Phosphatidylinositol 4-OH Kinase Is a Downstream Target of Neuronal Calcium Sensor-1 in Enhancing Exocytosis in Neuroendocrine Cells*

Manisha RajebhosaleDagger , Sam GreenwoodDagger , Jolanta Vidugiriene§, Andreas Jeromin, and Sabine HilfikerDagger ||

From the Dagger  University of Manchester, School of Biological Sciences, Oxford Road, Manchester M13 9PT, United Kingdom,  Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada, and § Research and Development, Promega Corporation, Madison, Wisconsin 53711

Received for publication, May 14, 2002, and in revised form, December 3, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neuronal calcium sensor-1 (NCS-1), the mammalian orthologue of frequenin, belongs to a family of EF-hand-containing Ca2+ 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 Ca2+ ionophore that bypasses K+ and Ca2+ 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 Ca2+ 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neuronal calcium sensor-1 (NCS-1),1 the mammalian homologue of frequenin, belongs to the EF-hand family of Ca2+-binding proteins (1, 2), which includes recoverin/S-modulin, visinin, visinin-like proteins, neurocalcin, and hippocalcin. Some members of this family, such as recoverin/S-modulin and visinin, are expressed only in photoreceptor cells and are thought to function in the control of visual transduction pathways, whereas the functional roles of the other members are largely unknown. Most family members are solely expressed in neuronal and neuroendocrine cells, but a frequenin homologue has been identified in yeast, suggesting a more general role for this protein in mediating Ca2+ responses.

All members of the NCS family share four EF-hand motifs and a myristoylated N terminus. For NCS-1, three of the four EF-hands are functional Ca2+-binding motifs, whereas the EF-hand closest to the N terminus (EF1) is non-functional (2). Biochemical and structural analysis of recoverin have led to the calcium/myristoyl switch model in which Ca2+ binding to the NCS proteins may trigger their translocation from the cytosol to intracellular membranes (3, 4). This model suggests that Ca2+ binding to the NCS proteins induces a large conformational change, resulting in the exposure of the myristoyl group believed to allow membrane attachment. In addition, the movement of the myristoyl group is thought to expose a hydrophobic pocket within the protein that could then interact with target proteins (4). However, recent studies suggest that the calcium/myristoyl switch is not a general feature of all members of this family, because the localization of NCS-1 was found to be independent of Ca2+, indicating that the myristoyl group may be freely accessible in the absence of Ca2+ binding to NCS-1 (5).

Overexpression of frequenin in Drosophila facilitates evoked neurotransmission at the neuromuscular junction (6), and injection of frequenin into Xenopus spinal neurons enhances both spontaneous and evoked neurotransmission (7). NCS-1 is also present in adrenal chromaffin and PC12 neuroendocrine cells, where its overexpression has been shown to increase Ca2+-regulated exocytosis from dense-core granules (8), analogous to the effect reported for Drosophila frequenin in enhancing synaptic vesicle exocytosis. However, the mechanism(s) by which NCS-1/frequenin regulates vesicular release is not known.

A wide array of binding partners has been identified for NCS-1/frequenin, only some of which may be of physiological relevance. NCS-1/frequenin has been shown to activate membrane-bound guanylate cyclase and to inhibit rhodopsin kinase (6, 9). However, these in vitro effects on photoreceptor proteins are unlikely to be relevant to the function of NCS-1/frequenin in vivo in neurons and neuroendocrine cells. NCS-1 also seems to activate various calmodulin targets, such as cyclic nucleotide phosphodiesterase, calcineurin, and nitric-oxide synthase (10, 11), and to modulate a variety of ion channels such as voltage-gated Ca2+ channels (12-15) and A-type K+ channels (16). Despite these interactions, the binding partners for NCS-1 in vivo are not known for certain, and thus the molecular downstream targets mediating NCS-1 function remain to be determined.

Recently, yeast frequenin was demonstrated to genetically and biochemically interact with, and regulate the activity of, the yeast phosphatidylinositol 4-OH kinase (PI4K) Pik1 (17), which itself has been shown to be essential for Golgi-to-cell surface vesicular trafficking (18, 19). Similarly, the mammalian Pik1 homologue PI4Kbeta (20) has been shown to interact with NCS-1 in vitro and if immunoprecipitated upon co-expression in non-neuronal cells (21, 22), suggesting that PI4Kbeta may be an evolutionarily conserved and functionally important downstream target of NCS-1/frequenin.

Inositol phospholipids have recently emerged as important regulators of exo- and endocytosis (23, 24). Regulated exocytosis of dense core vesicles has been shown to require a phosphatidylinositol transfer protein (PITPalpha ) (25) and a PI(4)P-5-kinase (26). In addition, the presence of a dense core vesicle-associated PI4K activity essential for exocytosis (27) has led to the hypothesis that generation of PtdIns(4,5)P2 at the plasma membrane (28) is an important step in secretion. PtdIns(4,5)P2 is a substrate for phospholipase C (PLC) activated by G-protein-coupled receptors, and changes in PtdIns(4,5)P2 levels are thus anticipated to lead to changes in secretion evoked by G-protein-coupled receptor agonists. However, in both neuronal and neuroendocrine cells, PtdIns(4,5)P2 has been shown to play an important role in exocytosis independent from its PLC-mediated cleavage (29, 30), and PtdIns(4,5)P2 is likely to act directly on a specific target (31).

In the present study, we provide evidence that NCS-1 enhances secretion from PC12 cells through regulating the activity of PI4K. Overexpression of NCS-1, but not of its myristoylation mutant, led to an enhancement of ATP- and ionomycin-evoked release. NCS-1 expression also decreased the sensitivity of the cells to phenylarsine oxide (PAO), an inhibitor of PI4K, and increased the levels of PtdIns(4,5)P2 at the plasma membrane. Finally, three mutations in NCS-1 equivalent to those that interfere with the binding of recoverin to rhodopsin kinase had lost their enhancing effect on exocytosis, suggesting that NCS-1 acts through activating PI4K, leading to enhanced PtdIns(4,5)P2 levels and a downstream enhancement of regulated secretion.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Expression Vectors-- The NCS-1 construct was generated as described previously (32). The PI4Kbeta , PI4Kbeta (D656A), and PI4Kbeta N terminus-GFP constructs were kindly provided by Rachel Meyers and have been described previously (33). The INP51-GFP and PH-EGFP/PH(R40L)-EGFP constructs were donated by John York and Tamas Balla, respectively.

