INTRODUCTION

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Proteins belonging to the nuclear factor of activated T cells (NFAT)¹ family are a family of transcription factors that regulate the expression of many inducible genes during the immune response (1-5) (reviewed in Rao et al. (6)). NFAT proteins are expressed in a variety of immune system cells as well as in endothelial cells, certain neuronal cells, and other cells outside the immune system (6-12). NFAT proteins contain two adjacent ~300-amino acid regions that are conserved within the NFAT family, a regulatory domain located near the N terminus and a strongly conserved DNA binding domain (4-6, 13-18). In resting cells, NFAT proteins are phosphorylated and reside in the cytoplasm; upon stimulation of cells with calcium-mobilizing agents, they become dephosphorylated and translocate rapidly to the nucleus (6, 7, 13, 15, 16, 19, 20). In the nucleus, they interact cooperatively with Fos-Jun proteins and possibly other transcription factors to modulate gene transcription (6, 21-24).

The activity of NFAT transcription factors is directly regulated by calcineurin, a calcium- and calmodulin-dependent phosphatase also known as protein phosphatase 2B (6, 25). Calcineurin controls multiple functions of NFAT proteins, including their nuclear import, their DNA-binding affinity, and their transactivation (reviewed in Rao et al. (6)). Both the phosphorylated and dephosphorylated forms of NFAT1 bind to calcineurin immobilized on calmodulin-Sepharose beads (13, 26). The binding has been mapped to the N-terminal regulatory domains of NFAT proteins using recombinant NFAT1 and NFAT2 expressed in bacteria (15, 27), as well as recombinant NFAT1 and NFAT4 expressed in mammalian cells (13, 16, 17, 26).

Calcineurin is a heterodimer of a catalytic A subunit and a calcium-binding regulatory B subunit (25). The A subunit contains the catalytic domain at its N terminus, followed by binding regions for the B subunit and for calmodulin that were defined by deletion and mutational analysis (28-31). An autoinhibitory domain, whose removal renders calcineurin constitutively active, is located near the C terminus of the calcineurin A chain (29-31). Determination of the calcineurin structure showed that the autoinhibitory domain forms an -helix that is positioned in the active site of the calcineurin catalytic domain, thus preventing the access of substrates to the active site (32). Both in human and in bovine calcineurin, the B-chain binding region is also -helical (32, 33), while the region between the B-chain-binding helix and the autoinhibitory domain is disordered in the crystal structure (32). Calmodulin binding to this disordered region is presumed to displace the autoinhibitory domain from the calcineurin active site, thus permitting the access of substrates to the active site.

The clinically important immunosuppressive drugs, cyclosporin A (CsA) and FK506, have long been known to inhibit T lymphocyte activation and NFAT activation, primarily because of their ability to inhibit the phosphatase activity of calcineurin toward phosphoprotein substrates (reviewed in Refs. 34-37). CsA and FK506 do not inhibit calcineurin activity directly, but form inhibitory complexes with intracellular receptors, known as immunophilins, that are present at high abundance in all cells; the immunophilin receptors for CsA are known as cyclophilins (CyPs), while those for FK506 are known as FK-binding proteins or FKBPs. The structures of human and bovine calcineurin, in complex with FK506·FKBP12, have been solved: the drug-immunophilin complex binds to the calcineurin B-binding -helix of the calcineurin A chain and inhibits calcineurin by an indirect mechanism (32, 33).

Here we show that NFAT proteins interact more strongly with calmodulin-activated than inactive calcineurin. Two regions of calcineurin are implicated in the interaction, both of which are located on the calcineurin catalytic domain. One of these is a targeting region complementary to a region we have recently defined as the docking site for calcineurin on NFAT (27), while the second is a region overlapping the active site that is preferentially exposed in activated calmodulin-bound calcineurin. Based on our data, we suggest that NFAT proteins are targeted to calcineurin in resting cells, and that they make an additional interaction with the active site of calcineurin in activated cells. Our results have implications for the regulation of NFAT by calcineurin in resting and activated cells, and for the development of new types of immunosuppressive drugs.

