18:0/C20:4 and C18:0/C18:1 molecular species of PtdInsP2 (a) and PtdIns(3,4,5)P3 (b) in human neutrophils were determined at a range of times following addition of fMLP. The data are expressed as either response ratios (as defined in fig. 2) or, through use of the calibration curve presented in Fig. 2d, and protein assays, pmol mg−1 protein (Y-axis, right) and are means ± SEM (n= 4). (c) The ratio of the quantity of each molecular species of PtdIns(3,4,5)P3 divided by that of their respective PtdInsP2 species.

These results suggest that the two common species of PtdInsP2 and PtdIns(3,4,5)P3 that we detect in human neutrophils, of which the C18:0/C20:4-containing molecules are the most abundant, are both available and recognized by the relevant PtdInsP2 and PtdIns(3,4,5)P3 metabolising enzymes. However, a careful analysis of the conversion of each molecular species of PtdInsP2 into its respective PtdIns(3,4,5)P3 (Fig. 3c), indicated that C18:0/C20:4-PtdIns(3,4,5)P3 accumulated selectively on fMLP-stimulation (p<0.0001, 2 way ANOVA, factors being species and time for 6-120 s).

Quantifying PtdIns(3,4,5)P3 species in EGF-stimulated MCF10a cells

MCF10a cells are an immortalised human breast epithelial cell line that has been used to create a panel of homologously-targeted variants relevant to the study of PI3K signalling pathways25-26, including deletion of the PtdIns(3,4,5)P3 3-phosphatase PTEN. These cells are highly adherent and hence we first adapted the extraction protocol to avoid the need for chloroform contact with the tissue culture plastic, then attempted to detect PtdInsP2 and PtdIns(3,4,5)P3 molecular species. Neutral loss scans revealed five common fatty-acyl species of both PtdInsP2 and PtdIns(3,4,5)P3 (Fig. 4a,b; these species were also detected in neutral loss scans of PtdInsP and PtdIns, data not shown). We quantified these five species in extracts from 105 growing, starved or starved then EGF-stimulated wild-type or PTEN−/− MCF10a cells (Fig. 4c,d). The efficiency of the extraction process was determined using an internal spike of ISD and variations in cell-input were corrected using the quantity of methylated C18:0/C18:1-PtdSer we recovered from the same extracts. In a small number of parallel assays we also quantified phosphoinositide pools in these cells using conventional 3H-myo-inositol labeling and quantification of deacylated phosphoinositides by anion-exchange HPLC. The relative sizes of the total PtdInsP2 and PtdIns(3,4,5)P3 pools derived using either assay format were remarkably similar and exhibited similar changes upon EGF-stimulation (Supplementary Fig. 8), supporting the fidelity of the mass spectrometric analysis.

Figure 4

Identification and quantification of the molecular species of PtdIns(3,4,5)P3 in wild-type and PTEN−/− MCF10a cells. (a-b) Neutral loss scans of the common families of molecular species of PtdInsP2 (a) and PtdIns(3,4,5)P3 (b) in EGF-stimulated, wild-type MCF10a cells, Further fragmentation and analysis of the relevant daughter ions indicated they possessed the fatty acids indicated in c,d. Data was collected using a QTRAP 4000 mass spectrometer. (c-d) The levels of these species of PtdInsP2 (c) and PtdIns(3,4,5)P3 (d) in the indicated cell lines and conditions are presented as mean response ratios normalized for cell input via the recovered C18:0/C18:1-PtdSer (means ± SEM, n= 3). (e) The ratio of the quantity of each molecular species of PtdIns(3,4,5)P3 divided by that of their respective PtdInsP2 species. Full details of molecular species, and masses of respective parent ions detected in MCF10a cells can be found in Supplementary Table 1.

The concentrations of all five species of PtdIns(3,4,5)P3 were increased by EGF-stimulation in a manner augmented by loss of PTEN (Fig. 4d). Interestingly, although C18:0/C20:4-PtdInsP2 was the least abundant species of PtdInsP2, this fatty-acyl species accumulated selectively in the PtdIns(3,4,5)P3 pool upon EGF-stimulation (Fig. 4e; p<0.0001, 2 way ANOVA, factors being species and conditions).

Quantifying PtdIns(3,4,5)P3 species in fat and liver

Quantification of PtdIns(3,4,5)P3 in small samples of tissue remains an academically and commercially important challenge. We used our approach to measure Class I PI3K activity in vivo by studying the effects of insulin on mouse liver and human fat.

