The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, J.
Right arrow Articles by Arai, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, J.
Right arrow Articles by Arai, N.
The Journal of Immunology, 2001, 167: 2677-2687.
Copyright © 2001 by The American Association of Immunologists

Inhibition of NFATx Activation by an Oligopeptide: Disrupting the Interaction of NFATx with Calcineurin1

Jie Liu*, Ken-ichi Arai{dagger} and Naoko Arai2,*

* Department of Immunology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304; and {dagger} Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Calcium-dependent phosphatase calcineurin (CN) regulates the activation and nuclear translocation of NFAT. We identify here a novel CN-binding motif in one member of the NFAT family, NFATx, and a peptide based on this motif, Pep3. Pep3 binds CN and competes with wild-type NFATx for CN interaction. Amino acid mutations within Pep3 show that multiple amino acid residues are required for the effective functions of Pep3. Ectopic expression of Pep3 in a Th clone via a retrovirus-mediated gene transfer could selectively block the nuclear translocation of endogenous NFATx, whereas it had little effect on the nuclear translocation of another member of the NFAT family, NFATp. Furthermore, in transfection experiments, Pep3 also blocked the nuclear translocation of transfected NFATx, but not NFATp, in the B cell line M12, demonstrating specific inhibition of Pep3 for NFATx. Importantly, several cytokines produced by the T cell clone were severely repressed by ectopic Pep3, and indeed, the production of these cytokines was enhanced by the expression of wild-type NFATx. Our results show selective inhibition of NFATx activation and cytokine expression by Pep3 and suggest a new approach for studying the biology of each NFAT family member. This approach may provide an opportunity for pharmacological targeting of Ca2+-dependent signaling events.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The functional significance of the transcription factor NFAT was realized when it became apparent that the clinically important immunosuppressive agents cyclosporin A (CsA)3 and FK506 blocked NFAT responses by inhibiting a NFAT upstream activator, calcineurin (CN). NFAT was originally identified as a T cell-specific transcription factor that played a critical role in regulating IL-2 gene expression. Subsequently, functional studies revealed that many genes are regulated by NFAT, including IL-4 (1), IL-5 (2), GM-CSF (3, 4, 5), IFN-{gamma} (6), TNF-{alpha} (7), CD40 ligand (8, 9), Fas ligand (10, 11), CD25 (12), and CD5 (13). Moreover, the expression of NFAT has been detected in a variety of immune and nonimmune cells and tissues (see Discussion).

The NFAT family contains several members, includingNFATx/NFAT4/NFATc3, NFATp/NFAT1/NFATc2, NFATc/NFAT2/NFATc1, NFAT3/NFATc4, and the recently identified NFAT5 (14, 15, 16, 17, 18, 19). With the exception of NFAT5, NFAT family members share a certain degree of sequence similarity. The region of homology spans the Rel similarity domain (RSD) and the N-terminal homology domain, whereas the remaining C-terminal domains are more divergent. The RSD is responsible for NFAT DNA-binding and association with its nuclear partners, such as AP1, NF-AT-interacting protein of 45 kDa, c-maf, and GATA2/4 (20, 21, 22, 23). However, the N-terminal homology domain is important for many aspects of NFAT responses to Ca2+/CN signaling: interaction with CN, dephosphorylation, nuclear translocation, DNA-binding activity, and transcriptional activity. It has been demonstrated that NFAT resides in the cytoplasm of resting cells in a Ser/Thr-phosphorylated state. Upon cell activation with calcium ionophore or receptor cross-linking, a sustained increase of intracellular calcium activates phosphatase CN, which in turn dephosphorylates NFAT, allowing it to translocate into the nucleus (24, 25, 26). Once in the nucleus, NFAT can associate with its nuclear partner, target NFAT binding sites within the promoter/enhancer of NFAT-responsive genes, and modulate their transcription. In addition to triggering nuclear translocation, dephosphorylation of NFAT by CN also results in higher DNA-binding activity, thus enhancing NFAT transcriptional activity (24, 26). Because CN controls NFAT activities from different aspects via its phosphatase activity, the interaction between NFAT and CN has been intensively studied. We have previously identified two independent CN-binding regions, designated CN-binding region (CNBR)1 and CNBR2, located in the N-terminal domain of murine NFATx (mNFATx) (27). Although the interaction between CN and mNFATx can be mediated by either binding site, CNBR2 displayed a much higher CN-binding activity than that of CNBR1, and the nuclear translocation of mNFATx was disrupted only by deletion of CNBR2 (27). These results suggest that CNBR2 was the major CN target domain in mNFATx. Through interacting with CN, CNBR2 may recruit CN to mNFATx and initiate CN-mediated signal transduction to mNFATx. A CN-binding motif, termed CM2, was also mapped to CNBR1 in NFATp (28, 29).

Sequence similarities among NFAT family members may reflect some functional similarities. An in vitro DNA-binding assay and the data obtained from a reporter gene assay conducted in transfection systems both suggest that NFAT family members have overlapping DNA-binding and transcriptional activities. However, the phenotypes of mice harboring NFAT genetic mutations have demonstrated unique functions for each of these family members. Mice lacking NFATc in the lymphoid system, as evaluated by recombination-activating gene-2-/- blastocyst complementation, displayed hypoproliferation and selective impairment of IL-4 production (30, 31). Conversely, mice lacking NFATp or both NFATp and NFATx showed an increase in Th2 cytokine production (32, 33). It is likely that the opposing activities of NFATc and NFATp/NFATx activities achieve balanced cytokine production in the immune system. In addition to functioning in the immune system, individual NFAT is also differentially involved in developmental and physiological events outside the immune system. Lack of NFATc results in defective cardiac valve formation in gene-targeting mice (34, 35); NFAT3 was involved in cardiac hypertrophy (23), and NFATp appears to participate in chondrogensis (36). All of these studies indicate that individual NFAT isoforms may control the expression of distinct subsets of genes, and that they cannot functionally replace each other. Therefore, our knowledge of NFAT has grown rapidly as we have recognized the differential involvement of NFAT members in biological processes. However, many aspects of NFAT biology remain to be addressed, including the downstream targets of NFAT and the mechanisms associated with differential function(s) of each NFAT protein in diverse cell types.

To investigate these areas, one of the most promising approaches may be the use of NFAT isoform-specific inhibitors. Although CM2 peptide was shown to be able to inhibit NFATp activation, it also blocked other NFATs, including NFATc and NFATx, CN interaction and dephosphorylation (28, 29). These results suggest that CM2 is a common CN-binding motif found in most NFAT family members and might interfere with the functions of several, if not all, NFAT family members. Characterization of CNBR2 suggests that the CNBR2-CN interaction may be a potential target for the development of an isoform-specific NFAT inhibitor. In fact, expression of CNBR2 inhibited mNFATx-mediated gene transcriptional activity, possibly ascribable to its competition with mNFATx1 for CN binding (27). Therefore, an isoform-specific NFAT inhibitor can be developed based on investigating the interaction of individual NFAT with CN and then disrupting such an interaction.

