T Cell Receptor-induced Nuclear Factor κB (NF-κB) Signaling and Transcriptional Activation Are Regulated by STIM1- and Orai1-mediated Calcium Entry*

T cell activation following antigen binding to the T cell receptor (TCR) involves the mobilization of intracellular Ca2+ to activate the key transcription factors nuclear factor of activated T lymphocytes (NFAT) and NF-κB. The mechanism of NFAT activation by Ca2+ has been determined. However, the role of Ca2+ in controlling NF-κB signaling is poorly understood, and the source of Ca2+ required for NF-κB activation is unknown. We demonstrate that TCR- but not TNF-induced NF-κB signaling upstream of IκB kinase activation absolutely requires the influx of extracellular Ca2+ via STIM1-dependent Ca2+ release-activated Ca2+/Orai channels. We further show that Ca2+ influx controls phosphorylation of the NF-κB protein p65 on Ser-536 and that this posttranslational modification controls its nuclear localization and transcriptional activation. Notably, our data reveal that this role for Ca2+ is entirely separate from its upstream control of IκBα degradation, thereby identifying a novel Ca2+-dependent distal step in TCR-induced NF-κB activation. Finally, we demonstrate that this control of distal signaling occurs via Ca2+-dependent PKCα-mediated phosphorylation of p65. Thus, we establish the source of Ca2+ required for TCR-induced NF-κB activation and define a new distal Ca2+-dependent checkpoint in TCR-induced NF-κB signaling that has broad implications for the control of immune cell development and T cell functional specificity.

Activation of T cells following antigen binding to the T cell antigen receptor (TCR) 3 induces diverse lineage-and fate-specific proinflammatory and immune-modulatory responses. Central to these responses is the induction of quantitatively distinct intracellular Ca 2ϩ signals and their selective activation of the key transcription factors NFAT and NF-B (1-6). The mechanism by which Ca 2ϩ controls NFAT activation in lymphocytes is well established (7). In contrast, although Ca 2ϩ has been implicated in TCR-induced NF-B signaling (8 -10), how Ca 2ϩ regulates NF-B activity is largely unexplored and represents a significant gap in our understanding of transcriptional control of T cell development, activation, and functional specificity.
In this study, we sought to determine both the source and mechanism of Ca 2ϩ control of antigen receptor-induced NF-B activation in T cells. We show that influx of extracellular Ca 2ϩ via STIM1 and Orai is critical for TCR-but not TNFinduced IB␣ degradation and NF-B activation. Importantly, we also demonstrate that Ca 2ϩ -dependent, PKC␣-mediated phosphorylation of p65 critically regulates its nuclear localization and transcriptional activation following TCR engagement. Thus, our findings define important new proximal and distal Ca 2ϩ -dependent checkpoints in TCR-induced NF-B signaling that have broad implications for the control of immune cell development and functional specificity.

Materials and Methods
Cells and Cell Culture-Primary human T cells were obtained from the University of Pennsylvania Immunology Core facility. Jurkat T cells were from the ATCC, and Jurkat T cells stably expressing E106A Orai1 were a gift from Dr. Jonathan Soboloff (Temple University, Philadelphia, PA). All cells were cultured in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Thermo Scientific, Logan, UT), 2 mM L-glutamine, penicillin (50 units/ml), and streptomycin (50 units/ml).
Plasmids and Transfections-A cDNA construct expressing full-length p65 N-terminally tagged with EGFP was obtained from Addgene (Cambridge, MA). Mutant p65 constructs were generated using a site-directed mutagenesis kit (Stratagene, La Jolla, CA) to convert serine 536 to alanine or aspartic acid. Short hairpin STIM1 suppression and rescue constructs were generated in the laboratory of Dr. Dan Billadeau (Mayo Clinic, Rochester, MN), as were EGFP-shPKC␣ and EGFP-pCMS2 (control vector). For transfection, Jurkat T cells were suspended at 20 million cells/ml in RPMI 1640 medium, and 10 million cells were electroporated with 10 g of DNA (for overexpression or mutant expression) or 40 g of DNA (for suppression assays) at 315 V for 10 ms using a BTX ECM 830 electroporator (Harvard Apparatus, Holliston, MA). STIM1 and PKC␣ suppression assays were performed 48 h post-transfection, and EGFP-p65 and p65 mutant expression assays were performed 16 -24 h post-transfection.
Immunoblotting-Cells were harvested and lysed using Nonidet P-40 lysis buffer consisting of 50 mM Tris-HCl (pH 7.5), 20 mM EDTA, 1% Nonidet P-40, and complete inhibitors (1 mM sodium orthovanadate, 1 mM PMSF, 10 g/ml leupeptin, and 5 g/ml aprotinin). Protein concentrations in cell lysates were determined using Bio-Rad reagent and quantified in a Cary 50 Bio UV-visible spectrophotometer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (4 -15%, Bio-Rad) and then transferred onto PVDF membranes (Millipore, Billerica, MA). Membranes were probed with the respective primary anti-human antibodies and then incubated with protein A HRP secondary antibodies. Blots were developed with enhanced chemiluminescence using Pierce ECL Western blotting sub-strate. All immunoblots presented are from a single experiment representative of at least three independent experiments.
