Physiological Reviews, Vol. 80, No. 4, October 2000, pp. 1483-1521
Copyright ©2000 by the American Physiological Society

Calcineurin: Form and Function

Section of Hematology Research and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota; and Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, Massachusetts

I. INTRODUCTION
II. A BRIEF HISTORY AND OVERVIEW OF CALCINEURIN
    A.  Calcineurin: The Early Years
    B.  Calcineurin Properties
III. PHYSIOLOGICAL ROLES FOR CALCINEURIN
    A.  Lower Eukaryotes
    B.  Higher Eukaryotes
    C.  Inhibitors of Calcineurin
IV. CALCINEURIN STRUCTURE
    A.  A Dinuclear Metal-Binding Phosphoesterase Motif
    B.  Three-Dimensional Structure
    C.  Active Site Architecture
    D.  Metal Ion Requirements
V. ENZYMATIC MECHANISM
    A.  Mechanism of Phosphoryl Group Transfer: Evidence for Direct Transfer to Water
    B.  Catalytic Role of the Dinuclear Metal Center
    C.  Conserved Active Site Residues
    D.  A Model for the Calcineurin Catalytic Mechanism
VI. REGULATION

	ABSTRACT

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Rusnak, Frank and Pamela Mertz. Calcineurin: Form and Function. Physiol. Rev. 80: 1483-1521, 2000.Calcineurin is a eukaryotic Ca²⁺- and calmodulin-dependent serine/threonine protein phosphatase. It is a heterodimeric protein consisting of a catalytic subunit calcineurin A, which contains an active site dinuclear metal center, and a tightly associated, myristoylated, Ca²⁺-binding subunit, calcineurin B. The primary sequence of both subunits and heterodimeric quaternary structure is highly conserved from yeast to mammals. As a serine/threonine protein phosphatase, calcineurin participates in a number of cellular processes and Ca²⁺-dependent signal transduction pathways. Calcineurin is potently inhibited by immunosuppressant drugs, cyclosporin A and FK506, in the presence of their respective cytoplasmic immunophilin proteins, cyclophilin and FK506-binding protein. Many studies have used these immunosuppressant drugs and/or modern genetic techniques to disrupt calcineurin in model organisms such as yeast, filamentous fungi, plants, vertebrates, and mammals to explore its biological function. Recent advances regarding calcineurin structure include the determination of its three-dimensional structure. In addition, biochemical and spectroscopic studies are beginning to unravel aspects of the mechanism of phosphate ester hydrolysis including the importance of the dinuclear metal ion cofactor and metal ion redox chemistry, studies which may lead to new calcineurin inhibitors. This review provides a comprehensive examination of the biological roles of calcineurin and reviews aspects related to its structure and catalytic mechanism.

	I. INTRODUCTION

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The year 1999 marked the 20th anniversary of the isolation of the Ca²⁺- and calmodulin-dependent protein serine/threonine phosphatase calcineurin (206). During the past 20 years, the biological roles of calcineurin have progressed from a putative inhibitor of the calmodulin-dependent phosphodiesterase (444) to the ground-breaking discovery that it is the target of the immunosuppressant drugs cyclosporin A (CsA) and FK506, pharmacological reagents that have been used to demonstrate it as a major player in Ca²⁺-dependent eukaryotic signal transduction pathways (238). In recent years, several milestones regarding calcineurin structure have been achieved including the determination of the three-dimensional structure by X-ray diffraction methods (124, 197) and biochemical, spectroscopic, and physical studies that are beginning to unravel its catalytic mechanism (150, 259, 261, 262, 470, 471). Insight into its physiological functions include mapping its subcellular localization (10, 106, 175, 219, 286, 306); the discovery of its colocalization with other important signaling proteins (365); and, aside from Ca²⁺/calmodulin, the finding of possible endogenous regulators of its activity including redox and/or oxidative stress (45, 111, 345, 447, 470, 471) as well as interacting proteins (224, 234, 277, 359, 400). The next generation of studies, which includes the use of transgenic mouse technology, is beginning to reveal interesting yet sometimes subtle roles for this enzyme in the whole organism (181, 254, 281, 320, 460, 477).

It will not be the attempt of this review to provide an all-encompassing survey of calcineurin. In fact, several excellent review articles on calcineurin and other protein serine/threonine phosphatases are available, some quite comprehensive (60, 130, 203, 205, 207, 208, 316, 371). In addition, numerous specialized articles focusing on particular aspects of either calcineurin structure or function have been published. A list of these appears in Table 1 for the benefit of the reader who would prefer to be directed to specific calcineurin-related topics. Rather, we focus on a comprehensive treatise of some of the recent developments of calcineurin since the last major review was published (371) (ca. 1990 to present).

View this table: [in this window] [in a new window]   Table 1. Calcineurin reviews listed according to subject

	II. A BRIEF HISTORY AND OVERVIEW OF CALCINEURIN

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A.  Calcineurin: The Early Years

Calcineurin was first detected by Wang and Desai (444) as a column fraction that inhibited the calmodulin-dependent cyclic nucleotide phosphodiesterase. Independently, Watterson and Vanaman (452) also obtained highly purified fractions of calcineurin from bovine brain extract by use of calmodulin-affinity chromatography but erroneously referred to the 58- and 18-kDa subunits of calcineurin as "affinity-purified phosphodiesterase." Klee and Krinks (206) are credited with the first purification of calcineurin and hypothesized that it might be a regulatory subunit of phosphodiesterase since it was demonstrated to inhibit phosphodiesterase activity. Other groups subsequently showed that calcineurin inhibited the Ca²⁺/calmodulin-dependent isozymes of cyclic nucleotide phosphodiesterase and adenylate cyclase by competing for calmodulin in a Ca²⁺-dependent fashion, and they speculated that its function may be regulatory (435, 436, 445). Shortly thereafter, Klee et al. (204) coined the descriptive label "calcineurin" on the basis of its Ca²⁺-binding properties and localization to neuronal tissue (204), a popularized name which is widely used to date and which we will use throughout this review. At that time, the true function of calcineurin had yet to be revealed. It was not until pioneering work in the early 1980s in Philip Cohen's lab, investigating cellular extracts capable of dephosphorylating the - and -subunits of phosphorylase kinase, that a fraction represented as protein phosphatase 2B (PP2B) was demonstrated to be identical to Klee's calcineurin (390, 391).

B.  Calcineurin Properties

Biochemical studies during the 1980s continued and determined many of the physical properties listed in Table 2 (61, 130, 208). Purified calcineurin is a heterodimer consisting of a catalytic subunit, calcineurin A, and a "regulatory" subunit, calcineurin B. 

View this table: [in this window] [in a new window]   Table 2. Physical properties of calcineurin

Cloning efforts have provided evidence that all eukaryotic organisms possess one or more genes for each subunit; Table 3 is a compilation of known calcineurin A and calcineurin B gene sequences to date. Genes for calcineurin A and B subunits have been identified in yeast, filamentous fungi, protozoa, insects, and mammals. The -quaternary structure of calcineurin observed in mammals is conserved in lower eukaryotic organisms. These subunits are tightly associated and can only be dissociated by use of denaturants (271).

View this table: [in this window] [in a new window]   Table 3. A comprehensive list of published calcineurin A and calcineurin B gene sequences

1.  Calcineurin A

A) CLASSIFICATION. In addition to calcineurin, the serine/threonine protein phosphatase family members include protein phosphatases 1 (PP1), 2A (PP2A), and 2C (PP2C), phosphatases essential for a number of signal transduction pathways in eukaryotic cells (61, 371). The original classification of this family was proposed by Ingebritsen and Cohen (167), separating almost all the serine/threonine phosphatase activity in mammalian tissue extracts into two classes (60, 61, 167, 371). Type 1 protein phosphatases were found to dephosphorylate the -subunit of phosphorylase kinase, whereas type 2 protein phosphatases dephosphorylate the -subunit of phosphorylase kinase. Differences between the two types are also found with inhibitors; type 1 is inhibited by phosphopeptide inhibitors 1 and 2, whereas the type 2 class is not affected by these inhibitors.

