The Linker Region Joining the Catalytic and the Regulatory Domains of CnA Is Essential for Binding to NFAT*

Calcineurin (CN) is an important regulator of developmental processes and in adults controls the immune response through its regulation of nuclear factor of activated T cells (NFAT). The physical interaction between CN and NFATs is an essential step in the activation of NFAT-dependent genes by calcium signals. Using deletional and substitutional analyses, we have identified a 13-amino acid region within CN that is essential for the interaction with NFAT and with two other CN-binding proteins, AKAP79 and Cabin-1. The interaction of CN with these proteins is selectively disrupted by substitution of specific amino acid residues within this region, indicating that NFAT and other CN-interacting proteins bind differentially to CN. This selectivity suggests that the region identified in CN could be a potential molecular target for immunosuppressive and other therapeutic interventions in diseases involving the CN/NFAT pathway.

transcription factors comprises five members, and four of them, named NFATc1-NFATc4 (HUGO nomenclature) are regulated by CN, which dephosphorylates them to promote nuclear translocation and subsequent NFAT-dependent gene expression (5). The CN/NFAT signaling pathway is the target of the immunosuppressive actions of cyclosporin A and FK506. These drugs, once bound to their cellular partners, block the active site of CN, inhibiting its phosphatase activity (6,7). As a result, NFAT activation, essential for the immune response, is inhibited. However, treatment with these immunosuppressants is associated with severe side effects that are thought to be mediated through the inhibition of CN substrates other than NFATs (8). Although the motifs within the regulatory domain of NFAT mediate interaction with the catalytic subunit of CN (CnA) (4), the specific sequence(s) within CN involved in this interaction has not been described.
Plasmid Constructs-The plasmid constructs are detailed in the supplemental data.
GST Fusion Protein Expression-All the GST fusion proteins were expressed in Escherichia coli strain BL-21. The overnight culture was diluted 1/20 and grown until the A 600 nm was 0.6 -0.8. Production of fusion protein was induced by adding up to 1 mM isopropyl ␤-D-thiogalactopyranoside to the growth culture and incubating for 3 h at 37°C with vigorous shaking. The cell pellet was collected by centrifugation, suspended in 10 mM EDTA containing PBS, and sonicated. After addition of Triton X-100 up to 1%, cell debris was discarded by centrifugation, and the soluble proteins were purified with GSH-Sepharose 4B beads (Amersham Biosciences), and the bound GST fusion proteins were store at 4°C. The amount of purified GST protein was confirmed by running on an SDS-PAGE gel followed by Coomassie staining.
Cell Culture, Transient Transfections, and Cell Extracts-HEK-293 and HeLa cells were cultured in 10% fetal bovine serum containing Dulbecco's modified Eagle's medium medium supplemented with Lglutamine plus antibiotics (penicillin/streptomycin). The HEK transient transfections were performed by the modified calcium phosphate method (10). HeLa cells were transfected with Lipofectamine Plus reagents (Invitrogen) following the manufacturer's recommendations.
Transfected cells were washed with PBS, scraped from plates, and collected by centrifugation at 4°C. Cells were then lysed by adding 100 l of either binding buffer (20 mM  ‡ These two authors contributed equally to this work, and the order of authorship should be considered arbitrary.
§ Recipient of an FIS contract and is supported by FIS Grant PI031474.
¶ Recipient of a Centro Nacional de Investigaciones Cardiovasculares fellowship.
Immunofluorescence and Confocal Analysis-Transfected HeLa and HEK-293 cells plated onto coverslips were fixed with 4% formaldehyde in PBS with 4% saccharose for 10 min at room temperature. They were then washed three times in PBS and permeabilized with 0.25% Triton X-100 in PBS for 10 min at room temperature. Permeabilized cells were incubated in blocking buffer (10% bovine serum albumin [BSA] in PBS) for 20 min, followed by 1-h incubation with a 1:1000 dilution of rabbit polyclonal anti-NFATc2 antiserum (672) or 1:750 dilution of M2 anti-FLAG monoclonal antibody in blocking buffer. The subcellular localizations of NFATc2 and exogenous CnA were visualized by confocal analysis (Radiance 2100, Bio-Rad) after incubation with 1:1000 diluted goat anti-mouse IgG Alexa 488 and 1:1000 diluted goat anti-rabbit IgG Alexa Fluor 594 (Molecular Probes).
