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Regulation of Retinoid X Receptor Responsive Element-Dependent Transcription in T Lymphocytes by Ser/Thr Phosphatases: Functional Divergence of Protein Kinase C (PKC) and PKC in Mediating Calcineurin-Induced Transactivation¹

Laboratory of Molecular Cell Biology, Science Applications International Corporation-Frederick, Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, MD 21702

	Abstract

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T lymphocyte activation signals regulate the expression and transactivation function of retinoid X receptor (RXR) through an interplay of complex signaling cascades that are not yet fully understood. We show that cellular Ser/Thr protein phosphatases (PPs) play an important role in mediating these processes. Inhibitors specific for PP1 and PP2A decreased basal expression of RXR RNA and protein in T lymphocyte leukemia Jurkat cells and prevented activation-induced RXR accumulation in these cells. In addition, these inhibitors attenuated the RXR responsive element (RXRE)-dependent transcriptional activation in transient transfection assays. Inhibitors of calcineurin (CN), by contrast, did not have any effect on the basal RXR expression and even augmented activation-induced RXR expression. Expression of a dominant-active (DA) mutant of CN together with a DA mutant of protein kinase C (PKC), a novel PKC isoform, significantly increased RXRE-dependent transcription. Expression of catalytically inactive PKC or a dominant-negative mutant of PKC failed to synergize with CN and did not increase RXRE-dependent transcription. Expression of a DA mutant of PKC or treatment with PMA was found to attenuate PKC and CN synergism. We conclude that PP1, PP2A, and CN regulate levels and transcriptional activation function of RXR in T cells. In addition, CN synergizes with PKC to induce RXRE-dependent activation, a cooperative function that is antagonized by the activation of the conventional PKC isoform. Thus, PKC and PKC may function as positive and negative modulators, respectively, of CN-regulated RXRE-dependent transcription during T cell activation.

	Introduction

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Activation of T cells through interaction of cognate Ag with the TCR/CD3 complex, cross-linking with anti-CD3 Abs, or treatment with phorbol esters results in a cascade of signaling events. These events lead to IL-2 production and either T cell proliferation or inhibition of proliferation and activation-induced apoptosis (AICD),³ depending on the requirements of the specific signaling outcome (1, 2, 3, 4, 5, 6). Among the major biochemical pathways that are activated during T cell activation are protein kinase C (PKC), a family of Ser/Thr protein kinases that play a crucial role in IL-2 production and control of cellular growth (7, 8). Recent studies have identified a Ca²⁺ independent PKC isoenzyme as a novel PKC isoform that selectively associates with T cell synapse, a supramolecular activation complex that consists of TCR, CD28, LFA-1, other signaling molecules and associates with specialized domains called membrane rafts (9, 10, 11). PKC has been shown to synergize with calcineurin (CN) in activating a number of regulatory elements in the IL-2 promoter and induce IL-2 production (12, 13, 14, 15). In addition, the two enzymes have been shown to cooperate and induce Fas ligand expression during AICD (16).

Retinoid X receptors (RXRs) play a central role as nuclear transcription factors by homodimerization or through heterodimerization with many other members of the retinoid and steroid receptor family of transcription factors (17, 18, 19, 20, 21, 22, 23). The importance of RXRs in T lymphocyte signal transduction is beginning to be understood. We have recently shown that RXR levels are differentially regulated during activation signals that lead to T cell proliferation and signals that result in the inhibition of cellular proliferation and AICD (24, 25). These studies also uncovered the role of mitogen-activated protein kinase (MAPK) pathways in the regulation of RXR-dependent transcriptional activation of the RXR responsive element (RXRE)-containing promoters and identified extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK) pathways as positive and negative transcriptional regulators, respectively (24). From these studies, it became apparent that T lymphocyte activation modulates the expression and transactivation function of RXR through an interplay of complex kinase cascades that are not yet fully understood.

Activation of MAPKs is a transient process. Inactivation of these enzymes by protein phosphatases is highly regulated and has been shown to be conducted by phosphatases like protein phosphatase (PP) 1 and PP2A (26, 27, 28, 29). CN, also known as PP2B, is a Ca²⁺-calmodulin dependent PP which dephosphorylates NFAT for translocation to the nucleus and IL-2 transcription (30, 31). In the present study, we show that cellular Ser/Thr PPs play an important role in the regulation of RXR expression and RXR-dependent transcriptional activation in T lymphocytes. We also found that CN synergizes with PKC in regulating RXRE-dependent transcriptional activation, a cooperative function that is antagonized by activated PKC.

