From the Departments of Neuroscience, § Pharmacology and Molecular Sciences, and ¶ Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
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Calcineurin is a widely distributed protein phosphatase regulated by calcium and calmodulin. It mediates the immunosuppressive actions of drugs such as cyclosporin and FK506, and has been implicated in a number of calcium-sensitive pathways in the nervous system, including regulation of neurotransmitter release and modulation of long-term changes in synaptic plasticity. Calcineurin associates physiologically with other proteins, including calmodulin, FKBP12 (FK506-binding protein), the ryanodine receptor, and the inositol 1,4,5-trisphosphate receptor. We now report the identification, molecular cloning, and functional characterization of a novel protein, cain (calcineurin inhibitor), that interacts with and inhibits calcineurin. The full-length cain cDNA predicts a 240-kDa protein with no significant homology to any known protein. Cain associates with calcineurin both in vitro and in vivo, leading to a non-competitive inhibition of calcineurin activity. The putative calcineurin-binding domain of cain, a 38-amino acid region defined by mutational analysis, is highly basic. Like calcineurin, cain has a prominent neuronal expression and a wide tissue distribution. Cain's expression pattern in the brain closely resembles that of calcineurin, indicating a physiologic association between the two proteins.
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INTRODUCTION |
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Calcineurin is a serine/threonine protein phosphatase activated by calcium and the calmodulin-calcium complex (1, 2). This enzyme was first purified from the brain, where it was found in high concentrations in neurons (3). Calcineurin is a heterodimer consisting of a 60-kDa catalytic subunit (CnA)1 and a 19-kDa calcium-binding regulatory subunit (CnB). The discovery that immunophilins cyclophilin and FKBP12 (FK506-binding protein) form inhibitory complexes with calcineurin in the presence of immunophilin-binding drugs such as cyclosporin A and FK506 (4, 5) explained the mechanism of immunosuppression by these drugs (6), and established calcineurin as a mediator of T cell activation (7-9). Calcineurin activity is necessary for the synthesis of several cytokine genes through the dephosphorylation of a family of transcription factors known as NF-AT (nuclear factor of activated T cells) (10-13). By inhibiting calcineurin activity, cyclosporin A and FK506 prevent the nuclear translocation of NF-AT secondary to dephosphorylation, thereby suppressing T cell activation (14).
Although it is expressed in many tissues, calcineurin is most concentrated in neuronal systems, and comprises over 1% of total protein in brain (2, 15). Calcineurin has diverse roles in the nervous system, where it participates in many calcium-regulated pathways. Dynamin I, whose GTPase activity regulates synaptic vesicle recycling (16, 17), is a substrate of calcineurin (18, 19). Calcineurin modulates the GTPase activity of dynamin I by altering its phosphorylation state, thereby regulating the release of neurotransmitters such as glutamate (18, 20). Calcineurin regulates the activity of N-methyl-D-aspartate (NMDA) receptor channels by both altering their ion gating properties (21) and promoting desensitization (22). Elevation of intracellular calcium concentration following opening of the NMDA receptor channel activates several downstream proteins including neuronal nitric oxide synthase, which influences neuronal cell death secondary to glutamate NMDA neurotoxicity (23, 24). The activity of nitric oxide synthase is diminished by phosphorylation so that dephosphorylation and subsequent activation of nitric oxide synthase by calcineurin implicates this phosphatase in NMDA-mediated neurotoxicity (25).
Besides mediating glutamate neurotoxicity, NMDA receptors influence synaptic plasticity. A role for protein kinases in shaping synaptic plasticity is well established (26), as multiple experimental paradigms have demonstrated a close correlation between reduction of kinase activity and impairment of long-term potentiation in the hippocampus (27-29). More recently, phosphatases, especially calcineurin, have been implicated in regulating synaptic plasticity (15). Calcineurin has diverse, sometimes contradictory, effects on synaptic plasticity (30-32). Recently, Winder and colleagues (33) discovered that calcineurin inhibits a novel, intermediate phase of long-term potentiation, which influences the transition from short-term to long-term memory (34).