	EXPERIMENTAL PROCEDURES

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Reagents-- The glutathione S-transferase (GST) fusion proteins GST-LSF, GST-human (h) NFAT1 (1-415), GST-hNFAT2 (1-418), and GST-hNFAT4 (11-419) (abbreviated GST-NFAT1, GST-NFAT2, and so forth) have been described (27, 38). The hexahistidine-tagged NFAT1(1-415) was generated by subcloning the coding region for the 415 N-terminal amino acids of NFAT1 into the pQE32 vector (Qiagen). Hexahistidine-tagged NFAT1 truncation mutants were generated by a polymerase chain reaction and subcloned into pQE vectors (Qiagen). Recombinant bacterially expressed human calcineurin and human FKBP12 were prepared as described previously (32). The recombinant catalytic domain of human calcineurin (CnCAT), incorporating a hexahistidine tag at the C terminus, was made by expressing amino acids 1-347 in bacteria. The recombinant bacterially expressed His¹⁵¹  Ala mutant of human calcineurin A was coexpressed with calcineurin B in the pET-15b vector, and was a kind gift of Dr. Jun Liu (Massachusetts Institute of Technology) (39). Cyclophilin A (CyPA) was kindly provided by Dr. Christopher Walsh (Harvard Medical School). Bovine calcineurin, calmodulin, and myoglobin were obtained from Sigma. The RII peptide (DLDVPIPGRFDRRVSVAAE) (40), the calcineurin autoinhibitory peptide (YITSFEEAKGLDRINERMPPRRDAMPSD) (31), the control ovalbumin peptide (41), and the SPRIEIT-25 (KPAGASGPSPRIEITPSHELMQAGGY), SPRIEIT-13 (SGPSPRIEITPSH), and SPAIAIA-25 (KPAGASGPSPAIAIAPSHELMQAGGY) peptides from murine NFAT1 (27) were synthesized at the Tufts-New England Medical Center Peptide Synthesis Facility. For some experiments the RII peptide was purchased from Bachem Fine Chemicals, Switzerland. The polyclonal rabbit antiserum directed against the autoinhibitory peptide has been described (13). Protease and phosphatase inhibitors were purchased from Sigma and Calbiochem.

Iodination of Calcineurin-- Calcineurin or the calcineurin catalytic domain (CnCat) (~100 µg) was iodinated with 1 mCi carrier-free Na¹²⁵I (NEN Life Science Products) using IODO-BEADs (Pierce), then separated from free Na¹²⁵I on a Sephadex G-25 spin column Biospin 6 (Bio-Rad) and stored in aliquots at 80 °C. Specific activities ranged from 150 to 600 cpm/fmol. Identical results were obtained whether recombinant human calcineurin or bovine calcineurin (Sigma) were used in the binding assay.

Binding Assays-- In some experiments, calcineurin binding to GST proteins was assessed by Western blotting as described previously (13, 15). For ¹²⁵I-calcineurin binding to GST fusion proteins, equivalent amounts of GST-NFAT or GST-LSF fusion proteins were immobilized on gluthathione-Sepharose beads by incubation for 30 min at 4 °C in binding buffer (50 mM Tris phosphate, pH 8.0, 150 mM NaCl, 5 mM MgCl₂, 5 mM 2-mercaptoethanol, 1% Triton X-100, supplemented with 1 mM sodium orthovanadate, 20 µM leupeptin, 10 µg/ml aprotinin, and 2 mM phenylmethylsulfonyl fluoride). Beads were washed, resuspended in binding buffer, and incubated with ¹²⁵I-calcineurin for 30 min at 4 °C. Binding of calcineurin to hexahistidine-tagged NFAT proteins was assessed after binding them to nickel-nitrilotriacetic acid beads (Qiagen); in these experiments 30 mM imidazole was added to the binding reaction and to the washes to prevent nonspecific binding of calcineurin to the nickel beads. Background binding of calcineurin to the nickel beads is shown in all such experiments. Final concentrations of 200 µM CaCl₂, 5 mM EGTA, and 500 nM calmodulin were used unless otherwise noted. Where indicated, CsA·CyPA and FK506·FKBP12 complexes, formed by incubation of equimolar concentrations (40-400 µM) of the drugs with the immunophilins for 1 h on ice, were added at the indicated final concentrations to the binding reaction. The RII, autoinhibitory, and control peptides were used at final concentrations of 200 µM. SPRIEIT and SPAIAIA peptides were used at the indicated final concentrations. After incubation, individual binding reactions were filtered through a 5-µm hydrophilic polyvinylidene difluoride filter (Millipore) using a vacuum manifold (Millipore) to separate bound and unbound ¹²⁵I-calcineurin. Filters were washed rapidly under continuous suction with 24 ml of 50 mM Hepes, pH 7.0, 150 mM NaCl, 5 mM MgCl₂, 200 µM CaCl₂, 10% glycerol, 1% Triton X-100. Washed filters were transferred to tubes and ¹²⁵I-calcineurin retained by the immobilized GST or hexahistidine-tagged proteins was quantified in a Hewlett Packard Gammacounter.