Here we identify a 16-aa oligopeptide, Pep3, derived from CNBR2 of mNFATx, that blocked the interaction of NFATx CNBR2 with CN. When expressed in a T cell clone via a retrovirus-mediated gene transfer, Pep3 specifically blocked the nuclear translocation of NFATx, thus preventing NFATx from DNA binding. NFATp activation, in contrast, was unaffected. In addition to inhibiting the activation of endogenous NFATx, Pep3 also blocked the nuclear translocation of transfected NFATx, but not NFATp, in the murine B cell line M12, indicating that Pep3 has the specificity to block NFATx activation. Importantly, the expression of Pep3 impaired cytokine production in the T cell clone. Our studies provide the first evidence of the discovery of an inhibitor of an NFAT isoform. This specific effect of this peptide should provide an effective approach to the study of NFAT biology. It will also be of considerable interest to apply our studies to developing drugs with perhaps fewer side effects than CsA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of retroviral vectors, infection of retrovirus into T cell clones, and cell lines

pMX-IRES-EGFP was constructed as described previously (54). For making the retroviral vector encoding Pep3, cDNA covering the region encoding Pep3 was isolated from pME-mNFATx1 (15) and inserted into pCMV-Tag2A (Stratagene, La Jolla, CA). Then, the Flag-tagged Pep3 was isolated and ligated into pMX-IRES-EGFP to generate pMX-IRES-EGFP-Flag-Pep3. The cDNA-encoding Flag only was also ligated into pMX-IRES-EGFP to generate pMX-IRES-EGFP-Flag.

Retrovirus infections into D10 and M12 cells were conducted as described previously (54, 55). Green fluorescent protein (GFP)-positive cells were sorted and were >90% pure by reanalysis. The sorted cells were activated every 2 wk after Ag stimulation and cultured for further assay. Transfection into M12 cells was performed by electroporation (280V, 975 µF) with 2 x 106 cells.

T cell clone D10 and M12.4.1 cells, as well as the retrovirus packaging cell line of Phoenix-Eco provided by Dr. G. Nolan (Stanford University, Stanford, CA), were cultured as previously described (54, 56, 57, 58).

In vitro CN-binding assay, in vitro CN-competition assay, and peptide syntheses

The in vitro CN-binding assay was done as previously described (27). For the in vitro CN-competition assay, different amounts of the synthesized peptides were preincubated with purified CN (Sigma-Aldrich, St. Louis, MO) as indicated for 1 h at 4°C. Then, the glutathione-Sepharose 4B-bound GST-CNBR2 fusion protein was added, and the incubation was continued for 1 h in buffer containing 150 mM NaCl, 50 mM HEPES buffer, 10 µM CaCl2, 0.25% Nonidet P-40, 1 µg/ml of leupeptin, 1 µg/ml of aprotinin, 10 mM NaF, 1 mM NaV3O4, and 10 mM sodium pyrophosphate. After washing the beads, the glutathione-Sepharose 4B-bound fraction was eluted by boiling in Laemmli’s sample buffer and analyzed by SDS-PAGE followed by Western blots using anti-GST (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-A subunit of CN (CNA) Abs (Sigma-Aldrich).

All the peptides were synthesized by Research Genetics (Huntsville, AL).

Immunoprecipitation and dot blotting

For immunoprecipitation, total cell lysates from pMX-IRES-EGFP-Flag-Pep3- or pMX-IRES-EGFP-Flag-infected D10 cells were incubated in vitro with purified CN. After 2 h incubation at 4°C, 20 µl agarose-conjugated anti-Flag Ab (Kodak, Rochester, NY) was added to each reaction. The immunocomplexes were eluted by boiling the beads in reducing SDS sample buffer and detected by Western blot using anti-CNA Ab. Purified CN was subjected to the SDS-PAGE directly as a control.

For the detection of the Flag epitope tag in D10 cells infected with pMX-IRES-EGFP-Flag or pMX-IRES-EGFP-Flag-Pep3, dot blotting was performed by dotting different amounts of the total lysates directly on to nitrocellulose membrane (Amersham, Arlington Heights, IL). After air-drying, the Flag epitope was detected by Western blot using an anti-Flag Ab (Stratagene). The anti-Flag Ab was also dotted on the same membrane as the positive control.

Nuclear extract isolation and EMSA

Nuclear extracts from D10, pMX-IRES-EGFP-Flag-Pep3-, or pMX-IRES-EGFP-Flag-infected D10 cells were prepared as previously described (15) and assayed for NFAT or NF-{kappa}B DNA-binding in EMSA using 32P-labeled oligonucleotide corresponding to the distal NFAT-binding site or NF-{kappa}B site of the mIL-2 promoter as the probe. Excess amounts (100-fold) of unlabeled distal NFAT oligonucleotides or unlabeled AP1 oligonucleotides were used as the competitors as indicated. For the supershift assay, 1 µl anti-NFATx, anti-NFATp, or anti-mIgG was added to the EMSA reaction before the addition of the probe. After a 15-min incubation, the probe was added for further EMSA reaction.

Immunoassay for cytokine production

D10 cells at day 14 after Ag stimulation were either left unstimulated or stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) (P/I). The cells were harvested after 48 h of stimulation, and cytokine production was detected by ELISA (55).

Immunostaining assay and confocal microscopy

Immunofluorescence staining was performed as described previously (15). An affinity-purified polyclonal Ab, AP{alpha}DS, raised against a bacterially produced recombinant peptide of human NFATx extending from amino acid residues 387–728 was used to detect NFATx. An anti-NFATp mAb (Santa Cruz Biotechnology) was used for detecting NFATp. The secondary Abs used were tetramethylrhodamine isothiocyanate-conjugated Abs (Zymed Laboratories, San Francisco, CA). The stainings were examined under a Leica TCS SP laser scanning confocal imaging system (Leica, Deerfield, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pep3 present in the CNBR2 of mNFATx blocked CNBR2-CN interaction in vitro

CNBR2 has been mapped to the region between the SP boxes and the RSD in the N-terminal domain of mNFATx (Fig. 1GoA). It displayed higher CN-binding affinity than CNBR1 found at the N terminus of mNFATx (27). To map more precisely the region of CNBR2 that is responsible for direct CN binding, we synthesized a series of the overlapping peptides derived from CNBR2 (Fig. 1GoA) and tested whether these peptides could disrupt the interaction between CNBR2 and CN. The peptides were added as competitors to an in vitro GST-CNBR2/CN-binding assay. As previously demonstrated, NFATx CNBR2 efficiently bound purified CN as shown by anti-CN immunoblotting of the GST pull-down (Fig. 1GoB, upper panel, lane 1). The interaction between GST-CNBR2 and CN was not affected by the addition of the CNBR2-derived peptides Pep1, Pep2, or Pep4 (lanes 2, 3, and 5). However, in the presence of Pep3, the binding of GST-CNBR2 to CN was greatly diminished (lane 4). Western blotting with an anti-GST Ab revealed equivalent amounts of GST-CNBR2 fusion proteins used in each reaction (Fig. 1GoB, lower panel), and a GST control did not interact with CN (data not shown). This result suggests that Pep3 can compete with GST-CNBR2 fusion protein for CN interaction.



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 1. Mapping of CN-binding motif in CNBR2 of mNFATx. A, Amino acid sequences of CNBR2 and synthesized peptides with schematic representation of the N-terminal domain of NFATx. The function of each motif shown is as discussed in the introduction (27 28 ). B, Pep3 competed with GST-CNBR2 fusion protein for CN binding. An equal amount of synthesized peptide of 1, 2, 3, or 4 (represented as Pep1, Pep2, Pep3, and Pep4, respectively) was incubated with purified CN for 1 h at 4°C, followed by adding glutathione-Sepharose 4B-bound GST-CNBR2 to each reaction. After binding and purification, the eluted fraction was run on 10% SDS-PAGE and immunoblotted with anti-CNA Ab (upper panel). The same membrane was reprobed with anti-GST Ab to verify equal amounts of GST-CNBR2 used in each reaction (lower panel). In vitro CN-binding of GST-CNBR2 in the absence of a peptide was for a control (lane 1). C, The in vitro CN-competition assay was done under the same conditions as described for B, using increasing amounts of Pep3.

