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At least 19 recordsLinked to original sources
PubMed1995

Calcineurin subunit interactions: mapping the calcineurin B binding domain on calcineurin A.

Recombinant forms of the A and B subunits of the protein phosphatase calcineurin were produced in Escherichia coli, reconstituted into a heterodimer and purified to homogeneity. The reconstituted heterodimer exhibited properties like that of bovine brain calcineurin. This included calmodulin-stimulated activity and a subunit stoichiometry and Stokes radius consistent with native-like structure. In order to map the region on the A subunit where calcineurin B binds, a series of overlapping 20-residue peptides corresponding to this putative domain were synthesized. Using isolated calcineurin A and B subunits, an assay that relied upon peptide inhibition of calcineurin B stimulation of calcineurin A activity was developed. All five peptides, but not a control peptide, inhibited calcineurin B-dependent stimulation of calcineurin A although with different potencies. The three most effective inhibitory peptides spanned calcineurin A residues 338-377. These three peptides also altered the electrophoretic mobility of the isolated calcineurin B subunit during native polyacrylamide gel electrophoresis indicating a direct interaction between these peptides and calcineurin B. The peptide corresponding to residues 348-367 was also able to block binding of calcineurin B to the catalytic subunit.

Amino Acid Sequence↗
PubMed1996

Calcineurin A alpha (PPP3CA), calcineurin A beta (PPP3CB) and calcineurin B (PPP3R1) are located on human chromosomes 4, 10q21-->q22 and 2p16-->p15 respectively.

Calcineurin (also called protein phosphatase-2B) is a calmodulin-regulated protein phosphatase which plays an important role in signal transduction. The enzyme is a heterodimer of a 58-59 kDa calmodulin-binding catalytic subunit (calcineurin A) and a small (i.e. 19 kDa) Ca(2+)-binding regulatory subunit (calcineurin B). The highly conserved calcineurin B is encoded by a single gene in all tissues except testes, whereas there are three isoforms of calcineurin A (alpha, beta and gamma) encoded by genes on three different chromosomes. This enzyme can play a critical role in transcriptional regulation and growth control in T lymphocytes by a mechanism believed to involve dephosphorylation of the nuclear factor NF-AT which is essential for transcription of the interleukin-2 gene. To better evaluate the potential role of the calcineurin genes in human genetic disorders, we have studied their chromosome locations. Calcineurin B (PPP3R1) is located on human chromosome 2p16-->p15 and calcineurin A beta (PPP3CB, previous gene symbol CALNB) is present on 10q21-->q22. We confirm the localization of calcineurin A alpha (PPP3CA, previous gene symbol CALNA) to chromosome 4 without regional localization.

Base Sequence↗
PubMed2000

Low affinity Ca2+-binding sites of calcineurin B mediate conformational changes in calcineurin A.

Limited proteolysis of calcineurin in the presence of Ca(2+) suggested that its calmodulin-binding domain, readily degraded by proteases, was unfolded while calcineurin B was compactly folded [Hubbard, M. J., and Klee, C. B. (1989) Biochemistry 28, 1868-1874]. Moreover, in the crystal structure of calcineurin, with the four Ca(2+) sites of calcineurin B occupied, the calmodulin-binding domain is not visible in the electron density map [Kissinger, C. R., et al. (1995) Nature 378, 641-644]. Limited proteolysis of calcineurin in the presence of EGTA, shows that, when the low affinity sites of calcineurin B are not occupied, the calmodulin-binding domain is completely protected against proteolytic attack. Slow cleavages are, however, detected in the linker region between the calmodulin-binding and the autoinhibitory domains of calcineurin A. Upon prolonged exposure to the protease, selective cleavages in carboxyl-terminal end of the first helix and the central helix linker of calcineurin B and the calcineurin B-binding helix of calcineurin A are also detected. Thus, Ca(2+) binding to the low-affinity sites of calcineurin B affects the conformation of calcineurin B and induces a conformational change of the regulatory domain of calcineurin A, resulting in the exposure of the calmodulin-binding domain. This conformational change is needed for the partial activation of the enzyme in the absence of calmodulin and its full activation by calmodulin. A synthetic peptide corresponding to the calmodulin-binding domain is shown to interact with a peptide corresponding to the calcineurin B-binding domain, and this interaction is prevented by calcineurin B in the presence but not the absence of Ca(2+). These observations provide a mechanism to explain the dependence on Ca(2+) binding to calcineurin B for calmodulin activation and for the 10-20-fold increase in affinity of calcineurin for Ca(2+) upon removal of the regulatory domain by limited proteolysis [Stemmer, P. M., and Klee, C. B. (1994) Biochemistry 33, 6859-6866].