Despite the abundance of calcineurin and its role in many biologically important processes, few endogenous regulators of calcineurin have been identified. Calmodulin, bound to CnA, enhances its activity, while the autoinhibitory domain of CnA competitively inhibits the phosphatase activity (35). Superoxide dismutase prevents the in vivo inactivation of calcineurin secondary to oxidative damage (36), but there is no evidence for direct binding between the two proteins. FKBP12 associates with calcineurin, even in the absence of immunosuppressive drugs (37). This interaction involves residues distinct from those mediating the FK506-dependent association of FKBP12 and calcineurin, and allows FKBP12 to function as a calcineurin anchoring protein. FKBP12 targets calcineurin to intracellular calcium channels such as the ryanodine receptor (38-40) and the inositol 1,4,5-trisphosphate receptor (41). Calcineurin activity regulates the calcium flux through these channels secondary to changes in their phosphorylation states (42). Another well known anchoring protein for calcineurin is AKAP79 (A kinase anchoring protein), a scaffolding protein that also binds cAMP-dependent protein kinase and protein kinase C (43). Physical association between calcineurin and AKAP79 leads to inhibition of calcineurin activity, as well as its targeting to neuronal membranes (44).
It is unlikely that the above short list of proteins could adequately explain the diversity of calcineurin function. To better appreciate the role of calcineurin in multiple calcium-sensitive signaling pathways, we sought additional regulatory molecules for this unique phosphatase. We now report the identification and characterization of cain (calcineurin inhibitor), a novel, large protein that physiologically interacts with calcineurin and potently inhibits its catalytic activity.
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EXPERIMENTAL PROCEDURES |
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Materials-- FK506 was a gift from Dr. Joseph Steiner (Guilford Pharmaceutical). Glutathione-Sepharose and calmodulin-Sepharose beads were purchased from Pharmacia Biotech. Purified human FKBP12, purified bovine calcineurin, the RII peptide (calcineurin substrate), and cAMP-dependent protein kinase were purchased from Sigma. Anti-CnA antibody was obtained from Transduction Laboratories, and anti-CnB antibody was purchased from Upstate Biotechnology Inc. The rabbit polyclonal anti-cain antibody was raised against (His)6-cain (described below), and affinity-purified using the immunogen as described (45).
Plasmid Constructs for Yeast Two-hybrid Analysis and in Vitro
Binding Assays--
CnA was subcloned into SalI and
NotI sites of pPC86/97 (46) from the full-length human
calcineurin A open reading frame, using the following polymerase
chain reaction primers: 5'-acgcgtcgaccatggccgccccggag-3' and
5'-ttttgcggccgctcactgggcagtatggtt-3'. Cain deletion mutants were
generated from the cain fragment obtained from the two-hybrid screen
using the following polymerase chain reaction primers and an
appropriate vector primer: cain1518-1610,
5'-ttttgcggccgccaggtgatggcagcagcc-3'; cain1518-1683,
5'-ttttgcggccgcgcctccactggccagcag-3'; cain1518-1752, 5'-ttttgcggccgcgtaggtggtgtgacggtg-3'; cain1762-1810,
5'-acgcgtcgaccagcatctcagaggagac-3'; cain1715-1810,
5'-acgcgtcgactcaaggcaaaactgagtc-3'. Inserts were then digested
with SalI and NotI and cloned into corresponding sites in pPC86 and pGEX-4T-2 (Amersham Pharmacia Biotech). All of the
constructs were verified by sequencing.
Yeast Two-hybrid Screen--
The full-length human FKBP12 open
reading frame was cloned into yeast expression vector pPC97, containing
the GAL4 DNA-binding domain. This was used to screen a rat hippocampal
cDNA library cloned into pPC86 (47), containing the GAL4
transactivation domain. The plasmids were introduced sequencially by
LiAc-mediated transformation into FKBP12-deficient HF7C yeast strain,
constructed as described (48). A total of 1.5 × 106
independent clones were screened, and positive interacting proteins were identified by selecting for His+ growth phenotype with
a strict dependence on the presence of FK506. Positive clones were
further evaluated for -galactosidase expression by nitrocellulose
filter lift assays as described (46).