 
To assess whether the inhibition by Pep3 was specific, we performed an in vitro CN-binding assay in the presence of increasing concentrations of Pep3. As shown in Fig. 1GoC, Pep3 blocked GST-CNBR2 fusion protein binding to CN in a dose-dependent manner. This inhibition was specific, because other CNBR2-derived peptides, Pep1, 2, and 4, did not affect GST-CNBR2 fusion protein association with CN (data not shown). Pep3 was further narrowed down, and the CN contact core sequence was mapped to 8 aa within the middle portion of the Pep3 (J. Liu, unpublished observation). Overall, these studies provide evidence that Pep3, a 16-aa oligopeptide, is the CN-binding motif present in CNBR2 and is able to specifically block the binding of CNBR2 to CN.

Multiple amino acid residues are required for the effective function of Pep3

We next addressed which residue(s) within Pep3 are involved in direct binding to CN. Amino acid substitutions were introduced within the 8-aa CN contact core sequence that spans the middle portion of Pep3 (Fig. 2GoA). As expected, wild-type Pep3 disrupted GST-CNBR2 binding to CN (Fig. 2GoB, lane 2). Several single amino acid substitutions in the Pep3 sequence (D->A, F->A, L->A, and V->A) resulted in a partial loss of binding activity compared with wild-type Pep3 (compare lane 2 with lanes 3, 5, 6, and 8). When all four of these amino acids were replaced with alanine residues (represented as QFLV->AAAA mutant), Pep3 lost most of its inhibitory function (lane 9). Therefore, it is unlikely that Pep3 contacted CN through a single amino acid; it is more likely that several amino acid residues were involved. Alternatively, some of the amino acids may be required for folding an appropriate surface recognizable by CN. Mutations of these amino acids may disrupt the structure or lead to a conformational change, resulting in diminished CN-binding affinity of Pep3.



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 2. Effect of Pep3 mutations on CN binding. A, Sequences for wild-type Pep3 and each Pep3 mutant. Single amino acid mutation was done by replacing Asp, Gln, Phe, Leu, Ser, or Val with Ala, respectively (represented as D, Q, F, L, S, and V mutants, respectively). The QFLV mutant had all four amino acids replaced by alanine residues. B, The in vitro CN-competition assay was performed under the same conditions as described for Fig. 1Go, using wild-type and mutated Pep3 as the competitors.

 
Pep3 functions as a CN-binding motif

The direct association of Pep3 with CN was further analyzed in vivo. To stably express Pep3 in cells, we used a retrovirus infection system encoding Flag-tagged Pep3 (Flag-Pep3) or Flag only (mock) to infect the murine Th2 cell clone D10. Cell lysates from D10 cells expressing Flag-Pep3 or Flag alone were mixed with purified CN, immunoprecipated with an anti-Flag Ab, and immunoblotted using an anti-CN Ab. We observed CN binding specifically mediated by Pep3, because no CN was coprecipitated with the cell extracts isolated from D10 cells expressing Flag alone (Fig. 3GoA, lanes 2 and 3). The expression levels of Flag and Flag-Pep3 were monitored by dot blot using different amounts of the cell lysates from Flag-Pep3 or Flag-expressing D10 cells. The result showed approximately equal expression levels of both Flag and Flag-Pep3 (Fig. 3GoB). No Flag was detected in lysates from control vector (pMX-IRES-EGFP)-infected D10 cells under all indicated concentration conditions (Fig. 3GoB, top row). These data confirmed that Flag and Flag-Pep3 were expressed at comparable levels. Parallel experiments were also performed in another cell line, Phoenix-Eco. Likewise, we observed Flag-Pep3-CN binding in lysates from Phoenix-Eco cells (data not shown). The data, taken together, strongly support the idea that Pep3 is an important CN-binding motif that can bind CN both in vitro and when expressed in cells.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 3. Interaction between Pep3 and CN in D10 cell extracts. A, Cell extracts (500 µl) from D10 cells infected with a retrovirus encoding Flag epitope-tagged Pep3 or Flag epitope alone were incubated with 5 µl of purified CN (5 mg/ml; Sigma-Aldrich), followed by immunoprecipitation with anti-Flag Ab. Complexes were run on 10% SDS-PAGE and immunoblotted using anti-CNA Ab (lanes 2 and 3). The assay for complex formation was performed under the condition in which an excess amount of CN (5 µl of 5 mg/ml CN) and a limited amount of the expressed Pep3 protein (500 µl infected extracts) were added. Five microliters purified CN protein (5 mg/ml) was directly subjected to SDS-PAGE as an input control for Western blot using an anti-CN Ab (lane 1). B, Dot blot was performed as described in Materials and Methods.

 
Expression of Pep3 inhibited cytokine production in Th clones

Having demonstrated a direct interaction between Flag-Pep3 and CN, we were interested to see the functional consequences of Flag-Pep3 expression on cytokine production in murine Th clones. Therefore, RV-Flag-Pep3-infected D10 cells, which were >90% positive for GFP expression by FACS (data not shown), were evaluated for the effect of Flag-Pep3 on their cytokine profile. As shown in Fig. 4GoA, D10 cells produced large amounts of IL-5, IL-6, and IL-13 when stimulated with P/I. However, D10 cells expressing Flag-Pep3 showed markedly impaired production of IL-5, IL-6, and IL-13 compared with the parental D10 cells (Fig. 4GoA). This effect was specific for Pep3, based on the evidence that D10 cells expressing Flag alone displayed a cytokine expression pattern similar to that of the parental D10 cells. Furthermore, we have also constructed a retrovirus expressing Flag-tagged Pep2, in which Pep2 did not have the ability to interact with CN (Fig. 1Go), as a control. As expected, Pep2 had no effects on cytokine production when expressed in the cells (data not shown). However, the parental D10 cells we used failed to produce IL-4. We also tried other D10 cells obtained from American Type Culture Collection (Manassas, VA). Our preliminary data showed that Pep3 had less inhibitory effect on IL-4 production as compared with its effect on IL-5 and IL-6 production. The effects of Pep3 on cytokine expression in D10 cells were also confirmed with intracellular cytokine staining by flow cytometry, giving results similar to those obtained by ELISA (data not shown).



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 4. Effect of Pep3 expression on Th cytokine production. A, Parental D10 cells or D10 cells expressing Flag-Pep3 or Flag alone were either unstimulated or stimulated with P/I for 48 h. IL-5, IL-6, and IL-13 were measured by ELISA. B, Experimental conditions were the same as described for A, except that D10 cells were infected by retrovirus encoding wild-type mNFATx. The data are representative of three independent experiments.

 
We also infected a retrovirus encoding the wild-type mNFATx in D10 cells, showing that production of IL-5, IL-6, and IL-13 was enhanced by mNFATx (Fig. 4GoB). These data suggest that Pep3 may inhibit cytokine production through its ability to block mNFATx activation.

Expression of Pep3 selectively prevented the activation of endogenous NFATx

Because Pep3 blocked the interaction between CN and CNBR2 of NFATx (Fig. 1Go) and suppressed D10-produced cytokines (Fig. 4GoA), we then tried to determine the mechanism(s) associated with Pep3 inhibition. We first analyzed the subcellular localization of endogenous NFATx in D10 and in Flag-Pep3-expressed D10 cells by confocal microscopy. We used an anti-NFATx Ab that revealed that the subcellular localization of endogenous NFATx in D10 cells was similar to that in other types of cell lines, being sequestered in the cytoplasm of untreated D10 cells and entering the nucleus in response to stimulation with calcium ionophore (Fig. 5GoA, a and b). However, in D10 cells expressing Flag-Pep3, we found that NFATx could no longer translocate into the nucleus in response to calcium ionophore stimulation, suggesting that expression of Flag-Pep3 abolished the nuclear translocation of endogenous NFATx (Fig. 5GoB, a and b). This observation drove us to test the cellular localization of other possible NFAT family members expressed in D10 cells. Endogenous NFATp was detected in the cytoplasm of unstimulated D10 cells by an anti-NFATp Ab, and the addition of calcium ionophore to the cells triggered NFATp nuclear translocation (Fig. 5GoA, c and d). Notably, the same cellular distribution pattern of endogenous NFATp was also seen in D10 cells expressing Flag-Pep3, indicating that the nuclear translocation of endogenous NFATp was not affected by the presence of Pep3 (Fig. 5GoB, c and d).