Mice of either wild-type or Gnasxl genetic background (Gnasxl mice lack XLαS, the imprinted isoform of Gαs, and are hyper-sensitive to insulin27) were injected intra peritoneal with insulin or a saline control and 8 minutes later samples of liver were frozen rapidly. Primary lipid extracts were spiked with ISD and then processed as described. Neutral loss scans revealed that C18:0/C20:4-molecular species were by far the most abundant versions of both PtdInsP2 and PtdIns(3,4,5)P3 in these samples (Fig. 5a and Supplementary Fig. 9). We then quantified C18:0/C20:4-PtdIns(3,4,5)P3 and other phosphoinositides as described above using C18:0/C20:4-PtdSer to correct for sample size. In parallel, we prepared SDS lysates from the same frozen specimens to quantify phosphorylation of PKB, as a known marker of PI3K activation. Insulin stimulated a substantial increase in the levels of C18:0/C20:4-PtdIns(3,4,5)P3 that was augmented in the Gnasxl genetic background (Fig. 5b) but had no effect on the levels of C18:0/C20:4-PtdInsP2 (Supplementary Fig. 9). Insulin also increased phosphorylation of PKB in the liver samples, although this response appeared less strongly augmented in the Gnasxlm+/p− genetic background (Fig. 5c and Supplementary Fig. 10).

Figure 5

Detection and quantification of insulin-stimulated PtdIns(3,4,5)P3 responses in mouse liver and human adipose tissue. (a) Neutral loss scan, collected using a QTRAP 4000 mass spectrometer, of PtdIns(3,4,5)P3 species in wild-type (WT), insulin-stimulated mouse liver. (b) Levels of C18:0/C20:4-PtdIns(3,4,5)P3 in the livers of WT or Gnasxl mice, after injection of insulin or saline (means ± SEM, n= 4). (c) Phosphorylation status of S473 in PKB for parallel samples to those analyzed in b; data normalized for input material via immuno-blotting for β-COP (See Supplementary Fig. 10 for blot). (d) Neutral loss scan of PtdIns(3,4,5)P3 species in human adipose tissue following oral ingestion of glucose. (e) Levels of C18:0/C20:4-PtdIns(3,4,5)P3 in healthy human adipose tissue following overnight starvation either before (Fasting) or 90 mins after (Post-Glucose) oral ingestion of glucose, for three individuals (means ± SEM, technical replicates n= 4). (f) Phosphorylation status of S473 in PKB for parallel samples to those analyzed in e; data normalized for input material via immuno-blotting for actin (See Supplementary Fig. 10 for blot).

Human adipose tissue is highly responsive to changes in blood insulin. We starved healthy human volunteers overnight and removed and froze small biopsies of adipose tissue both before and 90 minutes after oral ingestion of glucose. We made a minor modification to our standard extraction procedure to pre-deplete the neutral storage lipids prior to extracting the polyphosphoinositides. This had no effect on recovery of ISD or endogenous PtdInsP2 and PtdIns(3,4,5)P3 species, but reduced recovery of less polar lipids such as PtdIns and PtdSer. Neutral loss scans revealed that the main molecular species of both PtdInsP2 and PtdIns(3,4,5)P3 was C18:0/C20:4 (Fig. 5d and Supplementary Fig.9). We quantified C18:0/C20:4-PtdIns(3,4,5)P3 as above, but using PtdInsP2 to correct for the amount of tissue (because recovery of PtdSer was poor under the modified conditions). In parallel, we prepared SDS lysates and quantified phosphorylation of PKB. These experiments showed the levels of C18:0/C20:4-PtdIns(3,4,5)P3 were increased following ingestion of glucose (Fig. 5e) and this was paralleled by an increase in PKB phosphorylation (Fig. 5f and Supplementary Figs. 9 and 10).

Discussion

Since PtdIns(3,4,5)P3 is the sole PtdInsP3 in cells it can be quantified directly by our approach without the need for further purification from other regio-isomers prior to infusion into a mass spectrometer. In principle however, this approach could be extended to the quantification of other phosphoinositides if it could be coupled to a method that could distinguish between positional isomers on the inositol ring e.g. distinguishing between the three biological isomers of PtdInsP2 (PtdIns(4,5)P2, PtdIns(3,5)P2 and PtdIns(3,4)P2 or PtdInsP (PtdIns3P, PtdIns4P and PtdIns5P).

In addition to providing a sensitive and reproducible route to quantifying PtdIns(3,4,5)P3 and related phosphoinositides, the methodology described here also identifies the fatty acyl complement of the molecular species measured. In all of the primary cells and tissues that we investigated, the most abundant species of PtdIns(3,4,5)P3, PtdInsP2, PtdInsP and PtdIns was, by far, the stearoyl/arachidonoyl form. This is consistent with much previous work indicating arachidonate is highly and selectively enriched in the sn-2 position of phosphoinositides7,9,12. The striking exception was MCF10a cells, where the stearoyl/arachidonoyl species of these lipids were amongst the least prevalent. Others have reported that cell lines grown in the presence of serum become “deficient” in arachidonate28, potentially explaining this observation. Strikingly, however, the stearoyl/arachidonoyl form of PtdIns(3,4,5)P3 selectively accumulated in both EGF-stimulated MCF10a cells and fMLP-stimulated neutrophils relative to its levels of enrichment in the total cell pool of its precursor, PtdInsP2. This suggests mechanisms must exist to selectively accumulate the stearoyl/arachidonoyl form of PtdIns(3,4,5)P3 in stimulated cells, raising important questions as to how this occurs and why.