Luciferase Reporter Analysis-Luciferase-based transcriptional analysis was performed on Jurkat T cells transfected with 2 g of total DNA (PBXII B firefly luciferase and pRL TK Renilla luciferase in a 20:1 ratio) per transfection (5 ϫ 10 6 cells in 500 l of medium) using a square-wave BTX electroporator at 315 V for 10 ms. Twenty-four hours after transfection, cells were treated with PMA (200 nM), PMA (200 nM) and ionomycin (1 g/ml), anti TCR (0.5 g/ml), and anti-CD28 (1:50) or TNF (10 ng/ml) for 4 h. Cells were then lysed in passive lysis buffer (Promega), and luciferase activity was measured using a Luminoscan 96-well automated luminometer (Thermo LabSystems, Franklin, MA). Firefly/Renilla luciferase ratios were calculated using Ascent software (Thermo LabSystems), and the mean ratio from at least three independent experiments (3-4 replicates/experiment) for each condition was compared.
Microarray Analysis-RNA was isolated using an RNeasy Plus kit (Qiagen). Biotin-labeled cRNA was generated using the Illumina TotalPrep RNA amplification kit, and a Bioanalyzer (Agilent Technologies, Wilmington, DE) was used to assess total RNA and cRNA quality. Illumina HumanHT-12 version 4 expression bead chips were hybridized with cRNA from two biological replicates per condition and scanned on an Illumina BeadStation 500GX. Scanned images were converted to raw expression values using GenomeStudio v1.8 software (Illumina). Data analysis was performed using the statistical computing environment R (v3.2.3), the Bioconductor suite of packages for R, and RStudio (v0.98). Raw data were background-subtracted, variance-stabilized, and normalized by robust spline normalization using the Lumi package (36). Differentially expressed genes were identified by linear modeling and Bayesian statistics using the Limma package (37,38). Probe sets that were differentially regulated (Ն1.5-fold change between all treatments, false detection rate Ϲ 5% after controlling for multiple testing using the Benjamini-Hochberg method (39,40)) were used for heatmap generation in R. Clusters of co-regulated genes were identified by Pearson correlation using the hclust function of the stats package in R. Differentially expressed NF-B-dependent genes were identified using a list of validated and putative NF-B target genes curated by the laboratory of Dr. Thomas Gilmore at Boston University. All microarray data have been deposited in the GEO database for public access (GSE76804).
Quantitation of p65 Nuclear Translocation-Jurkat T cells transfected with EGFP-shPKC␣ or EGFP-pCMS2 (48 h) or untransfected cells suspended in medium containing 2 mM Ca 2ϩ or Ca 2ϩ free equivalent solution were adhered to Cell-Tak-treated coverslips for 10 -15 min. Cells were then stimulated at 37°C as indicated. For PKC␣ suppression experiments, cells were stimulated in the presence of 2 mM Ca 2ϩ . At the indicated times, cells were fixed in formaldehyde (3.7%) for 30 min, permeabilized with 0.2% Triton-X-100 for 15 min, and blocked overnight in 2% BSA at 4°C. Fixed and blocked cells were incubated with rabbit anti-p65 primary antibody (Santa Cruz Biotechnology, catalog no. 372, 1 g/ml) for 1 h at 37°C or overnight at 4°C degrees, washed three times for 5 min each in 1% BSA in PBS, and incubated with Alexa 488 or 546 goat antirabbit secondary antibody (4 g/ml) for 1 h at 37°C. Nuclei were then labeled with Hoechst 33342 (Life Technologies, catalog no. H3570, 4 g/ml), washed three times for 5 min each in 1% BSA in PBS, and mounted in Fluoromount (Fisher). Images of p65 localization were obtained with a Yokagawa spinning disk confocal system (Tokyo, Japan) mounted on a Leica DMI4000 microscope (Leica Microsystems, Wetzlar, Germany), and imaging parameters were optimized independently for each channel to maintain fluorescence within the linear range while maximizing intensity resolution. Images of p65 and Hoechst were overlaid, and cytoplasmic/nuclear p65 localization was determined using the Multiwavelength Cell Scoring application (Molecular Devices, Downingtown, PA). Average nuclear and cytoplasmic p65 fluorescence intensities were quantified within cytoplasmic and nuclear compartments, and intensity ratios were determined for each cell.
Real-time Localization of WT and Mutant p65-Jurkat T cells expressing WT and p65 Ser-536 mutants (16 -24 h) were adhered to Cell-Tak-coated (BD Biosciences) coverslips and maintained in culture medium (RPMI 1640 medium, 10% FBS, 1% Glutamax) in a temperature-and CO 2 -controlled chamber for 1 h during imaging. GFP-WT and GFP-p65 mutants were visualized every 10 s after stimulation with PMA (200 nM) with ionomycin (1 M) and/or PMA (200 nM) with and without the delayed addition of ionomycin (1 M).