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 5. Inhibition of endogenous NFATx nuclear translocation by expression of Pep3 in D10 cells. Cellular localization patterns of endogenous NFATx (a and c) and NFATp (b and d), either in parental D10 cells (A) or in D10 cells expressing Flag-Pep3 (B), were detected using anti-NFATx and anti-NFATp Abs, respectively. Cells were either unstimulated (a and b) or stimulated with 0.5 µM A23187 (c and d) for 1 h.

 
To further confirm that NFATx nuclear translocation was affected by Pep3, we assessed NFAT DNA-binding activity by EMSA using the NFAT binding site as a probe with nuclear extracts from parental, mock-, and Flag-Pep3-expressed D10 cells stimulated with PMA (50 ng/ml) and calcium ionophore/A23187 (0.5 µM) (P/A). In nuclear extracts from parental D10 cells, an inducible complex comprised of NFAT proteins was detected (Fig. 6Go, lane 1). The formation of the complex was specifically competed by the addition of excess amounts of either unlabeled NFAT oligonucleotides or unlabeled AP1 oligonucleotides (Fig. 6Go, lanes 2 and 3), suggesting that the complex formed in the activated D10 cells was represented, at least, by NFAT and AP1. Nuclear extracts from mock-infected D10 cells likewise exhibited similar NFAT DNA-binding activity after treatment with P/A. Although this NFAT complex was detected in nuclear extracts from P/A-stimulated D10 cells expressing Flag-Pep3, the intensity of the complex was much weaker than that observed in either D10 or D10 cells expressing Flag (compare lane 5 with lanes 1 and 4 of Fig. 6Go). Considering that expression of Flag-Pep3 abolished the nuclear translocation of NFATx, but not NFATp, it seems likely that in D10 cells expressing Flag-Pep3, the impaired nuclear translocation of NFATx may account for the reduced NFAT binding activity, whereas the remaining NFAT binding activity was contributed to, if not caused entirely, by the nuclear translocated NFATp upon cell activation.



View larger version (76K):
[in this window]
[in a new window]
  		FIGURE 6. Effect of expression of Pep3 on DNA-binding activity of NFAT. A, EMSA was conducted using nuclear extracts prepared from P/A-stimulated D10 cells (lane 1), D10 cells expressing Flag (lane 4), and D10 cells expressing Flag-Pep3 (lane 5), following treatment of the cells with P/A for 6 h. Competition experiments were done in the presence of 100-fold excess amounts of unlabeled oligonucleotides corresponding to the mouse IL-2 distal NFAT binding site (lane 2) and the AP1 site (lane 3), respectively. Supershift experiments were conducted as described in Materials and Methods by using Abs to NFATx (lane 7), NFATp (lane 8), or to IgG (lane 6) as a control. NF-{kappa}B DNA binding activity was also measured in P/A-stimulated D10 cells expressing Flag (lane 9) and Flag-Pep3 (lane 10).

 
To dissect the nature of the NFAT complex formed in P/A-stimulated D10 cells expressing Pep3, we performed supershifts of the complex using Abs specific for NFATp and NFATx. We found that the NFAT complex formed in P/A-stimulated D10 cells expressing Pep3 contained NFATp but not NFATx, evidence that the band was shifted by the anti-NFATp, but not by the anti-NFATx Ab (compare lanes 5 and 6 with lanes 7 and 8 of Fig. 6Go). All the data above suggest that Pep3, acting as a competitor of NFATx for CN interaction, inhibited the nuclear translocation of NFATx,and in so doing, prevented NFATx from binding DNA, which subsequently led to impaired NFATx-dependent cytokine gene expression.

NF-{kappa}B has been suggested as a downstream target of CN (37). We measured NF-{kappa}B DNA-binding activity in Pep3-expressed D10 cells after stimulation by P/A that was indistinguishable from that in parental D10 cells and D10 cells expressing Flag (data not shown and Fig. 6Go, lanes 9 and 10).

Selective inhibition of Pep3 for NFATx

To further confirm that Pep3 inhibits NFATx activity in other cell types and to see the selective effect of Pep3 on NFAT family members more clearly, in vitro coexpression experiments were performed. We first transfected a murine B cell line, M12, with an expression plasmid encoding NFATx or NFATp, and we examined their subcellular localization states (Fig. 7GoA). Transfected NFATx, or NFATp translocated from the cytoplasm to the nucleus of the cell following treatment with calcium ionophore, exhibited behavior similar to that observed in other types of cells. We then infected M12 cells with RV-Flag-Pep3. Stable clones were obtained by cell sorting and were transfected with an expression plasmid encoding NFATx or NFATp. The subcellular localization of NFATx was examined at the single-cell level, in which both Pep3 and NFATx were expressed (Fig. 7GoB). By confocal microscopy, cells infected with Pep3 retrovirus were detected by GFP expression (Fig. 7GoB, b and e, shown in green), and cells expressing NFATx were detected by an anti-NFATx Ab (Fig. 7GoB, a and d, shown in red). In M12 cell expressing Pep3, transfected NFATx was localized in the cytoplasm of unstimulated cells; however, it remained in the cytoplasm after stimulation with A23187. In contrast, NFATp nuclear translocation was clearly seen in cells expressing ectopic Pep3 following treatment of the cells with calcium ionophore (Fig. 7GoC), confirming that the presence of Pep3 had little effect on NFATp activation. These results confirm the idea that Pep3 is a potent and specific inhibitor for NFATx.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 7. Effect of Pep3 on the nuclear translocation of transfected NFAT. A, M12 cells were transfected with the expression vector for either NFATx or NFATp, and the localization of NFATx or NFATp was measured in the cells either before stimulation or after stimulation with A23187. M12 cells stably expressing Flag-Pep3 were used for transfection as described for A. The trasfected NFATx (B) or NFATp (C) were detected in the cells either left unstimulated (a–c) or stimulated with A23187 for 1 h (d–f) using anti-NFATx and anti-NFATp Abs, respectively. GFP expression (b and e, shown in green) and the signal detected by using an Ab specific for NFAT (a and d, shown in red) in the same cell confirmed the colocalization of ectopically expressed Pep3 with exogenous NFAT. Overlay panels show the merged image between GFP and NFAT (c and f).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The importance of NFAT in the immune system has been established. Recent work has further documented the importance of each NFAT member in a wide variety of cells and tissues beyond the immune system, including, for example, various cells of the nervous system (38, 39) and muscle cells (20, 40, 41, 42). All the studies suggest that whereas NFAT members share some similarities, they participate differently in certain physiological events. The generation of mice deficient in expression of individual NFAT family members has provided insight into member-specific functions; however, some of the phenotypes of these mice conflicted with the in vitro data (16, 17, 18). The discrepancies may result from functional redundancy or compensatory changes in the knockout mice during development. We present data here to show that a different approach to studying individual NFAT is using a specific inhibitor for each NFAT. We identified a specific, short amino acid region within mNFATx that is important for the interaction of CN. We also showed that a peptide of Pep3 derived from this region can selectively block the activation of NFATx.