Amino Acid Sequence↗
PubMed1994

Dual calcium ion regulation of calcineurin by calmodulin and calcineurin B.

The dependence of calcineurin on Ca2+ for activity is the result of the concerted action of calmodulin, which increases the turnover rate of the enzyme and modulates its response to Ca2+ transients, and of calcineurin B, which decreases the Km of the enzyme for its substrate. The calmodulin-stimulated protein phosphatase calcineurin is under the control of two functionally distinct, but structurally similar, Ca(2+)-regulated proteins, calmodulin and calcineurin B. The Ca(2+)-dependent activation of calcineurin by calmodulin is highly cooperative (Hill coefficient of 2.8-3), and the concentration of Ca2+ needed for half-maximum activation decreases from 1.3 to 0.6 microM when the concentration of calmodulin is increased from 0.03 to 20 microM. Conversely, the affinity of calmodulin for Ca2+ is increased by more than 2 orders of magnitude in the presence of a peptide corresponding to the calmodulin-binding domain of calcineurin A. Calmodulin increases the Vmax without changing the Km value of the enzyme. Unlike calmodulin, calcineurin B interacts with calcineurin A in the presence of EGTA, and Ca2+ binding to calcineurin B stimulates native calcineurin up to only 10% of the maximum activity achieved with calmodulin. The Ca(2+)-dependent activation of a proteolyzed derivative of calcineurin, calcineurin-45, which lacks the regulatory domain, was used to study the role of calcineurin B. Removal of the regulatory domain increases the Vmax of calcineurin, as does binding of calmodulin, but it also increases the affinity of calcineurin for Ca2+. Ca2+ binding to calcineurin B decreases the Km value of calcineurin without changing its Vmax.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

A cluster of basic amino acid residues in calcineurin b participates in the binding of calcineurin to phosphatidylserine vesicles.

Interactions between phospholipid membranes and the acyl chain and specific amino acid residues of myristoylated proteins are necessary for membrane association. In the present study we tested the effects of mutations of calcineurin B subunit amino acid residues K(20)K(21), K(24)R(25), K(27)K(28) to Glu on the interactions between calcineurin and phosphatidylserine vesicles. Calcineurin-phosphatidylserine interactions were measured using binding assays and assays of phosphatidylserine-stimulated calcineurin phosphatase activity. The reverse-charge calcineurin B subunit mutant had a slower mobility in SDS-PAGE relative to wild-type calcineurin B. In addition, the myristoylated calcineurin B reverse-charge mutant had a slower mobility in SDS-PAGE compared to the non-myristoylated form, in contrast to the faster mobility of myristoylated wild-type calcineurin B relative to non-myristoylated calcineurin B. The reverse-charge mutations had no apparent effect on N-terminal myristoylation, Ca(2+)-binding, or calcineurin heterodimer formation and stimulation of Ca(2+)/calmodulin-dependent phosphatase activity. However, in contrast to the results obtained using native calcineurin, phosphatidylserine vesicles did not bind to or activate the phosphatase activity of calcineurin containing the calcineurin B reverse-charge mutant. These results indicate that calcineurin B contains an amino terminal basic residue cluster that is involved in the binding of calcineurin to acidic phospholipids.

Amino Acids↗
PubMed2001

Ca2+ binding site 2 in calcineurin-B modulates calmodulin-dependent calcineurin phosphatase activity.

