Differential Roles of PKC-θ in the Regulation of Intracellular Calcium Concentration in Primary T Cells

Differential Roles of PKC-θ in the Regulation of Intracellular Calcium Concentration in Primary T Cells

doi:10.1016/j.jmb.2005.10.043 J. Mol. Biol. (2006) 355, 347–359 Differential Roles of PKC-q in the Regulation of Intracellular Calcium Concentration...

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doi:10.1016/j.jmb.2005.10.043

J. Mol. Biol. (2006) 355, 347–359

Differential Roles of PKC-q in the Regulation of Intracellular Calcium Concentration in Primary T Cells Santhakumar Manicassamy1, Maureen Sadim1 Richard D. Ye2 and Zuoming Sun1* 1

Department of Microbiology and Immunology, College of Medicine, University of Illinois Chicago, IL 60612, USA 2 Department of Pharmacology College of Medicine, University of Illinois, Chicago, IL 60612 USA

Activation of T lymphocytes requires protein kinase C theta (PKC-q) and an appropriately elevated free intracellular Ca2C concentration ([Ca2C]i). Here, we show that phorbol 12 myristate 13-acetate (PMA) inhibited Ca2C influx in wild-type but not PKC-qK/K T cells, suggesting that PKC-q plays a role in PMA-mediated inhibition of Ca2C influx. In contrast, T cell receptor (TCR) crosslinking in the same PKC-qK/K T cells did result in significantly decreased [Ca2C]i compared to wild-type T cells, suggesting a positive role for PKC-q in TCR-mediated Ca2C mobilization. In PKC-qK/K mice, peripheral mature T cells, but not developing thymocytes, displayed significantly decreased TCR-induced Ca2C influx and nuclear factor of activated T cells (NFAT) translocation upon sub-optimal TCR crosslinking. The decreased intracellular free Ca2C was due to changes in Ca2C influx but not efflux, as observed in extracellular and intracellular Ca2C mobilization studies. However, these differences in Ca2C influx and nuclear factor of activated T cells (NFAT) translocation disappeared with increasing intensity of TCR crosslinking. The enhancing effect of PKC-q on Ca2C influx is not only dependent on the strength of TCR crosslinking but also on the developmental stage of T cells. The underlying mechanism involved phospholipase Cg1 activation and inositol triphosphate production. Furthermore, knockdown of endo-genous PKC-q expression in Jurkat cells resulted in significant inhibition of TCR-induced activation of NFAT, as evidenced from NFAT reporter studies. Forced expression of a constitutively active form of calcineurin in PKC-qK/K Jurkat cells could readily overcome the above inhibition. Thus, PKC-q can both positively and negatively regulate the Ca2C influx that is critical for NFAT activity. q 2005 Elsevier Ltd. All rights reserved.

*Corresponding author

2C

Keywords: Ca

influx; PKC-theta; T cells; NFAT

Introduction Protein kinase C theta (PKC-q) mediates the critical T cell receptor (TCR) signals required for T Present address: M. Sadim, Medicine-Hematology and Oncology, Northwestern University 710 N. Fairbanks, Olson 8370, Chicago, IL 60611, USA. Abbreviations used: PKC-q, protein kinase C theta; TCR, T cell receptor; DAG, diacylglycerol; PLCg1, phospholipase Cg1; IP3, inositol triphosphate; IL-2, interleukin 2; siRNA, small interfering RNA; S.D., standard deviation. E-mail address of the corresponding author: [email protected]

cell activation.1–3 Engagement of TCR induces activation of phospholipase Cg1 (PLCg1), which catalyzes the hydrolysis of inositol phospholipids to produce diacylglycerol (DAG) and inositol triphosphate (IP 3). IP 3 induces Ca2C influx whereas DAG activates PKCs.4 Ionomycin (a Ca2C mobilizer) alone induces T cell anergy or apoptosis, whereas in combination with phorbol esters (PKC activators) it mimics the signals required for T cell activation.5 Thus, IP3-induced Ca2C influx and DAG-mediated PKC activation synergize, resulting in fully fledged T cell proliferation. Although DAG activates multiple isoforms of PKC, only PKC-q is selectively required for T cell activation in vivo.2,3 Mature

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

348 PKC-qK/K T cells failed to proliferate and produce interleukin 2 (IL-2) upon TCR stimulation due to defective activation of NF-kB and AP1. These observations are also substantiated by several in vitro studies.6–9 Mice deficient in other isoforms of PKC do not have a defect a in T cell activation, thereby reinforcing the importance of PKC-q in T cell activation. Appropriate elevation of intracellular Ca2C concentration induced by the engagement of TCR is an essential signal for T cell activation, since it is required for stimulation of the nuclear factor of activated T cells (NFAT).10,11 The amplitude and dynamics of [Ca2C]i changes carry the critical signals that determine whether TCR engagement results in T cell activation, anergy or death.5,12,13 TCR-mediated elevation in [Ca2C]i is characterized by an initial rapid increase followed by a decrease that eventually leads to relatively stabilized [Ca2C]i, which is higher than the level observed in resting T cells. Insufficient increase in the [Ca2C]i prevents T cell activation,14 whereas persistently high levels of [Ca2C]i, such as those seen in ionomycin or thapsigargin-treated cells, often result in anergy or death.5,15 Therefore, fine tuning of [Ca2C]i to an appropriate level is critical for eliciting appropriate response from T cells. However, little is known about the molecular mechanisms that regulate [Ca2C]i required for T cell activation. Here, we show that PKC-q mediated elevation in [Ca2C]i is mainly due to changes in influx but not efflux. In addition, we demonstrate that this effect is seen in mature peripheral T cells but not in immature thymocytes, and only upon sub-optimal TCR crosslinking. The underlying mechanism involves PLCg1 activation and IP3 generation. The results obtained using PKCqK/K murine T cells, and also siRNA generated PKC-q deficient Jurkat cells are shown.