Pep3 was originally derived from CNBR2, a major CN-binding region for mNFATx. Pep3 worked as a CN-binding motif both in vitro and in vivo, acting as a competitor to specifically block GST-CNBR2-CN interaction by binding directly to CN. Several lines of evidence suggest that Pep3 uniquely inhibited NFATx activation. Endogenous NFATx, which underwent nuclear translocation in a Th cell clone, D10, upon calcium ionophore stimulation, failed to complete this process under the same stimulation conditions in D10 cells that had been stably infected with retrovirus containing Pep3. However, the subcellular distribution of endogenous NFATp was not affected whether in the presence or absence of Pep3 in the cells. Inhibition of NFATx nuclear translocation resulted in reduced NFAT DNA-binding activity in D10 cells expressing Pep3. We have further dissected the nature of the NFAT complex formed in P/A-stimulated D10 cells expressing Pep3, and we found that this complex contained NFATp but not NFATx, as evidenced by supershift experiments on the complex using Abs specific for NFATp and NFATx (Fig. 6Go). Consistent with the immunostaining data described above, it was the nuclear-translocated NFATp that contributed, possibly in part, to NFAT DNA-binding activity remaining in Pep3-expressed D10 cells. Also, nuclear translocation of transfected NFATx, but not NFATp, was inhibited by Pep3 in the murine B cell line M12. Importantly, the expression of Pep3 impaired cytokine production in D10 cells, and, indeed, these cytokines were up-regulated by expression of wild-type mNFATx in the cells. The expression of Pep3 had no effect on other CN targets such as NF-{kappa}B. Taking these results together, the mechanism of action of Pep3 is unlikely to be mediated by inhibition of CN activity, because Pep3 had little effect on other NFAT members or other CN downstream targets. Instead, Pep3 prevented CN from recognizing NFATx as a substrate without altering the ability of CN to activate other substrates. Additional experiments, such as overexpression of CN, should be able to oppose the inhibitory effect mediated by Pep3.

We have examined from a different aspect whether the CN-binding motif corresponding to Pep3 is present in other proteins. None of the molecules outside the NFAT family, including other CN targets, showed sequence homology to that of Pep3. Furthermore, alignment of the sequences of all the NFAT family members showed only partial primary sequence conservation in the Pep3 region (Fig. 8Go). Based on the sequence information, we synthesized peptide derived from NFATp in the region corresponding to Pep3 and used it for the in vitro CN-competition assay. We found that this putative NFATp Pep3 was unable to disrupt the interaction between GST-CNBR2 fusion protein of mNFATx and CN, suggesting that this region of NFATp may not have CN-binding activity. Yet, it is possible that the sequence derived from NFATp for encoding the peptide corresponding to Pep3 shifted elsewhere. If this motif exists, then the question is whether it has characteristics similar to those of Pep3 of NFATx. Although we were unable to detect the influence of Pep3 on the direct interaction of NFATp with CN, from all the data on biological effects of Pep3 discussed above, NFATp activation was not affected by Pep3. Alternatively, until now, no additional CN-binding motif other than CM2 has been claimed to exist in NFATp. Despite the fact that NFATp displayed less sequence identity to that of NFATx within either the Pep3 region or the CN target core sequence, which we have mapped to 8 aa in the middle portion of Pep3, as we noted, the CN target core sequence is a hydrophobic amino-acid-rich sequence in all NFAT members. The peptide encoding this 8-aa CN target core sequence derived from Pep3 disrupted GST-CNBR2-CN interaction, whereas this short version of Pep3 was less effective than the wild-type Pep3 (J. Liu, unpublished observation, and Fig. 8Go). This suggests that the residues outside the CN target core sequence may also be involved, probably for assisting an appropriate conformation formation for Pep3. This possibility correlates with our amino acid substitution data, indicating that it was not a single amino acid, but multiple residues, participating in the function of Pep3. Park et al. (43) have reported identifying a CN-binding site in NFATc that was located in the region corresponding to Pep3 in NFATc. When the peptide encoding this binding site was used to compete NFATc for CN interaction, it inhibited such interaction. However, it was less effective in inhibiting NFATx, NFAT3, or NFATp interaction with CN (43). Similarly, we have also synthesized peptides derived from the sequence corresponding to Pep3 in NFATc and performed CN-competition assays using this peptide. This peptide showed a weak inhibition on CNBR2 of NFATx interaction with CN, as compared with that of Pep3 (J. Liu, unpublished data). This suggests that the CN-binding sites mapped by our group and by Park et al. (43) in NFATx and NFATc, respectively, have different CN-binding strengths.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 8. Sequence comparison in the corresponding region of Pep3 among different NFAT family members. The CN target core sequence is boxed.

 
What is the model for NFATx and CN interaction? The findings suggest that CN may also provide two possibly different sites or areas available for two independent CNBRs of NFATx. As for NFATx, our previous in vitro data show that mNFATx bound CN via either CNBR1 or CNBR2, and deletion of either binding site individually did not impair mNFATx association with CN (27). Furthermore, the alignment of the CN-binding motifs in CNBR1 and CNBR2 (compare the sequence of CM2 with the sequence of Pep3) did not imply a common or similar motif or feature shared by CM2 and Pep3. These data suggest that CNBR1 and CNBR2 may not compete with each other for binding the same site on CN. As for CN, it is a heterodimer composed of CNA and a B subunit (CNB) (44). We found that both GST-CNBR1 and GST-CNBR2 fusion proteins bound CNA. However, interestingly, only the interaction between GST-CNBR2 and CNA was greatly enhanced by the addition of CNB (J. Liu, unpublished observations). It seemed that CNBR1 and CNBR2 associated with CN via different sites or surfaces present in CN. CNBR1 binds a site in CNA, and CNBR2 may contact a surface area probably composed of both CNA and CNB. An alternative example came from the studies of the interaction between the immunosuppressive drug FK506 and CN. The complex of FK506 and its intracellular receptor protein, the immunophilin known as FK506-binding protein (FKBP), physically interacts with CN. The crystal structure of the FKBP12-FK506-CN complex has shown that FKBP12-FK506 contacts two distinct areas on CN. At the larger site, the composite surface formed by FK506 and surrounding residues of FKBP12 contacts residues on the exposed side of the CNB-binding helix of CNA and surrounding residues from both lobes of CNB. Another interaction is seen between FKBP12 and the catalytic domain of CNA near one side of the substrate-binding cleft (45, 46). It is still not clear why NFATx harbors two distinct CN binding sites. Nevertheless, it is less likely that two CNBRs of NFATx would be functionally redundant. In support of this idea, we found previously that CNBR2 has much higher CN-binding activity than that of CNBR1, although we have not compared the CN-binding activity between CM2 and Pep3 directly. Moreover, only deletion of CNBR2 from mNFATx abolished the most nuclear translocation event of mNFATx (27). Furthermore, in the presence of ectopically expressed Pep3, the nuclear translocation of NFATx was blocked. In this situation, despite the fact that CNBR2 failed to function due to competition with expressed-Pep3, CNBR1 was assumed to be able to bind CN. So it likely reflects that CNBR2 was the major CN-binding region for NFATx and might function as a docking site for CN, recruiting CN to NFATx, and thus initiating CN-mediated signaling on NFATx.