	RESULTS AND DISCUSSION

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Activated Calcineurin Binds More Strongly than Does Inactive Calcineurin to NFAT-- We measured the binding of ¹²⁵I-calcineurin to GST fusion proteins containing the first 415 and 418 amino acids of NFAT1 and NFAT2, respectively. We have previously shown that these N-terminal regions of NFAT proteins are sufficient for calcineurin binding (13, 15, 27). ¹²⁵I-Calcineurin was incubated with GST-NFAT1 or GST-NFAT2 immobilized on glutathione beads, and radioactivity bound to the beads was rapidly separated by filtration. Preliminary experiments (not shown) indicated that peak levels of ¹²⁵I-calcineurin binding were attained by 30 min, and that binding was linear with the amount of GST-NFAT protein used.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Calmodulin enhances the binding of calcineurin to NFAT. 125I-Calcineurin (Cn) (4 nM) was incubated with GST-NFAT2 or GST-LSF in the presence or absence of calcium and/or calmodulin (CaM) as indicated. Reactions lacking calcium contained 5 mM EGTA. The beads were rapidly washed by filtration to remove unbound calcineurin, and bound radioactivity was quantified. Reactions lacking calmodulin contained an equivalent concentration (500 nM) of myoglobin. In A, the average and standard deviation of a representative experiment, performed in triplicate, is shown. Note that error bars for some triplicates are too small to be apparent. In B, a second experiment using a different calcineurin preparation, with a greater calmodulin-dependent increase in binding, is shown.

Identification of a Short Fragment of NFAT1 That Binds to Calcineurin-- To determine the region of NFAT1 involved in binding calcineurin, we tested a series of systematic truncations of the NFAT1 regulatory domain, expressed as hexahistidine-tagged proteins in bacteria. These experiments identified an 86-amino acid fragment, NFAT1(97-183), which bound calcineurin as effectively as the original NFAT1(1-415) recombinant protein (Fig. 2). A shorter fragment, NFAT1(97-144), was also able to bind calcineurin in a calcium- and calmodulin-dependent fashion, but binding was decreased by comparison with NFAT1(1-415) or NFAT1(97-183).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Identification of a short fragment of NFAT that binds to calcineurin. A, 125I-Calcineurin (10 nM) was incubated with the indicated hexahistidine-tagged truncated derivatives of murine NFAT1 (1 µg, immobilized on nickel beads) in buffer containing 30 mM imidazole and EGTA, Ca2+, or Ca2+ and calmodulin (CaM) as indicated. The level of binding to the nickel beads alone under the same conditions is indicated. One of three representative experiments is shown. B, schematic summary of the results. The NFAT1(97-183) fragment binds calcineurin as well as NFAT1(1-415).