	Materials and Methods

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Cells and treatments

The T lymphocyte leukemia Jurkat cell line (clone E6-1) was obtained from American Type Culture Collection (Manassas, VA). Jurkat cells were maintained in RPMI 1640 medium (BioWhittaker, Frederick, MD) supplemented with 10 mM HEPES buffer, 2 mM L-glutamine, 60 µg/ml gentamicin, and 10% FBS (HyClone Laboratories, Logan, UT). PHA, PMA, okadaic acid (OA), norkadone (NA), and 9-cis retinoic acid (9-CRA) were obtained from Sigma-Aldrich (St. Louis, MO), and were used at 2.5 µg/ml, 50 ng/ml, 100 nM, 100 nM, and 1 µM, respectively. Cyclosporin A (CsA) (Biomol, Plymouth Meeting, PA), rottlerin, cantharidin (CA) (Calbiochem, San Diego, CA), tautomycin (Tau), and endothal thioanhydride (ET) (Alexis Biochemicals, San Diego, CA) were used at 2.5 µg/ml, 25 µM, 50 µM, 500 nM, and 5 µM, respectively.

RNase protection assay and semiquantitative RT-PCR

RNase protection assay and semiquantitative RT-PCR were performed as described previously (25).

Western blot

Protein extracts were electrophoresed in a 10% NuPAGE Bis Tris gel using NuPAGE MOPS-SDS running buffer (NOVEX, San Diego, CA), and transferred to a polyvinylidene difluoride membrane using an XCell Blot module (NOVEX). The membrane was blocked with Blocker Blotto (Pierce, Rockford, IL) and incubated overnight at 4°C with appropriate Ab. The protein was detected using the ECL Western blotting detection system from Amersham Pharmacia Biotech (Piscataway, NJ).

Nuclear run-on transcription assay

Nuclear run-on was performed with Jurkat cells by the procedure described previously (24).

Transfections

Transcriptional activity of RXR was studied by transfection using RXRE-containing luciferase reporter plasmid, TKCRBPII-Luc as described earlier (24). TKCRBPII-M-Luc was generated by cloning a DNA fragment containing mutations in the RXR binding site, in the TK-Luc vector. The plasmid pCMX-hRXR has been described earlier (24). Dominant-active (DA) PKC (A148E), dominant-negative (DN) PKC (K409R), DA PKC (A25E)-expressing plasmids, and the pEF4 HisA empty vector were provided by Dr. A. Altman (La Jolla Institute of Allergy and Immunology, San Diego, CA). The plasmid encoding the hemagglutinin (HA)-tagged DA mutant of calcineurin (HA-CnCaM-AI) was obtained from Dr. A. Altman with permission from Dr. M. Karin (University of California, San Diego, CA). Jurkat cells (10⁷) were transfected by electroporation using a Gene Pulser II (Bio-Rad, Hercules, CA) at 0.250 kV and 975 µF as described (24). After transfection, the cells were incubated in the medium for 24 h. Cells were then incubated with the indicated reagents and time periods before harvest and determination of luciferase activity using the luciferase assay system (Promega, Madison, WI). Transfection efficiency was normalized to protein concentrations in the extracts as described (24).

	Results

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Regulation of RXR expression in T cells by Ser/Thr PPs