A difference in divalent metal ion dependence led to the resolution of the type 2 enzymes into PP2A, PP2B (calcineurin), and PP2C (60). PP2A was originally described as having no requirement for divalent metal ion, calcineurin is regulated by Ca²⁺/calmodulin, and PP2C is Mg²⁺ dependent.¹ A more detailed discussion regarding the importance of metal ion cofactors of these phosphatases is described in section IVD. Differences among type 2 members are also found with regard to sensitivity to inhibition by macrolide inhibitors (see sect. IIIC1). PP2A and PP1 are inhibited by okadaic acid, whereas calcineurin is specifically inhibited by the immunosuppressant drugs CsA and FK506, in the presence of cyclophilin and FK506-binding protein (FKBP), respectively (238, 361).

Although different in metal ion dependence, sensitivity to inhibitors, and substrate specificity, PP1, PP2A, and calcineurin have homologous amino acid sequences and are evolutionarily related (26, 61, 371). In fact, the PP1/PP2A/calcineurin superfamily ranks among one of the most highly conserved enzyme families encountered (61). Within the active site domain, PP1 shares 49% amino acid identity with PP2A and 39% identity with calcineurin. Recent work has found homologs of this family in cyanobacteria (373, 473) and the archea (229, 251, 386). With the determination of the amino acid sequences of these phosphatases, it was found that the original classification of PP2C along side PP1, PP2A, and calcineurin does not hold at the primary sequence level. Thus PP2C does not share any homology with PP1/2A/calcineurin and is considered to be in a separate superfamily (33).

B) DOMAIN STRUCTURE. The active site of calcineurin is located on the A subunit which, in mammals, is 57-59 kDa depending on the isoform. The size of the catalytic subunit can be up to ~20% larger in lower eukaryotic species [e.g., Saccharomyces cerevisiae, 63 and 69 kDa (72, 242, 467); Schizosaccharomyces pombe, 64 kDa (327, 468); Drosophila melanogaster, 62 and 65 kDa (38, 132, 158); Cryptococcus neoformans and Dictyostelium discoideum, 71 kDa (75, 310)]. Nevertheless, there is strict conservation throughout all eukaryotic organisms such that all calcineurin A genes encode for a polypeptide consisting of a catalytic domain homologous to other serine/threonine protein phosphatases and three regulatory domains at the COOH terminus that distinguish calcineurin from other family members (Fig. 1). These domains have been identified as the calcineurin B binding domain (56, 132, 164, 379, 451), the calmodulin-binding domain (132, 191), and the "autoinhibitory" domain (137, 326), which binds in the active site cleft in the absence of Ca²⁺/calmodulin (197) and inhibits the enzyme, acting in concert with the calmodulin binding domain to confer calmodulin regulation.

View larger version (86K): [in this window] [in a new window]   Fig. 1. Primary sequence and domain structure of calcineurin A. The amino acid sequence represents the rat calcineurin A -isoform reported by Saitoh et al. (354). CaM, calmodulin; CNB, calcineurin B; AI, autoinhibitory.

These domains have been identified through standard biochemical mapping procedures including primary sequence comparisons, partial proteolysis experiments, peptide interaction studies, site-directed mutagenesis studies, and cross-linking studies. The X-ray structures of calcineurin (see sect. IVB) confirm the identification of residues involved in these regulatory domains and also indicate that the autoinhibitory domain forms an -helix that binds to the substrate-binding cleft of the enzyme (197). It has been shown that when Ca²⁺/calmodulin binds to the enzyme, inhibition ceases and likely involves a conformational change that exposes the active site. Interestingly, Perrino (324) has provided recent evidence for additional autoinhibitory elements within residues that are situated between the calmodulin-binding and autoinhibitory domains noted in Figure 1.

The NH₂ and COOH termini are highly variable between species as well as between calcineurin A genes within the same organism (130, 188, 208). Particularly striking are unusual amino acid compositions and sequences of particular isoforms. For example, the mammalian calcineurin A-isoform contains the sequence MAAPEPARAAPPPPPPPPPPPGAD... at the NH₂ terminus (117, 129, 220). The COOH terminus of the canA gene product from Dictyostelium contains a fourfold repeat of the sequence RXNSX(G/A)(E/D)LX as well as the repetitious sequence ..TTNNINPNSITTNENNSNEQLQQQQQQQQQQQPPTTTSTTT.. and, along with the NH₂ terminus, is enriched in glutamine and asparagine (75). The COOH terminus of calcineurin A from C. neoformans is highly enriched in proline, serine, threonine, and glycine (310). The function of these variable domains is unknown, but they may play a role in substrate recognition and/or localization.

C) CALCINEURIN PHOSPHORYLATION. Purified calcineurin from bovine brain contains up to 0.6 equivalents of phosphate (195), suggesting that it may be phosphorylated in vivo. Calcineurin can be phosphorylated by protein kinase C (138, 420), casein kinase I (382), and casein kinase II (136, 138, 257) in vitro. The site of phosphorylation by casein kinase II has been determined to be the serine in the sequence -RVFS(p)VLR- near the COOH terminus of the calmodulin-binding domain (Fig. 1) (138, 257). Although phosphorylation could be blocked by Ca²⁺/calmodulin, the kinetic properties of the phosphorylated and dephosphorylated forms are similar (136, 138). Furthermore, phosphorylated calcineurin still binds and is activated by calmodulin. Whether phosphorylation represents a means of regulating calcineurin activity in vivo remains to be demonstrated.

2.  Calcineurin B

A) SEQUENCE DIVERSITY AND ISOFORMS. The calcineurin B subunit is also highly conserved throughout evolution, with mammalian calcineurin B showing 86% amino acid sequence identity with insect calcineurin B (i.e., Drosophila) and 54% identity with calcineurin B from S. cerevisiae (Fig. 2). This high degree of conservation allows functional interchange of calcineurin B subunits between mammalian and N. crassa catalytic subunits (423). The gene for mammalian calcineurin B encodes a protein of 170 amino acids containing four Ca²⁺-binding EF-hand motifs (Fig. 2) (2).

View larger version (75K): [in this window] [in a new window]   Fig. 2. Multiple sequence alignment of calcineurin B sequences from diverse organisms. Calcineurin B sequences of Saccharomyces cerevisiae (221), Neurospora crassa (213), rat brain (48), rat testes (289), human (131), bovine (2, 304), Drosophila melanogaster (132, 450), and Naegleria gruberi (346) were aligned using the multiple sequence alignment editor of the Wisconsin Package version 9.0, Genetics Computer Group (Madison, WI). The four Ca2+-binding EF-hand motifs are indicated. The residues that participate in Ca2+ coordination are noted by an asterisk. The consensus sequence is defined in which a residue is conserved in all 8 sequences.

In mammals, there are two calcineurin B genes, one which is ubiquitously expressed, while mRNA for the second gene is found only in testes (48, 289, 424).

B) NH₂-TERMINAL MYRISTOYLATION. The mature calcineurin B protein is missing the initiator methionine, and the new -amino group of glycine at position 2 is acylated with myristic acid (1). This modification has been conserved throughout evolution from yeast to mammals, suggesting a crucial physiological role (71). To explore possible biological roles for calcineurin myristoylation, Heitman and colleagues (483) generated a mutant of calcineurin B in which glycine at position 2 was mutated to alanine, thereby preventing myristoylation. Surprisingly, expression of the wild-type and mutant proteins in S. cerevisiae demonstrated that myristoylation was not required for membrane association nor for interaction with immunosuppressant drug complexes. Indeed, the nonmyristoylated protein exhibited full biological function. These results were subsequently confirmed in biochemical experiments with purified myristoylated and nonmyristoylated calcineurin heterodimer which showed equivalent enzymatic activities, inhibition by the CsA/cyclophilin immunosuppressant drug complex, and interactions with a synthetic phospholipid monolayer (182). Interestingly, the myristoylated protein exhibited substantial thermal stability (~12°C) relative to the nonmyristolyated protein (182). At present, it is unknown whether the biological role of calcineurin B myristoylation is to impart increased stability to the protein or whether there is another role yet to be identified.

