OSBP-related protein 4L promotes phospholipase Cβ3 translocation from the nucleus to the plasma membrane in Jurkat T-cells

Phosphoinositide phospholipases C (PLCs) are a family of eukaryotic intracellular enzymes with important roles in signal transduction. In addition to their location at the plasma membrane, PLCs also exist within the cell nucleus where they are stored. We previously demonstrated that OSBP-related protein 4L (ORP4L) anchors cluster of differentiation 3ϵ (CD3ϵ) to the heterotrimeric G protein subunit (Gαq/11) to control PLCβ3 relocation and activation. However, the underlying mechanism by which ORP4L facilitates PLCβ3 translocation remains unknown. Here, using confocal immunofluorescence microscopy and coimmunoprecipitation assays, we report that ORP4L stimulates PLCβ3 translocation from the nucleus to the plasma membrane in Jurkat T-cells in two steps. First, we found that ORP4L is required for the activation of Ras-related nuclear protein (RAN), a GTP-binding nuclear protein that binds to exportin 1 and eventually promotes the nuclear export of PLCβ3. Second, we also observed that ORP4L interacts with vesicle-associated membrane protein–associated protein A (VAPA) through its two phenylalanines in an acidic tract (FFAT) motif. This complex enabled PLCβ3 movement to the plasma membrane, indicating that PLCβ3 translocation occurs in a VAPA-dependent manner. This study reveals detailed mechanistic insight into the role of ORP4L in PLCβ3 redistribution from storage within the nucleus to the plasma membrane via RAN activation and interaction with VAPA in Jurkat T-cells.

cation of PLC␤3 from the nucleus to the plasma membrane; and regulates its activation to promote IP 3 production. In this manner, ORP4L serves to regulate IP 3 -induced Ca 2ϩ signaling, mitochondrial respiration, and ATP generation and thereby maintains the survival of acute T-lymphoblastic leukemia (T-ALL) cells (24). However, the detailed pathway through which ORP4L mediates the translocation of intranuclear PLC␤3 remains to be identified.
Ras-related nuclear protein (RAN), also known as the GTPbinding nuclear protein RAN, is involved in the transport of proteins across the nuclear envelope by interacting with karyopherins and changing their ability to bind or release cargos. Cargo proteins containing a nuclear localization signal (NLS) are transported into the nucleus by binding importins. Inside the nucleus, RAN-GTP binds to importin and releases the import cargo. Cargo that needs to shift from the nucleus into the cytoplasm binds to exportin in a ternary complex with RAN-GTP. Upon hydrolysis of RAN-GTP to RAN-GDP outside the nucleus, the complex dissociates, and the export cargo is released (25,26). In this study, we reveal mechanistic insight into the role of ORP4L in PLC␤3 redistribution via RAN activation and VAPA involvement and offer a model for the shift of PLC␤3 from storage within the nucleus to its functional site.

PLC␤3 exists in the nucleus of unstimulated cells
The localization of PLC␤3 in Jurkat T-cells was investigated by confocal immunofluorescence microscopy. In unstimulated Jurkat T-cells, PLC␤3 exhibited an intranuclear localization (Fig. 1A). The fractionation of Jurkat T-cells analyzed by Western blotting further confirmed that a majority of PLC␤3 protein localized in the nucleus (Fig. 1B). Consistent with the observations in Jurkat T-cells, a similar intranuclear localization of PLC␤3 was apparent in K562, HeLa, and HepG2 cells (Fig. 1C). The Human Protein Atlas website (available from: https://www.proteinatlas. org/ENSG00000149782-PLCB3/cell#human) 5 also provided the PLC␤3 intranuclear localization images in MCF-7, A-431, and U-2 OS cells (Fig. S1). Conversely, our recent study suggested that PLC␤3 catalyzes IP 3 production in T-ALL cells as opposed to PLC␥1 in normal T-cells cells (24). When we compared the PLC␤3 protein level, we found significantly up-regulated expression of this protein in primary T-ALL cells and Jurkat T-cells as compared with normal T-cells (Fig. 1D). In contrast to intranuclear localization in T-ALL cells, confocal immunofluorescence showed that PLC␤3 is distributed at the peripheral plasma membrane in normal T-cells (Fig. 1E). These results indicated a pathway of T-ALL cells regulating PLC␤3 localization, which was different from normal T-cells.
It has been reported that the interaction between cargo molecules and the transport receptors is frequently mediated by short linear motifs on the cargo called NLSs or nuclear export signals (NESs) (27,28). Therefore, we dissected the sequence of PLC␤3 with two online platforms, NLStradamus (http://www. moseslab.csb.utoronto.ca/NLStradamus/) 5

