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The Journal of Immunology, 1999, 162: 4755-4761.
Copyright © 1999 by The American Association of Immunologists

Two Independent Calcineurin-Binding Regions in the N-Terminal Domain of Murine NF-ATx1 Recruit Calcineurin to Murine NF-ATx1

Jie Liu2,*, Esteban S. Masuda{dagger}, Lisako Tsuruta*, Naoko Arai3,{dagger} and Ken-ichi Arai*,{ddagger}

* Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Shirokane-dai, Minato-ku, Tokyo, Japan; {dagger} Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304; and {ddagger} Core Research for Evolutional Science and Technology (CREST), Hon-cho, Kawaguchi, Saitama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intracellular calcium regulates events controlling nuclear translocation of nuclear factor of activated T cells (NF-AT). Calcium-dependent phosphatase calcineurin (CN) plays a central role in this process. Structural and functional analyses of the N-terminal domain of murine NF-ATx1, a member of the NF-AT family, have defined two distinct CN binding regions (CNBRs), CNBR1 and CNBR2, which are located in the region preceding the SP boxes of serine/proline-rich sequences and the region between the SP boxes and Rel similarity domain, respectively. The binding of murine NF-ATx1 (mNF-ATx1) to CN was abolished by deletion of these two regions, yet was unaffected by the individual deletion. In contrast, the nuclear translocation of mNF-ATx1 was much reduced when only CNBR2 was removed. Luciferase assay revealed that both regions are required for mNF-ATx1-dependent activation of the murine IL-2 promoter. Most importantly, recombinant CNBR2 bound CN with a higher affinity, and when expressed in Jurkat cells, it functioned as a dominant negative mutant that prevented the transcription driven by exogenous mNF-ATx1, probably by interfering with the function of CN. We propose that activation of mNF-ATx1 can be modulated through two distinct CN target regions. Our findings provide a new opportunity for pharmacological intervention with Ca2+-dependent signaling events.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The calcium/calmodulin-dependent serine/threonine phosphatase, calcineurin (CN),4 is a heterodimeric protein composed of a calmodulin-binding catalytic subunit, CNA, and a Ca2+-binding regulatory subunit, CNB (1). CN is a target of the immunosuppressive drugs cyclosporin A (CsA) and FK506, which block T cell function by preventing transcriptional activation of cytokine genes. It has been suggested that CN plays an essential role in calcium-dependent dephosphorylation signal transduction pathways and subsequently leads to production of cytokines in T cells (2). Further studies have revealed that the potential of CN to regulate the expression of cytokine genes is largely due to effects on activation of a transcription factor termed nuclear factor of activated T cells (NF-AT) (3, 4).

Currently, four NF-AT family members (NF-AT1/NF-ATp, NF-ATc, NF-ATx/NF-AT4/NF-AT3c, and NF-AT3) have been identified, and they share functional and structural similarities (5, 6, 7, 8). The NF-AT complex is composed of at least two components. Both activation of the protein kinase C/Ras pathway and the elevated level of intracellular calcium are required for activation of this complex. The former is responsible for formation of the AP1 complex, as the nuclear components of NF-AT, while the latter leads to translocation of NF-AT from the cytoplasm to the nucleus, where it binds with AP1 at IL-2 promoter NF-AT sites (2, 9). Thus, nuclear transport is a critical step that allows NF-AT to function in the nucleus. CN has been shown to dephosphorylate NF-AT, the result being nuclear translocation of NF-AT (10), which can be inhibited by CsA and FK506 (11).

NF-AT protein is functionally divided into three domains. First, the Rel similarity domain (RSD) has a high sequence homology among different family members. It is responsible for DNA binding and cooperatively interacts with AP1 proteins (5, 7, 12). In addition, one of the two putative conserved nuclear localization signals (NLSs) present in NF-AT family members is located within RSD (5, 6, 7, 8). Second, a C-terminal domain eliciting less sequence homology has been reported to bear a transactivation motif (13). The third domain showing homology among NF-AT proteins is the N-terminal domain. Several conserved motifs, such as SP boxes that are rich in serines and prolines (6), CN-regulated inhibitory (CRI) sequence/serine-rich region (SRR) (14, 15), and another functional NLS (15, 16), have been identified within the N-terminal domain. The conserved serine residues in the SRR motif were found to be constitutively phosphorylated by cellular kinases and can be dephosphorylated by CN (15). Deletion of CRI in human NF-ATx1 (hNF-ATx1) or mutation of serines in the SRR motif of NF-ATc led to the constitutive nuclear translocation of either hNF-ATx1 or NF-ATc (14, 15). Furthermore, at least two conserved NLSs have been reported to be essential for the nuclear translocation of NF-ATc; one NLS located in RSD is associated with the majority of phosphorylated serines in SRR (15). Thus, NLS is probably masked by these phosphorylated serine residues. Both the domain interacting with CN and residues dephosphorylated by CN have been mapped within the N-terminus of NF-AT (6, 15, 16), suggesting that the N-terminal domain of NF-AT is a target of CN action involved in major activities of the Ca2+ signaling pathway and is important for the nuclear localization of NF-AT.