Calcium Imaging-Jurkat T cells (3 million cells/ml) were loaded with 3 M Fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR) in external solution containing 145 mM NaCl, 4.5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM glucose, 10 mM HEPES, 2 mM glutamine, and 2% fetal bovine serum (Hyclone, Thermo Scientific) for 10 min at 25°C. Cells were adhered to coverslips coated with Cell-Tak (BD Biosciences), mounted on the stage of a Leica DMI6000 microscope configured with a Photometrics Evolve 512 camera (Tucson, AZ) using an Olympus ϫ40 oil objective (Shinjuku, Tokyo, Japan), and images were acquired with MetaFluor software (Molecular Devices). During imaging, cells were perfused with Ca 2ϩ -free bath solution before activation with PMA (200 nM) and ionomycin (1 M), thapsigargin (1 M), anti-TCR (0.5 g/ml) and CD28 (1:50) antibodies, or TNF (10 ng/ml) to evaluate stimulus-dependent Ca 2ϩ release from the ER. The cells were then perfused with bath solution containing 2 mM Ca 2ϩ to assess Ca 2ϩ entry via activated Orai channels. In some experiments, cells were pretreated for 15 min with the Orai-1 inhibitor Synta66 (50 M, Aobious, Gloucester, MA) prior to stimulation. Ca 2ϩ mobilization was analyzed by plotting the emission ratio of 340/380-nm excitation for each cell. Each plot is the averaged ratio from at least 30 cells.
Statistical Analysis-Significance for all statistical tests was determined at p Ͻ 0.05 and is shown as *, p Ͻ 0.05; **, p Ͻ 0.01; and ***, p Ͻ 0.001 in all figures. Average firefly/Renilla luciferase ratios were calculated from three to four independent experiments and analyzed using two-tailed Welch's t test. Western blot protein intensities were quantified using ImageJ (http://imagej.nih.gov/ij/), and average protein intensity values were compared using two-tailed Welch's t test. p65 nuclear-tocytoplasmic fluorescence intensity ratios were assessed for normality using probability plots and Kolmogorov-Smirnov test for normality. Normal distributions were compared using twotailed Student's t test, and non-normal data were compared using Wilcoxon rank-sum test. Quantitative PCR relative quantification (RQ) values and percent input values were compared using two-tailed Welch's t test.

Extracellular Ca 2ϩ Is Required for TCR-induced NF-B
Signaling-Ca 2ϩ regulates proximal TCR signaling upstream of IKK activation (8 -10). However, the precise function of Ca 2ϩ

Novel Calcium-dependent Mechanisms of NF-B Activation
and the source of Ca 2ϩ required for NF-B activation are unknown. To address these questions, we first asked whether the initial release of Ca 2ϩ from ER stores was sufficient or whether sustained influx of extracellular Ca 2ϩ is required for NF-B activation in T cells. To distinguish between these pools of Ca 2ϩ , we activated T cells in the presence or absence of extracellular Ca 2ϩ with either anti-CD3 and anti-CD28 (3/28) to co-engage the TCR and CD28 or with the diacylglycerol analog PMA together with ionomycin (P/I), which activate PKC and release ER-stored Ca 2ϩ , respectively, and we compared these responses to those induced by the proinflammatory cytokine TNF. In Ca 2ϩ -free medium, 3/28 and P/I, but not TNF, induced a transient rise in cytoplasmic Ca 2ϩ concentration because of release from the ER. Reintroduction of extracellular Ca 2ϩ led to a sustained secondary increase in cytoplasmic Ca 2ϩ levels via entry through activated Orai1/Ca 2ϩ release-activated Ca 2ϩ channels in 3/28-and P/I-stimulated cells (35) (Fig. 1A). Thus, stimulating cells in the absence of extracellular Ca 2ϩ allows us to specifically determine whether release from ER stores alone is sufficient for NF-B signal activation.
As shown in Fig. 1B, top panels, all three stimuli induced the expected degradation and resynthesis of IB␣ in Jurkat T cells, consistent with activation of the IKK complex and the classical NF-B pathway. In contrast, neither 3/28 nor P/I induced IB␣ degradation in Ca 2ϩ -free medium, whereas IB␣ degradation and resynthesis in response to TNF remained intact (Fig. 1B,  bottom panels). Consistent with the effects on IB␣ degradation, 3/28-stimulated NF-B transcriptional activity was completely inhibited and P/I-induced transcriptional activation was significantly reduced in Ca 2ϩ -free medium (Fig. 1C). In contrast, TNF-induced NF-B reporter activity was unaltered in the presence or absence of extracellular Ca 2ϩ (Fig. 1C). A similar regulation of IB␣ expression was observed in primary human CD4ϩ T cells (Fig. 1D). Collectively, these findings reveal that transient release of Ca 2ϩ from ER stores is not sufficient and that extracellular Ca 2ϩ is required for TCR-induced NF-B activation.
TCR-induced NF-B Activation Requires STIM1 and Orai1-The extracellular Ca 2ϩ requirement for TCR-induced NF-B activation implies a crucial role for STIM1-operated , or TNF (10 ng/ml, right panel) to assess release from intracellular (ER) stores. Subsequent perfusion with Ca 2ϩ -containing (2 mM) medium was performed to assess the extent of Orai activation. B, Jurkat T cells bathed in Ca 2ϩ -containing (2 mM) (top panels) or Ca 2ϩ free (0 mM Ca 2ϩ , bottom panels) medium were activated by cross-linking CD3 and CD28 with P/I or TNF (10 ng/ml) for the times indicated, and IB␣ levels were determined by immunoblotting. C, NF-B transcriptional activity was measured under identical conditions as the immunoblot analyses in B in Jurkat T cells expressing an NF-B firefly luciferase reporter. Firefly luciferase activity is expressed relative to a Renilla luciferase control before (0 h) and after (4 h) stimulation with anti-CD3/28 (left), P/I (center), or TNF (right) in the presence (ϩ) or absence (Ϫ) of extracellular Ca 2ϩ . Mean firefly/Renilla luciferase ratios Ϯ S.E. from three independent experiments (4 replicates/experiment) are displayed, and statistical significance was evaluated using Welch's t test. ***, p Ͻ 0.001; N.S., not significant. D, primary human CD4ϩ T cells were stimulated in Ca 2ϩ -replete (0.4 mM) or Ca 2ϩ -free (0 mM) medium, and then IB␣ levels were determined by immunoblotting. Blots were probed with anti-␣tubulin as a loading control.
