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Regulate IgE Receptor-Triggered Exocytosis in Cultured Mast Cells 1
* Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel;
Allergene Ramat-Gan, Israel; and
Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030
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Abstract |
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(PI4K) and colocalize with the enzyme in the Golgi region. We show further that overexpression of NCS-1 in RBL-2H3 cells stimulates the catalytic activity of PI4K, increases IgE receptor (Fc
RI)-triggered hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), and stimulates FcRI-triggered, but not Ca2+ ionophore-triggered, exocytosis. Conversely, expression of a kinase-dead mutant of PI4K reduces PI4K activity, decreases FcRI-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis, and blocks FcRI-triggered, but not Ca2+ ionophore-triggered, exocytosis. Our results indicate that PI(4)P, produced by the Golgi-localized PI4K, is the rate-limiting factor in the synthesis of the pool of PI(4,5)P2 that serves as substrate for the generation of lipid-derived second messengers in FcRI-triggered cells. We conclude that NCS-1 is involved in the control of regulated exocytosis in nonneural cells, where it contributes to stimulus-secretion coupling by interacting with PI4K and positive regulation of its activity. |
Introduction |
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, the mammalian orthologue (5, 12).
Mast cells are professional secretory cells implicated in allergic and inflammatory responses. These cells release, in a Ca2+-triggered fashion, a number of allergic mediators, including vasoactive amines such as histamine, chemoattractants, and proteases. In addition, upon activation they produce and release metabolites of arachidonic acid and multiple cytokines, which together affect the early and late phases of the allergic reactions (13, 14). Physiologically, mast cell exocytosis is triggered by Ag-induced cross-linking of cell-bound IgE Abs and consequent aggregation of the high affinity IgE receptor (FcRI) (15). Alternatively, exocytosis can be triggered by receptor analogues, that directly activate G proteins (16, 17, 18, 19). The precise mechanisms by which secretion is brought about are still vague. However, a number of signaling pathways, including the activation of Src-like protein tyrosine kinases (PTKs) and phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing phospholipase C (PLC), are critical elements in stimulus-secretion coupling (reviewed in Ref.20).
In the present study we investigated the possible role and mechanism of action of NCS-1 in controlling Ca2+-regulated exocytosis in mast cells. We demonstrate that NCS-1 is endogenously expressed in cultured rat basophilic leukemia cells (RBL-2H3), where it interacts with and activates PI4K. We further show that overexpression of NCS-1 or a kinase-dead (KD) PI4K mutant stimulates or inhibits FcRI-induced PIP2 hydrolysis and exocytosis, respectively. Our findings therefore implicate NCS-1 as a novel regulator of FcRI-triggered exocytosis.
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Materials and Methods |
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The protease inhibitor mixture Complete was obtained from Roche (Indianapolis, IN). MTT, NO2Ph22-human serum albumin (HSA), p-nitrophenyl-N-acetyl-D-glucoseaminide, and phosphatidylinositol were purchased from Sigma-Aldrich (St. Louis, MO). The Ca2+ ionophore A23187 and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) were obtained from Calbiochem (San Diego CA). [
-32P]ATP (3000 Ci/mmol) and [3H]myo-inositol (18 Ci/mmol) were obtained from NEN-DuPont (Boston, MA).
Antibodies
Anti-NCS-1 Abs included affinity-purified polyclonal NCS-1 Abs made in chicken (serum 22), affinity-purified polyclonal NCS-1 Abs made in rabbit (serum 44162), and Abs directed against the N-terminal sequence of NCS-1. Rabbit anti-human PI4K was obtained from Upstate Biotechnology (Lake Placid, NY). Peroxidase-conjugated Affinipure goat anti-mouse, goat anti-rabbit, and donkey anti-chicken IgGs and FITC- or rhodamine/Cy2-conjogated donkey anti-mouse/rabbit/chicken IgGs were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
Isolation of mast cells
Rat peritoneal mast cells (RPMC) were obtained from Wistar rats by peritoneal lavage and purified as previously described (16). Briefly, a suspension of washed peritoneal cells was layered over a cushion of 30% Ficoll 400 (Amersham Pharmacia Biotech, Piscataway, NJ) in buffered saline and 0.1% BSA and centrifuged at 150 x g for 15 min. The purity of mast cells recovered from the bottom of the tube was >90%, as assessed by toluidine blue staining.