Here, we present data showing that the N-terminal domain of mNF-ATx1, an isoform of the murine NF-ATx (mNF-ATx), (17), is responsible for mNF-ATx1 interacting with CN. We also identified two CN binding regions within the N-terminus, each of which has the capacity to independently bind CN. The biological significance of these two separate CN binding regions was further assessed by monitoring their effects on the intracellular localization of mNF-ATx1 and mNF-ATx1 stimulated transcriptional activation of the murine IL-2 gene. Our studies revealed important features of the interaction of mNF-ATx1 with CN via the CN binding region, and light was shed on a structure-function model of mNF-ATx1 protein. Our finding that one of two CN binding regions acts as an inhibitor of mNF-ATx1 opens the way for development of immunosuppressive agents.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid constructions, expression, and purification of GST fusion proteins

The expression plasmid used for mNF-ATx1 (pME-mNF-ATx1) was previously described (17). pBJ5-CNA and pBJ5-CNB, which encode CNA and CNB subunits, were provided by N. A. Clipstone and G. Crabtree (Stanford University, Stanford, CA). pNF-AT72Luc containing the luciferase reporter gene under control of three copies of the murine IL-2 distal NF-AT sites (-290 to -261) (18) and pmoIL-2–321Luc containing the luciferase reporter gene under control of the IL-2 promoter that covers the position of -321 to +46 were constructed as previously described (18). pCMV-SEAP (Tropix, Bedford, MA), used as an internal control to evaluate transfection efficiency, is an expression vector of secreted alkaline phosphatase.

For a series of GST fusion constructs, DNA fragments derived from the N-terminal domain of mNF-ATx1 were inserted into either pGEX4T-1 or pGEX-5X-3, according to the reading frame (Pharmacia, Piscataway, NJ). All GST fusion proteins and GST protein expressed in Escherichia coli strain BL21DE3 were affinity purified according to the manufacturer’s instructions (Pharmacia).

pME-mNF-ATx1{Delta}R1, pME-mNF-ATx1{Delta}R2, and pME-mNF-ATx1{Delta}R1/R2 were obtained from pME-mNF-ATx1 (17) by deleting the N-terminal region preceding the SP boxes of mNF-ATx1 (nucleotide positions between 79–571, R1), the region between the SP boxes and the RSD (nucleotide positions between 966-1227, R2), and the region covering both R1 and R2 of mNF-ATx1, respectively. pcDNA-His-CNBR2 was constructed by inserting the CNBR2 cDNA fragment derived from mNF-ATx1 into pcDNA3.1/His vector (Invitrogen, Carlsbad, CA).

Cells and luciferase assay

COS-7 cells were grown in DMEM medium containing 10% FCS, 50 U/ml of penicillin, and 50 µg/ml streptomycin. Jurkat cell lines were cultured in RPMI 1640 medium supplemented with 5% FCS, 2 mM L-glutamine, 50 µM 2-ME, 50 U/ml of penicillin, and 50 µg/ml streptomycin. Transfections into COS-7 and Jurkat cells were conducted as previously described (14, 17).

For luciferase assays performed in COS-7 cells, 6 µg of pmoIL-2–321Luc reporter plasmid and 6 µg of pME-mNF-ATx1, pME-mNF-ATx1{Delta}R1, pME-mNF-ATx1{Delta}R2, or pME-mNF-ATx1{Delta}R1/R2 with pBJ5-CNA plus pBJ5-CNB plasmid were used. pME-18S was used to adjust the total amount of DNA transfected, as required. In competition experiments, Jurkat cells were transfected with 1 µg of pNF-AT72Luc plus 0.5 µg of pCMV-SEAP and either 0.25 µg of pME-mNF-ATx1 or pME-18S alone or together with 0.75 µg of pcDNA-His-CNBR2. The pcDNA3.1/His empty vector was used to adjust the total amount of DNA transfected, as required. Luciferase activity was measured using the Luciferase Assay System (Promega, Madison, WI).