Results Thapsigargin treatment and TCR crosslinking mediated elevation in [Ca2C]i is inhibited by PMA We have demonstrated previously that PKCqK/K T cells exhibited defects in several critical signaling pathways essential for T cell activation.3 It was also reported recently that TCR-induced Ca2C influx was diminished in PKC-qK/K T cells.2,16 We therefore decided to determine whether activation of other isoforms of PKC can compensate for the defects observed in PKC-qK/K T cells. T cells were first treated with phorbol 12 myristate 13-acetate (PMA), a pharmacological activator of multiple isoforms of PKC including PKC-q. The TCRinduced Ca2C influx was then measured using a calcium dye, Indo-1 AM. Based on the previously proposed positive role of PKC-q in the regulation of Ca2C influx,2 we anticipated that PMA would further enhance TCR-induced Ca2C influx. However, PMA inhibited the Ca2C influx in naı¨ve

Regulation of Intracellular Calcium Level by PKC-q

T cells stimulated by either TCR crosslinking using anti-CD3 antibody (Figure 1(a)) or thapsigargin treatment (Figure 1(b)). Since PMA activates multiple isoforms of the PKC family, we reasoned that PKC-q may preferentially promote Ca2C influx, whereas other isoforms may be responsible for PMA-mediated inhibition of the Ca2C influx. If this is the case, inhibition of the Ca2C influx by PMA should also be observed in the absence of PKC-q. We therefore examined Ca2C influx in PKC-qK/K T cells. PMA treatment actually failed to inhibit Ca2C influx in PKC-qK/K T cells treated with anti-CD3 antibody (Figure 1(c)) or thapsigargin (Figure 1(d)), strongly suggesting that PKC-q in fact is capable of mediating inhibition of [Ca2C]i under some conditions such as PMA treatment. A role for PKC-q as a negative regulator of Ca2C is therefore evident. However, in the absence of PMA treatment, [Ca2C]i was significantly reduced in PKC-qK/K T cells treated with anti-CD3 antibody (1 mg/ml) as compared to PKC-qC/C T cells (Figure 1(a) and (c)), suggesting that PKC-q can also play a positive role in TCR-induced Ca2C influx. In addition to the kinetic changes in [Ca2C]i, we determined the average peak increase in [Ca2C]i in the experiments described above, and the results are summarized in Figure 1(e) and (f). These results clearly demonstrate that PKC-q is capable of both positively and negatively regulating [Ca2C]i, depending on the type and strength of stimulus. We therefore examined carefully the effect of PKC-q absence on TCR-induced calcium mobilization. PKC-q-mediated increase in [Ca2C]i is dependent on the developmental stage of the T cells and the strength of the stimulus To further analyze the PKC-q-mediated increase in [Ca2C]i, T cells were obtained from wild-type and PKC-q null mice. They were then treated with increasing concentrations of anti-CD3 antibody to promote proportionately increased TCR crosslinking and thus activation (Figure 2(a)–(d)). [Ca2C]i was consistently lower in PKC-qK/K T cells as compared to wild-type T cells at all concentrations of anti-CD3 antibody used. However, the most significant differences between wild-type and PKC-qK/K T cells were detected at relatively lower concentrations of antiCD3 antibody treatment, clearly suggesting the importance of optimal engagement of TCR. The average peak [Ca2C]i observed in PKC-qK/K T cells was about one-third that of the wild-type cells when 0.5 mg/ml of anti-CD3 antibody was used (Figure 2(e)). As the amount of anti-CD3 antibody used for TCR crosslinking increased, the differences between wild-type and PKC-qK/K T cells concomitantly diminished. To exclude differences due to variations in uptake of the Ca2C dye, T cells were lysed by detergent treatment and maximum fluorescence (an indicator of the amount of dye taken up by cells) was determined. The differences in peak fluorescence between wild-type and mutant cells were not significant (data not shown), therefore

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Figure 1. PMA-mediated activation of PKC-q inhibits Ca2C influx. (a) and (b) PMA inhibited Ca2C influx induced by TCR stimulation or thapsigargin treatment. T cells were first loaded with Indo 1-AM. Prior to stimulation, wild-type T cells were treated with different concentrations of PMA as indicated for 2 min. The [Ca2C]i was then recorded by a spectrofluorimeter after (a) crosslinking anti-CD3 antibody or (b) thapsigargin treatment. (c) and (d). PMA failed to inhibit Ca2C influx induced by TCR stimulation or thapsigargin treatment. Prior to stimulation, PKC-qK/K T cells were treated with different concentrations of PMA as indicated for 2 min. The [Ca2C]i was then recorded after stimulation with (c) crosslinking anti-CD3 antibody or (d) thapsigargin. (e) and (f). The average peak [Ca2C]i values with error bars denoting the standard deviation (S.D.) were obtained from three independent experiments when T cells were stimulated by (e) crosslinking CD3 or (f) thapsigargin.

