Novel Function of Phosphoinositide 3-Kinase in T Cell Ca2+ Signaling

This study presents evidence that phosphoinositide (PI) 3-kinase is involved in T cell Ca2+ signaling via a phosphatidylinositol 3,4,5-trisphosphate PI(3,4,5)P3-sensitive Ca2+entry pathway. First, exogenous PI(3,4,5)P3 at concentrations close to its physiological levels induces Ca2+ influx in T cells, whereas PI(3,4)P2, PI(4,5)P2, and PI(3)P have no effect on [Ca2+] i . This Ca2+ entry mechanism is cell type-specific as B cells and a number of cell lines examined do not respond to PI(3,4,5)P3 stimulation. Second, inhibition of PI 3-kinase by wortmannin and by overexpression of the dominant negative inhibitor Δp85 suppresses anti-CD3-induced Ca2+response, which could be reversed by subsequent exposure to PI(3,4,5)P3. Third, PI(3,4,5)P3 is capable of stimulating Ca2+ efflux from Ca2+-loaded plasma membrane vesicles prepared from Jurkat T cells, suggesting that PI(3,4,5)P3 interacts with a Ca2+ entry system directly or via a membrane-bound protein. Fourth, although D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) mimics PI(3,4,5)P3 in many aspects of biochemical functions such as membrane binding and Ca2+ transport, we raise evidence that Ins(1,3,4,5)P4 does not play a role in anti-CD3- or PI(3,4,5)P3-mediated Ca2+ entry. This PI(3,4,5)P3-stimulated Ca2+ influx connotes physiological significance, considering the pivotal role of PI 3-kinase in the regulation of T cell function. Given that PI 3-kinase and phospholipase C-γ form multifunctional complexes downstream of many receptor signaling pathways, we hypothesize that PI(3,4,5)P3-induced Ca2+ entry acts concertedly with Ins(1,4,5)P3-induced Ca2+ release in initiating T cell Ca2+ signaling. By using a biotinylated analog of PI(3,4,5)P3 as the affinity probe, we have detected several putative PI(3,4,5)P3-binding proteins in T cell plasma membranes.

Engagement of the TCR 1 -CD3 complex stimulates an array of signaling cascades that culminate in the activation and proliferation of T lymphocytes. One of the early signaling events is a biphasic increase in intracellular Ca 2ϩ levels ([Ca 2ϩ ] i ), which is characterized by a high transient spike of [Ca 2ϩ ] i followed by a long-lasting plateau phase (1,2). It is believed that the initial phase of Ca 2ϩ response is attributable to the action of inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) that releases Ca 2ϩ from the endoplasmic reticulum (ER). Next, depletion of the ER Ca 2ϩ store signals an influx of Ca 2ϩ across the plasma membrane to sustain the wave of Ca 2ϩ signaling. Three discrete mechanisms have been proposed for the sustained inflow of Ca 2ϩ (2). First, Ins(1,4,5)P 3 receptors are present on the plasma membranes of T lymphocytes (3)(4)(5). Thus, Ins(1,4,5)P 3 may play a dual role of releasing Ca 2ϩ from ER stores and stimulating Ca 2ϩ influx across plasma membranes concurrently. Second, the capacitative Ca 2ϩ entry model (6) dictates that the emptying of the intracellular Ca 2ϩ store is coupled, either directly through conformational coupling or indirectly via diffusable factors, to the Ca 2ϩ release-activated Ca 2ϩ channel (7,8). Third, a TCR-operated Ca 2ϩ entry (TROCE) mechanism is activated in response to TCR-CD3 stimulation (2). However, this putative pathway is less well characterized. It is known to be independent of the depletion of intracellular Ca 2ϩ and inhibited by SKF96365, a Ca 2ϩ channel blocker, and phorbol esters (9).