Orai1 channel-mediated Ca 2ϩ influx. To explore this, we expressed a STIM1 shRNA construct or a bicistronic variant for concomitant re-expression of shRNA resistant STIM1 to normal levels in Jurkat T cells ( Fig. 2A). STIM1 suppression inhibited 3/28-and P/I-induced extracellular Ca 2ϩ influx, and this was rescued by re-expression of STIM1 (Fig. 2B). Consistent with the lack of TCR-induced NF-B signaling in Ca 2ϩ -free medium (Fig. 1, B and C), STIM1 suppression prevented 3/28and P/I-induced IB␣ degradation in T cells (Fig. 2C, left versus center panel). In contrast, IB␣ degradation and re-expression were normal in STIM1-rescued cells, confirming that the inhibition was due to loss of STIM1 (Fig. 2D, right panel). Furthermore, both 3/28-and P/I-induced NF-B transcriptional activity was reduced in STIM1-suppressed cells, and this was again rescued by concomitant STIM1 re-expression (Fig. 2C). Notably, TNF-induced IB␣ degradation and NF-B transcriptional activity were unaffected by suppressing STIM1 (Fig. 2, C  and D).
To confirm the role of Orai in Ca 2ϩ -dependent NF-B activation, we examined the consequence of inhibiting Orai-mediated Ca 2ϩ influx with the Orai inhibitor Synta66 (Fig. 3A) and by expressing a mutant Orai1 (glutamic acid at position 106 mutated to alanine, E106A) that exerts a dominant negative effect on the Ca 2ϩ permeability of endogenous Orai channels (Fig. 3C). In the presence of Synta66, the sarcoplasmic/endoplasmic reticulum Ca 2ϩ ATPase inhibitor thapsigargin triggered Ca 2ϩ release from the ER, evident as a small transient increase in cytoplasmic Ca 2ϩ (in Ca 2ϩ -free medium), but no subsequent sustained increase in cytoplasmic Ca 2ϩ (Fig. 3A, compare the top and bottom panels) following perfusion with Ca 2ϩ -containing medium. Consistent with the effects of STIM1 suppression (Fig. 2), Synta66 (Fig. 3B) inhibited 3/28but not TNF-induced IB␣ degradation. A similar block in stimulus induced Ca 2ϩ entry, and IB␣ degradation was observed in permeation-defective Orai1-E106A cells (Fig. 3, C and D) Taken together, these results reveal an obligate role for STIM1-operated Orai1-mediated Ca 2ϩ entry in TCR-but not TNF-induced IB␣ degradation and NF-B activation.
Ca 2ϩ Controls the Transcriptional Activity of TCR-induced NF-B-P/I treatment mimics TCR signaling upstream of IKK activation because PMA activates the strictly diacylglycerol-dependent and Ca 2ϩ -independent "novel" PKC isoform PKC (41), and ionomycin-mediated Ca 2ϩ release from the ER activates STIM1-dependent Orai activation (Figs. 1A and 2B). Thus, both 3/28 and P/I stimulation of T cells induce rapid PKC-mediated IKK-dependent degradation of IB␣, followed by resynthesis of IB␣ via NF-B-driven transcription (Figs. 1,  2, 3 and 4A). In seeking to determine the precise contribution of Ca 2ϩ release from the ER to NF-B activation, we found that PMA alone induces substantial IB␣ degradation (Fig. 4A, center panel), suggesting that strong pharmacological activation of PKC can circumvent the requirement for Ca 2ϩ upstream of IKK activation. However, treatment with ionomycin alone had no effect on IB␣ levels (Fig. 4A, bottom panel), indicating that Ca 2ϩ mobilization in the absence of PKC activation is not sufficient to activate the IKK complex.
Strikingly, although PMA in the absence of ionomycin induced IB␣ degradation, this was not followed by IB␣ resynthesis (Fig. 4A, compare IB␣ levels at 60 min). Moreover, the kinetics of PMA-induced IB␣ degradation were delayed compared with the response to P/I. We reasoned that the delayed IB␣ degradation following stimulation with PMA alone likely reflects a cooperative role previously identified for the Ca 2ϩ regulated phosphatase calcineurin A (CnA) in CBM complex formation, IKK activation, and IB␣ degradation (10,42). Indeed, overexpression of a Ca 2ϩ -independent, constitutively active CnA rescued the delay in PMA-induced IB␣ degradation so that the rate and extent of degradation were indistinguishable from those in P/I-stimulated cells (Fig. 4B). Importantly, although constitutively active CnA rescued the modulatory role of Ca 2ϩ in proximal steps of NF-B activation (i.e. IKK activation), it did not rescue IB␣ re-expression in PMA-stimulated cells, indicating that a separate Ca 2ϩ -dependent mechanism regulates the distal steps of NF-B activation.