Calcineurin is the Ca(2+)- and calmodulin-dependent Ser/Thr phosphatase. Human calcineurin-Aalpha and wild-type or mutated calcineurin-Bs were coexpressed in Escherichia coli and purified by calmodulin-Sepharose affinity chromatography. Four calcineurin-B mutants were studied. Each had a single conserved Glu in the 12th position of one EF-hand Ca(2+) binding site replaced by a Lys, resulting in the loss of Ca(2+) binding to that site. Phosphatase activities of the enzymes toward a (32)P-labeled phosphopeptide substrate were measured. Inactivating Ca(2+) binding sites 1, 2, or 3 in calcineurin-B reduced Ca(2+)-dependent phosphatase activity of the enzymes in the absence of calmodulin with the site 2 mutation being most effective. Inactivating Ca(2+) binding site 4 did not change enzyme activity or sensitivity to Ca(2+) in either the absence or presence of calmodulin. The calmodulin-dependent phosphatase activity of the enzymes containing site 1, 2, or 3 mutations in calcineurin-B was also decreased compared to enzyme with wild-type calcineurin-B. Of these enzymes, the one with the site 2 mutation was most profoundly affected as determined by the magnitude of the shift in Ca(2+) concentration dependence. Binding of a fluorescein-labeled calmodulin to the wild-type and the site 2 mutant enzymes was examined using fluorescence polarization measurements. The decrease in Ca(2+) sensitivity for the enzyme with calcineurin-B site 2 inactivated is apparently due to a decrease in the affinity of that enzyme for calmodulin at low Ca(2+) concentrations. These data support a role for Ca(2+) binding site 3 in the carboxyl half of calcineurin-B in transmitting the Ca(2+) signal to calcineurin-A and indicate that site 2 in the amino half of calcineurin-B is critical for enzyme activation.

Affinity Labels↗
PubMed1989

Characterization of a high-affinity monoclonal antibody to calcineurin whose epitope defines a new structural domain of calcineurin A.

Monoclonal antibodies have been raised against native calcineurin using conventional in vivo immunization and hybridoma procedures. The relatively high affinity of nonimmune IgG for the two subunits of calcineurin resulted in large nonspecific binding values for immunoassays of native, dissociated and denatured calcineurin, which complicated the antibody screening. Monoclonal aCn5, a high-affinity IgG1 that exhibits specific binding, was characterized. Other calmodulin-binding proteins tested were not recognized by aCn5. Simple binding properties were exhibited in solid-phase experiments, Kd = 26 (+/- 4) pM, but the stoichiometry was low. The loss of immunoreactivity after denaturation of calcineurin indicated that the aCn5 epitope is of the assembled topographic, not segmental, type. The epitope was located to the A subunit and affinity was unaffected by the presence of calcineurin B. The epitope remained intact after proteolytic removal of the amino-terminal 20 residues of calcineurin A essential for phosphatase activity, and the carboxyl-terminal inhibitory and calmodulin-binding domains. The calmodulin-binding peptide derived from calcineurin, cA8, was not recognized by aCn5. Addition of Ca2+, Mn2+, Ni2+, chelators or dithiothreitol did not influence the affinity of aCn5 for the holoenzyme. Phosphatase activity of calcineurin, in the presence and absence of calmodulin and after removal of the inhibitory domain, was little affected by aCn5. Thus, the aCn5 epitope defines a previously unidentified structural domain of calcineurin A located in a region of the proteolytically resistant core that is topologically distinct from the catalytic, inhibitory, calmodulin-binding and calcineurin-B-binding domains, and not functionally connected with calcineurin B or the putative metal-binding domain(s).

Animals↗
PubMed1999

Inhibition of calcineurin phosphatase activity by a calcineurin B homologous protein.

Calcineurin, a Ca(2+)/calmodulin-stimulated protein phosphatase, plays a key role in T-cell activation by regulating the activity of NFAT (nuclear factor of activated T cells), a family of transcription factors required for the synthesis of several cytokine genes. Calcineurin is the target of the immunosuppressive drugs cyclosporin A and FK506 complexed with their cytoplasmic receptors cyclophilin and FKBP12, respectively. In this study we report that calcineurin is also the target of a recently identified Ca(2+)-binding protein, CHP (for calcineurin homologous protein), which shares a high degree of homology with the regulatory B subunit of calcineurin and with calmodulin. In Jurkat and HeLa cells, overexpression of CHP specifically impaired the nuclear translocation and transcriptional activity of NFAT but had no effect on AP-1 transcriptional activity and only a small (<25%) inhibitory effect on the transcriptional activity of NFkappaB. Further study indicated that CHP inhibits calcineurin activity. In cells overexpressing CHP, the phosphatase activity of immunoprecipitated calcineurin was inhibited by approximately 50%; and in a reconstituted assay, the activity of purified calcineurin was inhibited up to 97% by the addition of purified recombinant CHP in a dose-dependent manner. Moreover, prolonged activation of Jurkat cells was associated with a decreased abundance of CHP, suggesting a possible regulatory mechanism allowing activation of calcineurin. CHP, therefore, is a previously unrecognized endogenous inhibitor of calcineurin activity.