ORP4L facilities PLC␤3 translocation and activation by binding PLC␤3
We previously identified that ORP4L in fact interacts physically with PLC␤3 (24). These previous results suggested that PLC␤3 may be sequestered in the nucleus under unstimulated conditions and upon anti-CD3 stimulation is translocated to the plasma membrane in the presence of ORP4L, thereby gaining access to its substrate, PIP 2 (Fig. S2, A and B).
A coimmunoprecipitation assay showed that ORP4L interacts specifically with PLC␤3 but not with other isotypes of PLC␤s in Jurkat T-cells (Fig. S2C). PLC␤3 knockdown ( Fig. 2A) significantly reduced IP 3 production and Ca 2ϩ release from the ER upon anti-CD3 stimulation (Fig. 2B), indicating its dominant role in Jurkat T-cells. ORP4L overexpression promoted IP 3 production and Ca 2ϩ release, effects reduced in PLC␤3 knockdown cells (Fig. 2, C and D). ORP4L binds PLC␤3 via its amino acid region 445-513 (31). To investigate the role of this physical interaction, we generated a truncated ORP4L protein lacking the PLC␤3-binding region (ORP4L ⌬445-513). ORP4L overexpression enabled PLC␤3 translocation from the nucleus to the plasma membrane, but ORP4L ⌬445-513 failed to do this (Fig. 2E). Phosphorylation at Ser 537 contributes to the basal activity of PLC␤3 (32). Consequently, ORP4L but not ORP4L ⌬445-513 increased PLC␤3 activity (Fig. 2, E and F; phosphorylation of PLC␤3), IP 3 production, and Ca 2ϩ release (Fig. 2G). Confocal imaging showed that p-PLC␤3 was present only at the plasma membrane, and not in the nucleus, when cells were stimulated with anti-CD3 (Fig. 2E), indicating a sequestering of PLC␤3 in the nucleus in its inactive state. These results suggested that PLC␤3 translocation and activation are dependent on its direct interaction with ORP4L.

ORP4L activates RAN in Jurkat T-cells for PLC␤3 exportation from the nucleus
The small GTPase RAN plays a central role in the coordination and trafficking of nuclear proteins (33,34). To investigate mechanisms of PLC␤3 export, we performed RAN activity assays. As expected, anti-CD3 stimulation increased the active form of RAN (RAN-GTP) in a time-dependent manner (Fig.  3A), whereas RAN activity declined in ORP4L knockdown cells (Fig. 3B), indicating that ORP4L is required for RAN activation in Jurkat T-cells upon anti-CD3 stimulation. Anti-CD3 stimulation led to phosphorylation of PLC␤3 in a time-dependent manner (Fig. 3C). Leptomycin B (LMB), a specific inhibitor of exportin 1 (34,35), was added simultaneously with anti-CD3 and blocked the nuclear export of PLC␤3 (Fig. 3E) as well as the phosphorylation of PLC␤3 at the plasma membrane (Fig. 3, D ORP4L regulates PLC␤3 translocation and E). Furthermore, LMB treatment abolished PLC␤3 activation (Fig. 4, A and B), IP 3 production, and Ca 2ϩ release (Fig. 4C) upon ORP4L overexpression. These results suggest that ORP4L enables transduction of CD3 signaling to RAN and PLC␤3 activation in Jurkat T-cells dependent on exportin 1.