C) CALCIUM BINDING PROPERTIES. Klee et al. (204) were the first to discover that calcineurin binds Ca²⁺. With the use of flow dialysis, it was demonstrated that four Ca²⁺ bind with high affinity [dissociation constant (K_d) 10⁶ M] and that the Ca²⁺-binding sites were localized to the calcineurin B subunit. The complete primary sequence determination of calcineurin B revealed homology with calmodulin (35% identity) and troponin C (29% identity) (2), most of which was confined to four Ca²⁺-binding "EF-hand" motifs. More detailed thermodynamic aspects of Ca²⁺ binding became possible when the recombinant calcineurin B subunit was obtained via heterologous expression in Escherichia coli. Using the purified recombinant protein, Burroughs et al. (40) studied the metal binding properties using Eu³⁺ and Tb³⁺ luminescence spectroscopy. Four Eu³⁺-binding sites were revealed, two with relatively low affinity (K_d values of 1 ± 0.2 and 1.6 ± 0.5 µM) and two with relatively high affinity (K_d values of 0.14 ± 0.020 and 0.020 ± 0.010 µM). Tb³⁺ also bound but with slightly weaker affinities (K_d values of 0.04 ± 0.01 and 0.17 ± 0.02 µM for the COOH-terminal sites and 1-3 µM for the NH₂-terminal sites). Direct Ca²⁺ binding to calcineurin B has also been studied by flow dialysis, which found one high-affinity (K_d = 0.024 µM) and three lower affinity sites (K_d = 15 µM) (176). The NMR-active isotope ¹¹³Cd has been used as a Ca²⁺ surrogate to identify four similar but distinct metal binding sites consisting of all-oxygen coordination of pentagonal bipyramidal geometry as expected for an EF-hand Ca²⁺-binding site (176), later confirmed in the X-ray structure (124, 197). Ca²⁺ binding to individual sites of calcineurin B has been studied using point mutants of this subunit altered in each of the four EF-hands (104). This study confirmed the higher Ca²⁺ affinity for COOH-terminal EF-hand sites and also found that Ca²⁺ binding at these sites is likely to be structural.

D) CALCINEURIN B HOMOLOGS. EF-hand proteins have been classified into 39 distinct subfamilies containing anywhere from two to eight EF-hand domains (178). Calcineurin B proteins represent one subfamily of EF-hand proteins based on sequence alignments and congruence of domain and interdomain regions (302). In recent years, a number of Ca²⁺-binding proteins containing EF-hand domains have been identified from cloning studies and shown to be homologous to calcineurin B. These include NCS-1, a neuronal calcium sensor that inhibits rhodopsin phosphorylation in a Ca²⁺-dependent fashion (81), modulates calmodulin targets (359), and may regulate exocytosis (264); a protein p22/CHP (for calcineurin homologous protein) that is required for constitutive membrane traffic (25) and inhibits serum and GTPase stimulation of the Na⁺/H⁺ exchanger NHE1 (234); CIB (for calcium- and integrin-binding protein), a 22-kDa Ca²⁺-binding protein that interacts with the cytoplasmic tail of the integrin _IIb portion of the GPIIb/IIIa fibrinogen receptor (297); and a protein product of the SOS3 gene of Arabidopsis thaliana involved in tolerance to the ionic component of salt stress in plants (239). Homology to calcineurin B ranges from 27 to 31% for NCS-1, CIB, and SOS3 and to up to 43% for p22/CHP. Like calcineurin B, p22/CHP is myristoylated while NCS-1, CIB, and SOS3 contain the requisite consensus sequences for myristoylation. The fact that a constitutively active form of yeast calcineurin in transgenic tobacco plants resulted in increased salt tolerance provides evidence for a possible functional overlap between SOS3 and calcineurin B (320). On the contrary, biochemical studies with NCS-1 suggested that it could replace calmodulin rather than calcineurin B in activating calcineurin and other calmodulin-dependent enzymes (359). Furthermore, although p22/CHP is completely congruent with the calcineurin B family of proteins, arguments have been forwarded that it is more appropriate to place it in a separate subfamily (25). Undoubtedly, further studies are required to determine whether any or all of these proteins can function in a fashion comparable to calcineurin B.

	III. PHYSIOLOGICAL ROLES FOR CALCINEURIN

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A.  Lower Eukaryotes

Genetic methods for the selective deletion of one or both calcineurin subunits to assess biological function by noting the phenotype of the mutant strain are now possible in several eukaryotic organisms. In addition, the immunosuppressant drugs CsA and FK506, as specific calcineurin inhibitors, have provided complementary tools for discerning the role of calcineurin in many eukaryotic organisms (71, 142, 303, 387). Some of the most thorough work investigating biological roles for calcineurin have used the yeast S. cerevisiae as a model system. There are two genes for the catalytic subunit of calcineurin in S. cerevisiae (CNA1/CMP1 and CNA2/CMP2) and only one gene for the B subunit (CNB1). Calcineurin is essential in CsA- and FK506-sensitive yeast strains (36). Recent work has begun to explore the role of calcineurin in slightly more complex organisms such as N. crassa (153, 213, 335) and D. discoideum (75, 159, 252). Furthermore, calcineurin has either been isolated or detected from the human pathogens C. neoformans (310), Leishmania species (21, 342), the malarial parasite Plasmodium falciparum (87), helminth parasites (347), and schistosomes (186). In some of these, growth can be inhibited by the immunosuppressive agents FK506 and its analogs as well as CsA (16, 87, 186, 309, 342), thus raising the possibility that novel calcineurin inhibitors might be developed as specific antifungal and antiparasitic agents. The following sections detail what has been learned regarding physiological roles for calcineurin in lower eukaryotic organisms and is summarized in Table 4.

View this table: [in this window] [in a new window]   Table 4. Physiological roles of calcineurin in lower eukaryotic organisms

1.  Saccharomyces cerevisiae

A) RECOVERY FROM PHEROMONE-INDUCED GROWTH ARREST. Haploid cells of S. cerevisiae produce one of two mating pheromones, a-factor and -factor. Exposure of haploid strains to the opposite mating pheromone prepares cells for mating by inducing cell cycle arrest in G₁. This is mediated by an elaborate signal transduction pathway involving a rise in intracellular Ca²⁺ and activation of calcineurin (47, 142). Growth arrest can be observed as a zone of clearing surrounding a source of -factor on a lawn of cells and occurs within 24 h at 30°C. Escape from -factor-induced cell cycle arrest involves three metabolic processes that have been referred to as recovery, adaptation, and survival (287). Recovery is defined as the ability of cells to resume growth after removal of the pheromone, whereas adaptation is a process in which cells eventually resume growth in the continuous presence of pheromone. Both recovery and adaptation may involve common signaling components and can be observed by a shrinking of the zone of clearing and increasing turbidity within it, usually within ~24 h at 30°C. Survival differs from recovery and adaptation in that it describes whether a cell remains viable after exposure to pheromone.

Coincident with the cloning of genes for the two yeast calcineurin A subunits (CNA1/CMP1 and CNA2/CMP2, Table 3), strains deficient in either subunit were viable but failed to recover from -factor-induced growth arrest (72, 73, 242). Mata strains containing a single CNA1 or CNA2 mutation were twice as sensitive as wild type to -factor-induced growth arrest, whereas the double mutant CNA1/CNA2 was four times as sensitive, as assessed by the size of the halo after 24 h at 30°C (72, 73). Furthermore, once arrested, the double mutant failed to resume growth. In contrast, the CNB1 mutant did not show an increased sensitivity compared with wild type, but like the CNA1/CNA2 double mutant, it failed to recover from growth arrest. In wild-type cells, the immunosuppressant drugs CsA and FK506 also inhibited recovery from -factor-mediated growth arrest, and these required the presence of their respective immunophilins cyclophilin and FKBP (107). In addition, expression of the CNA1/CMP1 gene increased in the presence of -factor, the result of 5'-noncoding sequences in the CNA1/CMP1 gene matching closely the consensus sequence for the -factor element (467).

As expected, the activator protein of calcineurin, calmodulin, has also been shown to be required for escape from cell cycle arrest after exposure to pheromone (287). Calmodulin mutants did not display increased sensitivity to -factor, nor did these mutant strains appear to be affected in either recovery or adaptation. Indeed, both calcineurin and calmodulin mutants adapted as well as a wild-type strain to low concentrations of pheromone, and both mutants recovered after pheromone removal with the same kinetics as the wild-type strain. The process that appeared to be affected was survival, a result consistent with previous work indicating that Ca²⁺ is also essential for survival after exposure to -factor (166). Interestingly, in addition to calcineurin, the Ca²⁺, calmodulin-dependent protein kinases (CMK1 and CMK2), yeast protein kinase C (PKC1), and a mitogen-activated protein (MAP) kinase (MPK1) are also required for recovery from growth arrest, thus indicating that enzymes of opposing function are required for surviving exposure to -factor (287, 301, 461).