In Vitro Binding Assays--
GST fusion proteins were prepared
according to manufacturer's recommendations (Pharmacia Biotech) and
coupled to glutathione-Sepharose beads. (His)6-cain
construct was generated by subcloning the cain fragment isolated from
the two-hybrid screen into the SalI and NotI
sites of pET-28c(+) vector (Novagen). The fusion protein was prepared
as per the manufacturer's protocol. In each binding assay, 2 µg of
purified (His)6-cain and/or 2 µg of purified bovine calcineurin were added to 20 µl of the GST-fusion protein-Sepharose slurry (50% (w/v) in phosphate-buffered saline) and brought to a final
volume of 200 µl with binding buffer (50 mM Tris-HCl, pH
7.4, 100 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.2% Triton X-100, 0.5 mg/ml bovine
serum albumin, 0.5 mM -mercaptoethanol). After binding
was allowed to proceed at 4 °C for 1 h with gentle mixing, the
samples were washed three times with binding buffer, boiled in SDS
sample buffer, and separated by SDS-PAGE. Proteins were transferred
onto nitrocellulose membrane and immunoblotted with anti-T7 antibody
(Novagen) at 1:10,000 dilution and anti-CnB antibody at 1:1,000
dilution, followed by a 1:5,000 dilution of anti-mouse Ig antibody
conjugated to horseradish peroxidase. Western blots were visualized
using an enhanced chemiluminescence system (ECL, Amersham).
Co-precipitation Assays Using Transfected Cells--
The
two-hybrid cain fragment was subcloned into a mammalian expression
vector with a cytomegalovirus promoter and an in-frame N-terminal
myc epitope tag. This myc-cain construct was
introduced into HEK293 cells by calcium phosphate-mediated
transfection. Cells were harvested 48 h following transfection and
lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.2% Triton X-100, 0.5 mM -mercaptoethanol, 5 µg/ml aprotinin, 1 µg/ml
leupeptin, 6 µg/ml chymostatin, 0.7 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride). Equal amounts of cell
lysate were incubated with GST-Sepharose, GST-FKBP12-Sepharose, or
calmodulin-Sepharose beads (20 µl of 50% slurry) for 1 h at
4 °C with gentle agitation. Where indicated, GST-FKBP12-Sepharose
beads were preabsorbed with FK506 for 1 h at 4 °C and then
washed twice in lysis buffer prior to adding to the cell lysate. FK506
was also added to the cell lysate in co-precipitation assays as
indicated. Following the incubation, the Sepharose beads were washed
three times with lysis buffer and boiled in SDS sample buffer. SDS-PAGE
and Western blotting were performed as described above. The
anti-myc antibody was used at 1:3,000 dilution, and anti-CnA
antibody at 1:250 dilution.
Tissue Co-precipitation Assays-- Adult rat brain was homogenized in lysis buffer (described above) and spun at 20,000 × g for 20 min to remove insoluble materials. The resulting lysate was divided into equal aliquots and incubated with GST-FKBP12-Sepharose beads in the same manner described above. Bound proteins were analyzed by SDS-PAGE and Western blotting as described.
Cloning of Full-length Cain cDNA--
An adult rat brain
cDNA library in ZAPII vector (Stratagene) was screened using the
two-hybrid cain fragment, labeled with P in a nick
translation system (Boehringer Mannheim). A 5.4-kilobase open reading
frame was identified, and a probe from the N-terminal sequence
(nucleotides 41-1040) was generated for a second screen. A total of
4.0 × 106 clones were screened, yielding 33 overlapping inserts. 5'-Rapid amplification of cDNA ends was
performed using Marathon-Ready rat brain cDNA
(CLONTECH) according to manufacturer's
protocol.
Calcineurin Activity Assay-- The assay was carried out as described by Hubbard and Klee (49) unless otherwise indicated. A peptide corresponding to the phosphorylation site on the RII subunit of cAMP-dependent protein kinase was phosphorylated and used as substrate. Assays were performed in 50-µl aliquots containing 40 mM Tris-HCl, pH 7.4, 100 mM NaCl, 6 mM MgCl2, 0.1 mM CaCl2, 0.1 mg/ml bovine serum albumin, 0.5 mM dithiothreitol, 100 nM calmodulin, and 25 nM bovine calcineurin (Sigma). After 1 h preincubation with (His)6-cain at 4 °C, reactions were started by the addition of 2-150 µM substrate peptide. Assays were performed at 25 °C for 8 min and terminated by the addition of 5% trichloroacetic acid in 0.1 M KH2PO4.
Northern Blot Analysis-- A commercial rat multiple tissue Northern blot (CLONTECH) was hybridized according to the manufacturer's recommendations with the N-terminal cain probe described above.
Multiple Tissue Western Analysis-- Tissues from an adult male rat were harvested and homogenized in ice-cold phosphate-buffered saline + 5 µg/ml aprotinin, 1 µg/ml leupeptin, 6 µg/ml chymostatin, 0.7 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. Each homogenate was centrifuged at 16,000 × g for 20 min and the pellet discarded. Equal amounts (40 µg of proteins) from all samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membrane. Immunoblotting was performed using affinity-purified polyclonal anti-cain antibody, followed by anti-rabbit Ig at 1:5,000 dilution and detected with ECL reagents.
In Situ Hybridization-- RNA probes for FKBP12, CnA, and cain were generated by subcloning each open reading frame from pPC86 or pPC97 into the SalI and NotI sites of pBS(SK)II+ (Stratagene). Brain slices for in situ hybridization were prepared and probed as described (50).
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RESULTS |
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Identification of Cain, a Novel Calcineurin-binding Protein-- In initial experiments we utilized the yeast two-hybrid system to identify proteins that selectively bind FKBP12 in the presence of the drug FK506. We screened a rat hippocampal cDNA library and identified a 300-amino acid protein fragment without homology to any known protein sequence. This fragment constitutes the C-terminal portion of cain (calcineurin inhibitor).
In the yeast two-hybrid analysis, wild-type HF7c and FKBP12-deficient HF7c yeast strains were used to assay protein interactions based upon the activation of two reporters,
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Cloning and Mutational Analysis of Cain-- Utilizing nucleotide probes derived from the cain sequence, we screened a rat brain cDNA library and obtained 33 overlapping clones. A 5'-rapid amplification of cDNA ends polymerase chain reaction yielded no additional upstream sequence (data not shown). The full-length cDNA of cain assembled from overlapping clones contains 7272 nucleotides. The starting methionine was assigned to the first in-frame AUG codon (nucleotide 332). This AUG is preceded by two in-frame stop codons and exists in the context of a Kozak translation initiation consensus (51). The 6.5-kilobase open reading frame codes for a protein of 2182 amino acids with a predicted molecular mass of 243 kDa (Fig. 3). A search of sequence data bases revealed no significant homology with any previously characterized protein.
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Cain is a Potent, Non-competitive Inhibitor of Calcineurin-- To evaluate influences of cain on calcineurin catalytic activity, we assayed calcineurin activity in the presence of purified (His)6-cain. Cain potently inhibited calcineurin activity in vitro with half-maximal inhibition evident at about 0.4-0.5 µM and a calculated Ki value of 0.44 µM (Fig. 5A). A Dixon plot indicates that the inhibition is non-competitive (Fig. 5B).
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Cain Has a Wide Tissue Distribution-- Using Northern analysis, we detected a 7.5-kilobase transcript of cain enriched in brain, kidney, and testis but which also can be detected in all other tissues examined (Fig. 6A). This size transcript is consistent with our predicted full-length cDNA. In addition, a smaller and less abundant transcript, possibly resulting from alternative splicing, can be detected in all tissues examined.
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Cain Expression Closely Resembles Calcineurin in the Brain-- Calcineurin and FKBP12 share highly similar distribution patterns in the brain, supporting a functional interaction between these two proteins (52). In situ hybridization of cain, calcineurin, and FKBP12, revealed a striking similarity between cain and calcineurin expression (Fig. 7A). Both display low expression in the brainstem, but are prominent in the olfactory bulb, caudate, nucleus accumbens, olfactory tubercle, zona reticulata of the substantia nigra, and all layers of the hippocampus and dentate gyrus. The co-localization of cain and calcineurin transcripts appears even greater than that of FKBP12 and calcineurin. In the olfactory epithelium, where FKBP12 is virtually undetectable, both cain and calcineurin are present in great abundance (Fig. 7B). FKBP12 is localized predominantly to cerebellar Purkinje cells and not to granule cells. While cain and calcineurin are both enriched in granule cells, calcineurin also occurs in Purkinje cells (Fig. 7C).