ruling out such a possibility. In addition, expression levels of TCR on the surface of both wild-type and PKC-qK/K T cells were comparable (Figure 2(f)), indicating that the decreased Ca2C influx was not due to variations in TCR expression. Furthermore, ionomycin or thapsigargin, known promoters of Ca2C influx, that act independently of TCR, induced similar patterns of Ca2C influx in both wild-type and PKC-qK/K T cells (Figure 2(g), and data not shown), suggesting that the Ca2C mobilization pathways in both types of cells were comparable. It is therefore clear that the observed defects in Ca2C mobilization in PKC-qK/K T cells are due to lack of PKC-q and not due to any intrinsic defects in Ca2C mobilization. Thus, our results clearly demonstrate that the enhancing effect of PKC-q on Ca2C influx is dependent on the strength of TCR stimulation. Since TCR engagement also induces Ca2C influx in developing thymocytes, we therefore carried out similar experiments using wild-type and PKC-qK/K thymocytes. In contrast to peripheral mature T cells, no significant differences in TCR-induced Ca2C influx

were detected between wild-type and PKC-qK/K thymocytes (Figure 2(h)), suggesting that the role of PKC-q in the regulation of [Ca2C]i also depends on the developmental stage of T cells in addition to the strength of stimulation. PKC-q regulates Ca2C influx from both the intracellular stores and extracellular milieu but not Ca2C efflux The balance between Ca2C efflux and influx determines the [Ca2C]i.13,17 TCR stimulation leads to elevated [Ca2C]i due to Ca2C influx from both the intracellular stores and extracellular milieu. The elevated [Ca2C]i levels activate plasma membrane Ca2C-ATPase (PMCA) which exports Ca2C to the extracellular space (Ca2C efflux).18 To further dissect the role of PKC-q in the regulation of [Ca2C]i, we measured the rates of both Ca2C influx and efflux. T cells were first subjected to TCR stimulation (1 mg/ml of anti-CD3 antibody) so as to elevate [Ca2C]i, followed by treatment with

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Regulation of Intracellular Calcium Level by PKC-q

Figure 2. PKC-q enhances TCR-induced Ca2C influx depending on the developmental stage of T cells and strength of the stimulus. (a)–(d) Spleen T cells purified from wild- type (black) and PKC-q null mice (gray) were stimulated by crosslinking various concentrations of anti-CD3 antibody as indicated. The [Ca2C]i at different times after stimulation was then recorded. (e) The peak [Ca2C]i values with error bars denoting S.D. were averaged from three independent experiments as shown in (a)–(d). (f) Flow cytometric analysis of surface TCR levels on T cells obtained from wild-type (grey) and PKC-q null mice (black). (g) Ca2C influx in mature T cells treated with 100 nM thapsigargin. (h) Ca2C influx in thymocytes stimulated by TCR crosslinking with 1 mg/ml of anti-CD3 antibody.

1,2-bis(2-aminophenoxy)ethane-N,N,N 0 ,N 0 -tetraacetic acid (BAPTA), which chelates the extracellular Ca2C. Due to the absence of further Ca2C entry from extracellular space, the [Ca2C]i drops to

basal levels due to Ca2C efflux (Figure 3(a) and (b)). The rate at which elevated [Ca2C]i decrease to the basal level represents the efflux rate (Figure 3(c)). At the baseline, the extracellular Ca2C was then

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Figure 3. Impaired Ca2C influx from extracellular and intracellular Ca2C stores but not Ca2C efflux in PKC-qK/K T cells. (a) Wild-type (black) and PKC-qK/K T cells (gray) were stimulated by anti-CD3 antibody (1 mg/ml) to trigger Ca2C influx, and the [Ca2C]i was then recorded. BAPTA was added to the medium to chelate extracellular Ca2C, resulting in a rapid decrease in the [Ca2C]i to basal levels due to Ca2C efflux. Ca2C was then replenished to the extracellular medium, which resulted in a rapid increase in [Ca2C]i again. (b) A magnified portion of [Ca2C]i after addition of BAPTA, representing Ca2C efflux. (c) The efflux rate was averaged from three independent experiments as shown in (b). (d) A magnified portion of [Ca2C]i after addition of extracellular Ca2C, representing the Ca2C influx from extracellular sources. (e) The influx rate was averaged from three independent experiments as shown in (d). (f) and (g) The [Ca2C]i was recorded in T cells stimulated with (f) crosslinking 1 mg/ml of anti-CD3 antibody or (g) 100 nM thapsigargin in the presence of 3 mM of EGTA, which chelates the extracellular Ca2C, representing the Ca2C influx from the intracellular stores.