One downstream signaling component in S. cerevisiae regulated by calcineurin is the yeast transcription factor Crz1p/Tcn1p. Crz1p/Tcn1p is required for calcineurin-dependent induction of genes for the vacuolar and secretory Ca²⁺ pumps, Pmc1p and Pmr1p, respectively; one of two genes encoding -1,3 glucan synthase, FKS2; and the gene for the plasma membrane Na⁺ pump, PMR2 (Fig. 3) (263, 388). In addition, calcineurin has been shown to regulate the high-/low-affinity state of the plasma membrane K⁺ channel, Trk1p (269), and inhibit the vacuolar H⁺/Ca²⁺ exchanger Vcx1p (69) by posttranlational mechanisms. Some of these are presented below in more detail.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Physiological roles of calcineurin in Saccharomyces cerevisiae. Calcineurin has numerous roles in budding yeast including recovery from -factor-induced growth arrest, salt and temperature tolerance, Ca2+ homeostasis, and Mn2+ tolerance (387). Many of these are mediated by Crz1p/Tcn1p, a calcineurin-dependent transcription factor necessary for the transcriptional induction of Pmc1p, Pmr1p, Pmr2p, and Fks2 (heavy arrows) (263, 388). In addition, calcineurin inhibits the activity of the vacuolar H+/Ca2+ exchanger (Vcx1p) and causes conversion of the K+ channel, Trk1p, to the high-affinity state. The latter occur independently of Crz1p probably by posttranslational processes (narrow arrows) (69). Cch1p, Ca2+ channel (313); Crz1p, calcineurin-responsive zinc finger transcription factor, product of the CRZ1 gene; Pmcp1, high-affinity vacuolar Ca2+ pump, product of the PMC1 gene (69); Pmr1p: secretory Ca2+ pump, product of the PMR1 gene; Tcn1p: alternative name for Crz1p protein; Vcx1p: low-affinity vacuolar H+/Ca2+ exchanger (69). Mid1p is an alternative name for the plasma membrane Ca2+ channel.

B) ADAPTATION TO SALT STRESS. The search for additional phenotypes found that calcineurin-deficient yeast exhibited decreased tolerance to the monovalent cations Na⁺ and Li⁺, but not K⁺, Ca²⁺, and Mg²⁺ (269, 300). The role of calcineurin in Na⁺/Li⁺ tolerance is thought to be mediated by transcriptional and posttranslational mechanisms. Adaptation to high salt stress requires the presence of a plasma membrane Na⁺-ATPase involved in Na⁺ and Li⁺ efflux, Pmr2p. Cells deficient in calcineurin accumulate Na⁺ and Li⁺ due to decreased expression of Pmr2p (269). Although no changes in intracellular Ca²⁺ have been observed after induction of the high-salt response, evidence indicates that Ca²⁺ mediates this response. Ca²⁺, via calmodulin activation of calcineurin, regulates adaptation to high salt stress by induced expression of Pmr2p (76, 154, 268), mediated by the transcription factor Crz1p/Tcn1p (Fig. 3) (263, 388). The activity of Pmr2p is also stimulated by Ca²⁺/calmodulin, thereby providing both transcriptional and posttranslational regulation of Na⁺ efflux mediated by Ca²⁺ (349, 454).

Cells deficient in CNB1 are unable to convert the K⁺ transport system (Trk1p, a K⁺ channel) to a high-affinity state. In the high-affinity state, this pump has increased affinity for K⁺, but the Michaelis constant (K_m) for Na⁺ or Li⁺ is unaffected, thereby resulting in increased Na⁺ uptake in calcineurin-deficient cells. The mechanism of this regulation has been hypothesized to be direct or indirect dephosphorylation of Trk1p by calcineurin (269).

Other proteins in addition to calcineurin are required for salt tolerance such as the gene products of PDE1, a low-affinity cAMP-dependent phosphodiesterase (154); URE2, a regulator of nitrogen catabolite repression (462); PMA1, the plasma membrane H⁺-ATPase (462); HAL3, a protein involved in cell cycle control and ion homeostasis (105); and STD1, a protein that interacts with the SNF1 protein kinase in two-hybrid and in vitro binding studies (113). Thus multiple parallel pathways are necessary for full induction of this response.

C) CALCIUM HOMEOSTASIS. Calcineurin is involved in the regulation of Ca²⁺ pumps and exchangers responsible for Ca²⁺ homeostasis in yeast (Fig. 3). These maintain cytoplasmic [Ca²⁺] in the range of 100-300 nM (68, 78). In addition, other ion transporters indirectly influence intracellular [Ca²⁺]. One of these is the vacuolar H⁺-ATPase, which provides the driving force for Ca²⁺ sequestration by the Ca²⁺/H⁺ exchanger encoded for by the VCX1 gene (114, 144, 408). Two Ca²⁺-ATPases, Pmc1p and Pmr1p, are responsible for depleting the cytosol of Ca²⁺. The former is localized to the vacuole (70), while the latter is important in the secretory pathway and localizes to the Golgi (349). Mutants deleted in either Pmc1p or Pmr1p cannot grow in media containing high Ca²⁺. Deletion of the gene for either calcineurin subunit, or treatment of cells with CsA or FK506, restores growth to either single PMC1 or double PMC1/PMR1 mutants in high Ca²⁺ media (70), indicating that calcineurin activation can have a negative effect on growth. As noted above, activation of calcineurin leads to transcriptional induction of the PMC1 and PMR1 genes via Crz1p/Tcn1p (263, 388).

Calcineurin mutants are also sensitive to extracellular Mn²⁺. Wild-type strains are able to prevent Mn²⁺ entry, whereas mutants exhibit an increased uptake phenotype (100), therefore indicating that the regulation of Mn²⁺ homeostasis by calcineurin follows a different mechanism than monovalent cation transport, in which export is regulated by a P-type ATPase (269, 300). An alternative hypothesis has been proposed in which Pmr1p, the Golgi-localized Ca²⁺ pump, plays a role in Mn²⁺ tolerance by sequestering Mn²⁺ to late compartments in the secretory pathway (263). Mn²⁺ may also be transported into the vacuole via the Ca²⁺/H⁺ exchanger Vcx1p (332).

D) -GLUCAN SYNTHASE AND CELL WALL SYNTHESIS. Calcineurin is responsible for transcriptional regulation of FKS2, one of two genes encoding -1,3-glucan synthase (Fig. 3) (95, 480). Calcineurin-dependent regulation occurs through Crz1p/Tcn1p (263, 388). The Fks1p protein is the predominant synthase expressed during optimum growth, but expression of Fks2p is induced upon treatment of cells with mating pheromone, high Ca²⁺, or growth on poor carbon sources. Deletion of the FKS1 and CNB1 genes results in lethality due to the inability to induce FKS2 (114). In fact, FKS1 mutants are hypersensitive to FK506 (95). These results suggest that calcineurin plays a role in regulating cell wall structure.

2.  Schizosaccharomyces pombe

Like the budding yeast, treatment of S. pombe with FK506 or deletion of the ppb1+ gene, encoding for the calcineurin A subunit (Table 3), is not lethal. However, calcineurin in fission yeast appears to have distinct functions. Calcineurin-deficient S. pombe cells exhibit drastic Cl-sensitive growth (399) and are defective in cytokinesis, transport, nuclear and spindle pole body positioning, cell shape (468), and sporulation (327). One function for calcineurin in S. pombe that appears to overlap with S. cerevisiae is the mating process, although the roles for calcineurin in mating appear to be distinct in these two organisms. In S. cerevisiae, calcineurin is required for the cell to recover from or survive growth arrest after exposure to pheromone. It thus may function to assist cells to reenter the cell cycle if they respond to -factor but fail to mate (see sect. IIIA1A). In S. pombe, calcineurin is required for the mating response, and calcineurin mutants in this organism are sterile (327, 468). Northern analysis indicates that the transcript for calcineurin varies during the cell cycle and can be induced by nitrogen limitation, a condition that favors mating in S. pombe (327). The latter effect was dependent on the transcription factor ste1.

3.  Neurospora crassa

N. crassa has been widely used as a model system for studying eukaryotic gene expression. In this fungus, calcineurin is thought to play a major role in hyphal extension during mycelial growth and in determining apical orientation. Thus calcineurin mRNA exhibited the highest expression during early mycelial logarithmic growth but was repressed before conidiation upon entry into stationary phase (153). A similar pattern of protein expression was observed but with about a 12-h lag behind message expression. Expression of antisense mRNA to the catalytic subunit, treatment with CsA and FK506, or disruption of the calcineurin B gene caused growth arrest preceded by aberrant and increased hyphal branching and entry into conidiation, consistent with a role in apical growth (213, 335). Growth on two different carbon sources, glutamate and sucrose, did not influence the level of expression, indicating that calcineurin is not involved in mechanisms related to catabolite repression (153).

4.  Aspergillus nidulans

Similar to N. crassa, calcineurin A mRNA levels vary during the cell cycle in A. nidulans, and disruption of the cnaA⁺ gene resulted in growth arrest (303, 343). After growth arrest in metaphase upon treatment with nocodazole followed by resuspension in fresh media to allow for synchronous growth, it was shown that calcineurin A message levels peak after the end of mitosis before DNA replication, with the highest expression appearing at the G₁/S boundary. Inducible gene disruption by homologous recombination revealed that the calcineurin A gene was essential for normal growth, and disruption was lethal. Further results indicated that the cnaA⁺ gene was required for early cell cycle events before DNA replication. Taken together with other data, this study suggested that calcineurin as well as other calmodulin targets may be required during different periods of the cell cycle.

5.  Cryptococcus neoformans

Fungal diseases are becoming an increasing health problem, most notably in individuals infected with human immunodeficiency virus (HIV); C. neoformans represents one of these life-threatening infectious agents (6). Heitman and colleagues (5, 67, 309, 310) have begun to explore the role of calcineurin in this pathogen with the goal of using novel CsA and FK506 analogs that have increased specificity toward the fungal calcineurin compared with the host (human) enzyme. Such compounds may eventually prove useful as antifungal agents with reduced toxicity and immunosuppressive effect toward the host. One such candidate is the FK506 analog L-685,818 (18-hydroxy, 21-ethyl-FK506). L-685,818 is toxic to C. neoformans, mediated by binding and inhibiting the fungal calcineurin, but has reduced immunosuppressive activity in humans (309). Growth studies in the presence of CsA and FK506 indicate that these drugs are growth inhibitory at 37°C but not at 24°C and that they inhibit a common target. Disruption of the calcineurin A gene resulted in mutant strains that are viable at 24°C but do not survive under conditions that mimic the host environment including elevated temperature, 5% CO₂, or alkaline pH (310). These mutant strains are no longer pathogenic, thus indicating that calcineurin is necessary for virulence in this organism.

6.  Dictyostelium discoideum

Calcineurin in the slime mold D. discoideum exhibited the familiar developmental pattern of expression as noted above for the filamentous fungi, with the highest level of expression during vegetative growth and decreasing expression during multicellular development (75). CsA and FK506 had no effect on growth, a process that can be separated from development in this organism (159). However, these drugs do inhibit developmental processes such as stalk cell spore formation and expression of prestalk and prespore developmental markers.

7.  Other lower eukaryotic organisms

A gene for calcineurin B has been isolated in the amoeboflagellate N. gruberi (346). mRNA levels are detectable in the amoebae and are cyclic, with peak abundance during flagellar formation, followed by a gradual decline. In the unicellular organism Paramecium tetraurelia, calcineurin localization was investigated by use of a specific antibody and immunocytochemical methods (209). Calcineurin was largely localized to the cilia and cell membrane, with only a diffuse staining pattern observed within the cell body. Further staining indicated that there was no difference in either localization or abundance in cells prepared either in logarithmic or stationary phase. Thus calcineurin abundance does not appear to change during the cell cycle as it does in the simple fungi. To further explore calcineurin's role in P. tetraurelia, anticalcineurin antibody or Ca²⁺/calmodulin-calcineurin was microinjected into cells. Anticalcineurin antibody blocked exocytosis after treatment with the exocytosis trigger agent, aminoethyldextran, while microinjection of a complex of Ca²⁺/calmodulin-calcineurin induced exocytosis. These results implicate calcineurin as the phosphatase previously shown to dephosphorylate a 63-kDa protein hypothesized to be involved in trichocyst exocytosis (198).

Recently, calcineurin has been isolated from Leishmania major (342) and Leishmania donovani (21). Calcineurin was isolated by chromatographic separation of cytosol from promastigotes where it was hypothesized to be a key regulatory component in the life cycle of this parasite. Interestingly, in L. major, extracellular growth is not inhibited by CsA, and in fact, a high-affinity complex of CsA with L. major cyclophilin forms [inhibitory constant (K_i) = 5.2 nM] but does not inhibit or form a tight complex with calcineurin from that organism, suggesting a possible mechanism for this organism's resistance to CsA (342). Interestingly, a complex between CsA, recombinant human cyclophilin, and L. major calcineurin was formed indicating that the parasitic calcineurin is functionally and structurally equivalent to mammalian calcineurin. A similar phenomenon was observed with calcineurin from the tapeworms Hymenolepsis microstoma and Hymenolepsis diminuta such that calcineurin from both organisms was inhibited by CsA complexed with mammalian cyclophilin but not H. microstoma cyclophilin (347). This was not the case with calcineurin from Schistoma mansoni (186) and Plasmodium falciparum (87). One hypothesis to explain the lack of complex formation with calcineurin is that parasitic cyclophilins are structurally different from mammalian cyclophilins, such that cyclophilin residues surrounding the CsA binding site that interact with calcineurin are not conserved in parasitic cyclophilins. Further studies are necessary to resolve these interesting findings.

B.  Higher Eukaryotes

1.  Calcineurin in plants

Evidence for a plant homolog of calcineurin was first obtained by Luan et al. (246) who demonstrated, using patch-clamp techniques, that CsA and FK506 blocked Ca²⁺-dependent inactivation of K⁺ channels in Vicia faba. A partially proteolyzed and constituitively active form of calcineurin also inhibited K⁺ channel activity. Furthermore, both CsA and FK506 inhibited a Ca²⁺-dependent phosphatase activity in cellular extract. Subsequent studies have provided additional evidence for calcineurin function in plants (reviewed in Ref. 245).

To date, however, calcineurin has not been successfully purified to homogeneity from plant tissue nor have bona fide genes for either subunit been cloned. The closest contenders are two EF-hand Ca²⁺-binding proteins encoded for by the SOS3 and AtCBL genes that are homologous to the calcineurin B subunit (218, 239). The protein encoded by the SOS3 gene is 30% identical to calcineurin B from various organisms, and mutations in SOS3 render A. thaliana sensitive to Na⁺ (239). The SOS3 protein is also homologous to NCS-1 (30% identity), a neuronal Ca²⁺ sensor in the recoverin family of EF-hand proteins (see sect. IIB2D). The AtCBL proteins are most homologous to calcineurin B (32% identity to rat calcineurin B) and can complement a yeast calcineurin B mutation, indicating a calcineurin B-like physiological function (218). Recent work, however, indicates that the AtCBL proteins interact with a novel group of protein kinases in a Ca²⁺-dependent fashion (372). A. thaliana contains at least six AtCBL genes. The AtCBL and SOS3 proteins clearly play different roles since they are unable to complement each other (218). It is intriguing that SOS3 and AtCBL encode for Ca²⁺-binding proteins, indicating that salt stress in plants may be regulated by Ca²⁺-dependent signaling pathways (possibly via calcineurin) as has been found in S. cerevisiae (see sect. IIIA1B). Further evidence for this hypothesis was obtained in a study demonstrating that overexpression of an activated form of yeast calcineurin conferred salt tolerance in transgenic tobacco plants (320). Similarly, genes for three of the AtCBL isoforms appear to be stress regulated. Whether SOS3 or the AtCBL proteins represent plant calcineurin B homologs or just close relatives will hopefully be resolved if a protein corresponding to plant calcineurin can be isolated and/or cloned and shown to be a functional phosphatase.

2.  Calcineurin in mammals

A) TISSUE DISTRIBUTION. Calcineurin is widely distributed in mammalian tissues, with the highest levels found in brain (168, 175, 216, 437). In addition, calcineurin A and B subunits have been observed in adipose tissue, adrenal cells (318, 319), bone osteoclasts (19), heart, hindbrain and spinal cord (394), kidney (42, 418, 419), liver (135), B and T lymphocytes (4, 50, 193), lung, medulla, olfactory bulb, pancreas (112), placenta (314), platelets (406, 438), retina (66), skeletal muscle (168), smooth muscle, spleen, testis and sperm (278, 286, 396, 409), thymus (42, 193), and thyroid (121).

Distinct tissue distribution is observed for the various isoforms of each subunit (42, 175, 219). An isoform of the catalytic subunit encoded for by the PPP3CC gene (-isoform, see Table 3) is testes specific (291, 292), as is the product of the PPP3R2 gene encoding an isoform of the regulatory subunit (48, 424). With the use of polyclonal antibodies that distinguish between the - and -isoforms of calcineurin A (encoded by the PPP3CA and PPP3CB genes, respectively; Table 3), it was found that calcineurin A was more abundant than A in the rat brain and heart, but the relative abundance is reversed in spleen, thymus, and lymphocytes (175, 219). These results partly explain the recent finding that PPP3CA knock-out mice produce T and B cells that mature normally, respond to mitogenic stimulation, and remain sensitive to both CsA and FK506, but are defective in in vivo antigen-specific T-cell responses (477). These PPP3CA-deficient mice also accumulate hyperphosphorylated tau protein and exhibit cytoskeletal changes in the hippocampus as a result of reduced calcineurin A activity (181). More recent studies indicate that synaptic depotentiation is completely abolished, indicating that calcineurin A may play a role in the learning and memory process (181, 485).

B) SUBCELLULAR DISTRIBUTION. Using a radioimmunoassay, Cheung and colleagues (10) measured the subcellular distribution of calcineurin in chick forebrain homogenate. In that study, calcineurin was highly enriched in the cytoplasmic and microsomal fractions as well as synaptosomes. Subsequent studies have confirmed its predominance in the cytoplasm and synaptosomal cytosol (219, 306). Politino and King (329) explored the physical association of calcineurin with synthetic phospholipid vesicles and showed that calcineurin binds small, acidic, unilamellar vesicles in a Ca²⁺-dependent fashion. Furthermore, the phosphatase activity of calcineurin was profoundly affected by phosphatidylglycerol or phosphatidylserine, with up to a 23-fold increase in activity toward phosphorylated histone, but inhibition using p-nitrophenylphosphate (p-NPP) or tyrosine phosphate. In a subsequent study it was hypothesized that the phospholipid-binding site was located on the calcineurin B subunit (330). Using synthetic phospholipid monolayers, Kennedy et al. (183) investigated the factors that contributed to calcineurin-phospholipid interactions and found that calcineurin binding is myristoyl independent, mediated by anionic phospholipids and/or diacylglycerol, and also affected by the presence of calmodulin.

There is overwhelming evidence for calcineurin in the nucleus along with other calmodulin-binding proteins such as casein kinase-2 and myosin light-chain kinase (34, 286, 336, 376, 426). In spermatids, calcineurin was localized to the nucleus, and its levels were most abundant during the initial stage of nuclear elongation, with almost no signal present in the cytoplasm (286). In the context of signaling pathways that activate nuclear factor of activated T cells (NF-AT), Shibasaki et al. (376) have shown that calcium induces an association between calcineurin and NF-AT that results in colocalization of both molecules to the nucleus.

Calcineurin has also been shown to be associated with the cytoskeleton (106). The latter finding is of interest given that several substrates of calcineurin are colocalized to the cytoskeleton including tau (115, 122, 181), microtubule-associated protein 2 (122, 284), tubulin (122), dystrophin (275, 440), and dynamin (90, 240).

C) CALCINEURIN FUNCTION. Numerous functions have been identified for calcineurin in higher eukaryotic organisms, and it is beyond the scope of this review to cover all of them comprehensively. Klee et al. (208) have provided a recent update and cited a number of specific reviews regarding calcineurin function (208). Table 5 is an attempt to summarize a number of tissues, systems, and specific substrates that are implicated to be regulated by calcineurin. In sections IIIB2D and IIIB2E, we provide a short review of the role of calcineurin on two key systems of importance in modern biology, apoptosis and cardiac hypertrophy.

View this table: [in this window] [in a new window]   Table 5. Physiological roles of calcineurin in higher eukaryotic organisms

D) CALCINEURIN AND APOPTOSIS. It has been recognized for some time that calcineurin plays a role in programmed cell death of T and B lymphocytes (32, 110, 116, 481). Recently, it has also been shown that calcineurin plays a role in apoptosis in neuronal cells via the cytochrome c/caspase-3 pathway (17). In T-cell hybridomas, apoptosis can be stimulated by ligation of the T-cell receptor/CD3 complex and has provided a useful in vitro model to investigate signaling pathways responsible for this biological phenomenon. Both CsA and FK506 inhibit this process, implicating calcineurin in the signaling pathway of apoptosis which is known to involve a rise in intracellular Ca²⁺ (110). Similarly, in the B-cell lymphoma cell lines WEHI-231, B104, and BL60, apoptosis induced by cross-linking of surface immunoglobulin receptors was inhibited by these immunosuppressant drugs (32, 116).

In lymphocytes, calcineurin and NF-AT appear to participate in apoptosis, in part by mediating the induction of Fas and Fas ligand which then interact and transduce the apopototic signal after T-cell receptor ligation (157, 226, 243, 421). Using a constituitive (Ca²⁺- and calmodulin-independent) form of calcineurin, Shibasaki and McKeon (375) demonstrated that calcineurin functions in calcium-induced apoptosis in mammalian cells deprived of growth factors and that this was a direct consequence of calcineurin's phosphatase activity. Interestingly, coexpression of Bcl-2 blocked calcineurin-induced apoptosis. At least one mechanism for how this occurs was provided subsequently by experiments which showed that Bcl-2 forms a complex with calcineurin that targets it to the cytoplasmic membrane (374). Although still maintaining phosphatase activity, calcineurin bound to Bcl-2 is unable to promote nuclear translocation of NF-AT. Furthermore, BAD, a proapoptotic member of the Bcl-2 family, is a substrate of calcineurin. Dephosphorylation of BAD by calcineurin enhances BAD heterodimerization with Bcl-x_L and apoptosis (443).

However, another intriguing hypothesis is that apoptosis is linked to cellular redox homeostasis. Wolvetang et al. (463) showed that inhibitors of the plasma membrane NADH-oxidoreductase (PMOR) activity induce apoptosis through a signaling pathway involving calcineurin (463). It was proposed that PMOR serves as a redox sensor that can regulate the signals required for apoptosis (227). The finding that calcineurin activity is sensitive to redox state changes (45, 111, 345, 447, 470, 471) provides support for this hypothesis and a means by which apoptosis could be regulated by the cellular redox potential.

E) IMPORTANCE OF CALCINEURIN IN CARDIOVASCULAR FUNCTION. Recently, calcineurin and NF-AT have been implicated in transducing signals responsible for cardiac morphogenesis and inducing cardiac hypertrophy (82, 232, 233, 281, 337, 377, 401, 402, 417). Thus disruption of the NF-ATc gene in mice results in failure to develop normal cardiac valves and septa, and the transgenic mice die from congestive heart failure in utero (82, 337). Overexpression of calcineurin has also been shown to induce cardiac hypertrophy and heart failure in transgenic mice that could be blocked by the immunosuppressant drug CsA (281). Furthermore, a transgenic mouse model for hypertrophy in which tropomodulin-overexpressing transgenic mice develop progressive dilated cardiomyopathy has provided evidence for increased calcineurin protein levels before the onset of the hypertrophic phenotype, suggesting that calcineurin may play an early regulatory role in this process (402). Similar results were found in skeletal muscle from mice subject to overload (88) and confirmed later in skeletal muscle cells virally transfected with insulin-like growth factor I (295, 367). Some of these studies have even proposed that immunosuppressant drugs such as CsA and FK506 might be used to treat hypertrophy (312, 401). Indeed, in a subsequent study, Sussman et al. (401) utilized an aortic banding model to induce hypertrophy and showed that treatment with CsA, albeit an excessive dose, resulted in significantly less hypertrophy. However, although a few studies have confirmed this finding (267, 402), several other groups examining calcineurin's role in this process have failed to demonstrate any efficacy of CsA (86, 247, 290, 476) and, in fact, Molkentin (280) has responded by reporting that CsA protected against pressure-overload hypertrophy after 7 days but not after 21 days. Although the reason for some of these discrepancies may be due to the dose of immunosuppressant drug used, current hypotheses suggest that multiple signaling pathways might be recruited to participate in the hypertrophic response and that inhibition of one parallel pathway (i.e., calcineurin) might delay but not prevent hypertrophy (108, 397, 441). Nevertheless, they implicate a possible role for calcineurin and NF-AT in cardiac function.