NFAT Binds the Calcineurin Catalytic Domain-- To define the region of calcineurin involved in NFAT binding, we generated a recombinant calcineurin containing only the catalytic domain (residues 1-347) of human calcineurin A (CnCAT). Despite lacking the calcineurin B-binding helix, the calmodulin-binding region and the autoinhibitory domain, this protein showed substantial binding to NFAT (Fig. 3). Binding of ¹²⁵I-CnCAT to GST-NFAT was equivalent in the presence and absence of calcium (Fig. 3A), consistent with the expectation that CnCAT should not need calcium or the calcineurin B chain to assume a stable protein conformation. Binding of ¹²⁵I-CnCAT to GST-NFAT was also equivalent in the presence and absence of calmodulin (Fig. 3B), consistent with the fact that CnCAT lacks the autoinhibitory region and the calmodulin-binding domain. Finally, binding of ¹²⁵I-CnCAT to GST-NFAT proteins was unaffected by drug-immunophilin complexes (Fig. 3C), which are not expected to bind CnCAT since they bind to the CnB-binding helix of the calcineurin A-calcineurin B heterodimer, which is lacking in CnCAT.

View larger version (44K): [in this window] [in a new window]   Fig. 3.   NFAT binds to the calcineurin catalytic domain. A and B, 125I-CnCAT (4 nM) was incubated with GST-NFAT1, GST-NFAT2, and GST-LSF in the presence of Ca2+ or EGTA (panel A), or in the presence of EGTA or Ca2+ and calmodulin (CaM) (panel B). C, 125I-CnCAT was incubated with GST-NFAT2 in the absence () or presence of 1 µM CypA or CsA·CypA complexes. D, addition of autoinhibitory (AI) or RII peptides (200 µM) partially inhibited the binding of 125I-CnCAT to GST-NFAT2 when compared with binding in the presence of an ovalbumin peptide used as a negative control (). Results for panels A-D are shown as average and standard deviations of a representative experiment performed in triplicate. Note that error bars for some triplicates are too small to be apparent. E, the peptide SPRIEIT-25 spanning the calcineurin docking site of NFAT1 inhibited binding of 125I-CnCAT to NFAT in a dose-dependent manner, with IC50 ~ 10 µM, whereas the mutant SPAIAIA peptide had no effect at the highest concentration used.

Peptides Spanning the NFAT Docking Sequence Inhibit the Binding of Activated Calcineurin to NFAT-- The SPAIAIA mutation in the SPRIEIT docking sequence (R112A, E114A, T116A) impaired not only the basal binding of NFAT1 to inactive calcineurin in the absence of calmodulin (27), but also the calmodulin-dependent binding of activated calcineurin to NFAT (Fig. 4A). Moreover the peptides SPRIEIT-25 and SPRIEIT-13 spanning 25 and 13 residues of this docking sequence competed the binding of activated calcineurin to NFAT1(1-415), whereas the peptide SPAIAIA-25, which incorporated the inactivating mutations, did not (Fig. 4A). The concentration of SPRIEIT peptides required for half-maximal inhibition of the binding of activated calcineurin to NFAT1 was ~25 µM (Fig. 4A), compared with the ~12 µM required for half-maximal inhibition of the binding of inactive calcineurin to NFAT (27). The binding of activated calcineurin to NFAT2 and NFAT4 was also inhibited by the SPRIEIT peptides (Fig. 4B) with an IC₅₀ of ~50 µM, again a higher concentration than necessary to inhibit the binding of inactive calcineurin to these proteins (IC₅₀ of ~25 µM) (27). SPRIEIT peptides were also able to inhibit binding of active and inactive calcineurin to the short NFAT1 fragment (97-183) (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   The SPRIEIT peptides spanning the NFAT1 docking site for calcineurin inhibit the binding of activated calcineurin to NFAT. 125I-Calcineurin (14 nM) was incubated with (A) GST-NFAT1 or (B) GST-NFAT2 or GST-NFAT4 in the presence of Ca2+ and calmodulin and the indicated concentrations of the SPRIEIT-25, SPRIEIT-13, and SPAIAIA-25 peptides. A also shows the level of 125I-calcineurin binding to a mutant GST-NFAT1 bearing the SPAIAIA mutation (R112A, E114A, T116A) in the docking sequence. The level of nonspecific binding of calcineurin was monitored by its binding to GST-LSF (LSF).