Supporting this conclusion and consistent with the lack of resynthesis of IB␣ in the absence of Ca 2ϩ (Fig. 4A), analysis of IB␣ mRNA levels revealed a limited induction of IB␣ transcription when PKC was activated with PMA or in response to PMA and ionomycin under Ca 2ϩ free conditions. In contrast, in the presence of extracellular Ca 2ϩ , IB␣ mRNA expression was significantly induced by PMA and ionomycin (Fig. 4C).
Because IB␣ re-expression is driven by activated NF-B as a negative feedback loop to limit NF-B signaling (43), we ques-tioned whether Ca 2ϩ -dependent IB␣ protein re-expression reflects a global requirement for Ca 2ϩ in NF-B transcriptional activation. As shown in Fig. 4D, ionomycin alone failed to activate NF-B reporter activity. PMA alone triggered a small but significant increase in activity compared with baseline; however, PMA and ionomycin together significantly enhanced (ϳ4-fold) NF-B-driven transcriptional activation. Together, these data indicate that Ca 2ϩ controls the distal transcriptional activation of NF-B following P/I stimulation of T cells.
To extend these findings, we performed an unbiased transcriptional analysis to fully assess the extent of Ca 2ϩ control over NF-B-regulated gene expression. Microarray analysis was performed on T cells stimulated in the absence or presence of extracellular Ca 2ϩ with either PMA alone or PMA and ionomycin. Transcriptional analyses identified 20, 96, and 112 differentially expressed genes (false detection rate, Ͻ0.05, log 2 -fold change, Ͼ0.59) at 1, 4, and 8 h, respectively, between unstimulated, PMA-treated, and P/I-treated T cells (Fig. 4E,  blue). This list of differentially expressed genes was further refined to include only validated and putative NF-B target genes on the basis of known targets (44 -47). In total, we found Time-dependent changes in IB␣ were determined by immunoblotting, and anti-␣-tubulin was used as a loading control. C, Ca 2ϩ traces in Jurkat T cells stably overexpressing the dominant negative E106A Orai1 that were stimulated with P/I, 3/28, or TNF as shown. D, Jurkat T cells stably overexpressing either wild-type Orai (Orai-cyan fluorescent protein, Orai-CFP) or the dominant negative Orai1-E106A were stimulated with 3/28, P/I, or TNF for the times indicated, and immunoblot analysis was performed to quantify IB␣ and ␣-tubulin. APRIL 15, 2016 • VOLUME 291 • NUMBER 16 that 10 -20% of all Ca 2ϩ -regulated genes were NF-B targets (Fig. 4E, red). Strikingly, 11 of 12 of these NF-B target genes were dramatically increased by Ca 2ϩ entry (2-to 20-fold increase, false detection rate, Ͻ0.05) relative to PMA stimulation (no Ca 2ϩ mobilization) or stimulation with P/I in 0 mM Ca 2ϩ (ER release but no Ca 2ϩ entry). This analysis also revealed that, among this cohort, three classical NF-B regulated genes (IB␣, CXCL8, and TNF) were among the top differentially expressed Ca 2ϩ -dependent genes (Fig. 4, E and F). Together, these data reveal an entirely novel function for Ca 2ϩ in regulating TCR-induced, NF-B-dependent gene activation.

Novel Calcium-dependent Mechanisms of NF-B Activation
Ca 2ϩ Is Required for TCR-induced p65 Nuclear Localization-The failure of PMA alone to induce IB␣ resynthesis following its degradation (Fig. 4A) implies that Ca 2ϩ controls NF-B activity distal to IKK activation. To address the mechanism of this regulation, we first asked whether Ca 2ϩ entry was required for NF-B nuclear localization following IB␣ degradation. As expected, P/I triggered rapid nuclear translocation of p65, which peaked 30 min after stimulation (Fig. 5, A and B). PMA alone had an effect at 30 and 60 min of stimulation, but the extent was significantly less than that induced by P/I at all time points (Fig. 5B). In contrast, ionomycin alone did not induce p65 nuclear localization at any time point. To confirm this apparent role for extracellular Ca 2ϩ in p65 nuclear localization, we examined the effects of PMA and ionomycin in the presence and absence of extracellular Ca 2ϩ . Again, ionomycin alone failed to trigger p65 nuclear localization in either the absence or presence of Ca 2ϩ (Fig. 5, C and D). However, p65 was strongly driven to the nucleus by the combination of P/I in the presence of extracellular Ca 2ϩ , whereas, in Ca 2ϩ -free medium, ionomycin failed to synergize with PMA, and the extent of p65 nuclear localization was identical to that triggered by PMA alone (Fig. 5D). Taken together, these findings reveal an essential role for Orai-mediated entry of extracellular Ca 2ϩ in nuclear translocation of p65 following its release from IB␣ in response to TCR signaling.