In this paper, we present data suggesting a new function of phosphoinositide (PI) 3-kinase in T cell Ca 2ϩ regulation via a PI(3,4,5)P 3 -sensitive Ca 2ϩ entry mechanism. In response to TCR activation, PI 3-kinase and other signaling molecules such as PLC-␥1 are recruited to the plasma membrane to form multifunctional complexes (10 -12). Activation of PI 3-kinase results in a transient accumulation of M levels of PI(3,4,5)P 3 and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 ), both absent in quiescent T cells (13). To date, a clear consensus on the mode of action of these lipid messengers in regulating TCR signaling has yet to emerge. Putative downstream effectors for PI (3,4,5)P 3 and PI(3,4)P 2 in receptor-stimulated signaling include Ca 2ϩ -independent PKC isozymes (␦, ⑀, , ), PLC-␥, Akt, and so forth (14). The results of this study suggest that PI(3,4,5)P 3 mediates a novel Ca 2ϩ entry mechanism on plasma membranes. Given the intimate relationship between PI 3-ki-* This work was supported by National Institutes of Health Grants GM53448 (to C.-S. C.) and AI21490 (to S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Flow Cytometric Cell Sorting and Analysis of Intracellular Ca 2ϩ -Mouse spleen cells (10 7 ) were treated with 5 g of anti-Thy1.2 mAb-or anti-B-220 mAb-fluorescein (FITC) conjugates in 200 l of culture medium consisting of a 1:1 mixture of Iscove's modified Dulbecco's medium and Ham's F-12 nutrient mixtures, 10% fetal bovine serum, 2 mM glutamine, 50 nM 2-mercaptoethanol, 20 units/ml bovine insulin, 20 nM progesterone, 5 g/ml transferrin, and 1 g/ml gentamicin for 30 min on ice. Anti-Thy1.2 and anti-B-220 are antibodies against the cell surface markers of T and B cells, respectively. The suspension was washed three times and resuspended in 500 l of the same medium. For intracellular Ca 2ϩ analysis, these cells were loaded with 1 M indo-1 AM for 30 min at 37°C, washed twice, and suspended in 1 ml of assay buffer consisting of 4.3 mM Na 2 HPO 4 , 24.3 mM NaH 2 PO 4 , 4.3 mM K 2 HPO 4 , 113 mM NaCl, 5 mM glucose, pH 7.4. A FACStar plus cell sorter (Becton Dickinson) was used for cell sorting and to monitor indo-1 fluorescence. FITC-stained cells were analyzed by monitoring the emission at 530 nm with excitation at 488 nm. Intracellular Ca 2ϩ was measured by comparing the ratio of indo-1 emission at 405 nm and 520 nm with excitation at 350 nm as described previously (18).
Transient Transfection-The construct expressing hemagglutinin (HA)-tagged ⌬p85 was a kind gift from Professor Alex Toker (Harvard Medical School). ⌬p85 is a deletion mutant that lacks a region required for tight association with p110 but is still able to bind to appropriate phosphotyrosine targets. Thus, ⌬p85 can compete with native p85 for binding to essential signaling proteins and behaves as a dominant negative mutant (19). Jurkat T cells were grown to a density of 5 ϫ 10 5 cells/ml in RPMI 1640 medium containing 10% fetal bovine serum. Cells were harvested, washed with serum-free Opti-MEM (Life Technologies, Inc.), and suspended in the same medium (5 ϫ 10 5 cells/ml). Transfection was carried out according to a modification of the protocol supplied by the manufacturer. Aliquots containing 0.5 g, 1.5 g, and 3 g of the HA⅐⌬p85 expression vector or 3 g of a control pCMV/blue plasmid in 500 l of Opti-MEM were incubated with 30 -60 l of the Plus reagent from the LipofectAMINE Plus reagent kit at 25°C for 15 min, and the mixture was added to 40 l of the LipofectAMINE reagent in 500 l of Opti-MEM. The mixture was incubated at 25°C for 15 min and added to 5 ml of the cell suspension. After 5 h at 37°C, the transfection media were replaced with 5 ml of the RPMI 1640 -10% fetal bovine serum medium. The transfected cells were allowed to grow for 6 days with the medium changed every other day to express foreign DNA. The collected cells were analyzed for anti-CD3-induced Ca 2ϩ response by fluorescence spectrometry and for HA⅐⌬p85 expression by Western blot using anti-HA antibodies.