Drug-Immunophilin Complexes Partially Inhibit the Binding of Activated Calcineurin to NFAT-- To determine whether NFAT binding to active calcineurin involved the calcineurin active site, we tested the effect of FK506·FKBP12 complexes on the binding of active (calmodulin-dependent) and inactive calcineurin to NFAT1(97-183). In both cases binding was inhibited in a dose-dependent manner by the drug-immunophilin complexes, but the extent of inhibition was much greater in the case of active compared with inactive calcineurin (~80% versus ~50% inhibition at 10 µM FK506·FKBP12) (Fig. 5, compare panels A and B). Moreover, approximately 10-fold higher concentrations of FK506·FKBP12 were required to achieve equivalent inhibition of inactive versus active calcineurin (i.e. 0.1 µM FK506·FKBP12 sufficed to inhibit binding of active calcineurin by 30%, whereas 1 µM FK506·FKBP12 was required for equivalent inhibition of binding of inactive calcineurin to NFAT; compare Fig. 5, A and B). The immunophilins CyPA and FKBP12 had no effect on the NFAT-calcineurin in the absence of the relevant immunosupressive drugs (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Inhibition of the NFAT-calcineurin interaction by drug-immunophilin complexes. 125I-Calcineurin (Cn) (10 nM) was incubated with NFAT1(97-183) and the indicated concentrations of preformed FK506·FKBP12 complexes in the presence (A) or absence (B) of Ca2+ and calmodulin. The average and standard deviation for four independent experiments is shown. Note that error bars for some quadruplicates are too small to be apparent.

The Catalytic Activity of Calcineurin Is Not Required for NFAT-Calcineurin Binding-- Since the previous experiments implicated the calcineurin active site in NFAT-calcineurin binding, we asked whether the catalytic activity of calcineurin was required for binding to NFAT proteins (Fig. 6). Recombinant wild-type calcineurin or the inactive His¹⁵¹  Ala mutant were incubated with GST-NFAT2 in the presence or absence of calmodulin. The beads were then washed and the amount of bound calcineurin was assessed by Western blotting with an antibody to the autoinhibitory peptide of calcineurin A, exactly as described previously (15). Fig. 5 shows that there was equivalent binding of the catalytically active and inactive calcineurin to NFAT, indicating that the NFAT-calcineurin interaction does not require the phosphatase activity of calcineurin. Consistent with this result, catalytically inactive calcineurins, when overexpressed, inhibited the nuclear import of NFAT4 (16), indicating that these proteins remained capable of interaction with NFAT.

View larger version (45K): [in this window] [in a new window]   Fig. 6.   The phosphatase activity of calcineurin is not required for binding to NFAT. The binding of recombinant wild-type (Wt) calcineurin or the catalytically inactive His151  Ala mutant (H151A) to GST-NFAT2 in the presence or absence of calmodulin was assessed by Western blotting.

Characteristics of the NFAT-Calcineurin Interaction-- The NFAT-calcineurin interaction is central to the proper regulation of the immune response (6, 35), and also plays a role in the function of cells and tissues outside the immune system (11, 12). We show here that calmodulin-activated calcineurin binds significantly more strongly than does inactive calcineurin to NFAT. The interacting regions are located on the catalytic domain of the calcineurin A chain and on an 86-amino acid fragment of the NFAT regulatory domain. Our results suggest that the interaction of activated calcineurin with NFAT involves at least two functionally separable regions. One of these is the SPRIEIT sequence of NFAT1, which we have previously identified as the docking site for inactive calcineurin in resting cells (27), while the second involves the enzyme active site. The active site interaction is inhibited by the calcineurin autoinhibitory domain and the RII substrate peptide, which bind in the active site, as well as by drug-immunophilin complexes that partially occlude the active site. The active site contact appears to make a substantially stronger contribution to the NFAT-calcineurin interaction under stimulatory conditions, when the calcineurin has been activated with calcium and calmodulin.