Cell-based, bioluminescent assay for monitoring the interaction between PCSK9 and the LDL receptor.

Monitoring the expression of cell-surface receptors, their interaction with extracellular ligands, and their fate upon ligand binding is important for understanding receptor function and developing new therapies. We describe a cell-based method that utilizes bioluminescent protein complementation technology to interrogate binding of a cellular receptor with its extracellular protein ligand, specifically LDL receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9). Purified, full-length tagged PCSK9 is added to assay wells containing cells that stably express LDLR with an extracellular complementary tag. When the tagged PCSK9 binds the receptor, a bright luminescence signal is generated. The interaction is detected at the cell membrane with add-and-read simplicity, no wash steps, and flexibility, allowing data to be collected in endpoint format, kinetically, or with bioluminescent imaging. The assay is flexible, is rapid, and reports accurate biology. It is amenable to 96-well and 384-well formats, and the robustness allows for screening of new drug candidates (Z' = 0.83). The assay reports correct potencies for antibody titrations across a 50%-150% potency range and detects potency changes due to heat stress, suggesting that it may be useful during drug development. This assay technology can be broadly applied when studying other receptors with their extracellular ligands, whether protein or small-molecule binding partners.

A bioluminescent assay for measuring glucose uptake.

Identifying activators and inhibitors of glucose uptake is critical for both diabetes management and anticancer therapy. To facilitate such studies, easy-to-use nonradioactive assays are desired. Here we describe a bioluminescent glucose uptake assay for measuring glucose transport in cells. The assay is based on the uptake of 2-deoxyglucose and the enzymatic detection of the 2-deoxyglucose-6-phosphate that accumulates. Uptake can be measured from a variety of cell types, it can be inhibited by known glucose transporter inhibitors, and the bioluminescent assay yields similar results when compared with the radioactive method. With HCT 116 cells, glucose uptake can be detected in as little as 5000 cells and remains linear up to 50,000 cells with signal-to-background values ranging from 5 to 45. The assay can be used to screen for glucose transporter inhibitors, or by multiplexing with viability readouts, changes in glucose uptake can be differentiated from overall effects on cell health. The assay also can provide a relevant end point for measuring insulin sensitivity. With adipocytes and myotubes, insulin-dependent increases in glucose uptake have been measured with 10- and 2-fold assay windows, respectively. Significant assay signals of 2-fold or more have also been measured with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and skeletal myoblasts.

Self-immolative bioluminogenic quinone luciferins for NAD(P)H assays and reducing capacity-based cell viability assays.

Highly sensitive self-cleavable trimethyl lock quinone-luciferin substrates for diaphorase were designed and synthesized to measure NAD(P)H in biological samples and monitor viable cells via NAD(P)H-dependent cellular oxidoreductase enzymes and their NAD(P)H cofactors.

Impact of microsatellite instability on survival of endometrial cancer patients.

BACKGROUND AND OBJECTIVE: Endometrial cancer (EC) is the most commonly diagnosed gynecologic malignancy among women worldwide and may be classified on the basis of different molecular, pathologic and genetic alterations, including microsatellite instability (MSI). Although MSI is associated with a more favorable outcome in colorectal cancer, its relationship with prognosis in EC cancer is not yet clear. The aim of our study is to identify whether MSI correlates with survival of patients in EC. MATERIALS AND METHODS: We examined MSI status and survival of 109 women. MSI was detected by employing the Promega MSI Analysis System, which used 5 mononucleotides markers (BAT-25, BAT-26, NR-21, NR-24, and MONO-27) to identify MSI in a tumor and normal tissue DNA and 2 pentanucleotide markers (Penta C and Penta D) for specimen identification. Median follow-up of patients was 40.4 months (range 5.2-47.9). Survival was estimated by the Kaplan-Meier method and Cox regression analysis was used to assess the effects of different variables on patient survival. RESULTS: MSI-high was detected in 15.6% EC cases, all of which were associated with endometrioid type histology. Kaplan-Meier survival analysis showed no statistically significant differences between patients with MSI-high and MSI stable tumors (P=0.4) and multivariate analysis concluded that MSI status remained insignificant after stage, histology and tumor grade adjustment (P=0.5). CONCLUSIONS: Our study showed no statistically significant relationship between MSI-high and survival of endometrial cancer patients.