Calcineurin↗
PubMed2002

Calcineurin-dependent regulation of Crz1p nuclear export requires Msn5p and a conserved calcineurin docking site.

Calcineurin, a conserved Ca(2+)/calmodulin-regulated protein phosphatase, plays a crucial role in Ca(2+) signaling in a wide variety of cell types. In Saccharomyces cerevisiae, calcineurin positively regulates transcription in response to stress by dephosphorylating the transcription factor Crz1p/Tcn1p. Dephosphorylation promotes Crz1p nuclear localization in part by increasing the efficiency of its nuclear import. In this work, we show that calcineurin-dependent dephosphorylation of Crz1p also down-regulates its nuclear export. Using a genetic approach, we identify Msn5p as the exportin for Crz1p. In addition, we define the Crz1p nuclear export signal (NES) and show that it interacts with Msn5p in a phosphorylation-dependent manner. This indicates that calcineurin regulates Crz1p nuclear export by dephosphorylating and inactivating its NES. Finally, we define a motif in Crz1p, PIISIQ, similar to the PxIxIT docking site for calcineurin on the mammalian transcription factor NFAT, that mediates the in vivo interaction between calcineurin and Crz1p and is required for calcineurin-dependent regulation of Crz1p nuclear export and activity. Therefore, in yeast as in mammals, a docking site is required to target calcineurin to its substrate such that it can dephosphorylate it efficiently.

Active Transport, Cell Nucleus↗
PubMed1995

NMR identification of calcineurin B residues affected by binding of a calcineurin A peptide.

Triple resonance 3D NMR methods have been used to study the interaction between calcineurin B and a peptide fragment of calcineurin A for which it has high affinity (KD approximately 4 x 10(-7) M). Although calcineurin B aggregates at NMR concentrations of approximately 1 mM, in the presence of a target peptide fragment of calcineurin A it becomes monomeric and yields NMR spectra that are very similar to those reported previously for calcineurin B solubilized by the zwitterionic detergent CHAPS. Changes in chemical shifts between CHAPS- and peptide-solubilized calcineurin B are small which is indicative of no differences in secondary structure. Residues most affected by binding to target peptide are found primarily on the hydrophobic faces of the four helices, present in each of the two globular domains in calcineurin B, and in the loops connecting helices II and III, IV and V, and possibly in the C-terminal 12 residues, which also exhibit a change in mobility.

Amino Acid Sequence↗
PubMed1995

Only in the presence of immunophilins can cyclosporin and FK506 disrupt in vivo binding of calcineurin A to its autoinhibitory domain yet strengthen interaction between calcineurin A and B subunits.

The two immunosuppressants cyclosporin A (CsA) and FK506 exert their major therapeutic effect by inhibiting T-cell activation. It is believed that the drugs first bind to their cellular receptors, known as immunophilins, and then target the protein phosphatase calcineurin for inhibition. The catalytic activity of calcineurin is regulated by its autoinhibitory domain (AID) and by the calcium-binding proteins calcineurin B (CnB) and calmodulin. We have used the yeast two-hybrid system to show that AID, CnB and calmodulin can only bind to a truncated catalytic subunit of yeast calcineurin (i.e.,CnA1 delta), devoid of AID, but not to full-length CnA1. Both CsA and FK506 cause disruption of the CnA1 delta-AID interaction, whereas their presence permits CnA1 delta to bind more strongly to CnB. In contrast, the binding of CnA1 delta to calmodulin is unaffected by the immunosuppressants. Significantly, in the absence of its cognate cytosolic receptor, neither CsA nor FK506 inhibits or stimulates the CnA1 delta-AID, CnA1 delta-CnB interactions. These in vivo observations not only provide supportive evidence for the mechanism by which drug-receptor complexes could modulate calcineurin activity but also unveil the possibility of identifying novel immunophilin-independent calcineurin inhibitors which may disturb the association of CnA1 delta to AID.