352 replenished so as to allow influx of the Ca2C. The rate at which the [Ca2C]i increases from the baseline represents the influx rate (Figure 3(a), (d) and (e)). The efflux rates in wild-type and PKC-qK/K T cells were similar to those indicated in Figure 3(b) and (c). On the other hand, the absence of PKC-q resulted in a significant decrease in the influx rate (Figure 3(d) and (e)). Since the majority of the Ca2C influx in this case was from the extracellular milieu, our observation suggests that PKC-q positively regulates TCR-induced Ca2C influx through the plasma membrane. We then investigated the effect of PKC-q on TCR-mediated changes in the [Ca2C]i influx rate from intracellular calcium stores (endoplasmic reticulum). In this experiment, the extracellular Ca2C was chelated by the addition of 3 mM EGTA prior to anti-CD3 antibody-mediated TCR crosslinking (Figure 3(f)). The Ca2C influx from intracellular stores was significantly reduced in PKC-qK/K compared to wild-type T cells. However, there was no difference in ionomycin or thapsigargin-induced Ca2C influx in the presence of extracellular EGTA, once again demonstrating that intracellular Ca2C mobilization pathways are intact (Figure 3(g), and data not shown). From the above studies, it is clear that PKC-q-mediated TCR signals regulate [Ca2C]i by specifically targeting the Ca2C influx from both the intracellular and extracellular sources, but not the Ca2C efflux. We and others have shown that PKC-q is required for TCR-mediated activation of essential transcription factors such as NF-kB, AP-1 and NFAT;2,3,8 however, the connection to elevated levels of intracellular calcium was not clearly established. We therefore further examined the role of PKC-q in the activation of the transcription factors described above using reporter essays and EMSA. Differential requirement for PKC-q in TCRmediated activation of NF-kB/AP-1 and NFAT One of the functions of TCR-induced Ca2C influx is to activate calcineurin, Ca2C-dependent phosphatase, that in turn dephosphorylates NFAT, and triggers its translocation to nucleus.11,17 In our previous study, we failed to detect any defect in NFAT translocation in PKC-qK/K T cells;3 however, here we used a relatively high concentration of antiCD3 antibody (10 mg/ml) to crosslink TCR. Based on the results shown above (Figure 2), we do know that the strength of TCR crosslinking is directly proportional to the observed [Ca2C]i and the absence of PKC-q has a profound negative effect, especially at lower levels of TCR crosslinking (Figure 2). We, therefore, used both wild-type and PKC-qK/K T cells to examine in greater detail the effect of degree of crosslinking of TCRs on the activation of NFAT. NFAT translocation was analyzed by EMSA after treating with various concentrations of anti-CD3 antibody (Figure 4(a)). To demonstrate specificity, antibodies against the corresponding transcription factors were used to perform super-shift assays. Indeed, under

Regulation of Intracellular Calcium Level by PKC-q

conditions of TCR crosslinking with lower concentrations of anti-CD3 antibody, PKC-qK/K T cells displayed an obvious defect in translocation of NFAT as compared to wild-type T cells. This defect was corrected when higher concentrations of anti-CD3 antibody were used for crosslinking TCR. However, NFAT activation levels observed in PKC-q K/K T cells failed to match the levels observed in PKC-qC/C T cells. Significant NFAT translocation to the nucleus was readily observed in wild-type T cells when stimulated with crosslinking 0.5 mg/ml of anti-CD3 antibody, whereas achieving similar NFAT translocation in PKC-qK/K T cells, required stimulation with at least 4 mg/ml of antiCD3 antibody. Coincidentally, to achieve Ca2C influx equivalent to that seen in wild-type T cells stimulated with crosslinking 0.5 mg/ml of anti-CD3 antibody, the PKC-qK/K T cells also required stimulation with 4 mg/ml of anti-CD3 antibody (Figure 2). Interestingly, translocation of NF-kB and AP1 in PKC-qK/K T cells remained significantly decreased even at the maximum concentration of anti-CD3 antibody used (Figure 4(b) and (c)), suggesting that NF-kB and AP1 pathways are more tightly dependent on PKC-q-mediated signaling in contrast to NFAT. Translocation of OCT-1, another transcription factor, was not affected in PKC-q T cells, thereby demonstrating specificity in the PKC-q-mediated effect (Figure 4(d)). Our previous observation that NFAT activation is independent of PKC-q can now be explained by the fact that a relatively high degree of TCR crosslinking can mask the requirement of PKC-q for NFAT activation. It also demonstrates the calcium dependence for NFAT activation. In addition, the activation of NF-kB and AP-1 appears to require some additional factors that are PKC-q-dependent. Upstream events that are necessary for PKC-q activation are PLCg1 activation that results in the generation of two essential second messengers, DAG and IP3. Therefore we examined the status of PLCg1 and determined the relative levels of IP3 in PKC-qC/C and PKC-qK/K T cells after TCR crosslinking. Specific tyrosine phosphorylation of PLCg1 (Y783) is a hallmark of its activation status and can be determined by immunoblotting using anti-PLCg1 antibody that recognizes tyrosine- phosphorylated PLCg1.19,20 Using such an approach, in addition to probing with anti-phosphotyrosine antibody, we observed significantly less tyrosine-phosphorylated forms of PLCg1 in PKC-qK/K as compared to PKC-qC/C T cells (Figure 4(e)). In resting T cells, in both cases, the activated form was hardly detectable. The above result clearly indicates the dependence of PLCg1 activation on PKC-q, although the former is traditionally considered to be upstream of the later. We can overcome this dependence by increasing the degree of TCR crosslinking (data not shown). We also examined one of the early, essential mediators of T cell activation, ZAP-70, a tyrosine kinase.4 There was, however, no significant difference in ZAP-70 activation between wild-type

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Figure 4. Differential requirement of PKC-q for activation of NFAT and NF-kB/AP1. (a)–(d). Bandshift analyses of NFAT, NF-kB and AP1. Purified wild-type (PKCqC) and PKC-qK/K (PKCq-) T cells were stimulated for 5 h with the indicated concentrations of plate-bound anti-CD3 antibody, and nuclear extracts were subjected to bandshift analyses using specific probes for (a) NFAT, (b) NF-kB, (c) AP1 and (d) OCT-1. Antibodies (Ab lanes) specific for different transcription factors were employed in supershift assays when T cells were stimulated by crosslinking 10 mg/ml of antiCD3 antibody. (e) Impaired tyrosine phosphorylation of PLCg1 in PKC-qK/K T cells stimulated by crosslinking 1 mg/ml of anti-CD3 antibody. Purified wild-type (C) and PKC-qK/K (K) T cells were stimulated by crosslinking CD3 for the indicated times. PLCg1 was then immunoprecipitated and detected by Western blot analyses with antibodies specific for the Tyr783-phosphorylated form of PLCg1, phospho-tyrosine or PLCg1 (top three panels). Western blot analysis was used to detect PKC-q in wild-type and mutant T cells (PKC-q). As a control, tyrosine phosphorylation of ZAP-70 was determined with antibodies specific for the Tyr319 phosphorylated form of ZAP-70 and total ZAP-70 (bottom two panels). (f) Intracellular IP3 concentrations of wild-type (open bar) and PKC-qK/K T cells (black bar) at different times after CD3 crosslinking are shown. Assays were repeated three times and averaged, with error bars denoting S.D.