Cell culture
RBL-2H3 cells (hereafter referred to as RBL) were maintained as adherent cultures in DMEM supplemented with 10% FCS in a humidified atmosphere of 6% CO2 at 37°C.
PCR amplification of NCS-1 fragments
Total RNA was isolated using the TRIzol reagent (Life Technologies, Gaithersburg, MD). cDNA was synthesized for 60 min at 42°C, followed by 5 min at 99°C, using the Reverse Transcription System Kit (Promega, Madison, WI). PCR was performed using primers A and B (A: 5'-GGGGTACCCCAGGATCGAGTTCTCCGAATTCA-3'; B: 5'-ATGTAGCCATCGTTATCCAAGT-3') and 1 µl of the RT reaction as template. PCR was performed for 40 cycles of 1 min at 94°C, 1 min at 57°C, and 1 min at 72°C. The PCR product was purified by agarose gel electrophoresis and sequenced. Blast search against the National Center for Biotechnology Information gene bank revealed that it was 99% homologous to Norvegicos rattus NCS-1.
Cell transfections
Full-length cDNAs encoding the 22-kDa wild-type NCS-1 or the kinase-dead mutant of PI4K (KD-PI4KD656A) were subcloned into the KpnI/XbaI sites of the pcDNA3 expression vector (Invitrogen, San Diego, CA). These constructs have been described in detail previously (11, 21). RBL cells (8 x 106) were transfected with 20 µg of DNA by electroporation (0.25 V, 960 µF). The cells were immediately replated in tissue culture dishes containing growth medium (supplemented with DMEM). G418 (1 mg/ml) was added 24 h after transfection, and stable transfectants were selected within 14 days.
Western blot analysis
Samples of cell extracts (normalized according to protein content or number of cells) were separated by SDS-PAGE under reducing conditions using 7.5–12% polyacrylamide gels. They were then electrophoretically transferred to nitrocellulose or polyvinylidene difluoride membranes. Blots were blocked for 3 h in TBST (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20) containing 5% skim milk or 2% BSA, followed by overnight incubation at 4°C with the indicated primary Abs. Blots were washed three times and incubated for 1 h at room temperature with the secondary Abs (HRP-conjugated donkey anti-chicken or goat anti-rabbit or anti-mouse IgG). Immunoreactive bands were visualized by the enhanced chemiluminescence method according to standard procedures.
Determination of cell viability by MTT cell proliferation assay
Cells were washed twice in phenol red-free RPMI 164 and seeded at 2.5 x 104 cells/well in 96-well plates in a final volume of 100 µl. At 0, 18, 24, 48, and 72 h 10 µl of MTT (5 mg/ml) was added to each well, and incubation was continued for 2 h at 37°C. At the end of the incubation period, 100 µl of HCl (0.07 M) diluted in isopropanol was added, and the spectrophotometric absorbance of each sample was measured at 570 nm using a Spectra Microplate Reader.
Coimmunoprecipitation of PI4K and NCS-1
Cell extracts were prepared by lysing the cells in a lysis buffer comprising 20 mM HEPES (pH 7.4), 100 mM NaCl, 2.5 mM MgCl2, 2 mM EDTA, 40 mM -glycerophosphate, 1% Nonidet P-40, 0.5 mM vanadate, and 1 mM DTT supplemented with a protease mixture and 1 mM PMSF. Aliquots containing 500 µg of protein were incubated for 18 h at 4°C with anti-PI4K (1/100 dilution), or with anti NCS-1 (serum 44162; 1/50 dilution). Protein A was subsequently added, and immune complexes were collected and washed three times in lysis buffer. Immune complexes were then resolved by SDS-PAGE, transferred to nitrocellulose filters, and probed with either anti-PI4K or anti-NCS-1 (serum 22), followed by HRP-conjugated secondary Abs. Immunoreactive bands were visualized by the ECL method according to standard procedures.
Activation of secretion of RBL cells
Cells were plated in 24-well plates (2.5 x 105 cells/well) and incubated overnight with monoclonal dinitro-phenyl (NO2Ph)-specific IgE Ab. The cells were then washed three times in Tyrode buffer (10 mM HEPES (pH 7.4), 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% BSA) and stimulated in the same buffer with the desired stimuli (Ca2+ ionophore A23187, TPA, or NO2Ph22-HSA Ag). Secretion was allowed to proceed for 30 min at 37°C. Aliquots from the supernatants were taken for measurements of released -hexosaminidase activity. The adherent cells were then lysed by the addition of 0.1% Triton X-100 to determine the total enzyme content. -Hexosaminidase activity was determined as previously described (22). The amount of secretion is presented as the percentage of the total -hexosaminidase enzymatic activity present in the cells.