Ca 2ϩ Regulates TCR-induced Phosphorylation of p65 at Ser-536 -At least 12 p65 serine or threonine residues have been identified whose phosphorylation regulates its nuclear localization and/or transcriptional activation (13-15, 22, 23, 26 -28, 30, 48 -52). We therefore asked whether Ca 2ϩ regulates p65 nuclear translocation by controlling the phosphorylation of any of these residues. Among these, we focused on signal-induced phosphorylation of p65 Ser-536 (25,53) because this has been implicated in TNF-driven NF-B activation (15). We found that neither PMA nor ionomycin alone induces Ser-536 phosphorylation (Fig. 6A). However, activation with both together (P/I) induced a robust tran-sient increase in phosphorylation at this residue that peaked at 15 min (Fig. 6A). Furthermore, the synergistic effect of PMA and ionomycin on Ser-536 phosphorylation requires extracellular Ca 2ϩ because no phospho-p65 was detected in cells stimulated in Ca 2ϩ -free medium (Fig. 6B). Notably, TNF also induced transient Ser-536 phosphorylation, although this occurred more rapidly than the response to P/I (peak at 5 min), suggesting distinct regulatory mechanisms. Moreover, TNF stimulated robust Ser-536 phosphorylation in the absence of extracellular Ca 2ϩ (Fig. 6C), again consistent with an obligate role for Ca 2ϩ entry in TCR but not TNF-induced NF-B activation in T cells.
Phosphorylation of p65 at Ser-536 has been shown to alter the kinetics of p65 nuclear translocation (24,25). To determine whether Ca 2ϩ -dependent Ser-536 phosphorylation regulates p65 nuclear localization, we expressed wild-type p65, a nonphosphorylatable serine-to-alanine (S536A) mutant, and a serine-to-aspartate (S536D) phosphomimic mutant in T cells. We then visualized nuclear translocation in real time by confocal imaging. Cells were first stimulated for 30 min with PMA alone to induce IB␣ degradation. Then ionomycin was added, and cells were observed for an additional 30 min. Consistent with the role for Ca 2ϩ in p65 nuclear localization, in cells expressing WT p65, PMA alone did not promote p65 nuclear migration, whereas robust nuclear translocation was observed within 10 min of Ca 2ϩ mobilization by exposure to ionomycin (Fig. 6D, top panel, 40-min time point). In contrast, after PMA treatment, ionomycin did not induce nuclear localization of S536A, whereas S536D exhibited Ca 2ϩ -independent nuclear localization during the initial 30 min of PMA stimulation (Fig. 6D, center and bottom panels) without any requirement for Ca 2ϩ mobilization with ionomycin. Together, these results establish that Ca 2ϩ entry is required for TCR-induced phosphorylation of p65 at Ser- FIGURE 5. Ca 2؉ controls TCR-induced p65 nuclear localization. A, Jurkat cells were stimulated with PMA, ionomycin (Iono), or both for 15, 30, or 60 min, and then nuclear localization of p65 was determined by confocal imaging. B, the data are presented as a mean Ϯ S.E. ratio of nuclear:cytoplasmic p65 from three independent experiments and were compared using Student's t test. *, p Ͻ 0.05; **, p Ͻ 0.01. C, Jurkat T cells were stimulated as in A for 30 min in the absence or presence of extracellular Ca 2ϩ , and p65 localization was determined by confocal imaging. D, the ratio of nuclear:cytoplasmic p65 was determined from three independent experiments, are presented as the mean Ϯ S.E. ratio of nuclear:cytoplasmic p65, and were compared using Student's t test. *, p Ͻ 0.05; **, p Ͻ 0.01. 536, and our mutational analysis indicates that this phosphorylation licenses the nuclear localization of p65 following its release from IB␣.
PKC␣ Regulates Ca 2ϩ -dependent p65-Ser-536 Phosphorylation-Several kinases, including IKK␣, IKK␤, IKK⑀, TBK1, and PKA, have been implicated in the phosphorylation of p65 at specific serine residues that regulate its transcriptional activation (26,54), but none of these kinases are known to be Ca 2ϩ dependent. Because PKC␣ is a Ca 2ϩ -dependent Ser/Thr kinase (55,56) and is the predominant conventional PKC isoform in T cells, we used shRNA knockdown (Fig. 7) to determine whether PKC␣ plays a role in TCR-induced NF-B signaling and, specifically, whether it mediates Ca 2ϩ -dependent phosphorylation of p65 at Ser-536. PKC␣ suppression did not affect IB␣ degradation induced by P/I, indicating no apparent role for PKC␣ upstream of IKK activation (Fig. 7B). However, similar to incubating cells with PMA alone (Figs. 4A), PKC␣ suppression prevented the resynthesis of IB␣ normally observed 60 min after stimulation with P/I (Fig. 7B, compare lanes 5 and 10). In contrast, PKC␣ suppression did not affect TNF-induced IB␣ degradation or resynthesis. Consistent with a role in Ser-536 phosphorylation, PKC␣ suppression significantly reduced P/I-induced Ser-536 phosphorylation but had no significant effect on phosphorylation induced by TNF (Fig. 7, C and D). Thus, these data confirm that Ca 2ϩ -dependent activation of PKC␣ regulates TCR-induced phosphorylation of p65 at Ser-536.