and PKC-qK/K T cells as evidenced from its Y319phosphorylation status (Figure 4(e), two bottom panels), suggesting that PKC-q acts downstream of ZAP-70. Consistent with the diminished PLCg1 phosphorylation, the intracellular IP3 levels were also reduced in PKC-qK/K T cells relative to the wild-type T cells subjected to TCR crosslinking. The basal levels of IP3 in both groups were, however, comparable (Figure 4(f)). The above results clearly indicate that in naı¨ve T cells, PKC-q enhances Ca2C influx by promoting PLCg1 activation. TCRinduced increase in [Ca2C]i is known to activate calcineurin, a phosphatase that then dephosphorylates NFAT, thereby facilitating its translocation to the nucleus.4 We next investigated the effect of PKC-q on the downstream events such as activation of NFAT

and compared the results to the NF-kB and AP-1 activation status. In addition to NF-kB and AP-1 activation, PKC-q regulates NFAT transcriptional activity Jurkat T cells have been the model of choice to study T cell activation. We therefore abrogated endogenous levels of PKC-q in these cells using the small interfering RNA (siRNA) approach. The pSuper-based RNAi system21 was used to express siRNAs targeted to three different regions of human PKC-q. The siRNA targeted to the N terminus reduced exogenously expressed human PKC-q in 293T cells to undetectable levels (Figure 5(a), left panel), whereas the scrambled control siRNA had

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Figure 5. PKC-q is required for stimulating NF-kB and AP1 transcriptional activity. (a) Expression plasmids encoding human PKC-q (hPKCq), mouse PKC-q (mPKCq), PKC-d and PKC-3 were co-transfected into 293T cells with pSuper plasmids encoding either control scrambled siRNA(C) or PKC-q-specific siRNA (siRNA). At 48 h after transfection, expression of different isoforms of PKCs was detected by Western blot analysis (top panels). Expression of actin served as a control for equal loading (bottom panels). (b) Control pSuper or pSuper encoding PKC-q siRNA were transfected into Jurkat cells together with an expression plasmid for GFP. Sorted GFP-positive cells were lysed and subjected to Western blot analysis of PKC-q expression (top panel). The bottom panel shows the expression of actin serving as a control for equal loading. (c). Jurkat cells were transfected with control pSuper (C) or pSuper encoding PKC-q siRNA (siRNA) alone or together with expression plasmids for mouse PKC-q (mPKCq) or PKC-d (mPKCd). At 24 h after transfection, Jurkat cells were left in medium alone (open bars) or stimulated by anti-CD3 and CD28 antibodies (black bars). An AP1 luciferase reporter was used to monitor the AP1 transcriptional activity which is indicated as the fold of stimulation relative to the activity obtained from unstimulated cells. (d) Measurement of NF-kB-dependent luciferase activity was performed under the conditions similar to those described in (c), but with additional controls involving treatment with TNFa (gray bars). (e) Jurkat cells were transfected with a GFP expression plasmid together with control pSuper (C) or pSuper producing PKC-q siRNA (siRNA), and stimulated with anti-CD3 and CD28 antibodies. Surface CD69 levels on GFP-positive cells were assayed by flow cytometry. The numbers indicate the percentage of CD69 positive cells after stimulation. The gray area represents the CD69 levels on unstimulated cells.

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no effect on PKC-q expression. In the targeted region, the mouse PKC-q differs by two nucleotides as compared to human PKC-q. The siRNA specifically designed for human PKC-q had no effect on expression of mouse PKC-q (Figure 5(a)). In addition, PKC-q siRNA did not prevent the expression of PKC-d and PKC-3, two isoforms of novel PKC (Figure 5(a), middle and right panels), thus indicating the specificity of the designed

355 PKC-q siRNA. To determine whether the siRNA can inhibit the expression of endogenous PKC-q, the pSuper vector was co-transfected into Jurkat cells along with a green fluorescent protein (GFP) expression plasmid, which allowed us to sort the cells expressing siRNA. In contrast to the cells transfected with the vector expressing scrambled siRNA, the GFP positive cells sorted from the Jurkat cells expressing PKC-q siRNA had no detectable

Figure 6. PKC-q is required to stimulate NFAT transcriptional activity. (a) Jurkat cells were transfected with NFAT-dependent luciferase reporter together with control pSuper (C) or pSuper encoding PKC-q siRNA (siRNA) alone or with expression plasmids for mouse PKC-q (mPKCq) or PKC-d (mPKCd) or constitutively active calcineurin (Cn). At 24 h after transfection, Jurkat cells were left in medium alone (open bars) or stimulated by anti-CD3 and CD28 antibodies (black bars). NFAT-dependent luciferase reporter activity is indicated as fold induction relative to the activity obtained from unstimulated cells. Jurkat cells transfected with expression plasmids encoding active or inactive PKC-q were stimulated with medium alone (open bars) or anti-CD3 and CD28 antibodies (black bars). The activity of NF-kB (b), AP1 (c), and NFAT (d) were monitored with the corresponding luciferase reporters as indicated.