Determination of inositol phosphate formation
Cells (mock-, NCS-1-, or KD-PI4K-transfected) were incubated for 18 h in a low inositol medium (medium 199) containing [3H]myo-inositol (250 µCi). Inositol phosphate formation was determined as previously described (23).
Determination of PI4K catalytic activity
PI4K was immunoprecipitated using anti-PI4K Abs as described above. Immune complexes were resuspended in 80 µl of kinase buffer, comprising 40 µM ATP, 20 mM HEPES (pH 7.4), 0.4 mg/ml phosphatidylinositol, 10 mM MgCl2, and 1 µCi of [-32P]ATP, and incubated for 15 min at 37°C. Reactions were stopped by adding 90 µl of 5 N HCl, and the lipids were extracted with 900 µl of chloroform/methanol (1/1) and 435 µl of water. The organic phases were collected and separated by TLC in chloroform/methanol/H2O/NH3 (130/115/20/15). Plates were autoradiographed, and PIP formation was quantified by densitometric analysis. PIP was identified by running nonlabeled PIP on the TLC plates as a standard. The latter was visualized by subjecting the TLC plate to iodine vapors.
Indirect immunofluorescence
Cells were grown on coverslips and fixed for 30 min at room temperature with 3% paraformaldehyde in PBS. Cells were then permeabilized for 30 min at room temperature with 0.1% Triton X-100, 5% FCS, and 2% BSA diluted in PBS. After washing, cells were incubated with primary Abs for 1 h at room temperature, followed by three washes and 1-h incubation with the appropriate secondary Abs (FITC/rhodamine/Cy2-conjugated, 1/200). After washing, the cells were mounted (Biomeda, Foster City, CA) and analyzed by laser confocal microscopy.
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Results |
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PCR with primers corresponding to positions 236 (sense) and 347 (antisense) of the NCS-1 coding sequence (primers A and B, under Materials and Methods) on RBL cell cDNA yielded a product of the predicted size of 111 bp (Fig. 1A). A product of similar size was also obtained when the PCR reaction was conducted on RNA derived from rat peritoneal mast cells (RPMC; Fig. 1B). Sequencing of both PCR products revealed that they were 99% homologous to rat NCS-1, therefore confirming that both RBL cells and RPMC express endogenously mRNA encoding NCS-1.
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To begin exploring the role of NCS-1 in mast cells, we stably transfected RBL cells with either full-length cDNA encoding the 22-kDa NCS-1 protein or with empty vector to generate control cells. This transfection resulted in a number of clones, which expressed elevated levels of the 22-kDa NCS-1 protein. Three stable clones, which expressed comparable and significantly higher levels of NCS-1, were selected for all further analyses (Fig. 3). Notably, this transfection did not increase the level of the 50-kDa NCS-1-immunoreactive protein (not shown). NCS-1-overexpressing cells were viable (Fig. 4), and their morphology was unaltered (not shown).
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Overexpression of NCS-1 failed to affect exocytosis evoked by a Ca2+ ionophore alone (Fig. 5A) or in the presence of the phorbol ester TPA (Fig. 5B). In contrast, overexpression of NCS-1 significantly potentiated Ag-induced exocytosis elicited by the cross-linking of cell-bound IgE Abs (Fig. 5C). Hence, throughout the range of Ag concentrations used, release of -hexosaminidase, a marker enzyme for exocytosis, was 2-fold higher in the NCS-1-overexpressing cells compared with the control mock-transfected RBL cells (Fig. 5C).