PCMV5-hGH was generated by subcloning the coding region of hGH, amplified by PCR and incorporating 5' EcoRI and 3' XbaI sites, into the equivalent sites of pCMV5. NCS-1 mutants (G2A, E120Q, F22A, E26A, F55A, T92A) were generated using a QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The identity of all constructs was verified using automated DNA sequencing.

PC12 Cell Culture, Transfection, and Secretion Experiments-- Cell culture, transfection, and secretion experiments were performed as described (34) with some modifications. PC12 cells (Riken Cell Bank) were cultured on 100-mm dishes coated with collagen (rat tail collagen type I; BD Biosciences). Cells were grown at 37 °C in 5% CO2 in full medium (RPMI 1640 with heat-inactivated 10% horse serum and 5% fetal bovine serum, penicillin (50 units/ml), and streptomycin (50 units/ml)). Confluent cells (passage 6 throughout all experiments) were harvested using 0.125% trypsin, 0.5 mM EDTA in PBS without Ca2+ and Mg2+ and plated onto collagen-coated 12-well dishes at 80% confluency. The next day (90-95% confluency), 0.6 µg of pCMV5-hGH and 0.6 µg of plasmid of interest were co-transfected using 4 µl of LipofectAMINE 2000 (Invitrogen) according to the manufacturer's specification in Dulbecco's modified Eagle's medium without serum and antibiotics to increase transfection efficiency. Each LipofectAMINE 2000 tube was used only once, as transfection and co-transfection efficiencies decreased when tubes were re-used multiple times. After 4 h, the transfection mixture was replaced by full medium. PC12 cells from two wells transfected with the same plasmids were harvested and pooled the next day to compensate for possible slight differences in transfection and co-transfection efficiencies. Cells were re-plated into 24-well plates at a ratio of two 12-wells for four 24-wells. Two days after replating (90-95% confluency), secretion experiments were performed with all test and control conditions carried out on the same pool of transfected cells. Controls were treated with 0.5 ml of physiological saline solution (PSS; 145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, 15 mM Hepes-NaOH, pH 7.4). ATP-evoked secretion was stimulated by a 10 min incubation with ATP buffer (PSS containing 300 µM ATP), KCl-triggered secretion was stimulated by a 10-min incubation with high K+ saline solution (PSS containing 95 mM NaCl and 56 mM KCl), and ionomycin-triggered secretion was stimulated by a 10-min incubation with ionomycin (5 µM ionomycin (Sigma) in PSS, final Me2SO concentration 0.5% (v/v)). Cells were incubated with alpha -latrotoxin (final 1.5 nM) in PSS for 10 min. At the end of the experiment, dishes were transferred to ice, and the supernatant was removed and centrifuged for 10 min in an Eppendorf centrifuge. hGH in the supernatant from this centrifugation was taken as secreted hGH. The cells in the dishes were resuspended in 0.5 ml of ice-cold phosphate-buffered saline containing 1 mM EDTA and added to the pellet of the Eppendorf centrifugation of the medium. Cells were then lysed by five freeze-thaw cycles (in a dry ice/ethanol bath and a 37 °C heating block), and insoluble material was pelleted in an Eppendorf centrifuge. hGH in the supernatant from this centrifugation was taken as the cellular hGH that was not secreted. hGH levels in the various samples were measured using an enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. All experiments were performed in duplicates or triplicates, and the average percent of total hGH released was calculated. Statistical analyses were performed with the paired Student's t test.

Western Blot Analysis of Overexpressed Proteins in PC12 and COS-7 Cells-- PC12 cells were plated onto collagen-coated 6-well tissue culture plates at 80% confluency as described above. COS-7 cells were grown at 37 °C in 5% CO2 in COS cell medium (Dulbecco's modified Eagle's medium with 10% fetal bovine serum, penicillin (50 units/ml), and streptomycin (50 units/ml)) on 100-mm dishes. The next day (90-95% confluency), cells were transfected with the appropriate vector using 2.4 µg of vector/well of a 6-well plate for PC12 cells and 10 µg of vector/100-mm dish for COS-7 cells, respectively, using LipofectAMINE 2000 (Invitrogen) as described above. One day later, cells were lysed using 500 µl of hot 1% SDS, and cell lysates were centrifuged at 12,000 × g for 10 min. Protein concentration in the supernatant was determined by BCA assay (Pierce). Equal amounts of total protein were separated by SDS-PAGE and transferred to Hybond-N membranes (Amersham Biosciences). Membranes were blocked with 2% non-fat dry milk in TTBS (0.05% Tween 20, 150 mM NaCl, 20 mM Tris, pH 7.5) for 1 h at room temperature and hybridized with primary antibody (either anti-NCS-1 rabbit polyclonal or anti-PI4Kbeta rabbit polyclonal, respectively, at 1:1000 dilution) overnight at 4 °C. After washing in TTBS, blots were incubated with a horseradish peroxidase-tagged anti-rabbit antibody (DAKO A/S) at 1:2000 dilution for 1 h at room temperature, washed, and developed using an enhanced chemiluminescence system (Amersham Biosciences).

Inhibition of PI4K Activity-- Cells were co-transfected with either pCMV5-hGH and pcDNA3.1 or with pCMV5-hGH and pcDNA3.1-NCS-1, as described above. Cells were incubated in RPMI 1640 medium containing various concentrations of phenylarsine oxide (Sigma) (final Me2SO concentration in all conditions maintained at 0.5% (v/v)) for 15 min at 37 °C in 5% CO2, followed by secretion assays as described above.

Immunofluorescence-- To visualize localization of endogenous NCS-1 and PI4Kbeta , non-transfected cells were plated onto coverslips coated with poly-L-lysine (coated for 1 h with 50 µg/ml poly-L-lysine, Mr 30,000-70,000 (Sigma)) and grown for 3 days in either full medium (non-differentiated) or in serum-reduced (1%) medium containing 50 ng/ml NGF 2.5 S (Invitrogen). Cells were fixed for 20 min at 37 °C with 4% (w/v) paraformaldehyde in PBS containing 4% (w/v) sucrose. Cells were permeabilized in 0.5% Triton X-100 in PBS for 30 min and preincubated in blocking buffer (10% goat serum (Vector Laboratories) in 0.5% Triton X-100 in PBS) for 1 h at room temperature, followed by exposure to primary antibody (diluted in blocking buffer) for 1 h at room temperature. Cells were washed in 0.5% Triton X-100 in PBS for 30 min and incubated with secondary antibodies for 1 h at room temperature.