Given the role we have identified for p65 Ser-536 phosphorylation in p65 nuclear localization (Fig. 6D) and the role for PKC␣ in p65 Ser-536 phosphorylation, we investigated whether PKC␣ controls p65 nuclear localization. Consistent with the role of PKC␣-dependent p65 phosphorylation, PKC␣ suppression significantly decreased the extent of p65 nuclear localization 15, 30, 60, and 90 min post-stimulation with P/I in 2 mM Ca 2ϩ (Fig. 7, E and F). Importantly, p65 localization, expressed as the nuclear to cytoplasmic ratio, in unstimulated T cells was identical regardless of the level of PKC␣ expression (median nuclear to cytoplasmic ratio ϪPKC , 0.54; median nuclear to cytoplasmic ratio ϩPKC␣ , 0.53).
We next asked to what extent this phosphorylation of p65 impacts p65 binding to promotors of Ca 2ϩ -dependent genes identified in our transcriptional analysis. We performed ChIP analyses to assess p65 binding to three genes found by transcriptional analysis to exhibit the strongest Ca 2ϩ dependent induction (IB␣, CXCL8, and TNF) and to assess the role of PKC␣-dependent p65 phosphorylation in promotor binding FIGURE 6. Ca 2؉ controls the phosphorylation of p65 at Ser536. A, Jurkat T cells were stimulated with P/I, PMA alone, or ionomycin (Iono) alone for the times shown, and then lysates were immunoblotted to determine the amounts of total p65 and Ser-536 phosphorylation. B, cells were treated with P/I in the absence or presence of 2 mM Ca 2ϩ , and then p65 or Ser(P)-536 amounts were determined by immunoblotting. C, Jurkat cells were incubated with TNF for the times shown in either Ca 2ϩ -containing or Ca 2ϩ -free extracellular bath solution, and then lysates were prepared and immunoblotted using the antibodies indicated. D, WT p65-GFP, and p65-GFP with serine 536-to-alanine (S536A) and serine 536-to-aspartate (S536D) point mutations were expressed in Jurkat T cells to determine the role of Ser-536 phosphorylation in p65 nuclear localization. WT and mutant p65-GFP localization was visualized over a time course of 60 min in live cells by spinning disk confocal microscopy. In each instance, cells were first stimulated for 30 min with PMA alone to trigger IB␣ degradation and then were treated in the continued presence of PMA with ionomycin after 30 min to assess the role of Ca 2ϩ in p65 nuclear localization. E, Jurkat cells were treated for the times indicated with either P/I (left panel) or PMA alone, followed by addition of ionomycin after 30 min (right panel), and then the amounts of IB␣ were determined by immunoblotting. (Fig. 7G). Quantification of p65 binding to IB␣, CXCL8, and TNF promoters in Jurkat T cells demonstrated that PKC␣ suppression significantly reduced p65 binding to IB␣, TNF, and CXCL8 promoters (Fig. 7G). These results are also consistent with the observed reduction in IB␣ protein re-expression (Fig.  7B), confirming that reduced promoter binding directly impacted subsequent transcription and protein expression. Together, this comprehensive analysis establishes the critical importance of Ca 2ϩ dependent PKC␣ activation in p65 nuclear localization, promotor binding, and transcriptional activation of a cohort of key NF-B target genes.

Discussion
The need to precisely determine how Ca 2ϩ regulates distinct transcriptional responses in T cells is underscored by that fact that almost 60% of TCR-induced genes are subject to Ca 2ϩ -dependent control (57). The notion that Ca 2ϩ regulates NF-B activation in lymphocytes is rooted in decades-old work dem- FIGURE 7. PKC␣ mediates Ca 2؉ -dependent but not TNF-induced p65 nuclear localization and promotor binding. A, PKC␣ levels were suppressed Ͼ90% 48 h after transfection of Jurkat T cells with a PKC␣ suppression construct as measured by immunoblot analysis. The densitometry plot represents the mean Ϯ S.E. from five independent experiments. B, Jurkat cells transfected with either vector alone (Control) or shPKC␣ were stimulated with either P/I or TNF for the times shown. Lysates were prepared, and IB␣ or ␣-tubulin was measured by immunoblot analysis. A representative example of four separate experiments is shown. C, cells were treated as described in B, and then p65 and Ser(P)-536 levels were quantified by immunoblot analysis. D, densitometric analysis of Ser-536 phosphorylation relative to the total p65 amount. Each value represents the mean Ϯ S.E. of normalized values from four independent experiments. **, p Ͻ 0.01; Welch's t test. E, Jurkat cells were stimulated for the indicated time with PMA and ionomycin, and p65 nuclear and cytoplasmic localization in control and PKC␣-suppressed cells was analyzed by confocal microscopy. The distribution of nuclear:cytoplasmic ratios is plotted for the indicated time points (the example is representative of three separate experiments). F, box plot of the p65 nuclear to cytoplasmic ratio. Wilcoxon rank-sum test revealed significant inhibition of p65 nuclear localization at each time point after stimulation. G, chromatin immunoprecipitation analysis of p65 promotor binding in cells stimulated for 60 min with PMA and ionomycin. IB␣, TNF, and CXCL8 abundance relative to chromatin input was compared between WT and PKC␣suppressed cells. Promoter binding (mean Ϯ S.D.) relative to chromatin input is shown for IgG (control) and p65 immunoprecipitates (representative of three independent experiments. **, p Ͻ 0.01, Welch's t test. onstrating that NFAT and NF-B activity is tuned to distinct calcium dynamics (3,5,6). Specifically, NFAT activation requires steady-state Ca 2ϩ elevations (2,3,5,6), although the amplitude of steady-state Ca 2ϩ signals may further dictate which NFAT isoform is activated (58,59). In contrast, selective activation of NF-B has been linked to low-frequency spikes in cytoplasmic Ca 2ϩ (3,5). However, little is known about the nature of these Ca 2ϩ signals, and the source of Ca 2ϩ required to activate NF-B has not previously been explored. Thus, in comparison with the established role and mechanism of Ca 2ϩ -dependent NFAT activation, the mechanisms by which Ca 2ϩ regulates TCR-induced NF-B activation remain undefined.