Disruption of each NFAT gene in mice has been reported, but the results from these deficient mice and in vitro data show some discrepancies. NFATp deficiency has been reported to enhance immune responses. The levels of IL-2, IL-4, TNF-{alpha}, and IFN-{gamma} produced by NFATp-/- T cells in response to anti-CD3 mAb or Con A are similar to those produced by wild-type T cells (47). In contrast, a decreased IL-4 expression was detected after the administration of anti-CD3 mAb in vivo, whereas Th2 cell development and the late phase of IL-4 production in vitro were enhanced (33). A reduction of IL-4 production was seen in NFATp-/- T cells when treated with Con A in vitro (48). In a separate study, early IL-4 gene expression was not affected in NFATp-/- mice; however, the expression of IL-4 was more sustained (49). NFATc gene disruption caused a decrease in IL-4 production, thus impairing Th2 responses (30). Notably, neither NFATp- nor NFATc-deficient mice demonstrated changes in IL-2 gene expression (30, 33). However, in studies using a dominant-negative NFAT mutant, IL-2 promoter activity and IL-2 protein expression level were decreased in cultured T cells (50). Moreover, transgenic mice expressing this dominant-negative mutant also showed impaired IL-2 gene expression, supporting the original idea when NFAT was purified that NFAT is required for IL-2 gene expression (50). In the case of NFATx, although NFATx-knockout mice showed normal cytokine production (51), NFATx has been demonstrated to bind the NFAT binding sites in the promoters of several cytokine genes and to modulate their transciption (15, 16). The roles of the various NFAT family members in regulating cytokine production are puzzling because different results were obtained from different studies using different assay systems. The discrepancy between the results from the knockout mice and the in vitro studies remains unexplained. It might be due to functional redundancy or compensatory changes in the knockout mice during development; it might be due to different stages of development of these cells and/or to the different roles of NFATs in cytokine production in different stages of T cells. Therefore, it is important and useful to develop specific inhibitors for individual NFATs. We have tested the function of mNFATx in our systems by infecting the mD10 clone with a retroviral vector containing wild-type mNFATx (RV-mNFATx). Strikingly, expression of mNFATx protein enhanced the production of all Th2 cytokines examined thus far, IL-5, IL-6, and IL-13, in both unstimulated and P/I-treated cells. This suggests that mNFATx plays an important role in the production of IL-5, IL-6, and IL-13 in D10 cells. In agreement with this observation, D10 cells expressing Pep3 secreted significantly lesser amounts of IL-5, IL-6, and IL-13 in relation to parental or RV-Flag-infected cells. Therefore, although Pep3 had no effect on the nuclear translocation of NFATp, it could still block production of several cytokines. It is possible that expression levels of NFATx, NFATp, and NFATc may vary in different types of cells, leading to different contributions to cytokine production mediated by NFAT family members. However, whether the inhibition of production of these cytokines by Pep3 in D10 cells is due solely to inhibition of NFATx function remains to be elucidated. Coinfection of the retroviral vectors encoding NFATx and Pep3 may provide further insight. We also infected retrovirus encoding Pep3 into developing Th2 cells cultured under Th2 conditions. Unlike the results obtained from D10, a committed Th2 clone, we could not detect the inhibition of cytokine production by Pep3 in developing Th2 cells (J. Liu, unpublished data). It still remains unclear whether the difference between D10 cells and developing Th2 cells is due to different developmental stages of these two cells or to the different roles of NFATx in cytokine production in naive and committed T cells.

In contrast to CM2 and immunosuppressive drugs CsA and FK506, which inhibit all NFAT and CN downstream functions, respectively, Pep3 is a selective inhibitor of NFATx activation. Thus, it may be of considerable importance for developing a highly specific immunosuppressive drug having fewer side effects than CsA and FK506. Accordingly, our studies may also be suited for the development of small molecule inhibitors with strong therapeutic potential. For example, the clinical use of CsA and FK506 for the treatment of cardiac hypertrophy is ineffective. CsA and FK506 can reduce cardiac hypertrophy, but the doses of CsA and FK506 for prevention of hypertrophy are much higher than those required for immunosuppression and produce kidney damage (52, 53, 54). The severe renal toxicity of CsA and FK506 further induces hypertension, which in turn causes cardiac hypertrophy. Because NFAT3 has been reported to be heavily involved in cardiac hypertrophy, it will be worthwhile to develop a specific inhibitor for NFAT3, which would be useful in preventing cardiac hypertrophy. Finally, our assay systems are also applicable for studying other molecular families in the future.


   Acknowledgments
 
We thank Drs. Hirokazu Kurata, Yongke Zhang, Nicholas Cacalano, and Stephen Hurst for critical discussions and technical help and Drs. James Johnston, Yong-Jun Liu, Robert Coffman, Anne O’Garra, and Dovie Wylie for critical review of the manuscript. We thank Dr. James Cupp, Eleni Callas, Jennifer Maskrey, Jesse Vargas, and Tim Brigman for cell sorting, and we thank Maribel Andonian for graphic assistance.


   Footnotes
 
1 DNAX Research Institute is supported by the Schering Plough Corporation. Back

2 Address correspondence and reprint requests to Dr. Naoko Arai, Department of Immunology, DNAX Research Institute of Molecular and Cellular Biology, 901 California Avenue, Palo Alto, CA 94304-1104. E-mail address: arai{at}dnax.org Back

3 Abbreviations used in this paper: CsA, cyclosporin A; CN, calcineurin; RSD, Rel similarity domain; CNBR, CN-binding region; m, murine; GFP, green fluorescent protein; CNA, the A subunit of CN; P/A, PMA (50 ng/ml) and calcium ionophore/A23187 (0.5 µM); CNB, the B subunit of CN; FKBP, FK506-binding protein. Back