In vitro CN binding assay

For the CN binding assay, cell lysates isolated from COS-7 cells transfected with the wild-type CN (pBJ5-CNA and pBJ5-CNB) were incubated with glutathione-Sepharose 4B-bound GST-mNF-ATx1 mutants, glutathione-Sepharose-bound GST, or glutathione-Sepharose 4B alone 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 Na 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-CNA (Sigma) mAbs.

Immunofluorescence staining

mNF-ATx1{Delta}R1 and mNF-ATx1{Delta}R2 expressed in COS-7 cells were visualized by immunofluorescence staining as previously described (17). An affinity-purified polyclonal Ab, AP{alpha}DS, raised against a bacterially produced recombinant peptide of human NF-ATx (hNF-ATx) extending from amino acid residues 387–728 (6), was used to detect mNF-ATx1 and its mutants. The secondary Ab used was FITC-labeled goat anti-rabbit IgG (Zymed, South San Francisco, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CN interacts independently with two distinct regions within the N-terminal domain of mNF-ATx1

To determine whether the N-terminal domain of mNF-ATx1 could mediate the association with CN, the construct encoding the fusion protein (GST-XND) of the N-terminal domain of mNF-ATx1 (amino acids 25–406) (17) with GST was made and expressed in E. coli. The purified GST-XND fusion protein or GST protein immobilized on glutathione-Sepharose 4B beads was incubated with cell lysates isolated from COS-7 cells that had been transfected with the expression vectors for the wild-type CN (pBJ5-CNA and pBJ5-CNB). After extensive washing, the glutathione-Sepharose 4B-bound fraction was eluted and analyzed by SDS-PAGE followed by Western blot analysis, using anti-CNA and anti-GST Abs, respectively. As shown in Fig. 1Go, CN was detected when GST-XND was used (lane 3); however, no CN was observed when glutathione-Sepharose 4B-bound GST or glutathione-Sepharose 4B alone was used under the same conditions (lanes 4 and 5), suggesting that CN binding depended on the presence of the N-terminal domain of mNF-ATx1.



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  		FIGURE 1. CN interacts with the N-terminal domain of mNF-ATx1. Equal amounts of cell lysates isolated from pBJ5-CNA and pBJ5-CNB transfected COS-7 cells were incubated in the presence of glutathione-Sepharose 4B-bound GST-XND fusion protein (lane 3), glutathione-Sepharose 4B-bound GST alone (lane 4), and glutathione-Sepharose 4B alone (lane 5). Following binding and washing, the bound fraction was eluted and analyzed by SDS-PAGE and examined by Western blotting using either anti-CNA (upper panel) or anti-GST Ab (lower panel). In lanes 1 and 2, purified GST-XND fusion protein and the cell lysate isolated from CN-transfected COS-7 cells were subjected to SDS-PAGE directly for Western blotting assay, as the controls. Size markers are shown in kilodaltons on the left.

 
To locate precisely the CN binding portion within the N-terminal domain of mNF-ATx1, we prepared a series of GST fusion proteins of mNF-ATx1 mutants having the N-terminal truncations (Fig. 2GoA). These fusions were analyzed by SDS-PAGE followed by either Coomassie brilliant blue staining (data not shown) or Western blots using an anti-GST Ab (Fig. 3Go).



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  		FIGURE 2. Schematic representation of deletion mutants of GST-mNF-ATx1 fusion protein. Numbers indicate amino acid positions in mNF-ATx1 (17) of different mutants. The SP boxes and the RSD are indicated.

 


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  		FIGURE 3. CN binding activities of different mNF-ATx1 GST fusion proteins. Equal amounts of COS-7 cell lysates that had been transfected with pBJ5-CNA and pBJ5-CNB were incubated with glutathione-Sepharose 4B-bound GST fusion proteins of XND (lane 1), XN{Delta}R2 (lane 2), XN{Delta}R1 (lane 3), or XN{Delta}R12 (lane 4). CN binding assays were performed under the same conditions as those described in Fig. 1Go. Size markers are shown in kilodaltons on the left.