Metabolic priming of GD2 TRAC-CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence.

Manufacturing chimeric antigen receptor (CAR) T cell therapies is complex, with limited understanding of how medium composition impacts T cell phenotypes. CRISPR-Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant (TRAC) gene resulting in TRAC-CAR T cells with an enriched stem cell memory T cell population, a process that could be further optimized through modifications to the medium composition. In this study we generated anti-GD2 TRAC-CAR T cells using "metabolic priming" (MP), where the cells were activated in glucose/glutamine-low medium and then expanded in glucose/glutamine-high medium. T cell products were evaluated using spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays in vitro, and potency against human GD2+ xenograft neuroblastoma models in vivo. Compared with standard TRAC-CAR T cells, MP TRAC-CAR T cells showed less glycolysis, higher CCR7/CD62L expression, more bound NAD(P)H activity, and reduced IFN-γ, IL-2, IP-10, IL-1ß, IL-17, and TGF-ß production at the end of manufacturing ex vivo, with increased central memory CAR T cells and better persistence observed in vivo. MP with medium during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo, which could lead to better responses against solid tumors in vivo.

Metabolic priming of GD2 TRAC -CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence.

Manufacturing Chimeric Antigen Receptor (CAR) T cell therapies is complex, with limited understanding of how media composition impact T-cell phenotypes. CRISPR/Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant ( TRAC ) gene resulting in TRAC -CAR T cells with an enriched stem cell memory T-cell population, a process that could be further optimized through modifications to the media composition. In this study we generated anti-GD2 TRAC -CAR T cells using "metabolic priming" (MP), where the cells were activated in glucose/glutamine low media and then expanded in glucose/glutamine high media. T cell products were evaluated using spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays in vitro and potency against human GD2+ xenograft neuroblastoma models in vivo . Compared to standard TRAC -CAR T cells, MP TRAC -CAR T cells showed less glycolysis, higher CCR7/CD62L expression, more bound NAD(P)H activity and reduced IFN-γ, IL-2, IP-10, IL-1ß, IL-17, and TGFß production at the end of manufacturing ex vivo , with increased central memory CAR T cells and better persistence observed in vivo . Metabolic priming with media during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo , which could lead to better responses against solid tumors in vivo .

A bioluminescence assay for aldehyde dehydrogenase activity.

The aldehyde dehydrogenase (ALDH) family of enzymes is critical for cell survival and adaptation to cellular and environmental stress. These enzymes are of interest as therapeutic targets and as biomarkers of stem cells. This article describes a novel, homogeneous bioluminescence assay to study the activity of the ALDH enzymes. The assay is based on a proluciferin-aldehyde substrate that is recognized and utilized by multiple ALDH enzyme isoforms to generate luciferin. A detection reagent is added to inactivate ALDH and generate light from the luciferin product. The luminescent signal is dependent on the ALDH enzyme concentration and the incubation time in the ALDH reaction; moreover, the luminescent signal generated with the detection reagent is stable for greater than 2 h. This assay provides many advantages over standard NADH fluorescence assays. It is more sensitive and the signal stability provided allows convenient assay setup in batch mode-based high-throughput screens. The assay also shows an accurate pharmacological response for a common ALDH inhibitor and is robust, with a large assay window (S/B=64) and Z'=0.75.

HaloTag-based purification of functional human kinases from mammalian cells.

Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification. Since the binding of HaloTag to the HaloLink resin is essentially irreversible, it overcomes the equilibrium-based binding limitations associated with affinity tags and enables efficient capture and purification of target protein, even at low expression levels. The target protein is released from the HaloLink resin by specific cleavage using a TEV protease fused to HaloTag (HaloTEV), leaving both HaloTag and HaloTEV permanently attached to the resin and highly pure, tag-free protein in solution. HaloTag fluorescent ligands enable fluorescent labeling of HaloTag fusion proteins, providing a convenient way to monitor expression, and thus facilitate the identification of optimal transient transfection conditions as well as the selection of high expression stable cell lines. The capabilities of this method have been demonstrated by the efficient purification of five functional human kinases from HEK293T cells. In addition, when purifications using FLAG, 3xFLAG, His(6)Tag and HaloTag were performed in parallel, HaloTag was shown to provide significantly higher yields, purity and overall recovery of the expressed proteins.

Time- and Dose-Dependent Toxicity Studies in 3D Cultures Using a Luminescent Lactate Dehydrogenase Assay.

Three-dimensional (3D) in vitro systems closely resemble tissue microenvironments and provide predictive models for studying cytotoxic drug responses. The ability to capture the kinetic profiles of such responses in a dynamic and noninvasive way can further advance the utility of 3D cell cultures. Here, we describe the use of a luminescent lactate dehydrogenase (LDH) toxicity assay for monitoring time- and dose-dependent effects of drug treatment in 3D cancer spheroids. HCT116 spheroids formed in 96-well ultralow attachment plates were treated with increasing drug concentrations. Medium samples were collected at different timepoints, frozen, stored, and analyzed at the end of experiments using the luminescent LDH-Glo™ Assay. High assay sensitivity and low volume sampling enabled drug-induced toxicity profiling in a time- and dose-dependent manner.

A Luminescence Assay to Quantify Cell Viability in Real Time.

Comprehensive understanding of cellular responses to changes in the cellular environment or by drug treatment requires time-dependent analysis ranging from hours to several days. Here, we describe a sensitive, nonlytic live-cell assay that allows continuous or 'real-time' monitoring of cell viability, growth, and cytotoxicity over an extended period of time. We illustrate the use of the assay for small drug molecule and antibody-dependent cytotoxicity studies using cancer cells in 384-well plates. We show that the ability to measure changes in live cells over time provides instantaneous information on the biological status of the cells, information about the mode of action of the drug, and offers an added advantage of preserving the cells for multiplexing with downstream applications.

A bioluminescent-based, HTS-compatible assay to monitor G-protein-coupled receptor modulation of cellular cyclic AMP.

We have developed a novel assay for monitoring changes in intracellular cyclic AMP (cAMP) concentration with high sensitivity (30 +/- 5 fmol [mean +/- standard error of the mean] of cAMP per well) and reproducibility (Z' of > 0.8). The assay is of format amenable to high throughput screening (HTS) in 96-, 384-, and 1,536-well plates, and as a bioluminescent assay is potentially less prone to interferences originating from fluorescent compounds. Because of its high sensitivity, fewer numbers of cells (1,000 cells per well) in low-volume 384-well plates are required to screen for changes in cAMP concentrations. The assay does not rely on the use of antibodies, and thus it does not suffer from changes in the affinity or quality of the antibodies. The assay is based on the fact that cAMP is a potent activator of cAMP-dependent protein kinase (PKA), and activation of PKA can be monitored by measuring ATP utilization in a kinase reaction. The amount of ATP consumed can be measured using a luciferase/luciferin luminescent reaction. Since the amount of relative luminescence units (RLU) generated is a measure of the remaining ATP, a reciprocal relationship between RLU and both the activity of PKA and the intracellular concentration of cAMP is observed. Thus, the functional activity of agents that modulate the activity of Galpha(s) or Galpha(i) forms of G-protein-coupled receptors (GPCRs), which cause change in intracellular cAMP, can be monitored by the change in the activity of PKA and the amount of RLU readout. The assay can be performed in two steps and requires only 30 min after cell lysis for completion. The assay has been successfully used to generate 50% effective concentration (EC(50)) values for forskolin, a known direct activator of cellular adenylate cyclases, and EC(50) values for agonists and 50% inhibitory concentration values for antagonists modulating GPCRs that alter adenylate cyclase activity (Galpha(s) and Galpha(i)). Finally, adherent, suspension, and frozen cells have been successfully used in this assay, thus offering flexibility and convenience for many HTS applications.