Pull-down Experiments-GST fusion proteins were employed as baits in pull-down experiments. The GST-containing beads were washed with binding buffer and incubated for 30 min rocking at 4°C in 60 l of lysates of HEK cells transfected with FLAG-tagged hCnA␣ constructs. Beads were then washed five times with 60 l of binding buffer, and the bound protein was eluted by boiling samples for 10 min in the presence of 1ϫ Laemli buffer. Samples were loaded onto an SDS-PAGE gel and transferred to nitrocellulose membranes, and the proteins were detected by Western blotting.
Co-immunoprecipitation Experiments-Transfected HEK-293 cells were lysed with calcium-depleted binding buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 6 mM MgCl 2 , 0.2% Triton X-100), and FLAG-tagged proteins were purified from 100-l extracts by adding 10 l of preequilibrated FLAG-agarose beads with 0.2% BSA-containing binding buffer described above and incubating with rocking for 1h at 4°C. Beads were collected by centrifugation at 4°C and washed five times with binding buffer, and the bound proteins analyzed by Western blotting after loading and running the samples on SDS-PAGE.
Immunoblotting-Laemmli buffer was added to samples up to 1ϫ, and samples were boiled for 10 min and separated by electrophoresis under reducing conditions by SDS-PAGE (6% for NFATc2 protein or 10% for FLAG-tagged or GFP proteins). Proteins were transferred to nitrocellulose membranes and then incubated in blocking solution (5% (w/v) skimmed milk in PBS) for 30 min at room temperature. After washing with PBS-T (0.1% Tween 20 in PBS), membranes were incubated with the mouse monoclonal anti-HA antibody 12CA5 (0.05% (v/v)) in PBS-T with 1% BSA or the rabbit polyclonal anti-GFP serum (0.1% (v/v)) in PBS-T with 5% skimmed milk for 2 h. Membranes were then washed three times for 5 min each with PBS-T, incubated with peroxidase-labeled goat anti-mouse IgG (Pierce) or anti-rabbit IgG (Pierce) for 1 h at room temperature, and washed three times with PBS-T and once with H 2 O. To detect FLAG-tagged proteins, membranes were incubated with the mouse monoclonal M5 anti-FLAG antibody (0.05% (v/v)) in TBS-T (0.05 M Tris, pH 7.5, 0.15 M NaCl, 0.1% Tween 20) for 30 min at room temperature, washed three times for 2 min each with TBS-T, and incubated with peroxidase-labeled goat anti-mouse IgG (Pierce) for 30 min at room temperature. Membranes were washed six times for 10 min each with TBS-T and once with TBS. Membrane-bound antibody was visualized with the ECL (enhanced chemiluminescence) detection reagent (Amersham Biosciences).
Phosphatase Activity-The enzymatic activity of the different human CnA␣ constructs was analyzed with the Biomol green cellular calcineurin assay kit (Biomol) according to the manufacturer's instructions.

RESULTS AND DISCUSSION
To identify the region of CN that interacts with NFAT, we generated deletion constructs of the ␣ isoform of human CnA ( Fig. 1A and supplemental Fig. 1) and expressed them in HEK-293 cells. Only the construct bearing residues 2-389, which conserves the structure of the full-length wild-type protein and encodes a constitutively active isoform (11), specifically interacted with the NFATc2-regulatory domain in pull-down experiments (Fig. 1B). This result suggests that amino acid residues 269 -389 are necessary for interaction between these two mol-ecules. To test whether this region mediates specific interaction (rather than simply maintaining conformation), we co-expressed residues 267-388 (CnA 267-388 ) with NFATc2 and confirmed their in vivo interaction in co-immunoprecipitation experiments (Fig. 1C). Constitutively active CN bound and dephosphorylated NFATc2, while full-length and CnA 267-388 bound to phospho-NFATc2 to the same extent. CnA 267-388 contains the carboxyl end of the catalytic domain, the CnB binding domain, and the linker sequence joining them. The x-ray structure of the CN heterodimer (12,13) indicated that the only accessible amino acids in the 269 -389 region lie within the 13-amino acid linker sequence (residues 335-347). Mutational analysis in yeast has shown that three specific residues within the linker region of yeast CnA are necessary for stress-induced CN activity in vivo (14). Although yeasts lack NFATs, these residues are conserved in all known CN sequences, so we analyzed their potential involvement in the interaction between human CnA and NFATc2. We substituted each of these three amino acids (S337P, H339L, and L343S), alone or in combination, within the sequence encoding residues 2-389 of human CnA␣. Substituted CnA proteins were transiently expressed in HEK-293 cells, and cell extracts were pulled down with GST-NFATc2. Mutation of any of these residues abolished the interaction between CnA␣ and the regulatory domain of NFATc2 ( Fig. 2A). Equivalent amounts of wild-type and mutant CN proteins from HEK-293 transfected cell lysates were used in all these pull-down experiments (see supplemental Fig. 2). In addition, the ability of all mutant CnA proteins to bind CnB was similar to that of wild-type CnA protein (data not shown).