In the present study, we have dissected the role of Ser/Thr phosphatases PP1, PP2A, and CN in the expression of RXR in T cells. When Jurkat cells were treated with 100 nM OA, a PP1 and PP2A specific inhibitor, there was a marked loss of the basal RXR mRNA levels within 8 h of treatment (Fig. 1A). Treatment with NA, an inactive analog of OA, did not have any effect on the RXR expression. Inhibition of RXR expression was also observed with PP2A-specific inhibitors CA and ET, as well as PP1-specific inhibitor Tau (Fig. 1B). These results indicate that active PP1 and PP2A are necessary to maintain the basal RXR levels in T cells. We next studied the effect of OA on the activation-induced up-regulation of RXR in these cells. Consistent with our previous findings (24), activation of Jurkat cells with PMA + PHA resulted in induction of RXR expression. However, when the treatment was conducted in the presence of OA, there was nearly complete inhibition of RXR mRNA (Fig. 1A) up-regulation. OA did not show any inhibitory effect on the activation-induced expression of IL-2 mRNA, but instead enhanced its expression (Fig. 1A). We have shown previously that treatment of Jurkat cells with CsA, a specific inhibitor of CN, does not change the basal levels of RXR expression, but enhances activation-induced RXR expression (24). We next studied the effect of CsA on the OA-induced loss of RXR expression during T cell activation by treating the cells with PMA + PHA + CsA for 8 h in the presence or absence of OA. Fig. 1A shows that addition of CsA did not prevent the OA-induced inhibition of RXR up-regulation by PMA + PHA.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. OA induces loss of basal and activation-induced RXR expression in Jurkat cells. A, Jurkat cells were treated as indicated for 8 h. Total RNA was isolated and subjected to RNase protection analysis using RXR and IL-2-specific probes as described (24 ). GAPDH was used as internal control for normalizing the RNA concentrations. For clarity, a shorter x-ray exposure time is shown for GAPDH at the bottom of the figure. RNA concentration was also normalized to the ribosomal RNA content in the samples (data not shown). B, Inhibition of RXR expression with PP2A-specific inhibitors, CA and ET, and PP1-specific inhibitor, Tau. Total RNA was extracted from Jurkat cells treated as indicated for 8 h and RXR RNA was quantitated using semiquantitative RT-PCR. PI, phosphoimager units. C, Nuclear extracts were prepared and 25 µg of protein were subjected to SDS-PAGE. After transfer to a membrane, RXR protein was detected by RXR (D-20) Ab using an ECL Western blotting detection system as described. PC, in vitro translated human RXR protein used as positive control. This experiment is a representative of three independent experiments. D, Nuclear run-on was performed with Jurkat cells as described (24 ). Equivalent amounts of radioactive RNA were hybridized to nylon membranes on which 10 µg of linearized and denatured indicated plasmids were slot-blotted. The membranes were washed and the transcriptional activity was quantitated using a bio-imaging analyzer (Bas 1000; Fuji, Stamford, CT).

To define the transcriptional or posttranscriptional mechanisms involved in the loss of RXR mRNA with OA, we measured the transcription of RXR mRNA 8 h after treatment with OA, using the nuclear run-on transcription assay (Fig. 1D). The levels of RXR mRNA synthesis did not show any significant change after the treatment with OA. However, under similar conditions, the mRNAs of two AP-1 binding proteins, c-jun and c-fos, and RXR mRNA from PMA + PHA-treated cells showed significant increase in transcription. These results indicate that the loss of RXR expression during OA treatment may involve posttranscriptional mechanisms.

Together, these data indicate that PP1 and PP2A inhibitors not only decrease basal RXR expression, but also inhibit RXR levels that are induced during activation of T lymphocytes. Unlike PP1 and PP2A inhibitors, CN inhibitors do not influence basal RXR levels and show an additive effect on the levels of RXR induced during activation. When used together, OA not only inhibits activation-induced RXR induction but also neutralizes CsA action.

Inhibition of PP1 and PP2A attenuates RXRE-dependent transcription independent of RXR levels

To investigate whether PP1 and PP2A inhibition-induced loss of RXR expression reflected in the corresponding loss of RXRE-dependent transcription, transcriptional activity was studied in Jurkat cells by transient transfection assay using the TKCRBP-II-Luc reporter (24). Fig. 2A shows the dose-dependent loss of endogenous RXRE mediated transcription by OA. At 10 nM OA, the concentration which inhibits only PP2A, the inhibition of RXRE-dependent transcription was significant (33%). At 100 nM, the concentration at which both PP2A and PP1 are inhibited, transcription was inhibited by 90%. PP1-specific inhibitor Tau and PP2A specific inhibitors CA and ET also inhibited RXRE-dependent transcription (Fig. 2B). These data are consistent with the dependence of RXRE-mediated transcription on active cellular PP1 and PP2A in Jurkat cells.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Loss of RXRE-dependent transcription by inhibitors of PP1 and PP2A. A, Jurkat cells were transfected with 5 µg TKCRBPII-Luc for 24 h and then treated for 8 h with indicated concentrations of OA. B, Jurkat cells were transfected with 5 µg TKCRBPII-Luc for 24 h and then treated for 8 h with PP2A-specific inhibitors, CA and ET, and PP1 specific inhibitor, Tau. Cells were harvested and luciferase activity was measured as described in Materials and Methods. The values represent the mean of three independent experiments with SE calculated for each value. C, Twenty-five micrograms of nuclear extract, prepared from TKCRBPII-Luc and TKCRBPII-Luc + pCMX-hRXR-cotransfected cells after 8 h of OA treatment was subject to Western blot analysis using RXR-specific Abs as described in Materials and Methods. PC, in vitro-translated human RXR protein used as positive control. D, Jurkat cells were transfected with 5 µg TKCRBP-II-Luc or TKCRBPII-M-Luc either in the presence or absence of 2.5 µg of pCMX-hRXR plasmid. After 24 h, cells were treated for 8 h with OA either in the presence or absence of 9-CRA. Cells were harvested and luciferase activity was measured as described in Materials and Methods. The values represent the mean of three independent experiments with SE calculated for each value.