Preparation of Jurkat T Cell Plasma Membranes-Two different methods were employed for the plasma membrane preparations. For the Ins(1,3,4,5)P 4 receptor binding assay, the membrane fraction was prepared according to a method described by Khan et al. (5). For the Ca 2ϩ release assay, the plasma membrane was purified by a modification of the method described by Neville (20). In brief, Jurkat T cells (4 ϫ 10 8 cells) were washed with phosphate-buffered saline and suspended in 5 ml of PM buffer consisting of 20 mM Hepes, pH 7.2, 110 mM KCl, 10 mM NaCl, 2 mM MgCl 2 , 5 mM KH 2 PO 4 , 1 mM dithiothreitol, 1 mM EGTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 20 g/ml leupeptin. The cell suspension was homogenized in a Dounce homogenizer using the loose pestle with 5 strokes up and down. The homogenate was centrifuged at 1,500 ϫ g for 10 min. The pellet was suspended in 3.125 ml of PM buffer and mixed with 5.5 ml of 69% (w/w) sucrose to make a final 44% (w/w) sucrose-membrane mixture. The mixture was overlaid with 42.3% (w/w) sucrose, and the two-phase suspension was subjected to centrifugation at 90,000 ϫ g for 2 h in a swinging bucket rotor. The membrane material at the interface of the phases contained the greatest enrichment in plasma membranes based on the activity of (Na ϩ -K ϩ )-ATPase. This fraction was collected, suspended in 5 ml of 10 mM Hepes, pH 7.5, containing 1 mM dithiothreitol, 1 mM 4-(2-aminoethyl-)benzenesulfonyl fluoride, and 20 g/ml leupeptin and centrifuged at 25,000 ϫ g for 10 min. The pellet was suspended in 1 ml of the same buffer. (1,3,4,5)P 4 Binding to Jurkat T Cell Plasma Membranes by di-C 8 -PI (3,4,5)P 3 and Inositol Polyphosphates-Since PI(3,4,5)P 3 contains Ins(1,3,4,5)P 4 as its head group, it is plausible that this inositol phospholipid shares the binding site on the plasma membrane with Ins(1,3,4,5)P 4 . To assess this possibility, we examined the displacement of [ 3 H]Ins(1,3,4,5)P 4 binding by di-C 8 -PI(3,4,5)P 3 , a water-soluble derivative, Ins(1,3,4,5)P 4 , and Ins(1,4,5)P 3 . The freshly prepared membrane preparation (200 g of protein) was incubated with 2 nM [ 3 H]Ins(1,3,4,5)P 4 (30 Ci/mmol) in 10 mM Hepes, pH 7.5, containing 100 mM KCl, 20 mM NaCl, and 1 mM EDTA in the presence of various concentrations of the competitive ligand, with a final volume of 0.3 ml. The mixture was incubated at 4°C for 15 min, and the reaction was terminated by centrifugation at 16,000 ϫ g for 5 min. The membranebound radioactivity was analyzed by liquid scintillation spectrometry. Nonspecific binding was measured in the presence of 30 M Ins(1,3,4,5)P 4 .

Displacement of [ 3 H]Ins
Fluorescence Spectrophotometric Measurement of Intracellular Ca 2ϩ -[Ca 2ϩ ] i was monitored by the change in the fluorescence intensity of fura-2-loaded cells. Jurkat T cells (1 ϫ 10 7 cells/ml), suspended in the aforementioned assay buffer containing 0.5% bovine serum albumin and 2 mM probenacid, were incubated with 10 M fura-2 AM in the dark for 1 h at 37°C. The cells were then pelleted by centrifugation at 1,000 ϫ g for 10 min, washed with assay buffer twice, and resuspended at approximately 8 ϫ 10 5 cells/ml in the same buffer containing 1 mM Ca 2ϩ . The effect of anti-CD3 mAb or various inositol lipids on [Ca 2ϩ ] i was examined by fura-2 fluorescence in a Hitachi F-2000 spectrofluorimeter at 37°C with excitation and emission wavelengths at 340 and 510 nm, respectively. The maximum fura-2 fluorescence intensity (F max ) in Jurkat cells was determined by adding A23187 (1 M), and the minimum fluorescence (F min ) was determined following depletion of external Ca 2ϩ by 5 mM EGTA. The [Ca 2ϩ ] i was calculated according to the equation where K d denotes the apparent dissociation constant (ϭ224 nM) of the fluorescence dye-Ca 2ϩ complex (21).