The only functional CN-interacting sequence present in NFATc2 is the PxIxIT motif (PxIxITc2), present in all CN- regulated NFAT proteins. A number of endogenous CN-binding proteins contain a PxIxIT-like motif within the region that binds to CN (15, 16). We analyzed the interaction of the CnA mutant proteins with the CN-interacting sequence from two of these, Cabin-1 (17) and AKAP79 (18) (Fig. 2C). Only the CnA␣ protein mutated at position 343 was able to interact with the Cabin-1 and AKAP79 domains as efficiently as was the wildtype CnA sequence (Fig. 2D). The linker region of CN thus appears to be critical in its interaction with these two proteins, and single amino acid substitutions differentially affect the binding of CN to different PxIxIT-containing proteins.
To investigate the impact of these amino acid substitutions on the in vivo protein-protein interaction, we analyzed the dephosphorylation and nuclear import of NFAT. We performed Western blots on extracts of cells co-transfected with NFATc2 and the CnA-(2-389) constructs. Constitutively active wildtype CnA-(2-389) dephosphorylated NFATc2, and substitution of more than one of the identified CN-linker amino acids inhibited this (supplemental Fig. 3). This finding was confirmed and extended by immunostaining, which showed that proteins bearing more than one of the amino acid substitutions are unable to translocate NFATc2 to the nucleus (Fig. 2D). To rule out the possibility that double or triple substitutions were affecting the activity of the catalytic core of CnA, we tested the phosphatase activity of these mutant CnA proteins expressed in HEK-293 cells on the commonly used CN-specific substrate RII peptide (19). The phosphatase activities of lysates from these cells were even higher than those detected in lysates of cells expressing the wild-type protein (Fig. 2E). The failure of CnA mutants to interact with other CN inhibitors might underlie their high phosphatase activity toward the RII phosphopeptide.
To further confirm the role of the CnA linker region in the interaction with NFAT in vivo, we expressed the wild-type linker sequence in the presence of NFATc2 and the constitutively active CN wild-type protein and analyzed the phosphorylation state of NFATc2 by Western blotting. Although single substitutions of CnA had a dramatic impact on the in vitro binding to NFATs, double or triple substitutions were required to block dephosphorylation and nuclear translocation of NFATs in intact cells ( Fig. 2D and supplemental data). This is likely to be related to in vivo low affinity interactions due to the higher level of the CnA mutants expressed in transfected cells.
To verify the role of the CnA linker region in the interaction with NFATc2, we generated two more constructs by inserting a stop codon right after either the linker region (construct expressing amino acids 2-346) or the catalytic domain (construct containing amino acids 2 to 336; Fig. 3A). These constructs were transfected into HEK-293 cells, and the corresponding cell extracts were employed in pull-down experiments using a GST fusion protein expressing the PxIxITc2 motif as bait (GST-PxIxITc2). Only those constructs containing the linker region were able to interact with the PxIxITc2 motif (Fig. 3B, lanes 1  and 3). Furthermore, these interactions were specifically inhibited by the addition of the VIVIT peptide (lanes 2 and 4). We further tested whether these constructs were able to interact with NFATc2 in vivo. To this end, HEK-293 cells were cotransfected with the new constructs described above and the NFATc2 expression plasmid. In accordance with the results obtained in vitro, we found that only the constructs containing the linker region (389 and 346) were able to translocate NFATc2 to the nucleus (Fig. 3C, left and middle panels). In contrast, the construct of amino acids 2-336, which does not contain the linker sequence, failed to induce the nuclear translocation of NFATc2 (Fig. 3C, right panels). These results demonstrate the involvement of the linker region of CnA␣ in the interaction with NFAT in vivo.