 
XN{Delta}R2 was a deletion mutant lacking the region between the SP boxes and the RSD. Binding assay with GST-XN{Delta}R2 fusion protein and COS-expressed CN showed that such deletion had no significant effect on its CN binding activity, compared with that of GST-XND fusion protein (Fig. 3Go). Likewise, when expressed in E. coli as a GST fusion protein, XN{Delta}R1, in which the N-terminal region preceding the SP boxes was removed from XND, still bound to CN (Fig. 3Go). However, when further deletion was made in XN{Delta}R1 at its C-terminus to yield XN{Delta}R12, the CN binding activity of GST-XN{Delta}R12 was drastically reduced. The lack of CN binding activity of GST-XN{Delta}R12 fusion protein was not due to a lower amount of the protein used. Blotting of the filter by anti-GST Ab revealed that the amount of GST-XN{Delta}R12 fusion protein, which was less than the amount of GST-XND or GST-XN{Delta}R2 fusion, was still comparable to that of GST-XN{Delta}R1 fusion protein (Fig. 3Go, lower panel).

Taken together, these results showed that both GST-XN{Delta}R1 and GST-XN{Delta}R2 fusion proteins are capable of interacting with CN and that the individual deletion of either R1 or R2 did not abolish the potential of mNF-ATx1 to bind CN. Since GST-XN{Delta}R12, which contains the overlapping region of both GST-XN{Delta}R1 and GST-XN{Delta}R2, failed to bind CN, mNF-ATx1 is likely to contain two CN binding regions (CNBRs): R1, localized at the region preceding the SP boxes, contains the amino acid residues 25–188; R2, corresponding to the region between the SP boxes and RSD of mNF-ATx1, contains the amino acid residues 317–406.

Removal of the R2 region results in impairment of the nuclear translocation of mNF-ATx1

Since the CN binding of mNF-ATx1 seemed to be mediated via either R1 or R2, we next asked whether the subcellular localization of mNF-ATx1 would be affected by deleting R1 and R2. To address this question, we prepared expression constructs of pME-mNF-ATx1{Delta}R1 and pME-mNF-ATx1{Delta}R2, in which R1 and R2 were deleted, respectively (Fig. 4Go). As reported previously, in COS-7 cells, nuclear translocation of overexpressed mNF-ATx1 molecule depends on coexpression of the wild-type CN followed by stimulation of the cells with calcium ionophore (17). Therefore, pBJ5-CNA and pBJ5-CNB were cotransfected with pME-mNF-ATx1{Delta}R1 or pME-mNF-ATx1{Delta}R2 into COS-7 cells. Immunostaining using an Ab recognizing the RSD of hNF-ATx1 revealed that mNF-ATx1{Delta}R1 was present predominantly in the cytoplasm of unstimulated cells (Fig. 5GoA). Following activation of the cells by A23187, mNF-ATx1{Delta}R1 translocated to the nucleus of most transfected cells (Fig. 5GoB). In marked contrast, mNF-ATx1{Delta}R2 showed no significant redistribution to the nucleus in response to the activation of CN in immunostaining (Fig. 5Go, C and D); mNF-ATx1{Delta}R2 remained in the cytoplasm of 90% of transfected cells. Although the accumulation of mNF-ATx1{Delta}R2 in the nucleus was observed in some activated cells, it represented only a small population among the transfected cells compared with that of mNF-ATx1{Delta}R1.



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  		FIGURE 4. Schematic representation of the N-terminal deletion mutants of mNF-ATx1. mNF-ATx1{Delta}R1 and mNF-ATx1{Delta}R2 were prepared by deleting R1 and R2, respectively, in the N-terminal domain of mNF-ATx1, as described in Results. The SP boxes, NLSs, and RSD are indicated.

 


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  		FIGURE 5. Subcellular localization of mNF-ATx1 deletion mutants. COS-7 cells were cotransfected with 1.25 µg each of pBJ5-CNA and pBJ5-CNB together with 2.5 µg of the expression plasmid pME-mNF-ATx1{Delta}R1 (A and B) or pME-mNF-ATx1{Delta}R2 (C and D). The transfected cells were either unstimulated (A and C) or stimulated with 0.5 µM A23187 for 30 min (B and D). Immunostaining was performed using an affinity-purified polyclonal Ab, AP{alpha}DS.

 
These results indicate that R2 is a domain actively involved in the nuclear localization of mNF-ATx1 and is indispensable for this process, whereas R1 elicits less drastic effects and cannot by itself transport mNF-ATx1 to the nucleus.