356 PKC-q (Figure 5(b)), suggesting a successful knockdown of the endogenous PKC-q. We next determined the effects of loss of endogenous PKC-q expression in Jurkat cells upon activation of NF-kB and AP1 reporters. As expected, TCR-crosslinking resulted in significant induction of both NF-kB and AP1 reporters in Jurkat cells expressing scrambled siRNA. In cells expressing PKC-q siRNA, the induction of both the reporters was significantly reduced (Figure 5(c) and (d)), clearly demonstrating the requirement for PKC-q in the activation of both NF-kB and AP-1. A similar inhibition was also observed in the presence of inactive PKC-q (a kinase dead mutant) as shown later (Figure 6(b) and (c)). TNFa is a known potent activator of NF-kB; however, PKC-q is not required in this pathway.8 As shown in Figure 5(d), TNFainduced activation of NF-kB was not affected by the PKC-q siRNA (Figure 5(d)), thereby demonstrating the specificity of PKC-q siRNA used in the experiments described above. Since siRNA does not inhibit mouse PKC-q (Figure 5(a)), we performed rescue experiments with the expression plasmid encoding mouse PKC-q. Activation of NF-kB and AP1 were both restored by the expression of mouse PKC-q but not by PKC-d in PKC-q siRNA expressing Jurkat cells (Figure 5(c) and (d)), suggesting that the observed inhibition of NF-kB and AP1 resulted from specific knockdown of PKC-q. The activation of transcription factors ultimately leads to an altered phenotype in T cells. Enhanced surface expression of CD69 upon TCR crosslinking is considered as an end-point marker of T cell activation.3 Once again, as expected, a significant increase in CD69 expression following TCR crosslinking was apparent in Jurkat cells expressing scrambled siRNA; however, the expression was significantly reduced in cells expressing PKC-q siRNA (Figure 5(e)). The above results clearly demonstrate that PKC-q-mediated signaling pathways are conserved in both human and mouse. Finally, we examined the effect of PKC-q on the regulation of NFAT activity, using an NFAT reporter plasmid. Knockdown of PKC-q by siRNA significantly reduced TCR-mediated activation of NFAT reporter activity (Figure 6(a)). Expression of mouse PKC-q but not the PKC-d isoform in PKC-q siRNA expressing Jurkat cells restored NFAT activity. Forced expression of a constitutively active form of calcineurin whose activity, independent of intracellular Ca2C, restored NFAT activity (Figure 6(a)). These results suggest that endogenous PKC-q is specifically required for stimulation of NFAT transcriptional activity in a calcineurindependent manner. Expression of constitutively active and inactive forms of PKC-q in Jurkat cells triggered the activation and inhibition of NF-kB and AP-1, respectively. It is interesting to note that the expression of constitutively active PKC-q had hardly any effect on the NFAT activation status. TCR crosslinking did not have any further effect on the activation status of NF-kB and AP-1 in

Regulation of Intracellular Calcium Level by PKC-q

constitutively active PKC-q expressing cells, thereby indicating that active PKC-q alone is enough for maximal activation of NF-kB and AP-1. However, TCR crosslinking significantly increased the NFAT activation levels in cells expressing the active form of PKC-q compared to untransfected cells or cells expressing the inactive form of PKC-q (Figure 6(d)). Therefore, it is clear that NF-kB and AP-1 activation is solely dependent on PKC-q; however, NFAT activation requires additional factor(s) that can be mobilized via TCRcrosslinking.

Discussion The role of PKC in various cellular signaling processes is well established.22 To date, there are 11 known isoforms of PKC, most of which are expressed in T cells. However, only PKC-q is indispensable for T cell activation.2,3 The relationship between PKC-q, changes in [Ca2C]i and activities of various transcription factors in T cell signaling is not clearly established. Here, we have addressed the above relationship by employing not only PKC-q knockout mouse T cells but also human transformed T cells such as Jurkat whose endogenous PKC-q levels were abrogated using specific siRNA. Our results clearly demonstrate the ability of PKC-q to both positively and negatively regulate Ca2C influx. In addition, the stimulatory effect of PKC-q on Ca2C influx that is seen during the course of T cell activation depends on the developmental stage of T cells and strength of the TCR crosslinking (or engagement). It is interesting to note that the PKC-q-mediated increase in [Ca2C]i is due to enhanced Ca2C influx from the extracellular milieu as well as the intracellular stores; however, Ca2C efflux remains unaltered. PKC-q plays a crucial role in the activation of various transcription factors such as NF-kB, AP-1 and NFAT. The TCR-mediated increase in [Ca2C]i is known to activate calcineurin, which in turn, activates NFAT. The data presented here clearly demonstrate the absolute requirement of PKC-q for the activation of NF-kB and AP-1. Fullfledged activation of NFAT, however, requires not only PKC-q but also some additional signals generated by TCR crosslinking. Previous studies either reported no change or moderate change in Ca2C influx upon T cell activation using PKC-qK/K naı¨ve T cells.2,16 Our study, however, clearly demonstrates a positive role for PKC-q in the regulation of Ca2C influx that is triggered by TCR activation. The role played by PKC-q is subtle and can be appreciated only when TCR crosslinking is carried out with relatively low concentrations of anti-CD3 antibody (1 mg/ml) (Figure 2). As the degree of TCR crosslinking increases, the difference observed between PKCqC/C and PKC-qK/K T cells decreases and at concentrations of antibody used in previous studies, the difference becomes almost insignificant