ORP4L interacts with VAPA and functions in PLC␤3 transport
The next question is how PLC␤3 is transported to the cell periphery. Because yeast ORPs (Osh proteins) and VAPs assemble at ER-plasma membrane contact sites to control the metabolism of plasma membrane phosphoinositides (36) and VAPs interact with microtubules, which play crucial roles in the maintenance and organization of cellular organelles as well as in membrane trafficking (19), we hypothesized that the VAPA may be involved in the translocation of PLC␤3.
ORP4L, via bimolecular fluorescence complementation, was shown to form a complex with its ER receptor, VAPA, in mammalian cells (14), but there is no direct evidence for the endogenous ORP4L and VAPA interaction. We next sought to investigate whether endogenous ORP4L interacts with VAPA in Jurkat T-cells. Collectively, the confocal immunofluorescence revealed colocalization between endogenous ORP4L and VAPA (Fig. 5A); moreover, the coimmunoprecipitation experiments showed that ORP4L interacted with the integral membrane protein VAPA (Fig. 5B). The GST pulldown experiments further confirmed that WT ORP4L directly interacts with VAPA, whereas the ORP4L lacking the FFAT motif did not (Fig.  5C). These data suggest that ORP4L directly interacts with VAPA via its FFAT motif.
To further dissect the functional role of ORP4L in PLC␤3 translocation and activation, we transfected Jurkat T-cells with full-length ORP4L or truncated ORP4L lacking the FFAT motif (ORP4L ⌬FFAT). Confocal immunofluorescence microscopy images showed that p-PLC␤3 clearly occurred in the cell periphery in ORP4L-overexpressing cells, but this could not be observed in cells overexpressing ORP4L devoid of the FFAT motif ( Fig. 5D). Accordingly, the results of Western blot analysis confirmed the enhanced PLC␤3 phosphorylation in cells overexpressing WT ORP4L but not in cells expressing ORP4L that lacks the FFAT motif (Fig. 5, E and F).
Next, we measured the IP 3 production and Ca 2ϩ release from ER upon anti-CD3 stimulation in Jurkat T-cells. The results showed that ORP4L overexpression promotes IP 3 production and Ca 2ϩ release (Fig. 5, G and H). To our surprise, in contrast to its effect on PLC␤3 activation, the ORP4L ⌬FFAT construct was able to increase the IP 3 and Ca 2ϩ response (Fig. 5, G and H), although to a lower extent compared with full-length ORP4L. PIP 2 located in the plasma membrane acts as substrate for PLC catalysis (38). Recently, an important role of the ORD of ORPs as a transporter for PIP 2 has been explored (39). We hypothe-sized that the PLC␤3 reaction may require the ORD of ORP4L to transport the substrate PIP 2 . Although ORP4L ⌬FFAT fails to facilitate PLC␤3 translocation, the ORD remaining enhances the PIP 2 transport for plasma membrane (PM) PLC␤3 catalysis. To this end, we constructed truncated ORP4L lacking FFAT and ORD (ORP4L ⌬FFAT&ORD). Compared with full-length ORP4L, ORP4L ⌬FFAT&ORD could not increase PLC␤3 activation or evoke the IP 3 and Ca 2ϩ response (Fig. 5, G and H), indicating the requirement of the ORD for PLC␤3 catalysis.
To further investigate the potential dependence of PLC␤3 translocation on VAPA, RNAi was used to inhibit VAPA expression. The translocation of PLC␤3 to a peripheral location upon anti-CD3 treatment was prevented by knockdown of VAPA (Fig. 6A), indicating a role of the ORP4L-VAPA complex in controlling PLC␤3 localization and activity. Moreover, PLC␤3 could not be shifted to its phosphorylated form in VAPA knockdown Jurkat T-cells, even when ORP4L was overexpressed ( Fig. 6, B and C). These results suggested that the nuclear pool of PLC␤3 in unstimulated Jurkat T-cells is mobilized to the plasma membrane in a manner dependent on the ORP4L-VAPA complex.