We first asked whether the relatively infrequent Ca 2ϩ spikes that selectively activate NF-B in lymphocytes (3, 5, 6) could be generated from ER release without a need for extracellular influx (60). Unexpectedly, we found that ER release is insufficient and that Ca 2ϩ influx via Orai is required to activate NF-B. We then focused on the mechanism by which Oraimediated Ca 2ϩ entry regulates NF-B activation.
Engagement of the TCR triggers canonical NF-B activation by PKC-driven formation of the CBM complex (containing CARMA1, Bcl10, and MALT1) (8,9,61,62). During formation of the CBM complex, Ca 2ϩ has been implicated in CARMA1 and Bcl10 phosphorylation via Calmodulin kinase II (63)(64)(65) and Bcl10 dephosphorylation by Calcineurin A (10,42). Our data confirm this general modulatory role for Ca 2ϩ in steps proximal to IKK activation and IB␣ degradation. However, we also found that pharmacological activation of PKC using either PMA stimulation alone (Fig. 4A) or PMA plus ionomycin in Ca 2ϩ -free medium (Fig. 1B) triggers substantial IB␣ degradation in the absence of Ca 2ϩ mobilization. Thus, rather than exhibiting an absolute requirement for Ca 2ϩ , our data suggest that Ca 2ϩ cooperates with PKC to accelerate the rate and, possibly, extent of IB␣ degradation. Hence, although PKC activation is sufficient for CBM complex formation and IKK activation, Ca 2ϩ serves a modulatory role via CnA upstream of IKK activation. An additional finding here is the obligate role for Ca 2ϩ in NF-B activation distal to IB␣ degradation, where it controls p65 phosphorylation, nuclear localization, target gene promotor binding, and transcriptional activation.
This regulatory role for Ca 2ϩ in IKK-distal signaling is entirely novel and establishes Ca 2ϩ as a critical regulator at multiple checkpoints of NF-B activity. Notably, although TNF signaling involves IKK activation and induction of p65 phosphorylation, our data establish that this occurs independently of any requirement for Ca 2ϩ . Most importantly, we show that TCR-induced p65 phosphorylation on Ser-536 definitively involves Ca 2ϩ -dependent activation of PKC␣ and that the kinetics of this phosphorylation are distinct from the regulation of p65 phosphorylation in response to TNF. A number of separate kinases have been described that control TNF-driven and TNF-independent p65 phosphorylation (13, 15, 20, 22-28, 30, 49, 50, 66 -69). However, none of these require Ca 2ϩ , and our experiments show that PKC␣ plays no role in TNF signaling. Thus, we established pathway-specific nodes of control for TCR versus TNF-induced NF-B signaling in which Ca 2ϩ regulation of PKC␣ represents a novel but crucial regulatory step in TCR-induced transcriptional activation of NF-B in T cells.
Furthermore, we delineated two separate Ca 2ϩ -dependent checkpoints, one proximal and one distal to IKK activation, that modulate TCR-induced NF-B signaling.
Our results have far-reaching implications concerning the mechanisms controlling T cell development and cell fate specification. In this regard, recent work has highlighted fundamental roles for TCR-induced Ca 2ϩ entry in the development of immunity (34). For example, individuals with functional defects in STIM or Ca 2ϩ release-activated Ca 2ϩ /Orai are profoundly immune-deficient, and mice conditionally lacking STIM in T lymphocytes develop autoreactive disorders in part because of defective thymic natural regulatory T cell induction by highaffinity self-agonists (70). A similar and selective defect in natural regulatory T cell development occurs in mice selectively lacking either c-Rel (71)(72)(73)(74)(75)(76) or upstream mediators of NF-B activation, including BCL10, PKC, CARMA1, CnA␤, and IKK␤ (77)(78)(79)(80)(81). Although our study focuses on p65-dependent transcriptional activation, the dual sensitivity of proximal and distal Ca 2ϩ signals we identified and the role of c-Rel in natural regulatory T cell development raises the intriguing possibility that c-Rel transcriptional activation is also Ca 2ϩ -dependent. If this were the case, then one would speculate that p65 and c-Rel regulation, like NFAT isoforms, might be tuned to quantitatively or qualitatively distinct Ca 2ϩ dynamics. Thus, although we have shown that p65 nuclear localization and transcriptional activation are regulated by Ca 2ϩ -dependent PKC␣-mediated phosphorylation of p65 Ser-536, distinct Ca 2ϩ -activated kinases could control c-Rel activity. Hence, critical goals of future studies will be to quantify the Ca 2ϩ threshold of IKK activation and p65 phosphorylation, identify the range of Ca 2ϩdependent Ser/Thr kinases activated following TCR engagement, and elucidate the role of Ca 2ϩ in c-Rel activation.