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in mast cells
The finding that overexpression of NCS-1 stimulated FcRI-induced, but not Ca2+ ionophore-induced, exocytosis strongly suggested that NCS-1 was not functioning as a Ca2+ sensor, but, rather, was involved in stimulus-secretion coupling. Hydrolysis of PI(4,5)P2 plays a critical role in FcRI-induced exocytosis, as is essential for Ca2+ mobilization and activation of protein kinase C (24). PI4K may thus play an important role in the maintenance of PI(4,5)P2 homeostasis and as such represents a critical factor in FcRI-dependent signaling. The yeast homologue of NCS-1 associates with Pik1, the yeast homologue of PI4K, and stimulates its activity (10). Similarly, NCS-1 and PI4K physically associate in neuronal cells (25) and when coexpressed in COS-7 cells (26). Therefore, we investigated the possibility that overexpressed NCS-1 may stimulate FcRI-induced exocytosis by associating with PI4K and up-regulating its activity. To this end, we performed coimmunoprecipitation experiments using Abs to either PI4K or NCS-1 to examine whether NCS-1 and PI4K form a complex in vivo. Abs to PI4K immunoprecipitated a 110-kDa protein (Fig. 6A), which was also detected by these Abs on a Western blot of a total cell lysate (Fig. 6B). These results confirmed that PI4K was endogenously expressed in the RBL cells. Reprobing the blot with Abs to NCS-1 revealed the presence of both the 22- and 50-kDa NCS-1 immunoreactive proteins (Fig. 6C), indicating that both endogenous NCS-1 and the NCS-1-related protein were associated with PI4K in intact RBL cells. Notably, when the same experiments were repeated using the NCS-1-overexpressing RBL cells, the amount of NCS-1 in the immune complex was considerably increased, whereas that of the 50-kDa NCS-1-related protein was concomitantly decreased (Fig. 6C). In a similar fashion, immunoprecipitation with anti-NCS-1 resulted in the coimmunoprecipitation of PI4K from either the control or the NCS-1-overexpressing RBL cells (not shown).
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interact in vivo and to identify the cellular site of their interaction, we used indirect immunofluorescence and laser confocal microscopy to localize NCS-1 and PI4K in RBL cells. As illustrated in Fig. 7, PI4K showed a cytosolic distribution and a clear perinuclear location (Fig. 7A). Consistent with previous studies, PI4K staining overlapped with that of the Golgi marker GOS28, implying that PI4K was localized to the Golgi (not shown). NCS-1 was mainly cytosolic (Fig. 7B). However, a considerable fraction of the protein displayed a perinuclear location (Fig. 7B), where it clearly colocalized with PI4K (Fig. 7C).
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with NCS-1 affected the catalytic activity of the kinase, the ability of the kinase, immunoprecipitated from control or NCS-1-overexpressing cells, to phosphorylate exogenously added phosphatidylinositol was compared. These experiments demonstrated that the activity of PI4K immunoprecipitated from cells overexpressing NCS-1 was 
2-fold higher than that of enzyme immunoprecipitated under identical conditions from control cells (Fig. 8), thus suggesting that the interaction with NCS-1 positively regulated PI4K activity. Notably, the amount of PI4K immunoprecipitated from each cell type was the same (data not shown), confirming that the increased amount of PIP formed was indeed due to increased catalytic activity of the enzyme precipitated from NCS-1-overexpressing cells.
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is a critical factor in FcRI-induced exocytosis
To investigate whether PI4K was indeed an important factor in FcRI-induced exocytosis, we stably transfected RBL cells with cDNA encoding a hemagglutinin-tagged, kinase-dead mutant of PI4K (KD-PI4K). Cells expressing the hemagglutinin-tagged mutant manifested a slower growth rate, as indicated by the MTT test; however, their viability was not affected (Fig. 4).
First we examined whether expression of the kinase-dead mutant suppressed the catalytic activity of PI4K. Indeed, as shown in Fig. 8, PI4K immunoprecipitated from cells expressing the KD-PI4K mutant was 1.7- or 3.2-fold less active than the enzyme immunoprecipitated from control or NCS-1-overexpressing cells, respectively (Fig. 8).
Next, we examined the responsiveness of the KD-PI4K-expressing RBL cells to a Ca2+- or Ag-induced trigger. As shown in Fig. 5, exocytosis triggered by Ca2+ ionophore alone (Fig. 5A) or in combination with TPA (Fig. 5B) was unaltered in KD-PI4K-expressing RBL cells. However, these cells failed to respond and undergo exocytosis when triggered via FcRI aggregation (Fig. 5C). These results therefore indicated that expression of the KD-PI4K mutant did not impair the ability of RBL cells to undergo Ca2+-dependent exocytosis per se, but selectively suppressed receptor-mediated exocytosis. Taken together, these results established an association between the effects of NCS-1 or the KD-PI4K mutant on PI4K activity and exocytosis.