Discussion
We recently reported that ORP4L organizes a signaling complex involving the translocation and activation of nuclear, ER, mitochondrial, and plasma membrane molecular components (24). However, the detailed role of ORP4L in the shift of PLC␤3 from the nucleus to plasma membrane remained unclear. The present study offers evidence that the underlying mechanism of PLC␤3 translocation promoted by ORP4L is related to RAN activation and assistance by the ER receptor for ORP4L, VAPA (Fig. 6D).
PLCs perform a catalytic function at the plasma membrane where their substrate, PIP 2 , is localized. However, PLCs also exist at other subcellular locations such as the cytoplasm and the nucleus (40 -43). This separation of an enzyme from its substrates provides a regulatory mechanism, but understanding how the key molecules and translocation events control the function of PLCs requires new insights. In this study, we found that under unstimulated conditions PLC␤3 mainly exists within the Jurkat T-cell nucleus but translocates to the plasma membrane upon anti-CD3 stimulation. Colocalization of ORP4L and PLC␤3 was observed in Jurkat T-cells stimulated with anti-CD3, and the two proteins were found to interact physically, which prompted us to consider a role for ORP4L in the PLC␤3 plasma membrane translocation. Indeed, depletion of ORP4L significantly inhibited the shift of PLC␤3 to the plasma membrane, consistent with an important role of ORP4L in controlling the distribution and activity of PLC␤3 in T-ALL cells.

ORP4L regulates PLC␤3 translocation
We have previously provided evidence that ORP4L can specifically interact with CD3⑀, G␣ q/11 , and PLC␤3, and these interactions were modestly enhanced upon anti-CD3 stimula-tion, suggesting a dynamic nature of the complex formation that is under the control of CD3 signaling (24). Therefore, we hypothesize that formation of the CD3⑀-G␣ q/11 complex scaf-ORP4L regulates PLC␤3 translocation folded by ORP4L enhances G␣ q/11 activity for activating RAN and PLC␤3 upon anti-CD3 stimulation. The increase in active RAN (RAN-GTP) upon anti-CD3 stimulation and the reduction of the shift of PLC␤3 by leptomycin B, an inhibitor of exportin 1, suggest that PLC␤3 trafficking from the nucleus is mediated by the exportin system.
ORP4L plays an important role in controlling the distribution and activity of PLC␤3 in Jurkat T-cells and primary T-ALL cells (24). The specific nature of this inhibition was further corroborated by data showing that depletion of VAPA, the ER membrane receptor of ORP4L, had a similar effect as depletion of ORP4L. We envision that the deletion of VAPA results in the dysfunction of ORP4L in regulation of PLC␤3 translocation in Jurkat T-cells.
Moreover, our recent findings showing that ORP4L binds PIP 2 are consistent with a model in which an ORP4L-VAPA complex may target PLC␤3 to distinct PIP 2 -containing microdomains at the plasma membrane (44,45). The observed affinity of ORP4L for PIP 2 , the major plasma membrane phosphoinositide (46), together with an interaction with PLC␤3 suggests an association of ORP4L with components of the plasma membrane. In contrast, interaction with VAPA, an integral membrane protein mainly found in the ER (19), targets ORP4L to ER membranes. This duality in membrane targeting may indicate that ORP4L has the capacity to simultaneously associate with the ER and the plasma membrane. In fact, ORP4L was found to colocalize with PLC␤3 at punctate peripheral sites, which could represent ER-plasma membrane contacts (47). Such regions are commonly referred to as "puncta" (48), and it is within the puncta that key signaling occurs during T-cell activation (49). Such contact sites are also known to play crucial roles in Ca 2ϩ regulation during store-operated Ca 2ϩ entry and muscle excitation-contraction coupling (37). Our findings thus raise the possibility that ORP4L may operate to coordinate the activity of PLC␤3 at ER-plasma membrane contacts.