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(Figure 2). In vivo T cell activation requires engagement of TCRs by few ligands, and sometimes a single TCR–ligand interaction is sufficient to trigger T cell activation.23–26 Therefore, sub-optimal TCR stimulation is more in line with a model representing T cell activation in vivo. Altman et al. observed defective Ca2C influx and PLCg1 activation in pre-activated and re-stimulated PKCq K/K T cells,16 which mostly represents the memory or effector T cells undergoing activationinduced cell death. Here, however, we studied naı¨ve T cells, which most likely represent the T cells initially engaged with antigens in primary immune responses. Under such conditions, T cells undergo proliferation, unlike re-stimulated T cells that undergo apoptosis. Altogether, PKC-q appears to play an important role in the TCR-mediated Ca2C influx both in naı¨ve and effector T cells but not in immature thymocytes. An elevated level of IP3 as a result of the activation of PLCg1 is known to be responsible for triggering the Ca2C influx.27 In agreement with reduced Ca2C influx in PKC-qK/K T cells upon TCR activation, both PLCg1 activation and IP3 production were impaired in PKC-q T cells (Figure 4). These results suggest that activated PKC-q stimulates Ca2C influx by affecting PLCg1 activity. The data presented here, as well as by others,16 show that PKC-q is required for maximal activation of PLCg1. This is a very provocative result, because PLCg1 is an immediate upstream component to PKCq in the T cell activation pathway.4 If this were to be true, we expect very little or no change in PLCg1 activity upon TCR crosslinking in the absence of PKCq; however, the evidence indicates the opposite. Although initial activation of PLCg1 is triggered by TCR and most likely to be independent of PKC-q, we believe that activated PKC-q can either directly or indirectly contribute to maximal activation of PLCg1. Such a notion is supported by the fact that loss of PKC-q does not completely eliminate PLCg1 activity in the Tcell activation process. In the absence of PKCq, activation of NFAT is definitely compromised under conditions of low TCR crosslinking. The above finding is physiologically more relevant because it simulates TCR engagement encountered in vivo.23,25 The effect of PKC-q on TCR-mediated activation of Ca2C is also apparent under conditions of relatively low TCR crosslinking, thus linking enhanced Ca2C influx mediated by PKC-q to NFAT activation. Although PKC-q is required for NFAT activation, for maximal activation TCR signals other than PKC-q also appear to contribute (Figure 6(d)). In addition to translocation, we also demonstrated that knockdown of PKC-q in Jurkat cells reduced NFAT as well as NF-kB-and AP1-mediated transcriptional activity (Figure 6(a)). Reduced NFAT activity, but not NF-kB and AP1 activity, was restored by forced expression of a constitutively active calcineurin, thereby demonstrating the requirement of increased Ca2C influx mediated by PKC-q in the activation of NFAT. The mechanisms underlying PKC-q-mediated activation of PLCg1 are, however, not clear.

The Tec family kinase, Itk, can be a potential link between PKC-q and PLCg1. PLCg1 is reported to be a target for Itk, as overexpression of Itk strongly activates PLCg1 as well as NFAT, even without TCR crosslinking.28 Similar to findings made in PKCqK/K T cells, T cells from mice deficient in the Tec family kinases Itk and Rlk display defective IP3 production and Ca2C influx due to reduced PLCg1 activity.29–31 Altman et al. demonstrated that Tec mediates the activation of PLCg1 in re-stimulated PKC-qK/K T cells.16 In contrast to Itk, Tec is expressed at very low levels in naı¨ve T cells, but is upregulated in re-stimulated T cells.28 It is likely that PKC-q regulates Ca2C influx via Itk in naı¨ve T cells, whereas effector T cells may depend on Tec to regulate PKC-q-mediated Ca2C influx. In contrast to NFAT, activation of NF-kB and AP-1 transcription factors appears to be completely dependent on PKC-q. The fact that expression of the active form of PKC-q in PKC-q knockout cells is sufficient to maximally activate NF-kB and AP-1 reporter activities even without TCR engagement substantiates the above claim. In addition, maximal TCR crosslinking induced by treatment with relatively high concentrations of anti-CD3 antibody cannot overcome PKC-q deficiency. Therefore, the observation that efficient TCR crosslinking induces significant NFAT activation even in the absence of PKC-q but not AP1 activation could explain the previously observed induction of T cell anergy in PKC-qK/K mice.32 Also previous studies have shown that activation of NFAT but not its transcriptional partner AP1, results in T cell anergy.5,15 Extensive crosslinking of TCRs in PKC-qK/K T cells induces Ca2C influx, most likely due to clustering of other coreceptors. PKC-q plays an indispensable role in T cell activation. TCR activation leads to activation of PLCg1 and the DAG generated as a result activates PKC-q that in turn enhances PLCg1. Such a positive feedback loop appears to further enhance the intracellular Ca2C level. In addition, PKC-q can potentially negatively regulate calcium mobilization as evidenced from our PMA studies; however, their physiological relevance is not apparent. Considering the multiple functions of PKC-q in the regulation of [Ca2C]i, it would be interesting to examine whether PKC-q plays a role in vivo in directing T cells toward different fates such as activation, anergy, or death.