PKC synergizes with CN to induce RXRE-dependent transcription

We have previously shown that CN may have a role in the regulation of RXR levels during T cell activation (24). However, the role of this phosphatase in RXRE-mediated transcription is unknown. Recent studies have shown that activated CN synergizes with a novel member of PKC isoenzyme PKC and induces IL-2 gene transcription (10). To investigate the contribution of CN in the regulation of RXRE-dependent transcription, Jurkat cells were cotransfected with TKCRBP-II-Luc and DA-CN, either in the presence of wild-type PKC, DA-PKC, DN-PKC, or the empty vector. Fig. 3 shows that expression of CN alone did not influence the RXRE-mediated transcriptional activity. However, if the transfection was performed in the presence of DA-PKC and DA-CN, there was a significant up-regulation of transcriptional activity. This synergistic cooperation was observed with both ligand-dependent and -independent transactivation of both endogenous and exogenously expressed RXR (Fig. 3). Transfection with DA-PKC alone, in the absence of DA-CN, only marginally increased the RXRE-dependent transcription. Wild-type PKC, DN-PKC, or empty vector failed to synergize with DA-CN and did not induce transcription. In subsequent experiments, expression of DN-PKC was found to interfere with the DA-CN and DA-PKC cooperation and resulted in the inhibition of RXRE-dependent transcription (Fig. 4).

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Induction of RXRE-dependent transcription by CN and PKC synergism. Jurkat cells were treated for 12 h with 9-CRA, 24 h after transfection with 5 µg TKCRBP-II-Luc or TKCRBP-II-Luc + 2.5 µg pCMX-RXR plasmids in the presence of 5 µg of indicated plasmid constructs. Cells were harvested and luciferase activity was measured. The values represent the mean of three independent experiments with SE calculated for each value.

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Specificity of CN and PKC cooperation in inducing RXRE-dependent transcription. A, Jurkat cells were treated for 12 h with CsA or rottlerin, 24 h after transfection with 5 µg TKCRBP-II-Luc plasmid in the presence of 5 µg of indicated plasmid constructs. Cells were harvested and luciferase activity was measured. The values represent the mean of three independent experiments with SE calculated for each value. B, Cell extracts from empty vector (lane 1), and DA-PKC + DA-CN (lanes 2–4)-transfected cells were subject to Western blot analysis as indicated using target-specific Abs. Lanes 3 and 4 contained extracts from transfected cells treated with CsA and rottlerin, respectively.

Taken together, these data indicate that CN plays a pivotal role in modulating RXRE-dependent transcription in T cells by cooperating with PKC and this synergism leads to positive regulation of RXRE-dependent transcription. Our data, however, do not rule out the role of other PKC isoforms in CN-induced RXRE-dependent transcription.