ORP4L regulates PLC␤3 translocation
In conclusion, our results suggest that ORP4L facilitates PLC␤3 translocation in T-ALL cells. RAN activation under control of ORP4L regulates the export of inactive PLC␤3 from the nucleus, and interaction of VAPA with ORP4L mediates PLC␤3 transport to the cell periphery. Our study offers novel mechanistic insight into ORP4L functions in T-ALL cells. It also provides a model for a shift of PLC␤ from subcellular storage to its functional site via RAN activation and VAPA function.

Human leukocyte specimens and cell lines
Fresh leukocytes were isolated from peripheral blood of healthy human donors and T-ALL patients after obtaining written informed consent. Naive CD3 ϩ T-cells were isolated using an Enhanced Human T Cell Recovery Column kit (Cedarlane, Burlington, Ontario, Canada) according to the manu-
Jurkat T-cells and K562 cells were maintained in RPMI 1640 medium containing 10% FBS, 100 units ml Ϫ1 penicillin, and 100 g ml Ϫ1 streptomycin. HeLa and HepG2 cells were maintained in Dulbecco's modified Eagle's medium containing 10% FBS, 100 units ml Ϫ1 penicillin, and 100 g ml Ϫ1 streptomycin. All cells were cultured at 37°C in a humidified incubator with 5% CO 2 .

Isolation of nuclear and plasma membrane fractions
Nuclear fractions were isolated using a nuclear/cytosol fractionation kit (Biovision) according to the manufacturer's instructions. Briefly, aliquots of 2 ϫ 10 6 cells were collected by centrifugation followed by adding cytosol extraction buffer-A and homogenization. After cytosol extraction buffer-B was Figure 6. ORP4L loses the ability to enhance PLC␤3 transport in VAPA knockdown cells. A, PLC␤3 translocation to the PM in Jurkat T-cells transfected with nontargeting (siNT) or VAPA-specific (siVAPA) siRNA upon 5-min stimulation with 10 g ml Ϫ1 anti-CD3 at 37°C. Distribution of VAPA is displayed in red. Scale bars, 10 m. B, PLC␤3 phosphorylation at Ser 537 was analyzed by Western blotting in control and ORP4L-overexpressing cells in the presence or absence of VAPA knockdown. For anti-CD3 treatment, cells were stimulated for 5 min with 10 g ml Ϫ1 anti-CD3 at 37°C. C, the relative p-PLC␤3 protein content was quantified from Western blots (B) and normalized with the ␤-actin signal. The data represent mean Ϯ S.D. from an experiment performed in triplicate. **, p Ͻ 0.01; ***, p Ͻ 0.001, Student's t test. All error bars represent S.D. D, a schematic model for the PLC␤3 shift from the nucleus to the plasma membrane. DAG, diacylglycerol.

ORP4L regulates PLC␤3 translocation
added, the sample was centrifuged, and the supernatant fraction was obtained. The pellet was resuspended in nuclear extraction buffer, vortexed, and centrifuged, and the supernatant (nuclear extract) was prepared. For plasma membrane isolation, cells were collected and resuspended in 0.2 mM EDTA in 1 mM NaHCO 3 in an approximate ratio of 1 ml/10 8 cells and incubated on ice for 30 min to swell the cells. Cells were homogenized using a Dounce homogenizer. The homogenates were centrifuged for 10 min at 175 ϫ g at 4°C to remove unbroken cells and nuclei, and the supernatant was centrifuged at 25,000 ϫ g for 30 min at 4°C to prepare a plasma membraneenriched microsome fraction. The supernatant was discarded, and the pellets were resuspended in 0.2 M potassium phosphate buffer, pH 7.2. The resuspended membranes then were loaded onto the two-phase system with a polymer mixture containing 6.6% Dextran T500 (GE Healthcare), 6.6% (w/w) poly(ethylene glycol) 3350 (Fisher Scientific), and 0.2 M potassium phosphate, pH 7.2. The phases were separated by centrifugation at 1150 ϫ g for 5 min at 4°C. The upper phase containing primarily plasma membranes was collected.