Received for publication March 12, 2001. Accepted for publication June 18, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Casolaro, V., S. N. Georas, Z. Song, I. D. Zubkoff, S. A. Abdulkadir, D. Thanos, S. J. Ono. 1995. Inhibition of NF-AT-dependent transcription by NF-{kappa}B: implications for differential gene expression in T helper cell subsets. Proc. Natl. Acad. Sci. USA 92:11623.[Abstract/Free Full Text]
  2. Prieschl, E. E., V. Gouilleux-Gruart, C. Walker, N. E. Harrer, T. Baumruker. 1995. A nuclear factor of activated T cell-like transcription factor in mast cells is involved in IL-5 gene regulation after IgE plus antigen stimulation. J. Immunol. 154:6112.[Abstract]
  3. Cockerill, G. W., A. G. Bert, G. R. Ryan, J. R. Gamble, M. A. Vadas, P. N. Cockerill. 1995. Regulation of granulocyte-macrophage colony-stimulating factor and E-selectin expression in endothelial cells by cyclosporin A and the T-cell transcription factor NFAT. Blood 86:2689.[Abstract/Free Full Text]
  4. Cockerill, P. N., A. G. Bert, F. Jenkins, G. R. Ryan, M. F. Shannon, M. A. Vadas. 1995. Human granulocyte-macrophage colony-stimulating factor enhancer function is associated with cooperative interactions between AP-1 and NFATp/c. Mol. Cell. Biol. 15:2071.[Abstract]
  5. Masuda, E. S., H. Tokumitsu, A. Tsuboi, J. Shlomai, P. Hung, K. Arai, N. Arai. 1993. The granulocyte-macrophage colony-stimulating factor promoter cis-acting element CLE0 mediates induction signals in T cells and is recognized by factors related to AP1 and NFAT. Mol. Cell. Biol. 13:7399.[Abstract/Free Full Text]
  6. Brown, D. A., F. B. Nelson, E. L. Reinherz, D. J. Diamond. 1991. The human interferon-{gamma} gene contains an inducible promoter that can be transactivated by tax I and II. Eur. J. Immunol. 21:1879.[Medline]
  7. McCaffrey, P. G., A. E. Goldfeld, A. Rao. 1994. The role of NFATp in cyclosporin A-sensitive tumor necrosis factor-{alpha} gene transcription. J. Biol. Chem. 269:30445.[Abstract/Free Full Text]
  8. Schubert, L. A., G. King, R. Q. Cron, D. B. Lewis, A. Aruffo, D. Hollenbaugh. 1995. The human gp39 promoter: two distinct nuclear factors of activated T cell protein-binding elements contribute independently to transcriptional activation. J. Biol. Chem. 270:29624.[Abstract/Free Full Text]
  9. Tsytsykova, A. V., E. N. Tsitsikov, R. S. Geha. 1996. The CD40L promoter contains nuclear factor of activated T cells-binding motifs which require AP-1 binding for activation of transcription. J. Biol. Chem. 271:3763.[Abstract/Free Full Text]
  10. Latinis, K. M., L. L. Carr, E. J. Peterson, L. A. Norian, S. L. Eliason, G. A. Koretzky. 1997. Regulation of CD95 (Fas) ligand expression by TCR-mediated signaling events. J. Immunol. 158:4602.[Abstract]
  11. Latinis, K. M., L. A. Norian, S. L. Eliason, G. A. Koretzky. 1997. Two NFAT transcription factor binding sites participate in the regulation of CD95 (Fas) ligand expression in activated human T cells. J. Biol. Chem. 272:31427.[Abstract/Free Full Text]
  12. Schuh, K., T. Twardzik, B. Kneitz, J. Heyer, A. Schimpl, E. Serfling. 1998. The interleukin 2 receptor {alpha} chain/CD25 promoter is a target for nuclear factor of activated T cells. J. Exp. Med. 188:1369.[Abstract/Free Full Text]
  13. Berland, R., H. H. Wortis. 1998. An NFAT-dependent enhancer is necessary for anti-IgM-mediated induction of murine CD5 expression in primary splenic B cells. J. Immunol. 161:277.[Abstract/Free Full Text]
  14. Hoey, T., Y. L. Sun, K. Williamson, X. Xu. 1995. Isolation of two new members of the NF-AT gene family and functional characterization of the NF-AT proteins. Immunity 2:461.[Medline]
  15. Liu, J., N. Koyano-Nakagawa, Y. Amasaki, F. Saito-Ohara, T. Ikeuchi, S.-I. Imai, T. Takano, N. Arai, T. Yokota, K.-I. Arai. 1997. Calcineurin-dependent nuclear translocation of a murine transcription factor NFATx: molecular cloning and functional characterization. Mol. Biol. Cell 8:157.[Abstract]
  16. Masuda, E. S., Y. Naito, H. Tokumitsu, D. Campbell, F. Saito, C. Hannum, K. Arai, N. Arai. 1995. NFATx, a novel member of the nuclear factor of activated T cells family that is expressed predominantly in the thymus. Mol. Cell. Biol. 15:2697.[Abstract]
  17. McCaffrey, P. G., C. Luo, T. K. Kerppola, J. Jain, T. M. Badalian, A. M. Ho, E. Burgeon, W. S. Lane, J. N. Lambert, T. Curran, et al 1993. Isolation of the cyclosporin-sensitive T cell transcription factor NFATp. Science 262:750.[Abstract/Free Full Text]
  18. Northrop, J. P., S. N. Ho, L. Chen, D. J. Thomas, L. A. Timmerman, G. P. Nolan, A. Admon, G. R. Crabtree. 1994. NF-AT components define a family of transcription factors targeted in T-cell activation. Nature 369:497.[Medline]
  19. Lopez-Rodriguez, C., J. Aramburu, A. S. Rakeman, A. Rao. 1999. NFAT5, a constitutively nuclear NFAT protein that does not cooperate with Fos and Jun. Proc. Natl. Acad. Sci. USA 96:7214.[Abstract/Free Full Text]
  20. Musaro, A., K. J. McCullagh, F. J. Naya, E. N. Olson, N. Rosenthal. 1999. IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1. Nature 400:581.[Medline]
  21. Jain, J., Z. Miner, A. Rao. 1993. Analysis of the preexisting and nuclear forms of nuclear factor of activated T cells. J. Immunol. 151:837.[Abstract]
  22. Hodge, M. R., H. J. Chun, J. Rengarajan, A. Alt, R. Lieberson, L. H. Glimcher. 1996. NFAT-driven interleukin-4 transcription potentiated by NIP45. Science 274:1903.[Abstract/Free Full Text]
  23. Izumo, S., H. Aoki. 1998. Calcineurin–the missing link in cardiac hypertrophy. Nat. Med. 4:661.[Medline]
  24. Shaw, K. T., A. M. Ho, A. Raghavan, J. Kim, J. Jain, J. Park, S. Sharma, A. Rao, P. G. Hogan. 1995. Immunosuppressive drugs prevent a rapid dephosphorylation of transcription factor NFAT1 in stimulated immune cells. Proc. Natl. Acad. Sci. USA 92:11205.[Abstract/Free Full Text]
  25. Loh, C., K. T. Shaw, J. Carew, J. P. Viola, C. Luo, B. A. Perrino, A. Rao. 1996. Calcineurin binds the transcription factor NFAT1 and reversibly regulates its activity. J. Biol. Chem. 271:10884.[Abstract/Free Full Text]
  26. Loh, C., J. A. Carew, J. Kim, P. G. Hogan, A. Rao. 1996. T-cell receptor stimulation elicits an early phase of activation and a later phase of deactivation of the transcription factor NFAT1. Mol. Cell. Biol. 16:3945.[Abstract]
  27. Liu, J., E. S. Masuda, L. Tsuruta, N. Arai, K. Arai. 1999. Two independent calcineurin-binding regions in the N-terminal domain of murine NF-ATx1 recruit calcineurin to murine NF-ATx1. J. Immunol. 162:4755.[Abstract/Free Full Text]
  28. Aramburu, J., F. Garcia-Cozar, A. Raghavan, H. Okamura, A. Rao, P. G. Hogan. 1998. Selective inhibition of NFAT activation by a peptide spanning the calcineurin targeting site of NFAT. Mol. Cell 1:627.[Medline]
  29. Aramburu, J., M. B. Yaffe, C. Lopez-Rodriguez, L. C. Cantley, P. G. Hogan, A. Rao. 1999. Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A. Science 285:2129.[Abstract/Free Full Text]
  30. Ranger, A. M., M. R. Hodge, E. M. Gravallese, M. Oukka, L. Davidson, F. W. Alt, F. C. de la Brousse, T. Hoey, M. Grusby, L. H. Glimcher. 1998. Delayed lymphoid repopulation with defects in IL-4-driven responses produced by inactivation of NF-ATc. Immunity 8:125.[Medline]
  31. Yoshida, H., H. Nishina, H. Takimoto, L. E. Marengere, A. C. Wakeham, D. Bouchard, Y. Y. Kong, T. Ohteki, A. Shahinian, M. Bachmann, et al 1998. The transcription factor NF-ATc1 regulates lymphocyte proliferation and Th2 cytokine production. Immunity 8:115.[Medline]
  32. Ranger, A. M., M. Oukka, J. Rengarajan, L. H. Glimcher. 1998. Inhibitory function of two NFAT family members in lymphoid homeostasis and Th2 development. Immunity 9:627.[Medline]