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DISCUSSION |
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In the present study we have identified a novel protein, cain, which physiologically interacts with and inhibits calcineurin. Multiple experimental paradigms including the yeast two-hybrid system and several binding studies reveal that the interaction occurs both in vitro and in vivo. We also detected physiologic association of cain and calcineurin in both brain and liver tissues. Perhaps the most impressive evidence establishing a physiologic link between cain and calcineurin is their strikingly similar localizations in the brain, where both proteins occur exclusively in neurons. Previously, we reported a close similarity between localizations of calcineurin and FBKP12 in the brain, supporting a physiological relationship between the two proteins (52). In situ hybridization reveals an even greater similarity between cain and calcineurin expression than that between calcineurin and FKBP12.
Cain inhibits calcineurin with a Ki value of about 0.4 µM, and appears to be the most potent known endogenous inhibitor of calcineurin. AKAP79, a well established calcineurin inhibitor, is about 0.2% as potent in this regard (44). The autoinhibitory peptide of calcineurin itself has a Ki value of 10-50 µM (35). Whether the inhibitory action of cain is exerted upon CnA or CnB is unclear. In our experiments we observe binding of cain to both CnA and CnB. However, the two calcineurin subunits are so tightly associated that it is not unexpected that they would bind as a complex. Cain binds to calcineurin at location(s) distinct from the FKBP12/FK506-binding sites, as multiple experiments showed that the association between cain and calcineurin is unaltered by the addition of FKBP12/FK506. FKBP12/FK506 sterically blocks access to the substrate-binding site of calcineurin, thereby inhibiting calcineurin activity (53, 54). The binding and subsequent inhibition of calcineurin by cain defines an independent regulatory site on calcineurin. We have further shown that this site is most likely distinct from the calmodulin-binding domain of calcineurin, as changes in calmodulin concentration produced no alteration in cain's inhibition of calcineurin activity.
How might cain physiologically regulate calcineurin? Our limited subcellular fractionation experiments indicate that cain is a soluble, cytosolic protein. However, it could well translocate to the cytoskeleton to which it might be loosely bound. Cain may function as a scaffolding protein attached to numerous proteins besides calcineurin. Consistent with this is the fact that calcineurin binding to cain is restricted to a small area in the C-terminal portion of the protein.
One might conceptualize functions of cain in analogy to AKAP79, another large protein that binds and inhibits calcineurin. For both cain and AKAP79, the putative calcineurin-binding site contains a preponderance of basic amino acid residues (44). AKAP79 binds cAMP-dependent protein kinase and protein kinase C as well as calcineurin. Conceivably, cain could also bind kinases. Whether the kinase and phosphatase activities of proteins attached to cain are exerted upon cain itself is not clear, as phosphorylation patterns for cain have not yet been established. Although we have identified cain as an inhibitor of calcineurin, its physiological role may be to provide a docking site for calcineurin in its inactive form. In this regard, cain may target inactivated calcineurin to specific intracellular locations where its release would provide calcium-regulated phosphatase activity to specific signaling pathways.
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ACKNOWLEDGEMENTS |
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We thank Jenny Hong and Hyung Chun for expert
technical assistance and careful reading of the manuscript. We also
thank Roxanne Ashworth for sequencing the full-length cain cDNA,
Noam Cohen for help with multiple tissue Western, Dr. Claude Klee for
the generous gift of human CnA cDNA, Dr. Joseph Steiner for a
gift of FK506, David Sabatini and members of the Snyder laboratory for
helpful advice and insightful discussions.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant MH-18501 from the National Institute of Mental Health, Research Scientist Award DA-00074 (to S. H. S.) from NIDA, National Institutes of Health, and Training Grant GM-07309 (to M. M. L. and P. E. B.) from NIGMS, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF061947.
To whom all correspondence should be addressed. Tel.:
410-955-3024; Fax: 410-955-3623.
1 The abbreviations used are: CnA, 60-kDa catalytic subunit; CaB, 19-kDa calcium-binding regulatory subunit; NMDA, N-methyl-D-aspartate.
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REFERENCES |
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