Base Sequence↗
PubMed1992

Inhibition of calcineurin by cyclosporin A-cyclophilin requires calcineurin B.

The interaction of the immunosuppressive complex cyclosporin A-cyclophilin (CsA-CyP) with the Ca2+/calmodulin-dependent protein phosphatase calcineurin is investigated using a recombinant form of the A subunit of calcineurin (rCNA). Only in the presence of purified calcineurin B (CNB) does rCNA show the response of native calcineurin, i.e. 50% inhibition of rCNA phosphatase activity at 6 nM human cyclophilin B and 0.6 microM human cyclophilin A using [32P]casein as substrate, yet stimulation of activity with p-nitrophenyl phosphate as substrate. This study demonstrates that the B subunit is necessary to confer sensitivity of calcineurin to CsA-CyP.

Amino Acid Isomerases↗
PubMed2001

A novel calcineurin splice variant that modifies calcineurin activity.

Calcineurin is a Ca(2+)/calmodulin dependent phosphoprotein phosphatase implicated in a wide range of disorders. Here, we report the cloning of a novel calcineurin A alpha splice variant that lacks both the catalytic and calcineurin B binding domains. Biochemical analysis revealed a stimulating effect on calcineurin activity at low calcium concentrations as well as protein-protein interaction with the catalytic calcineurin holoenzyme. By Western blot analysis, expression of similar short splice variants could be seen in the spinal cord of an animal model of familial amyotrophic lateral sclerosis, suggesting a role of these new variants in motor neuron disease.

Amino Acid Sequence↗
PubMed1984

The calmodulin-dependent activation and deactivation of the phosphoprotein phosphatase, calcineurin, and the effect of nucleotides, pyrophosphate, and divalent metal ions. Identification of calcineurin as a Zn and Fe metalloenzyme.

Studies on the interaction of calcineurin with its activator, calmodulin, showed that the 1:1 complex is the activated species. Concomitant with activation, a time-dependent deactivation of the phosphatase was observed. The process followed first order kinetics and was dependent on the presence of both Ca2+ and calmodulin. The deactivation rate constant at pH 7.6 and 30 degrees C was 0.06 min-1, which was increased by the substrate, p-nitrophenylphosphate (Km = 11 mM), to 0.47 min-1. PPi and nucleotides inhibited the enzyme competitively and accelerated the deactivation. The first order rate constant was increased to 2.3 min-1 by PPi (Ki = 55 microM) and to 8.0 min-1 by ADP (Ki = 0.94 mM). A theory dealing with the deactivation (applicable to chemical modification, etc.) of an enzyme in the absence and presence of various ligands is presented. The deactivated enzyme remained bound to calmodulin and was not reactivated by dissociation-reassociation of the calcineurin-calmodulin complex. Calcineurin was found to contain covalently bound phosphate; however, no difference in its content was detected upon deactivation, indicating that self-dephosphorylation was not involved. The deactivation could be reversed, as well as prevented, by divalent metal ions such as Ni2+ and Mn2+. Atomic absorption spectroscopy revealed nearly stoichiometric amounts of tightly bound Fe and Zn (but little other ions) on purified calcineurin, which remained bound during the calmodulin-dependent deactivation; removal of tightly bound metals is, therefore, not the cause of deactivation. Our results indicate that calcineurin is a metallophosphatase and not simply a Ca2+- and calmodulin-stimulated enzyme. In addition to the nondissociable Zn and Fe and the Ca2+ bound to the B subunit and calmodulin, the enzyme requires a divalent metal ion for structural stability and full activity.

Adenosine Diphosphate↗
PubMed1999

Interactions of calcineurin A, calcineurin B, and Ca2+.