  33. Hodge, M. R., A. M. Ranger, F. C. de la Brousse, T. Hoey, M. J. Grusby, L. H. Glimcher. 1996. Hyperproliferation and dysregulation of IL-4 expression in NF-ATp-deficient mice. Immunity 4:397.[Medline]
  34. de la Pompa, J. L., L. A. Timmerman, H. Takimoto, H. Yoshida, A. J. Elia, E. Samper, J. Potter, A. Wakeham, L. Marengere, B. L. Langille, G. R. Crabtree, T. W. Mak. 1998. Role of the NF-ATc transcription factor in morphogenesis of cardiac valves and septum. Nature 392:182.[Medline]
  35. Ranger, A. M., M. J. Grusby, M. R. Hodge, E. M. Gravallese, F. C. de la Brousse, T. Hoey, C. Mickanin, H. S. Baldwin, L. H. Glimcher. 1998. The transcription factor NF-ATc is essential for cardiac valve formation. Nature 392:186.[Medline]
  36. Ranger, A. M., L. C. Gerstenfeld, J. Wang, T. Kon, H. Bae, E. M. Gravallese, M. J. Glimcher, L. H. Glimcher. 2000. The nuclear factor of activated T cells (NFAT) transcription factor NFATp (NFATc2) is a repressor of chondrogenesis. J. Exp. Med. 191:9.[Abstract/Free Full Text]
  37. Klee, C. B., H. Ren, X. Wang. 1998. Regulation of the calmodulin-stimulated protein phosphatase, calcineurin. J. Biol. Chem. 273:13367.[Free Full Text]
  38. Graef, I. A., P. G. Mermelstein, K. Stankunas, J. R. Neilson, K. Deisseroth, R. W. Tsien, G. R. Crabtree. 1999. L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in hippocampal neurons. Nature 401:703.[Medline]
  39. Ho, A. M., J. Jain, A. Rao, P. G. Hogan. 1994. Expression of the transcription factor NFATp in a neuronal cell line and in the murine nervous system. J. Biol. Chem. 269:28181.[Abstract/Free Full Text]
  40. Chin, E. R., E. N. Olson, J. A. Richardson, Q. Yang, C. Humphries, J. M. Shelton, H. Wu, W. Zhu, R. Bassel-Duby, R. S. Williams. 1998. A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type. Genes Dev. 12:2499.[Abstract/Free Full Text]
  41. Abbott, K. L., B. B. Friday, D. Thaloor, T. J. Murphy, G. K. Pavlath. 1998. Activation and cellular localization of the cyclosporine A-sensitive transcription factor NF-AT in skeletal muscle cells. Mol. Biol. Cell 9:2905.[Abstract/Free Full Text]
  42. Boss, V., K. L. Abbott, X. F. Wang, G. K. Pavlath, T. J. Murphy. 1998. The cyclosporin A-sensitive nuclear factor of activated T cells (NFAT) proteins are expressed in vascular smooth muscle cells: differential localization of NFAT isoforms and induction of NFAT-mediated transcription by phospholipase C-coupled cell surface receptors. J. Biol. Chem. 273:19664.[Abstract/Free Full Text]
  43. Park, S., M. Uesugi, G. L. Verdine. 2000. A second calcineurin binding site on the NFAT regulatory domain. Proc. Natl. Acad. Sci. USA 97:7130.[Abstract/Free Full Text]
  44. Kincaid, R.. 1993. Calmodulin-dependent protein phosphatases from microorganisms to man: a study in structural conservatism and biological diversity. Adv. Second Messenger Phosphoprotein Res. 27:1.[Medline]
  45. Kissinger, C. R., H. E. Parge, D. R. Knighton, C. T. Lewis, L. A. Pelletier, A. Tempczyk, V. J. Kalish, K. D. Tucker, R. E. Showalter, E. W. Moomaw, et al 1995. Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex. Nature 378:641.[Medline]
  46. Griffith, J. P., J. L. Kim, E. E. Kim, M. D. Sintchak, J. A. Thomson, M. J. Fitzgibbon, M. A. Fleming, P. R. Caron, K. Hsiao, M. A. Navia. 1995. X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex. Cell 82:507.[Medline]
  47. Xanthoudakis, S., J. P. Viola, K. T. Shaw, C. Luo, J. D. Wallace, P. T. Bozza, D. C. Luk, T. Curran, A. Rao. 1996. An enhanced immune response in mice lacking the transcription factor NFAT1. Science 272:892.[Abstract]
  48. Schuh, K., B. Kneitz, J. Heyer, F. Siebelt, C. Fischer, E. Jankevics, E. Rude, E. Schmitt, A. Schimpl, E. Serfling. 1997. NF-ATp plays a prominent role in the transcriptional induction of Th2-type lymphokines. Immunol Lett. 57:171.[Medline]
  49. Kiani, A., J. P. Viola, A. H. Lichtman, A. Rao. 1997. Down-regulation of IL-4 gene transcription and control of Th2 cell differentiation by a mechanism involving NFAT1. Immunity 7:849.[Medline]
  50. Chow, C. W., M. Rincon, R. J. Davis. 1999. Requirement for transcription factor NFAT in interleukin-2 expression. Mol. Cell. Biol. 19:2300.[Abstract/Free Full Text]
  51. Oukka, M., I. C. Ho, F. C. de la Brousse, T. Hoey, M. J. Grusby, L. H. Glimcher. 1998. The transcription factor NFAT4 is involved in the generation and survival of T cells. Immunity 9:295.[Medline]
  52. Sussman, M. A., H. W. Lim, N. Gude, T. Taigen, E. N. Olson, J. Robbins, M. C. Colbert, A. Gualberto, D. F. Wieczorek, J. D. Molkentin. 1998. Prevention of cardiac hypertrophy in mice by calcineurin inhibition. Science 281:1690.[Abstract/Free Full Text]
  53. Sugden, P. H.. 1999. Signaling in myocardial hypertrophy: life after calcineurin?. Circ. Res. 84:633.[Free Full Text]
  54. Hemenway, C. S., J. Heitman. 1999. Calcineurin: structure, function, and inhibition. Cell Biochem. Biophys. 30:115.[Medline]
  55. Kurata, H., H. J. Lee, A. O’Garra, N. Arai. 1999. Ectopic expression of activated Stat6 induces the expression of Th2-specific cytokines and transcription factors in developing Th1 cells. Immunity 11:677.[Medline]
  56. Ferber, I. A., H. J. Lee, F. Zonin, V. Heath, A. Mui, N. Arai, A. O’Garra. 1999. GATA-3 significantly downregulates IFN-{gamma} production from developing Th1 cells in addition to inducing IL-4 and IL-5 levels. Clin. Immunol. 91:134.[Medline]
  57. Kaye, J., S. Porcelli, J. Tite, B. Jones, Jr C. A. Janeway. 1983. Both a monoclonal antibody and antisera specific for determinants unique to individual cloned helper T cell lines can substitute for antigen and antigen-presenting cells in the activation of T cells. J. Exp. Med. 158:836.[Abstract/Free Full Text]
  58. Cherwinski, H. M., J. H. Schumacher, K. D. Brown, T. R. Mosmann. 1987. Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies. J. Exp. Med. 166:1229.[Abstract/Free Full Text]
  59. Kamogawa, Y., H. J. Lee, J. A. Johnston, M. McMahon, A. O’Garra, N. Arai. 1998. A conditionally active form of STAT6 can mimic certain effects of IL-4. J. Immunol. 161:1074.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
S. Mehta, H. Li, P. G. Hogan, and K. W. Cunningham
Domain Architecture of the Regulators of Calcineurin (RCANs) and Identification of a Divergent RCAN in Yeast
Mol. Cell. Biol., May 15, 2009; 29(10): 2777 - 2793.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Martinez-Martinez, A. Rodriguez, M. D. Lopez-Maderuelo, I. Ortega-Perez, J. Vazquez, and J. M. Redondo
Blockade of NFAT Activation by the Second Calcineurin Binding Site
J. Biol. Chem., March 10, 2006; 281(10): 6227 - 6235.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Kang, H. Li, A. Rao, and P. G. Hogan
Inhibition of the Calcineurin-NFAT Interaction by Small Organic Molecules Reflects Binding at an Allosteric Site
J. Biol. Chem., November 11, 2005; 280(45): 37698 - 37706.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. H. A. Roehrl, S. Kang, J. Aramburu, G. Wagner, A. Rao, and P. G. Hogan
Selective inhibition of calcineurin-NFAT signaling by blocking protein-protein interaction with small organic molecules
PNAS, May 18, 2004; 101(20): 7554 - 7559.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
P. G. Hogan, L. Chen, J. Nardone, and A. Rao
Transcriptional regulation by calcium, calcineurin, and NFAT
Genes & Dev., September 15, 2003; 17(18): 2205 - 2232.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, J.
Right arrow Articles by Arai, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, J.
Right arrow Articles by Arai, N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS