Catalytic Subunit Of Calcineurin Research Articles

Cutting edge: Suppressor of cytokine signaling 3 inhibits activation of NFATp.

Recent studies have suggested that signaling initiated by the activation of Ag receptors and signaling activated through cytokine receptors may be regulated by a common set of inhibitory proteins. Suppressor of cytokine signaling 3 (SOCS-3), which has previously been demonstrated to inhibit cytokine signaling, is induced on TCR ligation. Overexpression of SOCS-3 can inhibit transcription driven by the IL-2 promoter in response to T cell activation. This inhibitory activity correlates with the suppression of calcineurin-dependent dephosphorylation and activation of the IL-2 promoter binding transcription factor, NFATp. Infection of primary murine T cells with a retrovirus encoding SOCS-3 blocks their IL-2 production in response to activation. Interestingly, SOCS-3 was found to coimmunoprecipitate with the catalytic subunit of calcineurin. These studies suggest that SOCS-3 may regulate T cell function as part of a negative feedback loop.

Enhanced myocyte contractility and Ca2+ handling in a calcineurin transgenic model of heart failure.

Impaired myocyte Ca2+ handling is a common characteristic of failing hearts and increases in calcineurin activity, a Ca2+-sensitive phosphatase, have been implicated in heart failure phenotype. Transgenic mice with cardiac-specific expression of an active form of calcineurin display depressed function, hypertrophy and heart failure. We examined whether defects in cardiomyocyte Ca2+ handling properties contribute to the impaired cardiac function in calcineurin transgenic mice. The levels of SR Ca2+ handling proteins, SR Ca2+ transport function and cardiomyocyte mechanics, as well as Ca2+ kinetics were examined in mice overexpressing a constitutively active form of calcineurin. Transgenic expression of activated calcineurin catalytic subunit resulted in significant protein increases (66%) in SERCA2 and decreases (35%) in phospholamban, as well as enhanced (approximately 80%) phospholamban phosphorylation. These alterations in the SR Ca2+-transport proteins resulted in increased V(max) and Ca2+-affinity of SERCA2. The myofibrillar Mg-ATPase activity was also significantly increased at pCa>6.0. The enhanced SR Ca2+ handling and Mg-ATPase activity reflected significant elevation in myocyte contractile parameters (3-fold), Ca2+ transient amplitude (1.5-fold) and the rate of Ca2+ signal decay (2-fold). In contrast, in vivo cardiac function assessed by echocardiography, indicated severely depressed contractility in calcineurin hearts. The apparent disparity in contractile properties between the cellular and multicellular preparations may be partially due to tissue remodeling, including interstitial fibrosis and a marked reduction (45%), dephosphorylation (81%) and redistribution of the gap junctional protein connexin-43, which could compromise intercellular communication. Despite enhanced SR Ca2+ handling and contractility in myocytes, pathological remodeling and defects in intercellular coupling may underlie contractile dysfunction of the calcineurin hearts.

A serine/threonine kinase which causes apoptosis-like cell death interacts with a calcineurin B-like protein capable of binding Na(+)/H(+) exchanger.

We surveyed proteins capable of binding to the cytoplasmic domain of Na(+)/H(+) exchanger (NHE)1 in a rat brain cDNA library with the yeast two-hybrid system. One clone obtained coded for a protein reported previously as a human calcineurin homologous protein (CHP). Since CHP is homologous to the regulatory subunit B of calcineurin, we expected a possible interacting partner of CHP like the catalytic subunit of calcineurin (calcineurin A), and surveyed this putative partner again with the yeast two-hybrid system. A clone thus obtained coded for a kinase, which is basically the same as that reported for human DRAK2. Overexpression of the rat homologue of DRAK2 caused apoptosis-like cell death of NIH3T3 cells, which was dependent on the kinase activity, confirming the previous result for DRAK2. The purified CHP and rat DRAK2 proteins synthesized in Escherichia coli could bind in vitro. CHP and rat DRAK2 expressed in COS-7 cells were found to be localized in the Golgi apparatus and nucleus, respectively. Some of them was also found in the membrane peripheral region. When they were co-expressed in the same cells, most of CHP moved to the nucleus where rat DRAK2 is located, suggesting in vivo interaction of these proteins. However, minor but significant fractions of both proteins were also found in the membrane peripheral region. Rat DRAK2 is expressed highly in thymus, spleen, and testis, where the apoptosis plays an important role in physiology.

Calsarcins, a novel family of sarcomeric calcineurin-binding proteins.

The calcium- and calmodulin-dependent protein phosphatase calcineurin has been implicated in the transduction of signals that control the hypertrophy of cardiac muscle and slow fiber gene expression in skeletal muscle. To identify proteins that mediate the effects of calcineurin on striated muscles, we used the calcineurin catalytic subunit in a two-hybrid screen for cardiac calcineurin-interacting proteins. From this screen, we discovered a member of a novel family of calcineurin-interacting proteins, termed calsarcins, which tether calcineurin to alpha-actinin at the z-line of the sarcomere of cardiac and skeletal muscle cells. Calsarcin-1 and calsarcin-2 are expressed in developing cardiac and skeletal muscle during embryogenesis, but calsarcin-1 is expressed specifically in adult cardiac and slow-twitch skeletal muscle, whereas calsarcin-2 is restricted to fast skeletal muscle. Calsarcins represent a novel family of sarcomeric proteins that link calcineurin with the contractile apparatus, thereby potentially coupling muscle activity to calcineurin activation.

Role of HSP90 in salt stress tolerance via stabilization and regulation of calcineurin.

The role of HSP90 in stress tolerance was investigated in Saccharomyces cerevisiae. Cells showing 20-fold overexpression of Hsc82, an HSP90 homologue in yeast, were hypersensitive to high-NaCl or H-LiCl stresses. Hsc82-overexpressing cells appeared similar to calcineurin-defective cells in salt sensitivity and showed reduced levels of calcineurin-dependent gene expression. Co-overexpression of Cna2, the catalytic subunit of calcineurin, suppressed the hypersensitivity. Cna2 and Hsc82 coimmunoprecipitated from control cells grown under normal conditions but not from stressed cells. In contrast, coimmunoprecipitation was detected with Hsc82-overexpressing cells even after exposure to stresses. Cna2 immune complexes from stressed control cells showed a significant level of calcineurin activity, whereas those from stressed Hsc82-overexpressing cells did not. Treatment of extracts from Hsc82-overexpressing cells with Ca(2+)-calmodulin increased the calcineurin activity associated with Cna2 immune complexes. Geldanamycin, an inhibitor of HSP90 abolished the coimmunoprecipitation but did not activate calcineurin. When the expression level of Hsc82 decreased to below 30% of the normal level, cells also became hypersensitive to salt stress. In these cells, the amount of Cna2 was reduced, likely as a result of degradation. The present results showed that Hsc82 binds to and stabilizes Cna2 and that dissociation of Cna2 from Hsc82 is necessary for its activation.

Molecular cloning and expression profile of Xenopus calcineurin A subunit

We have cloned a cDNA encoding a catalytic subunit of calcineurin (CnA) expressed in Xenopus oocytes. The deduced amino acid sequence indicates 96.3% and 96.8% identities with the mouse and human CnAα isoforms, respectively. Xenopus CnA (XCnA) RNA and protein are expressed as maternal and throughout development. Recombinant XCnA protein interacted with calmodulin in the presence of Ca 2+. Deletion of calmodulin binding domain and auto-inhibitory domain revealed calcium independent phosphatase activity, thereby showing that XCnA is likely to be modulated by both calmodulin and calcium.

An Essential Role of the Nuclear Factor of Activated T Cells in the Regulation of the Expression of the Cyclooxygenase-2 Gene in Human T Lymphocytes

We have previously reported that transcriptional induction of cyclooxygenase-2 (COX-2) isoenzyme occurs early after T cell receptor triggering, suggesting functional implications of cyclooxygenase activity in this process. Here, we identify the cis-acting elements responsible for the transcriptional activation of this gene in human T lymphocytes. COX-2 promoter activity was induced upon T cell activation both in primary resting T lymphocytes and in Jurkat cells. This induction was abrogated by inhibition of calcineurin phosphatase with the immunosuppressive drug cyclosporin A, whereas expression of an active calcineurin catalytic subunit enhanced COX-2 transcriptional activation. Moreover, cotransfection of nuclear factor of activated T cells (NFAT) wild type protein transactivated COX-2 promoter activity. Conversely, dominant negative mutants of NFATc or c-Jun proteins inhibited COX-2 induction. Electrophoretic mobility shift assays and site-directed mutagenesis allowed the identification of two regions of DNA located in the positions -117 and -58 relative to the transcriptional start site that serves as NFAT recognition sequences. These results emphasize the central role that the Ca(2+)/calcineurin pathway plays in COX-2 transcriptional regulation in T lymphocytes pointing to NFAT/activator protein-1 transcription factors as essential for COX-2 promoter regulation in these cells.

Calcineurin Co-regulates Contractile and Metabolic Components of Slow Muscle Phenotype

Activation of the transcription factor nuclear factor of activated T cells by the calcium-sensitive serine/threonine phosphatase calcineurin has been proposed as one of the molecular mechanisms by which motor nerve activity establishes the slow muscle phenotype. To investigate whether the calcineurin pathway can regulate the large spectrum of slow muscle characteristics in vivo, we treated rats for three weeks with cyclosporin A (an inhibitor of calcineurin). In soleus (slow muscle), but not in plantaris (fast muscle), the proportion of slow myosin heavy chain (MHC-1) and slow sarcoplasmic reticulum ATPase (SERCA2a) was decreased, whereas that of fast MHC (MHC-2A) and fast SERCA1 increased, indicating a slow to fast contractile phenotype transition. Cytosolic isoforms of creatine kinase and lactate dehydrogenase (most abundant in fast fibers), as well as mitochondrial creatine kinase and citrate synthase activities (elevated in fast/oxidative fibers) were dose dependently increased by cyclosporin A treatment in soleus muscle, with no change in plantaris. Calcineurin catalytic subunit was more abundant in soleus muscle fibers compared with plantaris. Taken together these results suggest that the calcineurin pathway co-regulates a set of multigenic protein families involved in the transition between slow oxidative (type I) to fast oxidative (type IIa) phenotype in soleus muscle.

Guinea-pig enteric nerves contain calcineurin and predominantly express the AB isoform of the catalytic subunit of calcineurin

Guinea-pig enteric nerves contain calcineurin and predominantly express the AB isoform of the catalytic subunit of calcineurin

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.

Identification of a Novel Region Critical for Calcineurin Function in Vivo and in Vitro

Calcineurin is a Ca2+/calmodulin-regulated protein phosphatase that plays critical functional roles in T-cell activation and other Ca2+-mediated signal transduction pathways in mammalian cells. In Saccharomyces cerevisiae, calcineurin regulates the transcription of several genes involved in maintaining ion homeostasis (PMC1, PMR1, and PMR2) and cell wall synthesis (FKS2). In this paper, we report the identification and characterization of 11 single amino acid substitutions in the yeast calcineurin catalytic subunit Cna1p. We show that six substitutions (R177G, F211S, S232F, D258V, L259P, and A262P) affect the stability of calcineurin and that two substitutions (V385D and M400R) disrupt the interaction between Cna1p and the calcineurin regulatory subunit Cnb1p. We also identify three mutations (S373P, H375L, and L379S) that are clustered between the catalytic and the calcineurin B subunit-binding domains. These mutations do not significantly affect the ability of Cna1p to interact with Cnb1p, calmodulin, or Fkb1p (FK506-binding protein). However, these residue substitutions dramatically affect calcineurin activity both in vitro and in vivo. Thus, by using a random mutagenesis approach, we have shown for the first time that the linker region of the calcineurin catalytic subunit, as defined by the Ser373, His375, and Leu379 residues, is crucial for its function as a phosphatase.

Dynamic NMR studies of ligand-receptor interactions: design and analysis of a rapidly exchanging complex of FKBP-12/FK506 with a 24 kDa calcineurin fragment.

Dynamic NMR methods, such as differential line broadening and transferred NOE spectroscopy, are normally reserved for the study of small molecule ligand interactions with large protein receptors. Using a combination of isotope labeling and isotope edited NMR, we have extended these techniques to characterize interactions of a much larger protein/drug complex, FKBP-12/ FK506 with its receptor protein, calcineurin. In order to examine this multicomponent system by dynamic NMR methods, the 93 kDa, tightly bound FKBP-12/FK506/Cn complex was replaced with a lower affinity, rapidly exchanging system consisting of FKBP-12/FK506 (13 kDa), recombinant calcineurin subunit B (CnB) (20 kDa), and a synthetic peptide (4 kDa) corresponding to the B binding domain (BBD) of calcineurin catalytic subunit A (CnA). Analysis of 1H-13C HSQC data acquired for the FKBP-12/ 13C-FK506 and FKBP-12/13C-FK506/CnB/BBD complexes indicates that FKBP-12/FK506 and CnB/BBD are in fast exchange in the quaternary complex. Comparison of proton line widths shows significant broadening of resonances along the macrocycle backbone at 13-CH, 13-OMe, 15-OMe, 18-CH2, 20-CH, 21-CH, and 25-Me, as well as moderate broadening on the macrocycle backbone at 17-Me, 24-CH, and the pyranose 12-CH2 protons. The tri-substituted olefin and cyclohexyl groups also show moderate broadening at the 27-Me, 28-CH, and 30-CH2 positions, respectively. Unexpectedly, little line broadening was observed for the allyl resonances of FK506 in the quaternary complex, although 13C longitudinal relaxation measurements suggest this group also makes contacts with calcineurin. In addition, intermolecular transfer NOE peaks were observed for the allyl 37-CH2, 21-CH, 30-CH2, 13-OMe, 15-OMe, 17-Me, 25-Me, and 27-Me groups, indicating that these are potential sites on the FK506 molecule that interact with calcineurin.

1 alpha,25-dihydroxyvitamin D3-induced upregulation of calcineurin during leukemic HL-60 cell differentiation

Cyclosporin A and FK506, at concentrations that inhibited phosphatase activity of calcineurin in HL-60 cellular lysates, augmented the proliferation of leukemic HL-60 cells. These immunosuppressants did not affect 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced monocytic differentiation of HL-60 cells, but did abrogate the 1,25(OH)2D3-induced inhibition of HL-60 cell growth. Treatment with 20 nmol/L 1,25(OH)2D3 led to a progressive increase in calcineurin phosphatase activity in subcellular fractions from HL-60 cell extracts, the increase in this activity appeared to parallel the phenotypic and functional changes of HL-60 cells during monocytic differentiation induced by 1,25(OH)2D3. Immunoblot analysis indicated that increase in calcineurin activity was concordant with the increased expressions of calcineurin catalytic subunit isozymes, calcineurin A alpha (CNA alpha), and calcineurin A beta(CNA beta), and a regulatory calcineurin B subunit (CNB) proteins, which were preceded by a coordinate increase in the levels of CNA alpha, CNA beta and CNB mRNAs. The expression of calmodulin remained unaltered throughout 1,25(OH)2D3-induced monocytic differentiation. These results suggest that calcineurin activation has a net negative effect on HL-60 cell proliferation, and that the increased expression of calcineurin may be involved in 1,25(OH)2D3-induced inhibition of HL-60 cell proliferation.

Signal Requirements for Interleukin 4 Promoter Activation in Human T Cells

We have studied the signal requirements for human IL-4 promoter activation in Jurkat T cells by the use of DNA transfection assays with vectors carrying the IL-4 promoter linked to a reporter gene. Stimulation with calcium (Ca2+) ionophores (ionomycin), but not with phorbol esters (phorbol myristate acetate, PMA) or cyclic AMP elevating agents (prostaglandin E2, PGE2), induced the transcriptional activity of the IL-4 promoter by ∼3-fold. Costimulation with ionomycin and PGE2resulted in the same level of IL-4 promoter activity as the stimulation with ionomycin alone. In contrast, costimulation with ionomycin and PMA decreased the activity of the IL-4 promoter by ∼40% compared to stimulation with ionomycin alone. Induction of IL-4 promoter by ionomycin was partially inhibited (∼50% inhibition) in the presence of as high as 2 μg/ml cyclosporin A (CsA), an inhibitor of the Ca+/calmodulin-dependent phosphatase calcineurin. Under the same conditions, only 0.1 μg/ml of CsA inhibited by >95% the transactivation of the IL-2 promoter in response to ionomycin and PMA. Transfection of a deletion mutant of the calcineurin catalytic subunit (ΔCaM-AI) known to have Ca2+-independent, constitutive phosphatase activity increased IL-4 promoter activity by ∼14-fold. Stimulation with ionomycin of cells transfected with low doses of ΔCaM-AI, further induced IL-4 promoter activity by ∼2-fold. These results identify the Ca2+-signaling system as a key component of the signal transduction pathway leading to IL-4 promoter activation in Jurkat T cells and suggest a major role of calcineurin in its regulation.

Inhibition of NF-AT Signal Transduction Events by a Dominant–Negative Form of Calcineurin

An inhibitory, "dominant-negative," form of the calcineurin catalytic (A) subunit was prepared, which lacks the calmodulin-binding domain, autoinhibitory domain and most of its catalytic core but possesses the regulatory (B) subunit binding domain. When tested for its ability to block calcineurin-dependent signaling in Jurkat cells, expression of this "B-subunit knock-out" (BKO) construct suppressed reporter gene activity driven by NF-AT, the pivotal promoter element for interleukin (IL)-2 gene induction. Immunoprecipitation of epitope-labeled BKO demonstrated for the formation of a tight complex with endogenous B subunit in Jurkat cells, consistent with an inhibitory mechanism that involves the sequestration of the B subunit. Furthermore, the sharply reduced NF-AT activity produced by co-transfecting BKO could be "rescued" by overexpression of transfected B subunit, suggesting that depletion of this subunit was responsible for the inhibition. These data suggest the potential utility of agents that disrupt calcineurin-mediated signal transduction pathways by blocking formation of the catalytically active dimer of calcineurin A and B subunits.

Calcineurin activation protects T cells from glucocorticoid-induced apoptosis.

In T cell hybridomas, TCR/CD3 complex-mediated stimulation induces apoptosis but inhibits that induced by glucocorticoids. A combination of ionomycin (IM), a calcium ionophore, and PMA, a protein kinase C activator, mimics the effects of the TCR/CD3-mediated stimulation. Glucocorticoid-induced apoptosis is, however, markedly inhibited by IM alone, and less markedly by PMA alone. The immunosuppressant FK506 canceled the inhibition by IM but not that by PMA. As calcineurin (CN) is one of the target molecules of FK506, we examined whether CN activation might have an anti-apoptotic effect. BOG8, a T cell hybridoma, was stably transfected with a mutant CN catalytic subunit with Ca2+/calmodulin-independent, constitutive but FK506-sensitive phosphatase activity. The transfectant clones were fairly resistant to glucocorticoid-induced death. Their resistance, however, was hardly affected by FK506 when added simultaneously with glucocorticoid, but was lost after a prolonged preincubation with FK506. In the parent BOG8 cells, FK506 failed to cancel the inhibitory effect of IM on glucocorticoid-induced death when the addition of FK506 was delayed for 1 h or more. These results suggest that CN activation is required for the resistance only as an early event. The transfectant clones produced IL-2 but failed to undergo apoptosis upon stimulation with PMA alone, whereas apoptosis was induced by a combination of IM and PMA. These results suggest that activation-induced cell death may require a higher level of CN activity than IL-2 production or may require another Ca(2+)-dependent pathway.

Molecular cloning and characterization of the genes encoding the two subunits of Drosophila melanogaster calcineurin.

Genomic clones containing the full coding sequences of the two subunits of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin, were isolated from a Drosophila melanogaster genomic library using highly conserved human cDNA probes. Three clones encoded a 19.3-kDa protein whose sequence is 88% identical to that of human calcineurin B, the Ca(2+)-binding regulatory subunit of calcineurin. The coding sequences of the Drosophila and human calcineurin B genes are 69% identical. Drosophila calcineurin B is the product of a single intron-less gene located at position 4F on the X chromosome. Drosophila genomic clones encoding a highly conserved region of calcineurin A, the catalytic subunit of calcineurin, were used to locate the calcineurin A gene at position 21 EF on the second chromosome of Drosophila and to isolate calcineurin A cDNA clones from a Drosophila embryonic cDNA library. The structure of the calcineurin A gene was determined by comparison of the genomic and cDNA sequences. Twelve exons, spread over a total of 6.6 kilobases, were found to encode a 64.6-kDa protein 73% identical to either human calcineurin A alpha or beta. At the nucleotide level Drosophila calcineurin A cDNA is 67 and 65% identical to human calcineurin A alpha and beta cDNAs, respectively. Major differences between human and Drosophila calcineurins A are restricted to the amino and carboxyl termini, including two stretches of repetitive sequences in the carboxyl-terminal third of the Drosophila molecule. Motifs characteristic of the putative catalytic centers of protein phosphatase-1 and -2A and calcineurin are almost perfectly conserved. The calmodulin-binding and auto-inhibitory domains, characteristic of all mammalian calcineurins A, are also conserved. A remarkable feature of the calcineurin A gene is the location of the intron/exon junctions at the boundaries of the functional domains and the apparent conservation of the intron/exon junctions from Drosophila to man.

FK-506- and CsA-sensitive activation of the interleukin-2 promoter by calcineurin

Antigen recognition by the T-cell receptor (TCR) initiates events including lymphokine gene transcription, particularly interleukin-2, that lead to T-cell activation. The immunosuppressive drugs, cyclosporin A (CsA) and FK-506, prevent T-cell proliferation by inhibiting a Ca(2+)-dependent event required for induction of interleukin-2 transcription. Complexes of FK-506 or CsA and their respective intracellular binding proteins inhibit the calmodulin-dependent protein phosphatase, calcineurin, in vitro. The pharmacological relevance of this observation to immunosuppression or drug toxicity is undetermined. Calcineurin, although present in lymphocytes, has not been implicated in TCR-mediated activation of lymphokine genes or in transcriptional regulation in general. Here we report that transfection of a calcineurin catalytic subunit increases the 50% inhibitory concentration (IC50) of the immunosuppressants FK-506 and CsA, and that a mutant subunit acts in synergy with phorbol ester alone to activate the interleukin-2 promoter in a drug-sensitive manner. These results implicate calcineurin as a component of the TCR signal transduction pathway by demonstrating its role in the drug-sensitive activation of the interleukin-2 promoter.

Chromosomal mapping of the human genes for the calmodulindependent protein phosphatase (calcineurin) catalytic subunit

Multiple catalytic subunits of the Ca2+ and calmodulin (CaM)-dependent protein phosphatase (PrP) ("calcineurin" or PrP-2B) are derived from at least two structural genes, type 1 ("calcineurin A alpha") and type 2 ("calcineurin A beta "), each of which can produce alternatively spliced transcripts. To examine the possible linkage of these genes, we analyzed genomic DNA from human/hamster hybrid cell lines using probes of 122 base pairs that were designed to bind selectively to exon 3 of the open reading frame. In this region, the nucleotide sequence of the type 2 murine cDNA that we cloned was greater than 99% identical to the type 2 human cDNA but only 78% identical to the type 1 human cDNA. Hybridization to Southern blots containing DNA from all human chromosomes showed that gene 1 was found on chromosome 4, whereas gene 2 segregated to chromosome 10. These data suggest that expression of the two calcineurin genes is not physically linked.

Characterization of the fragmented forms of calcineurin produced by calpain I

Calcineurin, a calmodulin-stimulated phosphatase from bovine brain, was hydrolyzed by calpain I from human erythrocytes. In the absence of calmodulin, calpain rapidly transformed the 60-kilodalton (kDa) catalytic subunit of calcineurin into a transient 57-kDa fragment and thereafter a 43-kDa limit fragment. In the presence of calmodulin, the 60-kDa subunit was sequentially proteolyzed to a 55-kDa fragment and then a 49-kDa fragment. Upon proteolysis in the absence or presence of calmodulin, the p-nitrophenyl phosphatase activity (assayed in the presence of calmodulin) was increased by 300%. The 43- and the 49-kDa fragments were found to (i) remain associated with the small subunit (17 kDa), (ii) have lost the ability to bind and to be activated by calmodulin, and (iii) have phosphatase activity that was still stimulated by Mn2+ or Ni2+. The 43- + 17-kDa form had similar Km values for various substrates, but the Vmax values were increased compared with the native enzyme. It is proposed that (i) a 43-kDa core segment of the 60-kDa subunit of calcineurin contained the catalytic domain, the small subunit-binding domain, and the metal ion (Mn2+ and (or) Ni2+) binding site; and (ii) two distinct types of inhibitory domains exist near the end(s) of the large subunit, one of which is calmodulin regulated, while the other is calmodulin independent.