Finally, we found that the expression of the GFP-linker fusion protein, as well as that of the potent inhibitor GFP-VIVIT (20), blocked the phosphatase activity displayed by constitutively active CN, and hence, NFATc2 was maintained in a  lanes 2-7), and GST-AKAP79 (lanes 8 -13) was determined by immunoblotting with anti-FLAG. Same aliquots of HEK transfected cells were employed. D, NFATc2 is not translocated to the nucleus by mutant CN proteins. HeLa cells were transfected with HA-NFATc2 and wild-type FLAG-tagged CnA proteins or those bearing single, double, or triple amino acid substitutions within the linker region. Subcellular localization of transfected NFATc2 (top) and CnA (bottom) proteins are shown. E, amino acid substitutions do not affect the phosphatase activity displayed by constitutively active CnA. The phosphatase activity of lysates from cells expressing the CnA proteins indicated was tested against the RII phosphopeptide, a CN specific substrate. Phosphatase activity (picomole of free phosphate released per minute per microgram of protein loaded) is expressed as the increase above the base line of cells transfected with the empty FLAG vector. The results from one representative experiment are shown.
highly phosphorylated state (Fig. 3D). In this experimental system, the small amount of DNA encoding the constitutively active CN that can be employed was responsible for the incomplete dephosphorylation of NFATc2. This demonstrates that the linker peptide is able to disrupt the NFAT-CN interaction in vivo and suggests that the linker region could also act as a docking site for NFAT. Further experiments will be necessary to confirm this possibility. In this regard, a docking site for the exogenous VIVIT peptide has been recently described as a composite site that involves the ␤ strands 11 and 14 of the catalytic domain of CN (21). Since the structure of activated CN remains to be defined, particularly in the linker region, this sequence could not be considered in the modeling of the complex performed by these authors. Thus, whether the residues within the linker region may also be involved in this interaction cannot be ruled out. However, our in vitro and in vivo experiments clearly demonstrate that the linker region is essential for the CN and NFAT proteins to interact. Since the linker region is required for NFAT binding (Fig. 3B) and the CN constructs employed by Li et al. (21) contained this region, the interaction of the PxIxIT sequence with the reported docking site is probably only functional in the presence of an intact linker sequence. Taken together, these results indicate that additional regions of CN, other than that reported previously (21), are also involved in the interaction with NFAT, generating a more complex picture for the CN-NFAT interaction.
Mutational analysis of the linker region allowed us to identify residues that affect interaction via this NFAT region. All three substitutions (S337P, H339L, and L343S) severely impaired the in vitro interaction with NFATc2, which only contains a PxIxIT site. The PxIxIT-derived VIVIT peptide is a high affinity NFAT inhibitor (20) and has been proposed as an investigative and therapeutic tool. But the discovery that most identified CN binding proteins contain a PxIxIT-like motif within the region that interacts with CN suggests that this peptide might not be highly selective for NFAT. The finding that a single amino acid substitution (L343S) selectively blocks interaction with NFAT without affecting the interaction of CN with other proteins (Cabin-1 or AKAP79) is of special significance. Reagents that exploit the selective interaction of CN with NFAT through residues of the linker region would be of use in dissecting important biological processes in which the CN/NFAT pathway plays a key role. These include the activation and development of the immune system, patterning of the vasculature, morphogenesis of the heart valves, and muscle and neural development.
Moreover, most side effects of the immunosuppressive drugs CsA and FK506 are thought to be mediated through the inhibition of CN substrates other than NFAT. Specific inhibitors that selectively target the linker region of CN would enable discrimination between binding of NFAT versus other substrates. This fine mapping may thus support the design of less toxic and more specific treatments for diseases where the CN/NFAT pathway is involved, including the pathological situations related to angiogenesis or cardiac and skeletal muscle growth.