[ 3 H]Inositol Phosphate Turnover Analysis-The examination of phosphoinositol turnover was carried out according to a modification of the procedure reported by Sei et al. (9). In brief, Jurkat T cells were incubated with myo-[2-3 H]inositol (10 Ci/10 6 cells/ml) in inositol-free RPMI medium supplemented with 10% fetal bovine serum. The cells were then washed with 20 mM Hepes, pH 7.4, containing 285 mM NaCl, 11 mM KCl, 1.3 mM Na 2 HPO 4 , 1 mM KH 2 PO 4 , 8.3 mM NaHCO 3 , 1.6 mM MgSO 4 , 2.2 mM MgCl 2 , 2.2 mM CaCl 2 , and 5.6 mM glucose. Aliquots containing 1 ϫ 10 6 cells were each resuspended in 0.3 ml of the aforementioned assay buffer plus 1 mM CaCl 2 and 100 M EGTA and transferred to 1.5-ml microcentrifuge tubes. Each sample was incubated with 1.5 g of anti-CD3 mAb or 20 M PI(3,4,5)P 3 for the indicated times and quenched by adding 0.25 ml of 6% trichloroacetic acid. The tubes were centrifuged for 2 min at 12,000 ϫ g. The supernatant (200 l) was analyzed by high performance liquid chromatography on a 5-m Adsorbosphere Sax column (4. vidin beads (Roche Molecular Biochemicals) were added. The mixture was incubated for an additional hour and centrifuged at 12,000 ϫ g for 5 min. The beads were washed with 1 ml of each of the following solutions in tandem: 10 mM Tris, pH 7.5, containing 5 mM EDTA and 150 mM NaCl, phosphate-buffered saline, and 2 M urea. After being dialyzed against distilled water for 12 h, proteins eluted at 2 M urea were analyzed by SDS-polyacrylamide gel electrophoresis and visualized by silver staining.

PI(3,4,5)P 3 Induced Intracellular Ca 2ϩ Increase in Mouse
Splenic T Cells but Not in B Cells-Previously, we reported that treatment of washed platelets with exogenous PI(3,4,5)P 3 induced Ca 2ϩ influx across plasma membranes, resulting in immediate cell aggregation (22). This finding prompted us to study PI(3,4,5)P 3 -induced Ca 2ϩ response in other cell types. Using fluorescence-activated cell sorting, we examined the effect of PI(3,4,5)P 3 on indo-1-loaded mouse spleen cells stained with FITC-conjugates of anti-Thy1.2 or anti-B-220 mAb. As shown, 10 M PI(3,4,5)P 3 induced a significant increase in [Ca 2ϩ ] i in Thy1.2-positive mouse spleen cells as soon as 15 s after stimulation. This PI(3,4,5)P 3 -stimulated Ca 2ϩ increase was also confirmed in human peripheral T cells and Jurkat T cells by fluorescence spectrophotometry. In contrast, [Ca 2ϩ ] i in the B-220-positive cell population remained unaffected even in the presence of 20 M PI(3,4,5)P 3 after a prolonged exposure up to several minutes (Fig. 1).
In addition, a number of cell lines, including NIH3T3 fibroblast cells, PC-12 pheochromocytoma cells, Hep G2 hepatocarcinoma cells, LNCaP prostate adenocarcinoma cells, were examined. None of these cells showed appreciable Ca 2ϩ response following PI(3,4,5)P 3 stimulation (data not shown). This celltype specificity underscores a fundamental difference in the role of PI 3-kinase in Ca 2ϩ regulation in different cells.