Inhibition of CN and PKC synergism by PKC

PMA is known to synergize with ionomycin or DA-CN to induce IL-2 promoter and is also known to increase CN and PKC cooperation-induced NF-B and IL-2 promoter activities (35). To study whether PMA had a similar effect on CN- and PKC-induced RXRE-dependent transcription, Jurkat cells were treated with PMA 24 h after transfection with TKCRBP-II-Luc and DA-CN plasmids, either in the presence of DA-PKC or the empty vector. Fig. 5A shows that PMA, even at a concentration as low as 1 ng/ml, not only inhibited basal levels of RXRE-dependent transcription, but also inhibited transcription induced by CN-PKC cooperation. Treatment with PMA did not have any significant effect on the expression levels of DA-PKC and DA-CN proteins as shown by Western blot analysis of the transfected cell extracts (Fig. 5B). To understand the mechanism of transcriptional inhibition, it was reasoned that PMA might antagonize RXRE-dependent transcription through activation of a conventional PKC isoform, which may have an inhibitory effect on CN-PKC-induced RXRE-dependent transcription. To test this hypothesis, a DA-PKC expressing plasmid was transfected in Jurkat cells together with TKCRBPII-Luc and DA-CN plasmids either in the presence of DA-PKC or empty vector. Results show that expression of DA-PKC significantly inhibited the CN- and PKC-induced RXRE-dependent transcription in a concentration-dependent manner, in addition to its inhibitory effect on basal RXRE-dependent transcription (Fig. 6A). This inhibition was also observed with both ligand-dependent and exogenously expressed RXR (data not shown). Cotransfection with DA-PKC had no significant effect on the expression levels of DA-PKC or DA-CN (Fig. 6B). These data demonstrate that activated PKC has a negative regulatory role in modulating RXRE-dependent transcription induced by PKC and CN synergism. Inhibitory effect of PMA on this synergy may be a result of PKC activation induced by this compound.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Inhibition of CN and PKC synergism-induced RXRE-dependent transcription by PMA. A, Jurkat cells were treated for 12 h with indicated concentrations of PMA, 24 h after transfection with 5 µg TKCRBP-II-Luc plasmid in the presence of 5 µg indicated plasmid constructs. Cells were harvested and luciferase activity was measured. The values represent the mean of three independent experiments with SE calculated for each value. B, Cell extracts from empty vector (lane 1) and DA-PKC + DA-CN (lanes 2–4)-transfected cells were subject to Western blot analysis as indicated using target-specific Abs. Lanes 3 and 4 contained extracts from transfected cells treated with 1 and 20 ng/ml PMA, respectively.

View larger version (30K): [in this window] [in a new window]   FIGURE 6. CN and PKC synergism-induced RXRE-dependent transcription is inhibited by activated PKC. A, Jurkat cells were transfected with 5 µg TKCRBP-II-Luc plasmid in the presence of various concentrations of indicated plasmid constructs. Cells were harvested after 36 h and luciferase activity was measured. The values represent the mean of three independent experiments with SE calculated for each value. B, Cell extracts from empty vector (lane 1), DA-PKC + DA-CN (lane 2), and DA-PKC + DA-CN + DA-PKC-transfected cells were subject to Western blot analysis as indicated using target-specific Abs.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

PP1 and PP2A are known to be essential for cell cycle regulation, and a tight balance between kinase and phosphatase activities controls the decision between cell survival and apoptosis (26, 27, 28). We have previously shown that the levels of RXR are regulated in T cells during cell cycle transitions, proliferation, and also under conditions that lead to the inhibition of proliferation or AICD (24, 25). Because PP1 and PP2A inhibitors attenuate both the basal as well as activation-induced expression of RXR, it is evident that PP1 and PP2A are important physiological regulators of RXR expression during T cell signaling.

We have demonstrated that inhibition of PP1 and PP2A interfered with both ligand-independent and -dependent RXRE-mediated transactivation. This inhibition was observed with both endogenous RXRE-dependent transcription and exogenously expressed RXR-dependent transcription. Remarkably, the decrease in RXRE-mediated transactivation by OA correlated with the loss of RXR levels only in cells, which expressed endogenous RXR. OA did not inhibit CMV-driven RXR expression but was instead found to induce RXR levels in pCMX-RXR-transfected Jurkat cells, perhaps, through its direct induction of CMV promoter. These results suggest that loss of endogenous RXRE-dependent transcription by OA treatment may in part be a result of its inhibitory effect on the accumulation of endogenous RXR protein, and also through its direct or indirect effect on transcriptional activity of RXR protein. OA-induced inhibition of RXRE-dependent transactivation may involve posttranslational modification of RXR and/or a cofactor(s), which are essential in mediating the transactivation.