Materials and Methods Antibodies and reagents Antibodies against murine anti-CD33, CD28, PLC-g1, ZAP-70, and hCD69-PE were purchased from BD PharMingen (San Diego, CA). Antibodies against phosphotyrosine783-PLC-g1, phosphotyrosine319-ZAP-70 and phosphotyrosine were purchased from Cell Signaling Technology (Beverly, MA). Goat anti-mouse IgG-HRP

358 and goat anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Regulation of Intracellular Calcium Level by PKC-q

Bandshift analysis Bandshift and supershift analyses were performed as described.3

Plasmids Inositol 1,4,5-triphosphate (IP3) measurement The wild-type, constitutively active or dominantnegative Xpress-tagged human PKC-q expression vectors, flag-tagged mPKC-q, and mPKC-d were gifts from Drs Amnon Altman (Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA) and Xin Lin (M. D. Anderson Cancer Institute, Houston, TX). The siRNA constructs were made in pSUPER vector,21 a kind gift from R. Agami (The Netherland’s Cancer Institute, Plesmanlaan, The Netherlands). To suppress endogenous hPKC-q expression, a doublestranded oligonucleotide with the sequence of gatccccGAGTATGTCGAATCAGAGAttcaa gagaTCTCTGATTCGACATACTCtttttggaaaagct, was inserted into pSUPER. A pSuper construct expressing a scrambled siRNA served as a control.

Mature T cells were stimulated by cross-linking of antiCD33 antibody as described above. The reaction was terminated by the addition of ice-cold trichloroacetic acid followed by 15 min incubation on ice. The samples were centrifuged at 14,000 rpm for 15 min at 4 8C, and the supernatant extracted with 10! volume of water-saturated diethyl ether, and then neutralized with 1 M NaHCO3. Inositol 1,4,5-triphosphate (IP3) was measured in duplicate using a competitive [3H]IP3 binding assay (Amersham Biosciences) according to the manufacturer’s instructions. Controls included medium alone or mock-stimulated (no primary antibody was added) cells. Reporter assay

Intracellular calcium analysis Mouse Tcells were obtained from the spleen and lymph nodes of PKC-qK/K or wild-type mice and purified using mouse T cell enrichment columns (R&D Systems, Minneapolis, MN). Purified CD3 T cells (5!106/ml) were incubated with 2 mM Indo-1 acetoxymethyl ester (Molecular probes, Eugene, OR) and 2% (v/v) fetal calf serum in HBSS (Hanks balanced saline solution) for 50 min. After a brief wash with HBSS, the cells were resuspended in HBSS at a concentration of 2!106/ml and stored on ice. T cells were stimulated with different concentrations of biotin-conjugated anti-mouse CD33 antibody and crosslinked with streptavidin (15 mg/ml). The intracellular Ca2C concentration was recorded using a PTI (Photon Technology International, Monmouth Junction, NJ) spectrofluorimeter, at 405 nm and 485 nm, with an excitation wavelength of 340 nm. The intracellular Ca2C level was measured as relative fluorescence based on the ratio of Indo-1 fluorescence at 405 nm and 485 nm, and standardized for Indo-1 loading and cell responsiveness. Calcium concentrations were calculated as described.33

Immunoprecipitation and Western blotting Purified T-cells were incubated in serum-free Roswell Park Memorial Institute (RPMI) medium for 2 h at 37 8C before stimulation. Cells were typically stimulated at 1! 107 cells per 300 ml in RPMI medium for the indicated times, and stimulation was stopped by addition of an equal volume of 2!lysis buffer (2% (v/v) NP-40, 50 mM Tris–HCl (pH7.5), 5 mM EDTA, 150 mM NaCl, 5 mM NaF, 5 mM disodium pyrophosphate, 1 mM sodium orthovanadate, 1:50 (v/v) protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO)). Cell lysates were cleared by centrifugation at 15,340g for 10 min at 4 8C, and used for immunoprecipitation. Immunoprecipitation, immunoblotting, and SDS-PAGE were performed using standard procedures. For immunoprecipitation, the primary antibody was added to the cell lysates, and the immunocomplexes were isolated using goat anti-mouse IgG beads (Sigma) and goat anti-Rabbit IgG agarose beads (Sigma).

Jurkat or Jurkat-Tag cells (10!106/ml) were transfected by electroporation with 5 mg of the NFAT or NF-kB or AP1 luciferase reporter plasmid together with 15 mg of the PKC-q siRNA or scrambled siRNA expression plasmids or 5 mg of different PKC expression plasmids. Identical amounts of the corresponding parental vectors were used as control. For normalization, 100 ng of the Renilla luciferase reporter vector, pTK-Renilla-LUC, was used. In the experiments in which the effects of overexpression of kinase-defective PKC-q (PKC-q-K409R) or a constitutively active PKC-q(A148E) were determined, the cells were co-transfected with either 15 mg of control vector or the indicated expression plasmids. After 24 h, cells were incubated for 6 h with OKT-3 (1 mg/ml) and anti-CD28 (2 mg/ml) and crosslinked with a secondary goat antimouse immunoglobulin (10 mg/ml). Cells were then lysed and assayed for dual luciferase activity (Promega, Madison, WI).

Acknowledgements We thank Drs Amnon Altman and Xin Lin for providing expression plasmids of various isoforms of PKCs, Dr Reuven Agami for the pSuper plasmid, Drs Prasad Kanteti and Bellur Prabhakar for critically reading the manuscript and helpful discussion. This work was supported by grants from American Cancer Society of Illinois Division, Schweppe Foundation, UIC Cancer center and UIC IRB and NIH R01-AI053147-01.

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Edited by J. Karn (Received 27 June 2005; received in revised form 10 October 2005; accepted 17 October 2005) Available online 8 November 2005