The direct introduction of micellar PI(3,4,5)P 3 to intact cells is also worth comment. Although how PI(3,4,5)P 3 permeates cell membranes remains unclear, published data from this and other laboratories show that exogenous PI(3,4,5)P 3 can readily fuse with cell membranes and exert cellular and biochemical responses in different cell types including platelets, NIH3T3 cells, and adipocytes (22)(23)(24). In line with our previous finding in platelets (22), the induction of Ca 2ϩ response displayed stringent specificity for PI(3,4,5)P 3 , underlying its second messenger role. Other phosphoinositides examined, including PI(3,4)P 2 , PI(4,5)P 2 , PI(3)P, failed to exert an appreciable change in [Ca 2ϩ ] i at 20 M (Fig.  2B). Moreover, fura-2 fluorimetry showed that the effect of PI(3,4,5)P 3 on [Ca 2ϩ ] i was also demonstrated in different subtypes of T cells, including mouse thymocytes, Jurkat T cells, and human peripheral T cells (data not shown). Taken together, these data indicate that exogenous PI(3,4,5)P 3 stimulated Ca 2ϩ influx in T cells regardless the stage of cell development.
PI (3,4,5)P 3 Did Not Perturb Membrane Permeability to Ca 2ϩ -Due to the extremely high charge density, it has been speculated that PI(3,4,5)P 3 might directly affect the properties of cellular membranes. Therefore, one might raise a concern that PI(3,4,5)P 3 facilitated Ca 2ϩ translocation across plasma membranes by acting like a detergent. To refute this possibility, we examined the effect of PI(3,4,5)P 3 on the permeability of liposomal vesicles to Ca 2ϩ . Fura-2 loaded multilamellar vesicles with a lipid composition similar to that of the plasma membrane were exposed to PI(3,4,5)P 3 vis à vis A23187 and 25-hydroxycholecalciferol, a sterol known to increase membrane permeability to Ca 2ϩ (26). As shown in Fig. 4, A23187 (1 M) caused a rapid and robust increase in fura-2 fluorescence, whereas 25(OH)D 3 (7 M) induced an immediate but more modest rise.
In contrast, 10 M PI(3,4,5)P 3 did not elicit any appreciable effect on fura-2 fluorescence. Taken together with the aforementioned cell-type and ligand specificity data, one could conclude that PI(3,4,5)P 3 does not perturb membrane permeability to Ca 2ϩ .

Role of PI 3-Kinase in TCR-mediated Ca 2ϩ
Signaling-The activation of Ca 2ϩ influx by PI(3,4,5)P 3 suggested a potential link between PI 3-kinase and T cell Ca 2ϩ signaling. To test this premise, a combination of pharmacological and molecular approaches was employed to characterize the role of PI 3-kinase in anti-CD3-mediated Ca 2ϩ response.
We first examined the effect of wortmannin, a potent PI 3-kinase inhibitor, on anti-CD3 mAb-induced Ca 2ϩ response in fura-2-loaded Jurkat T cells. Fig. 5 shows that ligation of the TCR-CD3 complex by anti-CD3 mAb provoked a 4-fold increase in cytosolic Ca 2ϩ (trace a). Subsequent exposure to exogenous PI(3,4,5)P 3 (10 M) only augmented the Ca 2ϩ response to a small extent. The anti-CD3-induced Ca 2ϩ response largely stemmed from Ca 2ϩ mobilization across the plasma membrane because deprivation of external Ca 2ϩ by EGTA inhibited 70% of the Ca 2ϩ signal (Fig. 5A, inset).
Pretreatment with wortmannin (1 M) attenuated the am- plitude of the anti-CD3-stimulated Ca 2ϩ influx by nearly 60%, which, however, could be rescued by the subsequent exposure to 10 M PI(3,4,5)P 3 (trace b). As shown, PI(3,4,5)P 3 could restore the [Ca 2ϩ ] i of wortmannin-treated cells to that of the control. Moreover, the extent of Ca 2ϩ increase greatly exceeded that elicited by PI(3,4,5)P 3 in anti-CD3-stimulated cells (trace a), suggesting that the reversal of wortmannin inhibition by PI(3,4,5)P 3 was not simply due to an additive effect.
We hypothesized that PI 3-kinase acted in concert with PLC-␥ in initiating Ca 2ϩ signaling following TCR activation. This premise is supported by the observation that when used alone, wortmannin and the PLC inhibitor U73122 (10 M) exerted 57% and 50% inhibition, respectively, on anti-CD3induced Ca 2ϩ increase, whereas a combination of these two inhibitors could virtually abolish the Ca 2ϩ response (Fig. 5B).