Deletion of either the R1 or R2 region abolishes mNF-ATx1-mediated transcriptional activation of the murine IL-2 gene

On the basis of the observation that removal of R2 reduced the nuclear translocation activity of mNF-ATx1, we then examined the roles of R1 and R2 in mNF-ATx1-dependent gene activation. As shown in Fig. 6Go, when expression vectors encoding the wild-type mNF-ATx1 (pME-mNF-ATx1) as well as CNA and CNB were transfected into COS-7 cells, exposure of the cells to PMA/A23187 enhanced transcription activity of the murine IL-2 promoter 4-fold over that of untreated cells. However, when pME-mNF-ATx1{Delta}R1 or pME-mNF-ATx1{Delta}R2 was used instead of pME-mNF-ATx1, mNF-ATx1-dependent transcription activity of the exogenous IL-2 promoter was decreased 1.8- or 1.9-fold following stimulation of the cells by PMA/A23187, respectively. Further deletion of both R1 and R2 resulted in the impairment of mNF-ATx1 transcription activity. Thus, both R1 and R2 are apparently essential for mNF-ATx1-dependent IL-2 promoter transcriptional activity.



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  		FIGURE 6. Effects of deletion mutations of mNF-ATx1 on transactivation of the IL-2 promoter. COS-7 cells were cotransfected with pmoIL-2–321Luc and pBJ5-CNA plus pBJ5-CNB along with the indicated expression plasmids. The transfected cells either were not stimulated or were stimulated for 8 h with PMA/A23187 as described in Materials and Methods. The relative luciferase unit (RLU) was normalized to the protein concentration by the bicinchoninic acid Protein Assay Reagent (Pierce, Rockford, IL). Data shown here are data derived from three independent transfection experiments.

 
CNBR2 of mNF-ATx1 exhibits potent CN binding activity

The results described above show that R1 and R2 have different roles in transmitting the CN-mediated signal to mNF-ATx1; i.e., removal of R2 abolished nuclear translocation of mNF-ATx1, but removal of R1 did not, although both are involved in the CN binding event. To clarify why they elucidate the different functions, we next examined the direct interactions of CN with these two regions. CNBR1, including amino acid residues of 25–143, is 45 amino acids shorter than R1; CNBR2, containing amino acid residues of 321–406, covers a region similar to that of R2 (Fig. 2GoB). Constructs encoding the GST fusion protein of CNBR1 or CNBR2 were expressed in E. coli. The purified GST-CNBR1 and GST-CNBR2 proteins were used to perform the binding assay under the same conditions as those described above. As expected, both GST-CNBR1 and GST-CNBR2 fusion proteins bound CN (Fig. 7Go), while no CN was observed when GST alone was used (data not shown). Remarkably, GST-CNBR2 fusion showed a much stronger CN binding activity than did the GST-CNBR1 fusion protein. This result was further confirmed using different amounts of purified CN, showing that the CN binding of GST-CNBR1 could be enhanced by the addition of increased amounts of purified CN. Nevertheless, its binding activity was weaker compared with that of GST-CNBR2 under the same conditions (data not shown). It suggested that the different functions of two CN binding regions might be due to different CN binding potentials.



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  		FIGURE 7. CN interacts with mNF-ATx1 at two distinct regions. CN binding assays were conducted under the same conditions as those described in Fig. 1Go, except that glutathione-Sepharose 4B-bound GST-CNBR1 and GST-CNBR2 fusion proteins were used. Glutathione-Sepharose 4B-bound GST-XND was used as a control.

 
CNBR2 protein exerts a dominant negative effect on mNF-ATx1-dependent transcription activity