NCS-1 and KD-PI4K modulate FcRI-induced hydrolysis of phosphoinositides
To further support the idea that the differences in PI4K activity monitored in vitro in the immune complexes indeed reflected the situation in vivo in intact cells, we investigated whether overexpression of NCS-1 or the KD-PI4K mutant also affected FcRI-induced inositol phosphate formation. Ag trigger of the control RBL cells increased the amounts of inositol phosphates formed by 6-fold (Fig. 9A). The identical stimulus resulted in a 9-fold increase in inositol phosphates formed in the NCS-1-overexpressing cells (Fig. 9A). In contrast, only a 2-fold increase was recorded in the KD-PI4K mutant-expressing cells (Fig. 9A). Notably, neither the overexpression of NCS-1, nor the introduction of the kinase-dead mutant exerted any effect on the basal rate of PI(4,5)P2 hydrolysis, as indicated by the equal amounts of inositol phosphates formed in the control and transfected cells in the absence of trigger (Fig. 9A). We also investigated the kinetics of Ag-triggered inositol phosphate formation in these three cell types. This analysis revealed that both the rate and the length of the period of accumulation of inositol phosphates formed were affected by overexpression of NCS-1 or the KD-PI4K mutant; NCS-1 accelerated the rate and increased the duration of inositol phosphate accumulation, whereas expression of the KD-PI4K mutant slowed down and shortened this period (Fig. 9, B and C).
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Discussion |
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NCS-1 was previously shown to affect constitutive secretory pathways by association with PI4K and regulation of its activity. In yeast, NCS-1 interacts with and activates PiK1, one of the yeast noninterchangeable isoforms of PI4K, which corresponds to the mammalian PI4K isoform (10). In Madin-Darby canine kidney cells, overexpressed NCS-1 inhibits the delivery of newly synthesized proteins from the trans-Golgi network to the apical cell surface and coimmunoprecipitates with PI4K (12). A similar inhibitory effect is achieved by expressing wild-type PI4K in Madin-Darby canine kidney cells, whereas expression of a KD mutant enhances this transport (21). These results, therefore, suggest that the mechanism by which NCS-1 affects the biosynthetic traffic involves modulation of PIP. Indeed, recent data indicate that PI4K also serves a downstream target for NCS-1 in neuroendocrine cells (5), therefore suggesting that the modulation of PIP is also the mechanism underlying the stimulatory activity of NCS-1 on regulated exocytosis in neuroendocrine cells. Because inositol phospholipids are also essential factors in the control of regulated exocytosis in nonneuronal cells, in the present study we investigated whether and how NCS-1 may control regulated exocytosis in the RBL mast cell line. Here we show that the mucosal mast cell line RBL expresses NCS-1 mRNA and protein as well as PI4K. We further show that NCS-1 and PI4K colocalize in the Golgi region and coimmunoprecipitate from intact cells, therefore suggesting that they associate in vivo. Such an association was previously shown to occur in cells overexpressing NCS-1, in COS-7 cells, or in cells of neuronal origin (25). Here we demonstrate that under physiological conditions, endogenously expressed NCS-1 and PI4K form a complex in nonneural secretory cells. Notably, consistent with previous results (26), Ca2+ is not required for this interaction.
RBL cells also express a 50-kDa protein that is recognized by anti-NCS-1 Abs. This protein is detected in reducing as well as denaturing gels, therefore excluding it from being a dimer or aggregated form of NCS-1. However, the relationship between this 50-kDa and the 22-kDa NCS-1 protein is presently unknown. It is interesting to note that the 50-kDa immunoreactive protein also coimmunoprecipitates with PI4K. Moreover, in cells overexpressing the 22-kDa NCS-1 protein, the amount of the 50-kDa protein that coprecipitates with PI4K is substantially reduced, thus suggesting that NCS-1 and the 50-kDa immune-related protein may compete for the same binding sites on PI4K. Taken together with the fact that the amount of NCS-1 expressed in RBL cells is significantly lower than that in neuronal cells such as PC12, it is tempting to speculate that NCS-1 and the 50-kDa related protein may fulfill a similar function. This idea awaits further clarification.