We also took an independent nonpharmacological approach to confirm the above results, in which Jurkat T cells were transiently transfected with a vector expressing HA epitopetagged ⌬p85. It is well documented that the deletion of the binding motif for the catalytic p110 subunit in ⌬p85 confers PI 3-kinase dominant negative activity (19). Overexpression of this dominant negative inhibitor in T cells has been shown to down-regulate TCR-mediated interleukin-2 gene expression (27), Erk2 (extracellular signal-regulated protein kinase) activation (28), and NFAT (nuclear factor of activated T cells) activation (29).
Western analysis using anti-HA antibodies verified the expression of HA⅐⌬p85 in transfected Jurkat T cells (Fig. 6A). It is noteworthy that the level of ⌬p85 expression displayed a direct correlation with the amount of cDNA used in transfection. Accordingly, transfected Jurkat T cells expressing varying levels of ⌬p85 were tested for Ca 2ϩ entry in response to anti-CD3 stimulation. In line with the wortmannin data, ⌬p85 suppressed anti-CD3-induced Ca 2ϩ response in a dose-dependent manner, ranging from 5% to 40% in accordance with the level of ⌬p85 expression (Fig. 6B, traces a-d). Since both wortmannin and ⌬p85 gave consistent results in inhibiting anti-CD3stimulated Ca 2ϩ response, these data strongly support the involvement of PI 3-kinase in TCR-mediated Ca 2ϩ signaling.
Moreover, the di-C 8 -PI(3,4,5)P 3 -or Ins(1,3,4,5)P 4 -stimulated Ca 2ϩ release was inhibited by SKF96365, which is consistent with that observed with the whole cell (Fig. 7). Also noteworthy is that Ca 2ϩ release induced by PI(3,4,5)P 3 or Ins(1,3,4,5)P 4 was not augmented by subsequent challenge with either agonist or Ins(1,4,5)P 3 (indicated by the arrows). The lack of Ca 2ϩ response was likely due to desensitization or saturation of the binding site instead of the depletion of Ca 2ϩ since the addition of 1 M A23187 following PI(3,4,5)P 3 treatment triggered the release of large amounts of Ca 2ϩ (Fig. 9, inset).
In contrast, for Ins(1,4,5)P 3 -stimulated Ca 2ϩ release, subsequent stimulation with PI(3,4,5)P 3 or Ins(1,3,4,5)P 4 caused additional release of Ca 2ϩ . Taken together with the binding data, this observation suggests that the putative PI(3,4,5)P 3 or Ins(1,3,4,5)P 4 receptor might be discrete from the Ins(1,4,5)P 3 receptor. Furthermore, the presence of Ins(1,4,5)P 3 receptors in T cell plasma membranes was confirmed by using two specific antibodies against the type I and type III receptors. Western blot analysis showed significantly more labeling of the plasma membrane with the type III receptor antibodies than with type I receptor antibodies (Fig. 10). It is noteworthy that this Ins(1,4,5)P 3 -receptor subtype distribution is similar to that reported for platelet plasma membranes (31).
Affinity Probing of PI (3,4,5)P 3 -binding Proteins in T Cell Plasma Membranes-We further prepared a biotinylated analog of PI(3,4,5)P 3 , Biotin-PIP 3 (Fig. 13A), to confirm the existence of PI(3,4,5)P 3 -binding proteins in T cell plasma membranes. This affinity ligand has been successfully applied to the purification of PI(3,4,5)P 3 -binding proteins even with a K d as high as 100 M (17). The plasma membrane fraction was treated with 5% CHAPS, and the solubilized proteins were incubated with Biotin-PIP 3 , followed by streptavidin beads. The adsorbed beads were spun down by centrifugation, washed with 150 mM NaCl, and eluted with 2 M urea. SDS-polyacrylamide gel electrophoresis analysis of the eluted proteins, visualized by silver staining, indicates two major protein bands with apparent molecular masses of 67 kDa and 59 kDa and several minor bands at and below 42 kDa (Fig. 13B). No protein band with a molecular mass greater than 70 kDa was detected.