Previous study has shown colocalization of CN with NF-AT4 in the nucleus of cells from the U2OS cell line (19). We also observed that CN migrates to the nucleus together with mNF-ATx, when coexpressed in COS-7 cells that had been stimulated with A23187 (J. Liu, unpublished observation). Based on the evidence of potent CN binding activity of CNBR2, we hypothesized that recombinant CNBR2 acts in a dominant negative manner by titrating out CN interacting with mNF-ATx1. To test this possibility, CNBR2 cDNA was constructed into the His-tagged expression vector to yield pcDNA-His-CNBR2. As shown in Fig. 8Go, when Jurkat cells were cotransfected with pNF-AT72Luc and pcDNA3.1/His empty vector, the promoter activity, which was very low in the cells before stimulation, increased following treatment of the cells with PMA/A23187. This activity is probably supported by the action of endogenous NF-AT. When pME-mNF-ATx1 was introduced into the cells, the promoter activity was further enhanced as much as 2.9-fold following stimulation. Interestingly, this enhancement by mNF-ATx1 was suppressed when the cells were transfected with pME-mNF-ATx1 along with pcDNA-His-CNBR2. Interestingly, we also found that expressed CNBR2 elicited an inhibitory effect on the reporter gene that was transactivated by the endogenous NF-AT; however, this effect was much less effective compared with that of mNF-ATx1-dependent reporter gene activity. Therefore, as predicted, CNBR2 protein, containing a CN binding region with high affinity but lacking the DNA binding domain and the transactivation domain, acts in a dominant negative manner in mNF-ATx1-dependent reactions.



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  		FIGURE 8. Recombinant CNBR2 suppresses mNF-ATx1-mediated transcription activity. Jurkat cells were transfected with pNF-AT72Luc reporter and pCMV-SEAP with pME-18S or pME-mNF-ATx1 alone or together with pcDNA-His-CNBR2. The empty expression vector pcDNA3.1/His was used to adjust the total amount of DNA transfected in each transfection, as required, and was used as a control. The transfected cells were either unstimulated or stimulated with PMA/A23187 for 8 h. In all transfections, pCMV/SEAP was included to monitor transfection efficiency. Luciferease activity values, given in relative luciferase units (RLU), were normalized to protein amounts in the lysates and to transfection efficiency. The data shown here were derived from three independent transfection experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Ca2+ signal- and NF-AT-dependent transactivation is a complex process. NF-AT rapidly translocates into the nucleus upon Ca2+ signaling, an event prevented by CsA and FK506 and hence requiring the action of CN. Association between CN and NF-AT1/NF-ATx/4 has been reported (14, 16, 20). In the present study we describe identification and characterization of the CN binding regions in mNF-ATx1. Using a series of truncated mNF-ATx1 proteins carrying a different N-terminal portion, we identified two distinct CN binding regions responsible for mNF-ATx1 interacting with CN. Removal of either region alone did not abolish CN binding of mNF-ATx1, but deletion of both regions did so (Fig. 3Go). These results are consistent with our reported data that multiple CN binding sites are present in the N-terminal domain of human NF-ATx1 (hNF-ATx1) and contribute to hNF-ATx1-CN interactions (14).

Like other NF-AT family members, the N-terminal domain of mNF-ATx1 is rich in serine/proline residues and appears to play an important role in controlling the subcellular localization of NF-AT (3, 4). As we noted in the CN binding assay, GST-XND fusion protein that had been incubated with the cell lysates of CNA/B-transfected COS cells migrated slightly slower than in the absence of the cell lysates. (Fig. 1Go, compare lane 1 with lane 3). It is likely that the change in mobility is due to a phosphorylation of this protein by kinase(s) existing in COS-7 cells.

Among the CNBRs of mNF-ATx1 we identified, CNBR2 (extending 85 amino acid residues located between the SP boxes and the RSD of mNF-ATx1) has unique properties. First, the CNBR2 fusion protein showed a strong binding activity to CN. Second, only when CNBR2 was removed from mNF-ATx1 was the nuclear translocation of mNF-ATx1 severely impaired (Figs. 4Go and 5Go), even although CNBR1 was present. Most recently, the CN binding site of C/CM2 sequence was mapped within the corresponding region of CNBR1 in NF-AT1 and NF-AT4 molecules, respectively (Fig. 9Go) (20, 21). The sequence of this putative CN binding site was also noted and conserved in CNBR1 of mNF-ATx1 protein, suggesting that CNBR1 might be commonly used among the different NF-AT family members for CN interaction. In contrast to our work, their results showed a constitutively cytoplasmic localization of NF-AT4 and NF-AT1 when this CN binding site was deleted. In our assay system, CNA and CNB were overexpressed during cotransfection with mNF-ATx1 deletion mutants, thus possibly overcoming the requirement of CNBR1 for mNF-ATx1 and transport mNF-ATx1{Delta}R1 to the nucleus through interaction with CNBR2. Likewise, Zhu et al. found that when coexpressed with CN, the NF-AT4 mutant, in which the C sequence (putative CN binding site) was deleted, translocated into the nucleus upon activation of Ca2+ signaling pathway (21). It is noteworthy that although the nuclear translocation of mNF-ATx1 was impaired dramatically by deleting CNBR2 (Fig. 5Go), it was not blocked completely; the mNF-ATx1{Delta}R2 molecule was present in the nucleus of approximately 10% of transfected cells upon activation (data not shown). It appears that CNBR1 may play a lesser role in mediating mNF-ATx1 nuclear translocation; the amount of mNF-ATx1 translocated with two CN contact points at both CNBR1 and CNBR2 may be greater than the amount elicited by single contact point at CNBR2. The requirement of CNBR2 for the nuclear translocation of mNF-ATx1 may mean that CNBR2 is an essential element for transducing CN-triggered signaling on mNF-ATx1. This idea is supported by the finding that when expressed in Jurkat cells, recombinant CNBR2 suppressed the transcriptional enhancing activity of wild-type mNF-ATx1 (Fig. 8Go). Compared with CNBR2, it seemed likely that expressed CNBR1 did so to a lesser extent under the same conditions as well as under the conditions in which different amounts of transfected CNBR1 were used (data not shown). Moreover, the level of inhibition of CNBR2 was comparable to that of the whole N-terminal portion of the hNF-ATx molecule (E. S. Masuda, unpublished observation).