Calcineurin B (CN-B) is the Ca(2+)-binding, regulatory subunit of the phosphatase calcineurin. Point mutations to Ca(2+)-binding sites in CN-B were generated to disable individual Ca(2+)-binding sites and evaluate contributions from each site to calcineurin heterodimer formation. Ca(2+)-binding properties of four CN-B mutants and wild-type CN-B were analyzed by flow dialysis confirming that each CN-B mutant binds three Ca2+ and that wild-type CN-B binds four Ca2+. Macroscopic dissociation constants indicate that N-terminal Ca(2+)-binding sites have lower affinity for Ca2+ than the C-terminal sites. Each CN-B mutant was coexpressed with the catalytic subunit of calcineurin, CN-A, to produce heterodimers with specific disruption of one Ca(2+)-binding site. Enzymes containing CN-B with a mutation in Ca(2+)-binding sites 1 or 2 have a lower ratio of CN-B to CN-A and a lower phosphatase activity than those containing wild-type CN-B or mutants in sites 3 or 4. Effects of heterodimer formation on Ca2+ binding were assessed by monitoring (45)Ca2+ exchange by flow dialysis. Enzymes containing wild-type CN-B and mutants in sites 1 and 2 exchange (45)Ca2+ slowly from two sites whereas mutants in sites 3 and 4 exchange (45)Ca2+ slowly from a single site. These data indicate that the Ca2+ bound to sites 1 and 2 is likely to vary with Ca2+ concentration and may act in dynamic modulation of enzyme function, whereas Ca(2+)-binding sites 3 and 4 are saturated at all times and that Ca2+ bound to these sites is structural.

Amino Acid Sequence↗
PubMed1995

Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex.

Calcineurin (CaN) is a calcium- and calmodulin-dependent protein serine/threonine phosphate which is critical for several important cellular processes, including T-cell activation. CaN is the target of the immunosuppressive drugs cyclosporin A and FK506, which inhibit CaN after forming complexes with cytoplasmic binding proteins (cyclophilin and FKBP12, respectively). We report here the crystal structures of full-length human CaN at 2.1 A resolution and of the complex of human CaN with FKBP12-FK506 at 3.5 A resolution. In the native CaN structure, an auto-inhibitory element binds at the Zn/Fe-containing active site. The metal-site geometry and active-site water structure suggest a catalytic mechanism involving nucleophilic attack on the substrate phosphate by a metal-activated water molecule. In the FKBP12-FK506-CaN complex, the auto-inhibitory element is displaced from the active site. The site of binding of FKBP12-FK506 appears to be shared by other non-competitive inhibitors of calcineurin, including a natural anchoring protein.

A Kinase Anchor Proteins↗
PubMed1995

Distribution and activity of calcineurin in rat tissues. Evidence for post-transcriptional regulation of testis-specific calcineurin B.

Calcineurin (CN), a Ca2+/calmodulin-regulated phosphatase 2B, plays an important role in many biological processes including T-cell signal transduction. In the present study, the distribution and activity of CN were investigated in rat tissues. CN has a wide tissue distribution, as measured by enzyme-linked immunosorbent assay. CN concentrations are 0.2-0.6 micrograms/mg protein in most tissues, while the brain contains 3-10-fold higher concentrations. Immunohistochemical analyses using a monoclonal antibody to CN B subunit reveals that CN is not evenly distributed but concentrated in specific cells, especially in the brain, kidneys and testis. The specific enzymic activity of CN in tissues is around 10 pmol.min.mg protein-1, except in brain and liver (60 pmol.min-1.mg protein-1 compared to 3.6 pmol.min-1.mg protein-1). The immunosuppressants cyclosporin A and tacrolimus, but not rapamycin, inhibit the phosphatase activity of CN derived from most tissues tested, while CN activity from liver was resistant to cyclosporin A. Furthermore, transcripts and protein of the common CN B subunit and of the testis-specific form of CN B subunit were analyzed. The common CN B subunit transcripts and protein are detected in all tissues. Transcripts for the 'testis-specific' CN B subunit are also found in brain, lung, thymus and heart, while the protein is only detected in testis. This indicates that the testis-specific CN B subunit gene expression is regulated at both transcriptional and posttranscriptional levels. The findings demonstrate that CN is a widely distributed protein phosphatase and that its activity is regulated in a tissue-specific manner.

Amino Acid Sequence↗
PubMed1999

Two independent calcineurin-binding regions in the N-terminal domain of murine NF-ATx1 recruit calcineurin to murine NF-ATx1.

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.

Animals↗