Gene transfer
For ORP4L knockdown, high-titer lentivirus carrying shRNA (TCAGAGTCAAGCTCAGGTGTA) prepared by Shanghai GenePharma (Shanghai, China) was used. 1 ϫ 10 6 cells were resuspended in 100 l of medium with lentivirus (multiplicity of infection, 100) and 5 g ml Ϫ1 Polybrene in a 24-well culture plate. Infections were carried out for 6 h at 37°C in 5% CO 2 . At the end of infection, 400 l of medium was added. Efficacy of knockdown of ORP4L was verified by Western blotting after 3-day infection. A 4D-Nucleofector TM System (Lonza, Basel, Switzerland) was used for transient transfection of ORP4L, ORP4L ⌬FFAT, and ORP4L ⌬FFAT&ORD constructs and VAPA siRNA (catalogue number sc-61768, Santa Cruz Biotechnology) in Jurkat T-cells.

Coimmunoprecipitation and GST pulldown assay
For the coimmunoprecipitation assay, 1 ϫ 10 7 Jurkat T-cells were washed twice with ice-cold PBS and incubated on ice for 30 min with 1 ml of lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 0.5 mM MgCl 2 , 10% glycerol, and 0.5% Triton X-100, pH 8.0) supplemented with protease inhibitor mixture (Roche Applied Science). Cell lysates were centrifuged for 15 min at 15,000 ϫ g. The supernatant was preabsorbed with 50 l of Protein G-agarose (Invitrogen) for 1 h at 4°C. The recovered supernatant was incubated overnight with VAPA or control antibody at 4°C. 50 l of Protein G-agarose was added to the lysate-antibody mixture and incubated for 2 h at 4°C on a roller. The beads were washed four times with lysis buffer and boiled in SDS-PAGE loading buffer. Samples were resolved on 10% SDSpolyacrylamide gels and subjected to Western blot analysis with antibodies.
For GST pulldown assay, pGEX-4T-1 (Addgene, Cambridge, MA), full-length ORP4L constructs (pGEX-4T-1-ORP4L), or the truncated ORP4L constructs lacking the FFAT motif (pGEX-4T-1-ORP4L ⌬FFAT) were transformed into E. coli Rosetta TM (DE3) (Novagen) and cultured at 37°C to A 600 0.5-1.0 followed by induction with 0.1 mM isopropyl 1-thio-␤-D-galactopyranoside for 16 -18 h at 18°C. The cells were collected, and crude bacterial lysates were prepared by sonication in lysis buffer 1 (50 mM Tris-Cl, 150 mM NaCl, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride, pH 8.0) in the presence of the protease inhibitor mixture (Roche Applied Science). Bacterial lysates were centrifuged for 20 min at 12,000 ϫ g, and the supernatants were used for fusion protein purification with GST-Bind beads (Novagen) according to the manufacturer's protocol. Jurkat T-cells were washed twice with cold PBS, lysed in lysis buffer, and shaken for 30 min on ice, and the lysate was cleared by a 10-min centrifugation at 12,000 rpm in a microcentrifuge. For pulldown, 10 g of GST/GST-ORP4L/GST-ORP4L ⌬FFAT and 30 l of GST-Bind beads were incubated for 30 min on ice followed by washing three times with PBS.
Jurkat T-cells lysates were then added and incubated at 4°C overnight. Then the beads were washed three times with lysis buffer, resuspended into 2ϫ SDS-PAGE loading buffer at 98°C for 5 min, and resolved on 10% SDS-polyacrylamide gels for Western blotting.