Bioluminescent Assays for Glucose and Glutamine Metabolism: High-Throughput Screening for Changes in Extracellular and Intracellular Metabolites.

Cancer cell metabolism is a complex, dynamic network of regulated pathways. Interrogation of this network would benefit from rapid, sensitive techniques that are adaptable to high-throughput formats, facilitating novel compound screening. This requires assays that have minimal sample preparation and are adaptable to lower-volume 384-well formats and automation. Here we describe bioluminescent glucose, lactate, glutamine, and glutamate detection assays that are well suited for high-throughput analysis of two major metabolic pathways in cancer cells: glycolysis and glutaminolysis. The sensitivity (1-5 pmol/sample), broad linear range (0.1-100 µM), and wide dynamic range (>100-fold) are advantageous for measuring both extracellular and intracellular metabolites. Importantly, the assays incorporate rapid inactivation of endogenous enzymes, eliminating deproteinization steps required by other methods. Using ovarian cancer cell lines as a model system, the assays were used to monitor changes in glucose and glutamine consumption and lactate and glutamate secretion over time. Homogeneous formats of the lactate and glutamate assays were robust (Z' = 0.6-0.9) and could be multiplexed with a real-time viability assay to generate internally controlled data. Screening a small-compound library with these assays resulted in the identification of both inhibitors and activators of lactate and glutamate production.

Measuring Activity of Phosphoinositide Lipid Kinases Using a Bioluminescent ADP-Detecting Assay.

Phosphatidylinositol (PI) and its phosphorylated derivatives, collectively called phosphoinositides, are important second messengers involved in a variety of cellular processes, including cell proliferation, apoptosis, metabolism, and migration. These derivatives are generated by a family of kinases called phosphoinositide lipid kinases (PIKs). Due to the central role of these kinases in signaling pathways, assays for measuring their activity are often used for drug development. Lipid kinase substrates are present in unique membrane environments in vivo and are insoluble in aqueous solutions. Therefore the most important consideration in developing successful lipid kinase assays is the physical state of lipid kinase substrates. Here we describe the preparation of lipid substrates for two major classes of lipid kinases, phosphatidylinositol 3-kinases (PI3Ks) and phosphatidylinositol 4-kinases (PI4Ks). Using PI4Ks as an example, we also provide a detailed protocol for small-scale kinase expression and affinity purification from transiently transfected mammalian cells. For measuring lipid kinase activity we apply a universal bioluminescent ADP detection approach. The approach is compatible with diverse lipid substrates and can be used as a single integrated platform for measuring all classes of lipid and protein kinases.

Endometrial cancer and microsatellite instability status.

Microsatellite instability (MSI) is an important factor in the development of various cancers as an identifier of a defective DNA mismatch repair system. The objective of our study was to define the association between microsatellite instability status and traditional clinicopathologic characteristics of endometrioid type adenocarcinoma. MATERIAL AND METHODS: MSI status of endometrial cancer was examined by employing the Promega MSI Analysis System. This system uses 5 mononucleotide markers to identify MSI in tumour and normal tissue DNA (BAT-25, BAT-26, NR-21, NR-24, and MONO-27), and 2 pentanucleotide markers (Penta C and Penta D) for specimen identification. In this study, we investigated MSI status in 109 endometrial carcinomas. RESULTS AND CONCLUSIONS: One hundred (92%) of 109 endometrial cancers showed endometrioid type histology and only 9 (8%) non-endometrioid type. MSI-high was found in 17% (17/100) of endometrioid type adenocarcinomas, in 0% (0/9) of non-endometrioid carcinomas. Selected clinicopathologic parameters for endometrioid type adenocarcinomas were compared to the MSI status which was separated into two groups - MSI-high and MSI stable. The results showed that MSI-high status was related to clinicopathologic parameters such as deep myometrial invasion and higher histologic grade in endometrioid type adenocarcinomas.

Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening.

Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z' = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.

Bioluminescent cell-based NAD(P)/NAD(P)H assays for rapid dinucleotide measurement and inhibitor screening.

Abstract The central role of nicotinamide adenine dinucleotides in cellular energy metabolism and signaling makes them important nodes that link the metabolic state of cells with energy homeostasis and gene regulation. In this study, we describe the implementation of cell-based bioluminescence assays for rapid and sensitive measurement of those important redox cofactors. We show that the sensitivity of the assays (limit of detection ∼0.5 nM) enables the selective detection of total amounts of nonphosphorylated or phosphorylated dinucleotides directly in cell lysates. The total amount of NAD+NADH or NADP+NADPH levels can be detected in as low as 300 or 600 cells/well, respectively. The signal remains linear up to 5,000 cells/well with the maximum signal-to-background ratios ranging from 100 to 200 for NAD+NADH and from 50 to 100 for NADP+NADPH detection. The assays are robust (Z' value >0.7) and the inhibitor response curves generated using a known NAD biosynthetic pathway inhibitor FK866 correlate well with the reported data. More importantly, by multiplexing the dinucleotide detection assays with a fluorescent nonmetabolic cell viability assay, we show that dinucleotide levels can be decreased dramatically (>80%) by FK866 treatment before changes in cell viability are detected. The utility of the assays to identify modulators of intracellular nicotinamide adenine dinucleotide levels was further confirmed using an oncology active compound library, where novel dinucleotide regulating compounds were identified. For example, the histone deacetylase inhibitor entinostat was a potent inhibitor of cellular nicotinamide adenine dinucleotides, whereas the selective estrogen receptor modulator raloxifene unexpectedly caused a twofold increase in cellular nicotinamide adenine dinucleotide levels.

A bioluminescent assay for the sensitive detection of proteases.

A bioluminescent general protease assay was developed using a combination of five luminogenic peptide substrates. The peptide-conjugated luciferin substrates were combined with luciferase to form a homogeneous, coupled-enzyme assay. This single-reagent format minimized backgrounds, gave stable signals, and reached peak sensitivity within 30 min. The bioluminescent assay was used to detect multiple proteases representing serine, cysteine, and metalloproteinase classes. The range of proteases detected was broader and the sensitivity greater, when compared with a standard fluorescent assay based on cleavage of the whole protein substrate casein. Fifteen of twenty proteases tested had signal-to-background ratios >10 with the bioluminescent method, compared with only seven proteases with the fluorescent approach. The bioluminescent assay also achieved lower detection limits (≤100 pg) than fluorescent methods. During protein purification processes, especially for therapeutic proteins, even trace levels of contamination can impact the protein's stability and activity. This sensitive, bioluminescent, protease assay should be useful for applications in which contaminating proteases are detrimental and protein purity is essential.

Evaluating the utility of a bioluminescent ADP-detecting assay for lipid kinases.

The lipid second messengers phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) and sphingosine 1-phosphate (S1P) are well recognized to play important roles in a variety of cellular processes, including cell proliferation, apoptosis, metabolism, and migration. Disruption of lipid signaling pathways often leads to human cancers, making lipid kinases attractive drug targets. In order to develop novel drugs against these enzymes, an assay that monitors their activity and amenable to high-throughput scale for screening large number of compounds is essential. The newly developed ADP-Glo assay is such an assay that measures kinase activity of lipid kinases by detecting the formation of ADP using a highly robust and sensitive bioluminescence approach. We evaluated this technology for studying lipid kinases, class I PI3 kinases, and sphingosine kinases and we show that the assay exhibits good tolerance to different lipids substrates. It generates kinetic parameters for substrates and inhibitors similar to those reported in the literature using other published assay formats. The sensitivity and robustness of this assay allow the detection of 5% of substrate conversion with Z' values >0.7 making it attractive for high-throughput screening (HTS) applications. It is noteworthy that ADP-Glo assay addresses the need for a single integrated platform to comprehensively measure all classes of lipid and protein kinases. The selected inhibitors of lipid kinases can be screened against the panel of desired protein kinases, making ADP-Glo assay a simple, inexpensive platform for HTS and profiling of lipid kinases.