PPI and PP2A inhibition by OA is known to induce the JNK pathway in Jurkat cells (37). Activation of the JNK pathway was recently shown to inhibit RXRE-dependent transcription in Jurkat (24) and COS-7 cells (38). OA-induced activation of the JNK pathway might contribute to the loss of RXRE-dependent transcription in these cells. PP1 and PP2A have been shown to inhibit PKC activity by inhibiting autophosphorylation (39), suggesting that OA-induced inhibition of PP1 and PP2A may interfere with RXRE-dependent transcription through an increase of PKC activation. This was supported by our data showing that activated PKC inhibited RXRE-dependent transcription (Fig. 6, see below).

CN and PKC have recently been shown to act as essential players in the activation of the IL-2 gene. A synergistic cooperation between the two has been shown to activate a number of IL-2 promoter-associated transcription factors such as NFAT, NF-B, and CD28RE (9, 10, 11, 12, 13, 14, 15). In addition, CN and PKC synergy has also been reported to induce Fas ligand expression in T cells during AICD (16). In this study, we have reported that this novel signaling cross-talk between CN and PKC is also operating to modulate RXR-mediated transcription in Jurkat cells. The activated CN plays an essential role as a positive transcriptional regulator of RXRE-dependent transcription in T cells through its synergy with PKC. This cross-talk between seemingly unrelated signaling cascades, involving CN, PKC, and RXR, represents a novel mechanism T lymphocytes use to modulate RXRE-dependent transcription. Our data, however, do not rule out the role of other PKC isoforms in CN-induced RXRE-dependent transcription.

PMA is known to synergize with DA-CN and induce NFAT and IL-2 promoters in transient transfection assays, and this effect has been attributed to the induction of PKC by PMA treatment (35). In this study, we have shown that PMA does not synergize with DA-CN to induce RXRE-dependent transcription but instead down-regulates basal and CN-PKC synergy-induced transactivation. To explore the molecular mechanism that might explain this inhibitory response to PMA, we hypothesized that PKC, a conventional PKC isoform activated by PMA in addition to the novel PKC isoenzyme, may act as a negative regulator of RXRE-dependent transcription. Our results have revealed that expression of a DA mutant of PKC not only inhibited the basal levels of RXRE-dependent transcription, but also attenuated CN-PKC synergy-induced RXRE-mediated transcription. Together, these data are consistent with the existence, in T cells, of a regulatory mechanism in which PKC and CN cooperate to activate RXRE-mediated transcription, a phenomenon that is negatively regulated by PKC. Whether PMA-induced inhibition of RXRE-dependent transcription is solely due to PKC activation remains to be studied. This study does not rule out the possibility that other PMA-induced PKC isoforms also contribute to this inhibition.

It is apparent that CN and PKC signaling cross-talk, which was initially discovered to be a hallmark of IL-2-promoter-dependent transcriptional activation, may also be involved in many other signaling pathways in T cells. We have provided direct evidence supporting the involvement of CN and PKC cooperation in RXRE-mediated signaling. The functional divergence of PKC and PKC, in regulating RXRE-mediated transcription, emphasizes the importance of multiple PKC isoforms in modulating RXR-dependent signaling in T lymphocytes.

	Acknowledgments

 
We thank Drs. M. Karin and A. Altman for their generous gifts of plasmid constructs. We also thank Allison Hazen for her help in DNA sequencing.

	Footnotes

 
¹ This project has been funded with federal funds from the Department of Health and Human Services under Contract Number NO1-CO-56000. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

² Address correspondence and reprint requests to Dr. Mohammad Ishaq, Laboratory of Molecular Cell Biology, Science Applications International Corporation-Frederick, Frederick Cancer Research and Development Center, National Cancer Institute, Building 550, Room 104, Frederick, MD 21702-1201. E-mail address: mishaq{at}nih.gov

³ Current address: Department of Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Durate, CA 91010.

⁴ Abbreviations used in this paper: AICD, activation-induced cell death; PKC, protein kinase C; CN, calcineurin; RXR, retinoid X receptor; MAPK, mitogen-activated protein kinase; RXRE, RXR responsive element; JNK, c-Jun N-terminal kinase; PP, protein phosphatase; OA, okadaic acid; 9-CRA, 9-cis retinoic acid; CsA, cyclosporin A; CA, cantharidin; Tau, tautomycin; ET, endothal thioanhydride; DA, dominant active; DN, dominant negative; NA, norkadone.

Received for publication June 12, 2001. Accepted for publication May 7, 2002.

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