Measurement of RAN activity
RAN activity was measured using a RAN activation assay kit according to the manufacturer's instructions (NewEast Biosciences). Briefly, anti-active RAN mouse mAb was incubated with cell lysates containing RAN-GTP. The bound active RAN was pulled down by Protein A/G-agarose, eluted, and detected by immunoblot analysis using polyclonal rabbit anti-Ran antibody.

Determination of phospholipase C activity
Phospholipase C activity was analyzed using the EnzChek Direct Phospholipase C Assay kit (Molecular Probes) according to the manufacturer's instructions.

Measurement of IP 3 production
IP 3 was measured using an ELISA kit for IP 3 (CEC037Ge, Cloud-Clone Corp.) according to the manufacturer's instructions. Briefly, 0.5 ϫ 10 6 Jurkat T-cells transfected with the indicated construct were lysed with lysis buffer and incubated with Detection Reagent A for 1 h at 37°C. After aspirating and washing three times, Detection Reagent B was added, incubated for 30 min at 37°C, then aspirated, and washed five times. Substrate Solution was added and incubated for 7 min at 37°C. Finally, the reactions were blocked with Stop Solution and read at 450 nm immediately.

Ca 2؉ imaging
Cells (0.5 ϫ 10 6 cells ml Ϫ1 ) transfected with the indicated construct were plated onto glass-bottomed dishes and incubated with 1 M Fluo-4-AM for 30 min at 37°C in ECB buffer (130 mM NaCl, 5 mM KCl, 1.5 mM CaCl 2 , 1 mM MgCl 2 , 25 mM Hepes, pH 7.5, 1 mg ml Ϫ1 BSA, and 5 mM glucose). The buffer was replaced, and incubation continued for 20 min at 37°C to permit dye de-esterification. Culture dishes were mounted on the stage of an inverted confocal microscope (Olympus FV3000 laser-scanning confocal microscope system) equipped with a 40ϫ objective. Calcium measurements were performed in fresh ECB buffer at 37°C with 5% CO 2 . The Ca 2ϩ images of ORP4L regulates PLC␤3 translocation cells excited with low-intensity 488-nm laser excitation were acquired at 2-s intervals alternately under time-lapse mode. Image data were subsequently analyzed using ImageJ (National Institutes of Health) and are presented as a ratio of F/F 0 in the final results where F 0 represents baseline fluorescence intensity in each cell.

Immunofluorescence microscopy
Cells were seeded onto coverslips, stimulated with anti-CD3 for the indicated times, and then fixed with 4% paraformaldehyde for 30 min at room temperature followed by permeabilization with 0.1% Triton X-100 for 5 min and blocking with 10% FBS for 30 min at room temperature. Cells were then incubated with primary antibodies in 5% FBS at 4°C overnight. After washing three times (10 min each) with PBS, cells were incubated with fluorescent secondary antibody conjugates at 37°C for 30 min followed by staining with Hoechst 33342 at room temperature for 10 min. Finally, the specimens were analyzed using a Zeiss LSM 510 Meta laser-scanning confocal microscope system or Olympus FV3000 laser-scanning confocal microscope system.

Western blot analysis
Cellular total protein samples were mixed with loading sample buffer, boiled for 10 min, and subjected to SDS-PAGE followed by transfer onto polyvinylidene difluoride membranes (Millipore, Life Science). After blocking and incubations of the membranes with primary antibodies and HRP-secondary antibody conjugates (Bio-Rad), the blots were developed by enhanced chemiluminescence (Millipore, Life Science). Proteins were quantified by densitometry using ImageJ (National Institutes of Health), and the data were normalized using the ␤-actin signal.

Statistical analyses
All data are expressed as mean Ϯ S.D. of at least three independent experiments. Differences between groups were analyzed by unpaired two-tailed Student's t test. p values of Ͻ0.05 were considered statistically significant.