Neuronal calcium sensor-1 facilitates neuronal exocytosis through phosphatidylinositol 4-kinase.

This work tested the theory that neuronal calcium sensor-1 (NCS-1) has effects on neurotransmitter release beyond its actions on membrane channels. We used nerve-ending preparations where membrane channels are bypassed through membrane permeabilization made by mechanical disruption or streptolysin-O. Nerve ending NCS-1 and phosphatidylinositol 4-kinase (PI4K) are largely or entirely particulate, so their concentrations in nerve endings remain constant after breaching the membrane. Exogenous, myristoylated NCS-1 stimulated nerve ending phosphatidylinositol 4-phosphate [PI(4)P] synthesis, but non-myristoylated-NCS-1 did not. The N-terminal peptide of NCS-1 interfered with PI(4)P synthesis, and with spontaneous and Ca(2+)-evoked release of both [(3)H]-norepinephrine (NA) and [(14)C]-glutamate (glu) in a concentration-dependent manner. An antibody raised against the N-terminal of NCS-1 inhibited perforated nerve ending PI(4)P synthesis, but the C-terminal antibody had no effects. Antibodies against the N- and C-termini of NCS-1 caused significant increases in mini/spontaneous/stimulation-independent release of [(3)H]-NA from perforated nerve endings, but had no effect on [(14)C]-glu release. These results support the idea that NCS-1 facilitates nerve ending neurotransmitter release and phosphoinositide production via PI4K and localizes these effects to the N-terminal of NCS-1. Combined with previous work on the regulation of channels by NCS-1, the data are consistent with the hypothesis that a NCS-1-PI4K (NP, neuropotentiator) complex may serve as an essential linker between lipid and protein metabolism to regulate membrane traffic and co-ordinate it with ion fluxes and plasticity in the nerve ending.

Phosphatidylinositol 4-OH kinase is a downstream target of neuronal calcium sensor-1 in enhancing exocytosis in neuroendocrine cells.

Neuronal calcium sensor-1 (NCS-1), the mammalian orthologue of frequenin, belongs to a family of EF-hand-containing Ca(2+) sensors. NCS-1/frequenin has been shown to enhance synaptic transmission in PC12 cells and Drosophila and Xenopus, respectively. However, the precise molecular mechanism for the enhancement of exocytosis is largely unknown. In PC12 cells, NCS-1 potentiated exocytosis evoked by ATP, an agonist to phospholipase C-linked receptors, but had no effect on depolarization-evoked release. NCS-1 also enhanced exocytosis triggered by ionomycin, a Ca(2+) ionophore that bypasses K(+) and Ca(2+) channels. Overexpression of NCS-1 caused a shift in the dose-response curve of inhibition of ATP-evoked secretion using phenylarsine oxide, an inhibitor of phosphatidylinositol 4-OH kinase (PI4K). Plasma membrane phosphatidylinositol 4,5-bisphosphate pools were increased upon NCS-1 transfection as visualized using a phospholipase C-delta pleckstrin homology domain-green fluorescent protein construct. NCS-1-transfected cell extracts displayed increased phosphatidylinositol-4-phosphate biosynthesis, indicating an increase in PI4K activity. Mutations in NCS-1 equivalent to those that abolish the interaction of recoverin, another EF-hand-containing Ca(2+) sensor, with its downstream target rhodopsin kinase, lost their ability to enhance exocytosis. Taken together, the present data indicate that NCS-1 modulates the activity of PI4K, leading to increased levels of phosphoinositides and concomitant enhancement of exocytosis.