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  		FIGURE 9. Functional structure of the N-terminal domain of mNF-ATx1. The mNF-ATx1 protein contains two distinct CNBRs (CNBR1 and CNBR2) and other potential motifs that show the sequence conservation to hNF-ATx1 and other family members. C/CM2 of NF-AT4/NF-AT1 (20, 21) shown as indicated, overlaps with CNBR1 of mNF-ATx1. The functions of these motifs are discussed in the text.

 
The mechanism of determination of the intracellular localization of the NF-AT family is a subject of considerable interest. It seems likely that phosphorylation/dephosphorylation of NF-AT is important for determining intracellular localization; NF-AT resides in the cytoplasm of resting cells in a phosphorylated state (19, 22, 23). Upon cell activation, CN dephosphorylates NF-AT directly and, in turn, induces NF-AT nuclear translocation. In this regard, CNBR2 forming a complex with CN probably makes CN accessible to phosphorylated residues, thereby inducing dephosphorylation of these residues, an event essential for the nuclear translocation of NF-AT.

We previously reported that an inhibitory sequence of 60 amino acid residues, termed CRI sequence, is located in the region preceding the SP boxes of hNF-ATx1 (Fig. 9Go). The deletion of this CRI sequence leads to nuclear translocation of hNF-ATx independent of Ca2+ signaling (14). Likewise, Beals et al. mapped an SRR motif in hNF-ATc with 23 amino acids located within the corresponding CRI region of hNF-ATx (Fig. 9Go). Mutation of serines in the SRR motif results in nuclear localization of NF-ATc (15). Therefore, it is reasonable to speculate that the NLS(s) is masked by phosphorylated serine residues in CRI/SRR (15). mNF-ATx1{Delta}R1, in which the deletion extends to the CRI/SRR, translocated to the nucleus in a stimulation-dependent manner. It is unclear whether CRI/SRR of mNF-ATx1 could function in the same manner as that of hNF-ATx or hNF-ATc.

Consistent with our previous reports, transcriptional activation of IL-2 promoter mediated by mNF-ATx1 increased markedly in PMA/A23187-stimulated COS-7 cells, when coexpressed with CNA and CNB. However, the transcriptional activity of mNF-ATx1 was reduced after either R1 or R2 was removed from mNF-ATx1 (Fig. 6Go). The reduction in transactivation activity of mNF-ATx1{Delta}R1 is probably due to the lack of the N-terminal transactivation domain (TAD) by deletion of the R1 region. Detailed analysis of the N-terminal TAD has been reported in the case of NF-AT1, in which the TAD was mapped within the first 100 amino acids (22). In hNF-ATx, we found that TAD localized within the first N-terminal 400 amino acids (H. Nishida, unpublished observation). Alternatively, the R1 binding site for CN has no functional significance. The remaining transactivation activity of mNF-ATx1{Delta}R1 is probably stimulated by translocated mNF-ATx1{Delta}R1, together with another TAD, which has been mapped at the C-terminus of hNF-ATx1 sharing sequence conservation with mNF-ATx1 (13). Similarly, PMA/A23187-induced transcription activity of mNF-ATx1 lacking R2 was reduced; however, this was due to impaired nuclear entry (Figs. 5Go and 6Go), although a low level of translocated molecules may have contributed to the activity to some extent. In fact, when both of two CNBRs were deleted, PMA/A23187-induced transcription activity of mNF-ATx1 was abolished, indicating that full interaction with CN is required for the activation of mNF-ATx1. Taken together, both R1 and R2 deletions caused the reduction of transactivation activity mediated by mNF-ATx1, however, probably through different mechanisms; R1, including a putative transactivation domain, is important for transcriptional activity of mNF-ATx1, while R2 plays an active role in nuclear localization of mNF-ATx1.