To determine the amount of overexpression of NCS-1, as well as the amount of co-expression of NCS-1 and hGH, 50% of cells were processed for secretion assays, whereas the remainder were replated onto poly-L-lysine-coated coverslips and fixed and processed for immunocytochemistry 2 days after replating. The amount of overexpressed protein was qualitatively assessed using antibody dilutions that barely visualized endogenous protein levels but easily detected overexpressed proteins. The amount of co-expression was calculated by counting the number of cells expressing both hGH and the protein of interest, as compared with cells expressing either one or the other protein only. Transfection efficiencies were calculated by counting the number of cells expressing either proteins, and 700-1200 cells were counted for each construct. Transfection efficiencies regularly amounted to around 15%, and all NCS-1 constructs reached co-transfection efficiencies of >= 90%.

The following antibodies were used: anti-NCS-1 rabbit polyclonal (44162) (35) or chicken polyclonal (22), anti-PI4Kbeta rabbit polyclonal (Upstate Biotechnology), anti-synaptobrevin/VAMP2 mouse monoclonal (Cl 69.1; Synaptic Systems), anti-syntaxin 1 mouse monoclonal (HPC-1; Sigma), and anti-hGH rabbit polyclonal (National Hormone and Peptide Program, NIDDK, National Institutes of Health). The specificities of the polyclonal anti-NCS-1 and anti-PI4Kbeta antibodies have been described previously (15, 33). Secondary antibodies included horse anti-mouse IgG conjugated to fluorescein isothiocyanate or Texas red (TR), goat anti-rabbit IgG conjugated to fluorescein isothiocyanate or TR (all from Vector Laboratories) and rabbit anti-chicken IgG conjugated to fluorescein isothiocyanate (Sigma). Cells were either examined on a confocal microscope (Leica TCS NT) under a ×40 oil-immersion objective, and data were acquired using Leica software or on an upright microscope (Leica) under a ×40 or ×100 oil-immersion objective, and data were acquired using IPLab.

PH Domain Imaging and Analysis-- Cells were co-transfected with NCS-1 or NCS-1(G2A) and either phospholipase C-delta PH domain-GFP construct (PH-EGFP) or PH(R40L)-EGFP, a mutant unable to bind PtdIns(4,5)P2 (36). Cells were either visualized live or after fixation and permeabilization as described above, 2 days after transfection, with identical results. For experiments measuring PH-EGFP intensities upon PAO treatment, cells were fixed but not permeabilized. To quantify intensities, rectangles of 80 × 110 pixels centered on each GFP-positive, well separated cell were collected from each random field acquired using a ×40 oil-immersion objective. Pixel intensities within each rectangle were calculated in IPLab, and a total of 100 cells were counted for each condition. Cells with the highest PH-EGFP expression levels were excluded, as they often gained a rounded appearance and formed membrane blebs (37). Experiments were done three times, and the data were analyzed using the paired Student's t test.

Electroporation of Cells, Endogenous Lipid Phosphorylation, and Thin Layer Chromatography-- PC12 cells were resuspended in RPMI 1640 medium (3 × 107 cells/ml). Cells (400 µl) were incubated with 15 µg of pcDCNA3.1-NCS-1, 15 µg of pcDNA3.1, or with 15 µg of pCMV5-GFP for 3 min and electroporated (330 V, 20 ms) using an ECM 830 Square Wave Electroporator (BTX; Genetronics Inc., San Diego, CA). Electroporated cells were immediately transferred into 10 ml of full medium, washed once, and counted. Under the indicated electroporation conditions, cell survival usually amounted to around 10-15%, and electroporation efficiency, as measured by counting the fraction of cells expressing GFP 2 days after electroporation, amounted to around 50%. Cells were plated onto collagen-coated 100-mm dishes at 5 × 106 cells/dish. Two days later (95% confluency), attached cells were harvested, washed into PBS, and counted, and 0.5 × 106 cells were pelleted in Eppendorf tubes. Pellets were quick-frozen in liquid N2 until further use.

Cells were resuspended in 100 µl of kinase buffer (20 mM Hepes/NaOH, pH 7.5, 100 mM NaCl, 10 mM MgCl2) and disrupted by passing five times through a 17-gauge needle syringe. The aliquot of the prepared lysate was used directly in an assay, or the resulting lysate was further fractionated by centrifugation at 10,000 × g for 15 min at 4 °C. The supernatant of this centrifugation was taken as cytosol fraction, and the pellet was resuspended in kinase buffer and taken as membrane fraction. PI4K activity was measured as described previously (61, 62). Briefly, to initiate phospholipid biosynthesis, cell lysate and membrane fractions were diluted with kinase buffer to 45 µl, and 5 µl of 0.5 mM ATP containing 10 µCi of [gamma -32P]ATP (3000-6000 Ci/mmol; PerkinElmer Life Sciences) was added. The reaction was incubated for 10 min at room temperature and stopped by adding 60 µl of 1 N HCl. A two-phase mixture was induced, and the phosphorylated lipids were extracted by adding 160 µl of chloroform:methanol, 1:1 (v/v). The extracted products were analyzed by thin layer chromatography (TLC) (silica gel 60 thin layer plates, using chloroform/methanol/15 N ammonium hydroxide/water (90:90:7:22) (v/v/v/v) as the solvent system). Radiolabeled PtdIns(4)P was identified by TLC mobility in comparison with purified PtdIns(4)P standard.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Localization of Endogenous NCS-1 and PI4Kbeta in PC12 Cells-- To identify the subcellular localization of NCS-1 and PI4Kbeta in PC12 cells, antibodies against NCS-1, PI4Kbeta , VAMP2, and syntaxin 1 were used to stain non-transfected PC12 cells. In both non-differentiated and NGF-differentiated cells, endogenous NCS-1 was mainly diffuse and cytosolic, with some additional perinuclear staining (Fig. 1, A and B). PI4Kbeta staining was observed in the cytosol and in a perinuclear area, as described for chromaffin cells (38), and upon NGF differentiation, PI4Kbeta staining was enriched in processes, consistent with its reported localization on secretory vesicles (27, 39) (Fig. 1, C and D). In comparison, VAMP2 staining was primarily observed in a perinuclear area in non-differentiated cells and was significantly enriched in processes in differentiated cells, in accordance with its localization on secretory vesicles of PC12 cells (40, 41) (Fig. 1, E and F). Syntaxin 1 was localized to the plasma membrane, with more intracellular staining observed upon NGF differentiation, suggesting that under conditions of intense membrane fusion, as occurs during neurite formation, syntaxin 1 undergoes recycling in differentiating PC12 cells (42) (Fig. 1, G and H). In all cases, no immunostaining was observed when incubation with primary antibodies was omitted (not shown). The comparative subcellular localization data suggest similar subcellular distributions for NCS-1 and PI4Kbeta , with both proteins being partially cytosolic and partially associated with perinuclear structures.