The association of NCS-1 with PI4K results in increased catalytic activity of the enzyme. This is indicated by the increased ability to phosphorylate exogenously added PI of PI4K immunoprecipitated from NCS-1-overexpressing cells compared with an equal amount of enzyme immunoprecipitated from control cells. Conversely, the catalytic activity of PI4K immunoprecipitated from cells expressing a KD mutant of PI4K is diminished. That these differences in kinase activity monitored in vitro indeed reflect the situation in intact cells is supported by the corresponding changes observed in PIP2 hydrolysis under stimulated, but not basal, conditions. Neither overexpression of NCS-1 nor expression of the KD-PI4K mutant had any effect on the rate of PIP2 breakdown under resting conditions. However, overexpression of NCS-1 or the KD-PI4K mutant, respectively, increased or reduced the rate and amount of inositol phosphate formation in response to a receptor trigger. These findings strongly imply that PI(4)P is a limiting factor in the process of FcRI-triggered PIP2 hydrolysis. Hence, under resting conditions, when the catalytic activity of PLC is low, the amount of PIP2 available in control or KD-PI4K-expressing cells suffices, and no differences in the amount of inositol phosphate formed are noticed. However, once the receptor is aggregated, and PLC activated, both the rate and the duration of inositol phosphate formation differ markedly, probably reflecting the different amounts of PIP2 available in the different cells, which, in turn, reflect the different amounts of PI(4)P. In this context, it is noteworthy that two isoforms of PLC have been identified in RBL cells and shown to participate in FcRI-triggered PIP2 hydrolysis (29, 30). PLC1 is cytosolic, but translocates to the plasma membrane after Ag trigger (29). PLC2 resides in the Golgi region (29) and yet is essential for FcRI-triggered inositol phosphate formation and degranulation (30). Taken together, these and our results are compatible with a model in which PLC1 is responsible for the initial phase of inositol phosphate formation by hydrolyzing a pre-existing pool of PIP2 at the plasma membrane. PLC2 then provides the additional amount of inositol phosphates required for cell degranulation by hydrolyzing PIP2 produced de novo following the activation of PI4K by NCS-1 in the Golgi region. The mechanism by which NCS-1 stimulates the catalytic activity of PI4K is presently unknown. A model has been proposed (10) in which the interaction of yeast NCS-1 with PiK1 removes an autoinhibitory constraint present within PiK1, thereby converting the enzyme into an active state. Although not proven here, our results are consistent with this model and implicate a similar mechanism for the mode of activation of mammalian PI4K by NCS-1.
In summary, our data clearly demonstrate that the regulatory role of NCS-1 in controlling regulated exocytosis is not restricted to neuronal cells, but includes nonneural professional secretory cells, such as mast cells. Our results further establish that NCS-1 does not serve as a Ca2+ switch, but, rather, is involved in stimulus-secretion coupling. Our results also suggest that the mechanism by which NCS-1 controls regulated secretion involves the stimulation of PI4K activity. Finally, our results indicate that PI(4)P, produced by the Golgi-localized PI4K is an important factor in the regulated secretory pathways, as it is required for synthesis of the pool of PI(4,5)P2 that serves as substrate for the generation of lipid-derived second messengers by receptor-activated PLC. These results therefore implicate NCS-1 as a critical factor for FcRI-dependent mast cell degranulation. The finding that primary mast cells (RPMC) also endogenously express NCS-1 lends further support to this idea.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ronit Sagi-Eisenberg, Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. E-mail address: histol3{at}post.tau.ac.il
3 Abbreviations used in this paper: NCS-1, neuronal Ca2+ sensor 1; HSA, human serum albumin; PI4K, phosphatidylinositol 4-kinase ; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PTK, protein tyrosine kinase; RBL, rat basophilic leukemia; TPA, 12-O-tetradecanoylphorbol-13-acetate.
Received for publication December 18, 2002. Accepted for publication September 3, 2003.
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References |
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and stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex. Nat. Cell. Biol. 1:280.[Medline]
RI: coupling form to function. Adv. Immunol. 76:325.[Medline]
interact in neuronal cells and are translocated to membranes during nucleotide-evoked exocytosis. J. Cell Sci. 115:3909. stimulates lipid kinase activity and affects membrane trafficking in COS-7 cells. J. Biol. Chem. 276:40183. activity and Ca2+ mobilization in antigen-stimulated mast cells. J. Leukocyte Biol. 65:321.[Abstract]
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) or triggered (
) for 20 min with DNP-HSA Ag (50 ng/ml). The inositol phosphates formed were then separated and quantified as described in Materials and Methods. The data presented are the means of 12 determinations and include three independent clones stably transfected with the empty pcDNA3 vector, three independent clones stably transfected with the NCS-1-pcDNA3 vector, and one clone stably transfected with the KD-PI4K