DISCUSSION
This study presents both pharmacological and molecular genetic evidence that PI 3-kinase plays an obligatory role in TCR-mediated Ca 2ϩ signaling via a PI(3,4,5)P 3 -sensitive Ca 2ϩ influx system on plasma membranes. This unique Ca 2ϩ entry mechanism connotes physiological significance considering the pivotal role of PI 3-kinase in the regulation of T cell function and may serve as a potential target for the modulation of T cell immunity.
Substantial evidence indicates that triggering of T cells through the TCR-CD3 complex leads to membrane recruitment of signaling proteins such as PI 3-kinase, PLC-␥1, and Grb2 to form multi-molecular signaling complexes (12). These proteins initiate distinct signaling cascades that culminate in cell proliferation and induction of effector functions like interleukin-2 secretion. However, in contrast to PLC-␥1 and Grb2, the precise role of PI 3-kinase in TCR signaling remains elusive. Recent evidence suggests that PI 3-kinase is required for Erk2 activation (28), NFAT activation (29), and interleukin-2 production (27) in stimulated T cells. The present data demonstrate that PI(3,4,5)P 3 , the primary output signal of PI 3-kinase, can generate Ca 2ϩ stimuli that synergize with Ins(1,4,5)P 3 -induced Ca 2ϩ release and capacitative Ca 2ϩ entry for sustaining elevated [Ca 2ϩ ] i , a driving force underlying many cellular responses.
Third, Ins(1,3,4,5)P 4 mimics PI(3,4,5)P 3 in many aspects of biochemical functions such as membrane binding and Ca 2ϩ release from plasma membrane vesicles. This in vitro crossreactivity, due to the largely shared structural motifs, raises an interesting question with regard to which species representing the physiologically relevant ligand responsible for the Ca 2ϩ entry. To date, published data on the role of Ins(1,3,4,5)P 4 in Ca 2ϩ mobilization across plasma membranes remain inconclusive. Although several reports implicated Ins(1,3,4,5)P 4 in mediating Ca 2ϩ entry in certain types of electrically nonexcitable cells such as sea urchin eggs (42), Xenopus oocytes (43), and platelets (44), other studies indicated that Ins(1,3,4,5)P 4 did not have a significant effect, if any, on potentiating Ca 2ϩ influx in other cells like mouse lacrimal acinar cells (45) and Jurkat T cells (33). The data obtained in this study support the latter view that Ins(1,3,4,5)P 4 does not play a role in anti-CD3-or PI(3,4,5)P 3 -elicited Ca 2ϩ influx.
Meanwhile, several research groups have isolated an Ins(1,3,4,5)P 4 -binding protein, GAP1 IP4BP , from platelet plasma membranes (44, 46 -48). GAP1 IP4BP was found to be a GTPase-activating protein with a molecular mass of 104 kDa. It remains enigmatic how this GAP protein is involved in Ca 2ϩ entry. However, our affinity ligand study indicates that although many PI(3,4,5)P 3 -binding proteins existed in the T cell plasma membrane, none of these proteins displayed a molecular mass in line with that of GAP1 IP4BP . This finding dampened the possibility that GAP1 IP4BP was involved in the PI(3,4,5)P 3induced Ca 2ϩ influx in Jurkat T cells.
In summary, although the mechanism by which PI(3,4,5)P 3 mediates Ca 2ϩ entry remains unclear, this PI(3,4,5)P 3 -sensitive pathway not only provides molecular insights into T cell Ca 2ϩ regulation but also represents a potential target for the modulation of cell function in T lymphocytes. Unlike inositol phosphates, PI(3,4,5)P 3 is membrane-permanent. Thus, it is plausible to design PI(3,4,5)P 3 analogues as antagonists of the putative receptors for therapeutic uses. However, outstanding questions that remain are as follows. What is its relationship with the Ins(1,4,5)P 3 receptor on plasma membranes? Is there cross-communication with other Ca 2ϩ channels (such as Ca 2ϩ release-activated Ca 2ϩ channels) on plasma membranes to regulate Ca 2ϩ entry? To address these questions, sequence analysis of the putative PI(3,4,5)P 3 -binding proteins is currently under way in this laboratory.