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Fig. 1.   Localization of endogenous NCS-1 and PI4Kbeta in PC12 cells. Non-differentiated and NGF-differentiated cells were stained as follows: A and B, NCS-1 (chicken anti-NCS-1, 1:50); C and D, PI4Kbeta (rabbit anti-PI4Kbeta , 1:50); E and F, VAMP2 (mouse anti-VAMP2, 1:1000); or G and H, syntaxin 1 (mouse anti-syntaxin 1, 1:100). Scale bars, 10 µm.

NCS-1 Enhances ATP-evoked Exocytosis from PC12 Cells-- To study the effects of NCS-1 and PI4Kbeta on exocytosis, we employed an assay developed by Holz and co-workers (43) in which plasmids encoding hGH and a second protein of interest are transiently co-transfected into PC12 cells. In transfected cells, hGH is packaged into vesicles of the regulated secretory pathway and serves as a reporter for exocytosis as a function of stimulation. A high probability of co-transfection of two distinct plasmids into the same cells makes it possible to investigate the effect of the protein of interest on hGH secretion. A change in hGH secretion upon co-transfection of a particular protein is taken as evidence for an involvement of the protein in exocytosis. This assay has been successfully used to implicate a variety of proteins in exocytosis (34, 44-46) and thus serves as a useful model system to study the role of NCS-1 and PI4Kbeta in secretion.

Extracellular ATP is thought to evoke secretion from PC12 cells by binding to two purinergic receptors: a P2Y receptor, which leads to the activation of PLC and concomitant inositol 1,4,5-trisphosphate-mediated release of Ca2+ from intracellular stores, and a P2X non-selective cation channel, which leads to Ca2+ entry across the plasma membrane (47, 48). ATP caused robust hGH secretion from PC12 cells co-transfected with control vector, which was enhanced when cells were co-transfected with NCS-1 (Fig. 2A), as reported previously (8). In the absence of secretagogue, the amount of hGH in the PC12 cell medium (~10%) did not increase upon NCS-1 transfection, indicating that NCS-1 does not induce a significant amount of constitutive secretion of hGH (Fig. 2A). Expression of NCS-1 did not alter the expression levels of hGH (Fig. 2B), indicating that the enhancement of exocytosis observed with NCS-1 is not because of a change in the relative amounts of expressed hGH. NCS-1 enhanced ATP-stimulated secretion by 31 ± 8% (n = 9, p < 0.01; see Fig. 2C). To test the functional relevance of the myristoyl group in NCS-1, cells were co-transfected with hGH and NCS-1(G2A), a point mutation that renders the myristoylation consensus sequence non-functional. This mutant was unable to enhance ATP-evoked exocytosis (3.6 ± 9.5% enhancement; n = 6; see Fig. 2C). In addition, a mutant in EF-hand EF3 (NCS-1(E120Q)) that has been shown to have impaired Ca2+-binding activity (12) still enhanced ATP-evoked secretion, albeit to a lesser extent as compared with wild-type NCS-1 (15.8 ± 1.9% enhancement; n = 5; p < 0.01; see Fig. 2C).


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Fig. 2.   NCS-1 enhances ATP-evoked secretion from PC12 cells. Cells were co-transfected in parallel either with pCMV5-hGH and empty control vector (pcDNA3) or with pCMV5-hGH and various test plasmids. hGH secretion was stimulated for 10 min with either control physiological solution (ctrl) or 300 µM ATP in physiological solution. The amount of hGH in the medium and in the cells was determined by enzyme-linked immunosorbent assay, and the percentage of secreted hGH and the total amount of hGH were calculated against an hGH standard curve. A and B, representative experiment comparing effects of control and NCS-1 vectors on hGH secretion and total amounts of hGH in cells co-transfected with pCMV5-hGH. C, to standardize results from the same test plasmids from repeated experiments, secretion observed in the control vector transfections was set to 100% for all experiments, and relative enhancement or inhibition of secretion of test plasmids was normalized to the control (normalized enhancement of release). Values shown represent means ± S.E. from multiple (n = 2-9) experiments. Statistically significant differences are marked by asterisks (*, p < 0.15; **, p < 0.01).

Double immunocytochemistry was performed on cells processed in parallel for secretion assays, using an anti-hGH antibody and an anti-NCS-1 antibody at a dilution that barely visualized endogenous protein but easily recognized overexpressed protein. For both mutant and wild-type NCS-1 constructs, transfection efficiencies were around 15%, and co-transfection efficiencies were comparable and >= 90% (data not shown). In addition, the level of overexpression of the different NCS-1 constructs was analyzed using both Western blotting and immunocytochemistry (Fig. 3, A and B). As assessed by immunocytochemistry and antibody concentrations that barely visualized endogenous protein, all three NCS-1 constructs were heavily overexpressed as compared with endogenous NCS-1 (8-10-fold), and no partial degradation of mutant NCS-1 constructs was detected by Western blotting (Fig. 3, A and B), indicating that the relative or total lack of an effect of the NCS-1 mutants on ATP-evoked secretion is not because of a difference in co-transfection efficiencies, expression levels, or partial degradation of expressed proteins. Overexpressed NCS-1 was found partially cytosolic and partially bound to the plasma membrane (Fig. 3, A and B). The myristoylation mutant NCS-1(G2A) was largely cytosolic (Fig. 3B), supporting the notion that the myristoyl anchor is required for membrane attachment. The Ca2+-binding mutant NCS-1(E120Q) was membrane-associated like wild-type NCS-1 (Fig. 3B), in agreement with the observation that the localization of NCS-1 is independent of Ca2+ (5).


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Fig. 3.   Localization and amounts of overexpressed NCS-1 and PI4Kbeta . A, extracts (60 µg) of PC12 cells transfected with various NCS-1 constructs were analyzed by Western blotting. B, overexpression and localization of NCS-1 constructs in NGF-differentiated cells was assessed by immunocytochemistry using anti-NCS-1 chicken polyclonal (22) at a 1:500 dilution to preferentially visualize NCS-1-overexpressing cells. C, extracts (60 µg) of PC12 or COS-7 cells transfected with various PI4Kbeta constructs were analyzed by Western blotting. D, expression of GFP-tagged N-terminal PI4Kbeta (PI4KN-term.) and GFP-tagged, palmitoylated/myristoylated 5'-PtdIns(4,5)P2-phosphatase (INP51) was visualized in live cells. Scale bar, 10 µm.

Next, we tested whether co-expression of PI4Kbeta , or mutants thereof, affect ATP-evoked hGH secretion. Neither wild-type PI4Kbeta , nor PI4Kbeta (D656A), a catalytically inactive mutant (33), nor a GFP-tagged N-terminal fragment (PI4KN-term.) changed ATP-evoked secretion, as compared with empty control vector (Fig. 2C). Immunocytochemistry with an anti-PI4Kbeta antibody (data not shown), as well as Western blot analysis, revealed that none of the three PI4Kbeta constructs were significantly expressed above endogenous protein levels, even though they could be overexpressed in COS-7 cells (Fig. 3C). Although not detectable above endogenous PI4Kbeta levels, expression of the GFP-tagged N-terminal fragment (PI4KN-term.) could be demonstrated (Fig. 3D). Finally, decreasing PtdIns(4,5)P2 levels presumably at the plasma membrane by co-expression of a GFP-tagged 5'-PtdIns(4,5)P2-phosphatase (INP51) predominantly plasma membrane-targeted by an N-terminal myristoylation/palmitoylation sequence (37) (Fig. 3D) significantly inhibited ATP-stimulated hGH secretion (24.9 ± 2.8% decrease; n = 2; p < 0.15; see Fig. 2C), indicating that PtdIns(4,5)P2 levels are important for regulated secretion. Thus, the lack of an effect of PI4Kbeta on ATP-evoked secretion may either be because of the endogenous enzyme not being present in rate-limiting concentrations or because of a lack of efficient overexpression of exogenous PI4Kbeta in this cell system.

Differential Effect of NCS-1 on hGH Secretion Evoked by Different Secretagogues-- As another means to address whether NCS-1 acts to enhance secretion by activating PI4Kbeta , we tested its effects on Ca2+-dependent secretion stimulated by secretagogues with distinct mechanisms of action. KCl at high concentrations induces Ca2+ influx by membrane depolarization, ionomycin acts as a Ca2+ ionophore, leading to global increases in [Ca]i, and alpha -latrotoxin triggers exocytosis by an unknown mechanism. Both KCl and ionomycin evoke secretion in a PLC-independent manner in PC12 cells (49). Although ATP-evoked hGH secretion was enhanced upon NCS-1 transfection (23.8 ± 2.7% increase, n = 5, p < 0.005; see Fig. 4, A and D), there was no effect on secretion when cells were stimulated with KCl (3.2 ± 4.4% increase, n = 5; see Fig. 4B) or alpha -latrotoxin (data not shown). There was a slight, variable NCS-1-dependent stimulation of hGH secretion induced by ionomycin (10 ± 6.7%; p < 0.25, n = 5; see Fig. 4, C and D).


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Fig. 4.   NCS-1 differentially modulates secretion evoked by different secretagogues. Cells were co-transfected in parallel either with pCMV5-hGH and empty control vector (pcDNA3) or with pCMV5-hGH and NCS-1. Representative experiments in which hGH secretion was stimulated for 10 min with control physiological saline (ctrl) and with 300 µM ATP in physiological saline (A), high K+ (56 mM KCl) solution (B), or with 5 µM ionomycin in physiological saline (C). D, secretion observed in cells co-transfected with pCMV5-hGH and empty vector was set to 100%, and relative enhancement of secretion with NCS-1 was normalized to control empty vector (normalized enhancement of release). Values shown represent means ± S.E. from five experiments. Statistically significant differences are marked by asterisks (*, p < 0.25; **, p < 0.005).

If NCS-1 enhances secretion through activating PI4Kbeta and concomitantly increasing PtdIns(4,5)P2 levels, the enhancement of ionomycin-stimulated release suggests that PtdIns(4,5)P2 levels may be directly relevant for exocytosis downstream from Ca2+ entry in a manner independent of PLC. In that case, it seems unclear why KCl-evoked release is not equally potentiated upon the expression of NCS-1. However, overexpressed NCS-1 was found enriched at the plasma membrane (Fig. 3) and thus may, at least in part, buffer depolarization-induced Ca2+ influx, which may mask its effect in enhancing exocytosis. Indeed, upon overexpression of NCS-1(E120Q), KCl-evoked secretion was slightly enhanced (10.3 ± 4.0; p < 0.1, n = 2), suggesting that decreasing the Ca2+ buffering activity of NCS-1(E120Q) at the plasma membrane unveils the stimulatory activity of this partially functional mutant.

Overexpression of NCS-1 Leads to a Shift in the Dose-response Curve Using Phenylarsine Oxide-- In chromaffin and PC12 cells, phosphorylation of phosphatidylinositols by PI4K is required for Ca2+-triggered exocytosis (25-27). In these cells, PI4K can be potently inhibited by PAO, resulting in a block of exocytosis (27, 50). Indeed, incubation of the cells with 3 µM PAO for 15 min prior to triggering Ca2+-dependent secretion with ATP resulted in a robust inhibition of hGH release in both vector and NCS-1-transfected cells (Fig. 5A). These PAO concentrations are similar to those reported to inhibit granule secretion and PtdIns(4,5)P2 production from synaptosomes (50). Treatment of the cells with PAO did not affect the total levels of hGH (data not shown), suggesting that it specifically blocks Ca2+-dependent secretion evoked by ATP, rather than interfering with hGH expression. Further, PAO blocked exocytosis in a dose-dependent manner in both control and NCS-1-transfected cells. When the levels of hGH release in the absence of PAO were normalized to 100%, inhibition of release was less pronounced in cells expressing NCS-1 as compared with cells expressing control vector (Fig. 5B). These data are consistent with our proposal that NCS-1 expression leads to enhanced levels of active PI4Kbeta and thus to a concomitant shift in the PAO dose-response curve of hGH secretion.


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Fig. 5.   NCS-1 expression causes a shift in the dose-response curve using phenylarsine oxide. Cells were co-transfected in parallel either with pCMV5-hGH and empty control vector (pcDNA3) or with pCMV5-hGH and NCS-1. A, experiment in which cells were incubated in medium containing either Me2SO (final 0.5% (v/v)) or 3 µM PAO in Me2SO (final 0.5% (v/v)) for 15 min before secretion assays were carried out using either control physiological saline (ctrl) or 300 µM ATP in physiological saline. B, experiment in which cells were incubated with 0, 0.3, 1, or 3 µM PAO for 15 min before secretion assays. The amount of secretion in the absence of PAO was set to 100% for both control vector and NCS-1-transfected cells. Results are shown from a single experiment performed in duplicate. Although the amount of inhibition with specific PAO concentrations was variable between experiments, the shift in the dose-response curve upon NCS-1 transfection was reproducible (n = 3 experiments, each performed in duplicate).

Increase in Plasma Membrane PtdIns(4,5)P2 Levels upon NCS-1 Transfection-- As another means to measure enhanced PI4Kbeta activity upon NCS-1 expression, we visualized plasma membrane PtdIns(4,5)P2 levels using a PLCdelta PH domain-GFP (PH-EGFP) construct. The PH domain of PLCdelta specifically binds PtdIns(4,5)P2 and has been shown to be predominantly associated with the plasma membrane (28, 37, 51). Indeed, PH-EGFP specifically labeled the plasma membrane, and to a lesser extent the cytosol, in live PC12 cells (data not shown). When cells were fixed and permeabilized, cytoplasmic staining was minimal, and PH-EGFP predominantly localized to the plasma membrane (Fig. 6A). The PLCdelta PH domain contains three critical basic residues that interact with PtdIns(4,5)P2, including Arg-40 (52). A mutated PH domain construct, PH(R40L)-EGFP (28), did not label the plasma membrane but was entirely cytosolic (Fig. 6A). These two constructs were used in co-transfection experiments with either NCS-1 or the non-functional NCS-1(G2A). The intensity of PH-EGFP staining in cells co-transfected with NCS-1 was counted from 300 individual cells well separated from each other and compared with the intensity of PH-EGFP staining in cells co-transfected with NCS-1(G2A) or cells co-transfected with empty control vector (pcDNA3) (Fig. 6B). NCS-1 expression led to a 25 ± 9.6% increase in PH-EGFP staining as compared with control vector and a 53 ± 7.4% increase as compared with NCS-1(G2A) (Fig. 6B). To exclude that the differences in PH-EGFP staining were because of differences in residual cytosolic PH-EGFP intensities upon NCS-1 transfection, we used the cytosolic PH(R40L)-EGFP as a control. NCS-1 expression did not lead to enhanced PH(R40L)-EGFP staining, suggesting that the observed increase in PH-EGFP staining is specific for PtdIns[4,5]P2 levels at the plasma membrane imaged by PH-EGFP. Co-expression of PH(R40L)-EGFP with either NCS-1 or NCS-1(G2A) led to a slight decrease in the averaged fluorescence intensities as compared with control empty vector (pcDNA3) (Fig. 6B), indicative of a competition for the transcriptional/translational machinery of the two co-expressed constructs. This competition was also visible upon co-expression of PH-EGFP and NCS-1(G2A), suggesting that the direct comparison between effects of NCS-1 and NCS-1(G2A) is most appropriate.


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Fig. 6.   Increase in plasma membrane PtdIns(4,5)P2 levels upon NCS-1 transfection as imaged with PH-EGFP. Cells were co-transfected in parallel with PH-EGFP and NCS-1, PH-EGFP and NCS-1(G2A), PH(R40L)-EGFP and NCS-1, or PH(R40L)-EGFP and NCS-1(G2A). Two days after transfection, cells were fixed and permeabilized, and EGFP fluorescence was imaged. Fluorescence intensities were quantified using rectangles of identical pixel size centered on 100 EGFP-positive cells for each condition. A, representative cells expressing NCS-1 and PH-EGFP (left) or NCS-1 and PH(R40L)-EGFP (right). Scale bar, 10 µm. B, averaged arbitrary fluorescence intensities (means ± S.E., n = 3) from a total of 300 cells each co-expressing empty control vector (pcDNA3) or NCS-1 or NCS-1(G2A) and either PH-EGFP or PH(R40L)-EGFP, respectively. *, p < 0.05. C, distribution of cells expressing identical plasma membrane-associated (PH-EGFP) or cytosolic (PH(R40L)-EGFP) GFP fluorescence (binned in 0.25 arbitrary units). Open squares and open diamonds, cells co-transfected with NCS-1. Closed circles and closed triangles, cells co-transfected with NCS-1(G2A).

Because expression levels in transfection experiments are highly variable, we also plotted PH-EGFP fluorescence intensities binned from the whole population of analyzed cells (Fig. 6C). NCS-1 expression led to a decrease in the number of cells with low amounts of plasma membrane PH-EGFP staining and a concomitant increase in the number of cells with larger amounts of plasma membrane PH-EGFP staining (Fig. 6C). These data suggest that NCS-1 overexpression leads to a specific increase of PtdIns(4,5)P2 levels at the plasma membrane, in agreement with its proposed role in activating PI4Kbeta .

PAO Treatment Leads to a Decrease in Plasma Membrane PtdIns(4,5)P2 Levels-- To confirm that PAO indeed inhibits the activity of PI4K, leading to a decrease of plasma membrane PtdIns(4,5)P2 levels, cells were transfected with the PH-EGFP construct before treatment with PAO. In live cells, PH-EGFP labeling of the plasma membrane could still be observed, but cytosol staining was increased (data not shown). The intensity of cytosolic PH-EGFP staining in cells transfected with PH-EGFP and treated for 15 min with PAO prior to fixation was counted from 300 individual cells and compared with the intensity of cytosolic PH-EGFP staining in transfected cells treated for 15 min with Me2SO (Fig. 7). PAO treatment led to a decrease in the number of cells with small amounts of cytosolic PH-EGFP staining and a concomitant increase in the number of cells with larger amounts of cytosolic PH-EGFP staining (Fig. 7A). However, the total amount of PH-EGFP staining in both cytosol and plasma membrane was not changed upon PAO treatment, indicating that the increase in PH-EGFP staining in the cytosol was accompanied by a decrease in PH-EGFP staining at the plasma membrane (Fig. 7, B and C). Overall, treatment of cells with PAO resulted in a 45 ± 6% increase in cytosolic PH-EGFP staining as compared with control Me2SO treatment, indicating that PAO treatment results in a partial knock-off of PH-EGFP staining from the plasma membrane because of a resultant decrease in plasma membrane PtdIns(4,5)P2 levels (Fig. 7C).


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Fig. 7.   PAO treatment leads to decrease in plasma membrane PtdIns(4,5)P2 levels. Cells were transfected with PH-EGFP, and 2 days after transfection, cells were treated with 5 µM PAO or Me2SO vehicle alone for 15 min prior to fixation. Fluorescence intensities were quantified using rectangles of identical pixel size centered within (for cytosol) or around (for total) 100 EGFP-positive cells for each condition. Distribution of cells expressing identical cytosolic (A) or total (B) PH-EGFP fluorescence (binned in 0.25 arbitrary units) is shown. Open squares, cells treated with Me2SO only. Closed circles, cells treated with 5 µM PAO in Me2SO. C, averaged arbitrary fluorescence intensities (means ± S.E., n = 3) from a total of 300 cells expressing PH-EGFP in either the absence or presence of PAO, measuring cytosolic (left) and total (right) GFP fluorescence, respectively. *, p < 0.001.

Overexpression of NCS-1 Leads to Increased PtdIns(4)P Production-- The effect of overexpressing NCS-1 on the phosphorylation of endogenous phosphatidylinositol (61, 62) was also examined in permeabilized PC12 cells. Expression of NCS-1 caused a significant increase in 32P labeling of PtdIns(4)P and PtdIns(4,5)P2 in cell lysates and membrane fractions, as compared with cells transfected with empty control vector (Fig. 8). The NCS-1-dependent increase in the labeling of PtdIns(4)P was abolished upon incubation with 10 µM wortmannin (data not shown), indicating that it was because of enhanced type III PI4Kbeta activity (Fig. 8). The substantial amount of radiolabeled PtdIns(4)P produced in the absence of NCS-1 overexpression was likely because of the activity of a type II enzyme, which is responsible for a large amount of PI4K activity in membranes of mammalian cells, as it was not sensitive to wortmannin and significantly stimulated with Triton X-100 (data not shown). These data are in agreement with a recent report indicating that endogenous PtdIns(4)P and PtdIns(4,5)P2 levels were increased in a stable NCS-1-expressing cell line as compared with control cells (63), which further validifies the present approach in permeabilized cells to measure PI4Kbeta -dependent changes in PtdIns(4)P and PtdIns(4,5)P2 levels upon transient overexpression of NCS-1.


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Fig. 8.   Effect of NCS-1 overexpression on phosphorylation of endogenous lipid substrate in permeabilized PC12 cells. Cells were electroporated with either pcDNA3.1 (C) or NCS-1 (N) constructs. Two days later, cell lysates or membrane fractions were analyzed for [32P]phosphate incorporation into phospholipids from [gamma -32P]ATP. A, representative TLC analysis of radiolabeled PtdIns(4)P and PtdIns(4,5)P2 production of entire cell lysates or membrane fractions. B, summary of quantitative data from several experiments (n = 6 for cell lysate, n = 3 for membrane fractions). *, p < 0.04. Phosphorylation of lipids was quantified by phosphorimaging analysis (PhosphorImager; Molecular Dynamics).

Novel NCS-1 Mutations Defective in Enhancing Ca2+-dependent Secretion-- NCS-1 is structurally similar to recoverin (53, 54) and can functionally replace the activating effect of recoverin on rhodopsin kinase in vitro (55). The residues within the N-terminal region of recoverin (Phe-22, Glu-26, Phe-55, Thr-92) implicated in making contacts with rhodopsin kinase (56) are all conserved in NCS-1. Moreover, the recently solved crystal structure of human NCS-1, which is identical in sequence to the rat homologue, revealed that three of these residues (Phe-22, Phe-55, Thr-92) are solvent-exposed and located in the wide hydrophobic crevice at the surface of NCS-1, which interacts with its protein ligand (54). The fourth, Glu-26, makes important side-chain contacts with helix J, the C-terminal region of NCS-1 that lies adjacent to the hydrophobic crevice and is thought to be important for ligand recognition (54). To determine whether these residues are essential for the function of NCS-1, we generated a series of novel NCS-1 mutations and tested their effects on hGH secretion (Fig. 9). Mutation E26A, which may interfere with the proper positioning of helix J, and mutations F22A and F55A, which may alter the ability of the hydrophobic crevice of NCS-1 to interact with its downstream target, abolished the ability of NCS-1 to enhance regulated secretion (Fig. 9, A and B). Mutation T92A was still functional in enhancing release, suggesting that the effects of F22A and F55A are specific and that not all solvent-exposed residues in the hydrophobic crevice interact with a protein ligand. In addition, the localization, overexpression levels, and co-transfection efficiencies of F22A, F55A, and E26A were equivalent to those of wild-type NCS-1 (Fig. 9C) (data not shown), and overexpressed mutant constructs were not degraded (Fig. 9C), indicating that their impaired ability to enhance secretion is not because of mislocalization or lowered expression levels. The in vivo effect of these mutants, together with the structural information mentioned above, suggests that residues Phe-22, Phe-55, and Glu-26 may function to allow protein ligand binding, which may directly or indirectly lead to activation of PI4Kbeta .


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Fig. 9.   Three novel NCS-1 mutants defective in enhancing Ca2+-dependent secretion. A, representative experiment comparing effects of control vector, NCS-1, and NCS-1 mutants on hGH secretion in cells co-transfected with pCMV5-hGH. B, to standardize results from the same plasmids from repeated experiments, secretion observed in the control vector transfections was set to 100% for all experiments, and relative enhancement of secretion of test plasmids was normalized to the control (normalized enhancement of release). Values shown represent means ± S.E. from multiple (n = 3) experiments. Statistically significant differences are marked by asterisks (*, p < 0.15; **, p < 0.05). C, extracts (60 µg) of PC12 cells transfected with various NCS-1 constructs were analyzed by Western blotting.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We examined the function of NCS-1, a neuronal Ca2+ sensor protein, in stimulus-secretion coupling in PC12 cells. Studies in Drosophila and Xenopus indicate that NCS-1 enhances synaptic efficacy (6, 7), and recent data demonstrate that NCS-1 regulates associative learning and memory in vivo in Caenorhabditis elegans (57). Although the effect of NCS-1 on enhancing transmitter release and/or synaptic plasticity is well established, the mechanism(s) of its action are poorly understood. In the present study, several independent lines of evidence indicate that NCS-1 enhances release by enhancing the activity of PI4K. 1) Secretagogues that activate PLC result in a pronounced NCS-1-dependent enhancement of secretion, whereas PLC-independent secretagogues have either none or only a slight effect on release. 2) Overexpression of NCS-1 leads to a shift in the dose-response curve of inhibition of release using PAO, a specific inhibitor of PI4K. 3) Overexpression of NCS-1 leads to an increase in plasma membrane-localized PtdIns(4,5)P2, as imaged with a PH-domain GFP construct. 4) Extracts from NCS-1-overexpressing cells display enhanced production of PtdIns(4)P in vitro, indicating increased PI4K activity. 4) Mutants of NCS-1 that are likely to be incapable of binding ligand or that are incapable of binding to membranes, have lost their ability to enhance secretion.

Overexpression of NCS-1 resulted in enhanced hGH secretion when cells were stimulated by ATP, a purinergic receptor agonist that activates PLC. Enhanced secretion using ATP as a secretagogue may thus reflect a PI4Kbeta -mediated increase in the amounts of PtdIns(4,5)P2 present at the plasma membrane, which act as substrate for PLC. However, the observation that NCS-1 also moderately enhanced ionomycin-triggered release suggests that increased levels of PtdIns(4,5)P2 have a more direct role in mediating exocytosis independent of PLC. Overexpression of NCS-1 has been reported to have no effect on Ca2+-dependent release from permeabilized PC12 cells (8), which is in contrast to the enhanced ionomycin-triggered release observed in this study. However, it seems possible that permeabilization would lead to the partial loss of NCS-1 and/or PI4Kbeta , which, at least in part, are both soluble in the cytoplasm.

The present study indicates that NCS-1 enhances large dense-core vesicle-mediated secretion from PC12 cells by activating PI4Kbeta . Because large dense-core vesicles contain high concentrations of intravesicular ATP, NCS-1 may elicit facilitation of neuropeptide and amine release by a purinergic receptor-mediated process. However, NCS-1 has also been reported to enhance secretion from small synaptic vesicles. Although the evidence for a requirement for PtdIns(4,5)P2 synthesis in synaptic vesicle secretion is conflicting (39, 50), NCS-1 may enhance exocytosis of synaptic vesicles by a similar mechanism. Alternatively, biochemical studies suggest that NCS-1 may have a diverse array of target proteins, some of which may be channel proteins (13-16). Thus, in addition to regulating PI4Kbeta activity, and dependent on the cell system, NCS-1 overexpression may lead to a combined effect on PtdIns(4,5)P2 levels and channel activity. Modulation of channel activity may be either direct or indirect through regulating the amount of channels expressed on the cell surface. For example, overexpression of NCS-1 leads to its localization in the cell periphery, in addition to a diffuse distribution throughout the cytoplasm, and to a concomitant increase in Kv4 channel localization at the plasma membrane (16), suggesting that NCS-1 may increase membrane trafficking of channel proteins, which by itself may be a PtdIns(4,5)P2-dependent process.

Activation of PI4Kbeta by NCS-1 will lead to concomitant de novo synthesis of PtdIns(4,5)P2, which has been shown to be essential for vesicle priming reactions in neuroendocrine cells (25, 26, 29, 58). During priming, PI4K and PtdIns(4)P-5-kinases may act in concert to modify the two newly juxtaposed membranes and contribute to the acquisition of fusion competence. Lipid polymorphism as exemplified by a localized increase in PtdIns(4,5)P2 may have a direct physiological role in the generation of membrane curvature as required during membrane fusion (59). Alternatively, the localized increase in PtdIns(4,5)P2 may allow recruitment of proteins involved in mediating secretion, such as CAPS (31, 60). In summary, our present study indicates that in neuroendocrine cells, the NCS-1-mediated enhancement of secretion is because of enhanced PI4Kbeta activity and concomitant increases in plasma membrane PtdIns(4,5)P2 levels.

    ACKNOWLEDGEMENTS

We thank A. Toker for providing the vector pCMV5, H. Weir (Astrazeneca) for providing a vector encoding human growth hormone, A. F. Parlow (National Hormone and Peptide Program, NIDDK, National Institutes of Health) for the anti-hGH antibody, A. Petrenko for providing alpha -latrotoxin, and L. Swanton for advice on COS-7 cell culture. We thank P. March for help with confocal image acquisition and P. Woodman and S. High for critical reading of the paper.

    FOOTNOTES

* This work was supported by MRC Grant G9722026 and a BBSRC David Phillips Fellowship (to S. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. Tel.: 44-161-275-5513; Fax: 44-161-275-5082; E-mail: sabine.hilfiker@man.ac.uk.

Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M204702200

    ABBREVIATIONS

The abbreviations used are: NCS, neuronal calcium sensor; PI4K, phosphatidylinositol 4-OH kinase; PLC, phospholipase C; PAO, phenylarsine oxide; GFP, green fluorescent protein; EGFP, enhanced GFP; PBS, phosphate-buffered saline; hGH, human growth hormone; PSS, physiological saline solution; NGF, nerve growth factor; VAMP, vesicle-associated membrane protein; PH, pleckstrin homology; PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PI(4)P-5-kinase, phosphatidylinositol 4-phosphate-5-kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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