In the signal transduction pathway, docking interactions are commonly used for facilitating enzymatic reactions, and the initial docking reaction is probably of higher affinity (24, 25, 26). It is possible that CNBR2 may function as a docking site, increasing the local concentration of CN next to CNBR1 and directing CN to the phosphorylated residues, thus facilitating mNF-ATx1 dephosphorylation. The lack of well-conserved amino acid sequences between CNBR1 and CNBR2 suggests a model in which two CNBRs interact with a single CN molecule, and each region makes different contact with the same CN. If so, the effects of two CNBRs on CN-mediated signaling to mNF-ATx1 may not be the same. Other possibilities including that a secondary or tertiary structure of CNBR1 and CNBR2 may be involved in recognition by CN.

A functional nuclear export signal (NES) has been reported to exist within the corresponding region of CNBR2 in NF-ATc (27). The sequence of the NES is not well conserved among the NF-AT family members, but the leucine-rich sequences, which characterize the NES motif, are found in CNBR2 of mNF-ATx1, suggesting that CNBR2 may contain the NES sequence in mNF-ATx1. Therefore, CN, via the interaction with CNBR2, probably masks the NES and protects it from being recognized by NES receptor(s) during the import process of mNF-ATx1.

CsA and FK506 are potent immunosuppressive agents that block T cell activation and lymphokine production, although other targets of action are also likely to exist (28, 29, 30, 31). Their serious side effects, including neurotoxicity and nephrotoxicity, have occurred in patients following systemic administration of CsA or FK506, probably related to their molecular target of CN (32). Other approaches to modulating the immune response with minimal side effects may include developing inhibitor(s) specifically interfering with the interaction between NF-AT and CN. Our findings may provide a useful information for developing a new category of immunosuppressants.

Another outcome presented here is that expressed CNBR2 suppressed the reporter gene that was transactivated by the endogenous NF-AT. However, the inhibitory extent was less than its negative effect on the reporter gene that was enhanced by mNF-ATx1 (Fig. 8Go). It has been demonstrated that although all NF-AT members can bind to the promoters of IL-2 and IL-4, NF-AT1 and NF-ATc account for the majority of the binding activity (3). Thus, expressed CNBR2 might act as a specific inhibitor of mNF-ATx1. This was further supported by the fact that the sequences within the corresponding region of CNBR2 among the different family members are not well conserved. Our studies shed light on an approach to identifying the unique function of each NF-AT family member.


   Acknowledgments
 
We are grateful to Dr. S. Imai (Keio University School of Medicine, Keio, Japan) for kindly providing the polyclonal Ab AP{alpha}DS. We thank Drs. S. Miyatake (University of Tokyo), R. Imamura (DNAX Research Institute), Y. Amasaki (DNAX Research Institute), M. Ohara, and D. Wylie for providing comments on the manuscript.


   Footnotes
 
1 This work was supported by Core Research for Evolutional Science and Technology. Back

2 Current address: Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, 901 California Ave., Palo Alto, CA 94304-1104. E-mail address: Back

3 Address correspondence and reprint requests to Dr. Naoko Arai, Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, 901 California Ave., Palo Alto, CA 94304-1104. E-mail address: Back

4 Abbreviations used in this paper: CN, calcineurin; CNA and CNB, subunits of wild-type calcineurin; CsA, cyclosporin A; NF-AT, nuclear factor of activated T cells; AP1, activating protein-1; RSD, Rel similarity domain; NLS, nuclear localization signal; CRI, calcineurin-regulated inhibitory; SRR, serine-rich region; h, human; m, murine; GST, glutathione S-transferase; CNBR, calcineurin-binding region; TAD, transactivation domain; NES, nuclear export signal. Back

Received for publication August 26, 1998. Accepted for publication January 27, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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