Oxidative stress is also thought to play a role in cardiomyopathy and heart failure (381). The possibility that calcineurin may be regulated by oxidative stress indicates that signaling pathways in which it is involved may be important in mediating processes that lead to cardiac dysfunction.

C.  Inhibitors of Calcineurin

1.  Natural product and synthetic inhibitors

A number of natural products have been isolated that are potent inhibitors of calcineurin and other serine/threonine protein phosphatases. The most potent, specific, and well-known inhibitors of calcineurin are the immunosuppressant drugs CsA and FK506 (Fig. 4), which inhibit calcineurin when complexed with their respective cytoplasmic receptors cyclophilin and FKBP (see Table 1 entry for a number of reviews on these drugs). Interestingly, in vitro calcineurin inhibition by these immunosuppressant drug complexes only occurs when a physiological substrate is used to assay the enzyme such as phosphocasein or phospho-R_II peptide, a peptide whose sequence represents the phosphorylation site of the regulatory subunit of cAMP-dependent protein kinase, a well-characterized and more physiological phosphopeptide substrate (31). The use of p-NPP as substrate results in activation of calcineurin by these immunosuppressant drug complexes (238, 403).

View larger version (32K): [in this window] [in a new window]   Fig. 4. Natural product and synthetic inhibitors of calcineurin. For the metal-ligating phosphonate inhibitors, n refers to the number of methylene units.

A number of other compounds have demonstrated inhibitory activity against calcineurin and other serine/threonine protein phosphatases. Okadaic acid, often used as a potent and specific inhibitor of PP2A, can also inhibit PP1 and calcineurin at higher concentrations. The ID₅₀ of okadaic acid for PP2A has been measured to be ~1 nM, while the ID₅₀ values for PP1 and calcineurin are ~300 nM and ~4 µM, respectively (29). The cyclic peptide microcycstin LR is a potent inhibitor of PP1 and PP2A, with a K_i value <0.1 nM. Although the inhibition of calcineurin by microcystin LR occurs at over 1,000-fold higher concentrations, microcystin LR still is a relatively potent inhibitor of calcineurin (IC₅₀ = 0.2 µM) (248). Dibefurin, a novel fungal metabolite, also has modest inhibitory activity against calcineurin (37).

Since the discovery of these natural product inhibitors, several new synthetic compounds have been found to be reasonable inhibitors of calcineurin and other phosphatases. Tatlock et al. (410) utilized computational docking experiments and synthetic derivatives of the exo,exo-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid ring system of endothall (Fig. 4) to search for enhanced ligand binding to calcineurin (410). Endothall is structurally related to the natural defensive toxin of blister beetles, cantharidin (210), a potent inhibitor of PP1 and PP2A (210) and a weak inhibitor of calcineurin (98). Substitution at the 5-endo position was hypothesized to provide reasonable binding interactions that mimicked the interaction between the active site of calcineurin and its autoinhibitory domain. Incorporation of a trans-cyclopropylphenyl group at this position afforded the most potent inhibitor, with an apparent K_i of 0.5 µM. Interestingly, the tethered dicarboxylic acid moiety and bridgehead oxygen atom of endothall and cantharidin derivatives were modeled to interact with the active site dinuclear metal center (see sect. IVC) (410).

A similar approach to inhibitor design incorporating pendant metal-coordinating groups that could anchor the inhibitor to the active site metal ions has been introduced by Widlanski and colleagues (296). A variety of alkylphosphonic acid derivatives containing an additional thiol or carboxylate group (Fig. 4) were explored as inhibitors of alkaline phosphatase and purple acid phosphatase. Assuringly, nearly all bound more tightly than substrate p-nitrophenol and up to 55-fold tighter than ethanylphosphonic acid, indicating that these additional function groups could improve binding affinity. Whether binding occurs via direct metal ligation for endothall and/or alkylphosphonic acid derivatives remains to be demonstrated by spectroscopic means. If correct, these compounds could provide a route to the design of more potent and selective metallophosphatase inhibitors.

Peptide inhibitors of calcineurin have been also been introduced. One of these, a 25-residue peptide based on the sequence of the autoinhibitory domain of the calcineurin A subunit from residues 457-481 (Fig. 1), is a relatively potent inhibitor of calcineurin phosphatase activity (137, 325). Recently, a high-affinity calcineurin-binding peptide was selected from a combinatorial peptide library based on the calcineurin docking motif of NF-AT (15). The peptide inhibited NF-AT activation and expression of NF-AT-dependent cytokine genes in T cells, but did not inhibit calcineurin phosphatase activity toward phospho-R_II peptide, and thus did not affect the expression of other cytokines that require calcineurin but not NF-AT. The latter point is significant because compounds such as this peptide that selectively interfere with calcineurin-NF-AT interaction without disrupting calcineurin phosphatase activity may prove to be less toxic immunosuppressants compared with CsA and FK506.

At least one other synthetic calcineurin inhibitor has been reported, PD 144795, a benzothiophene derivative shown to have anti-inflammatory and anti-HIV effects (126). Transcriptional activity mediated by p53 and NF-B were inhibited by both CsA and PD 144795. An in vitro assay of calcineurin activity from Jurkat cell lysate also indicates that PD 144795 led to dose-dependent inhibition of calcineurin.

It was previously concluded by Enan and Matsumura (93) that class II pyrethroid insecticides were potent inhibitors of bovine brain calcineurin, with IC₅₀ values of 10⁹ to 10¹¹ M. In that study, p-NPP and O-phospho-DL-tyrosine were used as substrates in the inhibition assay. However, in an independent study, none of the class II pyrethroids was able to inhibit purified bovine calcineurin using phospho-R_II peptide (97). Calcineurin activity in rat brain homogenate and in IMR-32 neuroblastoma cells in culture was also not affected by pyrethroids, indicating that these insecticides are not effective inhibitors of calcineurin (99).

The tyrphostins A8, A23, and A48, members of a family of tyrosine kinase inhibitors, inhibited calcineurin with IC₅₀ values of ~10⁵ M (258). However, the use of p-NPP as substrate in these studies should be questioned given the inhibition pattern of calcineurin noted above for CsA, FK506, and the pyrethroid insecticides. A follow-up study using phospho-R_II peptide or other suitable phosphoprotein substrate may confirm yet another class of calcineurin inhibitors.

2.  Endogenous regulators

In addition to synthetic and natural product inhibitors of calcineurin, a number of endogenous cellular proteins have emerged as inhibitors of calcineurin protein phosphatase activity and thus potential regulators of its in vivo function. One of the first to be identified was a 79-kDa protein kinase A anchoring protein (AKAP79) (58). AKAP79 associates with the regulatory subunit of the cAMP-dependent protein kinase and localizes it to postsynaptic densities. Using a yeast two-hybrid approach to search for proteins that interacted with AKAP79, Scott and colleagues (58) identified a positive clone encoding the calcineurin A subunit. Immunofluorescence studies demonstrated that calcineurin and the regulatory subunit of protein kinase A were colocalized in rat hippocampal neurons via AKAP79. Interestingly, AKAP79 contained a domain homologous to FKBP, hypothesized to be the calcineurin binding domain. A synthetic peptide based on this sequence was a noncompetitive inhibitor of calcineurin activity (58). A subsequent study, however, suggests that AKAP79 interacts with calcineurin through a site distinct from the FKBP-homologous region (177).

Another potential calcineurin regulatory protein is cain/cabin 1, a 2,220-residue phosphoprotein isolated by yeast two-hybrid screens of either rat hippocampal or mouse T-cell cDNA libraries (224, 400). Cain/cabin 1 binds to calcineurin and inhibits it in a noncompetitive fashion. The interaction between cain/cabin 1 and calcineurin was dependent on protein kinase C activation, and overexpression inhibited the transcriptional activation of the interleukin-2 gene and prevented dephosphorylation of the transcription factor NF-AT. Recently, cain/cabin 1 was found to regulate the transcription factor MEF2, itself regulated via calcineurin-dependent pathways, by binding to MEF2 and sequestering it in an inactive state (469).

In an expression library screen searching for proteins that interact with the ubiquitously expressed Na⁺-H⁺ exchanger NHE1, Lin and Barber (234) identified a novel protein, CHP (see sect. IIB2D), that specifically bound NHE1 and was critical for growth factor stimulation of exchange activity. Overexpression of CHP in Jurkat and HeLa cells resulted in inhibition of NF-AT nuclear translocation and transcriptional activity that was hypothesized to be the result of calcineurin inhibition (235). Indeed, the phosphatase activity of immunoprecipitated calcineurin was inhibited 50% in cells overexpressing CHP, whereas in a reconstitution assay, the activity of purified calcineurin was inhibited nearly quantitatively in a dose-dependent fashion. These results indicate that CHP could represent yet another member of this emerging class of endogenous calcineurin regulators.

Recently, a protein of the African swine fever virus, A238L, was found to display immunosuppressive activity by inhibiting NF-AT-regulated gene transcription in vivo (277). A238L coimmunoprecipitated with calcineurin after viral infection of Vero cells, and calcineurin phosphatase activity was inhibited in cellular extracts from viral-infected cells. It was hypothesized that A238L may enable the virus to evade host defense systems by preventing transcriptional activation of genes important for host immunity.

Although the classical mechanism for regulating calcineurin activity is via Ca²⁺/calmodulin, it is intriguing to speculate that these and possibly other proteins can interact with calcineurin to regulate subcellular targeting and/or activity toward specific substrates in novel yet undefined ways.

	IV. CALCINEURIN STRUCTURE

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A.  A Dinuclear Metal-Binding Phosphoesterase Motif

Before the availability of any structural data, Averill and colleagues (431, 432) predicted that the serine/threonine protein phosphatases were homologous to purple acid phosphatases and therefore might contain an active site dinuclear metal center. Their hypothesis was based on a comparison of the primary sequences of serine/threonine protein phosphatases with human, porcine, and bovine purple acid phosphatases, enzymes which were already well characterized and known to contain dinuclear iron centers. Their prediction was correct, and the authors were able to identify three of the metal ligands.

With increasing sequence data available, several groups have completed comprehensive sequence alignments of serine/threonine protein phosphatases and have identified a number of residues that are conserved in all members of this family (26, 132, 212, 244, 371, 486). These studies identified a "phosphoesterase motif" (Fig. 5) that is conserved not only in PP1, PP2A, and calcineurin, but in many other enzymes involved in the cleavage of phosphoester bonds, including acid and alkaline phosphatases, bacterial exonucleases, diadenosine tetraphosphatase, 5'-nucleotidase, phosphodiesterase, sphingomyelin phosphodiesterase, an enzyme involved in RNA debranching, and a phosphatase in the bacteriophage  genome,  protein phosphatase (244).

View larger version (13K): [in this window] [in a new window]   Fig. 5. The phosphoesterase motif as found in calcineurin and purple acid phosphatase. The original motif was identified by Koonin (212) and modified by Zhuo et al. (486). Residues that have been identified as metal ligands are shown in bold (for calcineurin A, these represent residues Asp-90, His-92, Asp-118, and Asn-150; cf. Figs. 7A and 10). Conserved, nonligand residues also found in the active site (Asp-121, Arg-122, and His-151) are underlined. The phosphoesterase motif of plant (200) and mammalian purple acid phosphatases (92, 185, 380) is also shown (bold residues are represented as Asp-135, Asp-164, Y-167, and Asn-201, while the underlined residues are represented as Asp-169 and His-202 in the kidney bean enzyme; cf. Figs. 7B and 11). The motif in the plant and mammalian purple acid phosphatases represents a variation of the phosphoesterase motif. PP1, protein phosphatase 1.

Four of the residues in the phosphoesterase motif (bold letters, Fig. 5) are ligands to a dinuclear metal cofactor in PP1 and calcineurin. Thus it has been hypothesized that this motif provides a scaffold for an active site dinuclear metal center in every member of the phosphoesterase family (244, 351). Support for this hypothesis was provided in studies of  protein phosphatase which demonstrated a spin-coupled dinuclear metal binding site by spectroscopic means (272, 352), and in the recent determination of the three-dimensional structure of E. coli 5'-nucleotidase, a distant member of the metallophosphatase superfamily that has an active site containing two Zn²⁺ separated by 3.3 Å (211). The conserved phosphoesterase motif suggests a common catalytic mechanism for enzymes involved in phosphotransfer reactions, a hypothesis that seems to be the case for at least two of these, calcineurin and  protein phosphatase (see below).

The phosphoesterase motif is also found in purple acid phosphatases, albeit in a slightly modified form (Fig. 5). Despite these differences, the phosphoesterase motif in purple acid phosphatase has a similar ---- fold accommodating the dinuclear metal center (199, 201, 395).

B.  Three-Dimensional Structure

The three-dimensional structures of several enzymes in the metallophosphatase family have been solved. X-ray structures (with highest resolution noted in parentheses) of PP1 (2.1 Å) (91, 120), calcineurin (2.1 Å) (124, 197), kidney bean purple acid phosphatase (2.65 Å) (199, 201, 395), mammalian purple acid phosphatase (1.55 Å) (128, 425), and the periplasmic 5'-nucleotidase from E. coli (1.7 Å) (211) have been solved. In these structures, the phosphoesterase motif described in the previous section is represented as a ---- scaffold for an active site dinuclear metal center. The three -strands of this motif form a parallel pleated sheet that is capped by intervening -helices. Two metal ions are positioned at the apex of this fold forming a dinuclear metal center with 3.0-4.0 Å between metal ions, with four of the metal ligands provided by residues in loops between -sheets and -helices.

A ribbon diagram representing the X-ray structure of phosphate-inhibited calcineurin, complexed with the immunosuppressant drug complex FK506·FKBP, is shown in Figure 6. The overall structure of the catalytic subunit (shown in gray) is ellipsoidal and consists of a mixture of -helices and -sheets. The metal ions of the dinuclear metal center are obscured in this diagram by the orange van der Waals spheres of phosphate that form a bridge between the two metal ions (see Fig. 10, below). The calcineurin B-binding domain (cf. Fig. 1) is evident in this structure as an -helix that protrudes from the core of the molecule, forming the binding site for the calcineurin B subunit (Fig. 6, yellow). Absent in this structure are the calmodulin-binding and autoinhibitory domains, since a truncated form of calcineurin missing these domains was the source of protein for crystallization studies (124). In a subsequent study that determined the structure of the holoenzyme, it was found that the autoinhibitory domain folds into an -helix that binds to the substrate-binding cleft of the catalytic domain, with one of the glutamate residues forming hydrogen bonds to metal-coordinated water molecules (197). Interestingly, the calmodulin domain was disordered in the X-ray structure, and therefore, our knowledge of how this domain interacts with the active site and autoinhibitory domains to confer calmodulin-regulation remains rudimentary.

View larger version (96K): [in this window] [in a new window]   Fig. 6. Ribbon diagram showing the three-dimensional structure of calcineurin complexed with FK506·FK506 binding protein (FKBP). The figure was created using the X-ray coordinates of Griffith et al. (protein data bank file 1TCO) (124), using MOLSCRIPT (214) and Raster3D (20). The catalytic subunit calcineurin A is colored in gray, the calcineurin B subunit is yellow, FKBP is green, and the immunosuppressant drug FK506 is shown in ball-and-stick fashion in gray. The four Ca2+ of the B subunit are represented as blue spheres, and the myristoyl group of the B subunit is colored as red van der Waals spheres. The phosphate molecule bound to the active site metal ions of the A subunit is represented as orange van der Waals spheres.

The calcineurin B subunit in Figure 6 is colored in yellow and is seen forming a complex with calcineurin A via the calcineurin B-binding helix. Four Ca²⁺, shown as blue spheres, are bound in the EF-hand domains of the B subunit. The NH₂-terminal myristoyl group of calcineurin B (red van der Waals spheres) is situated in a hydrophobic cleft between two amphipathic helices that are integral components of two EF-hands (124). In contrast, the X-ray structure of the nonmyristoylated protein shows the first five residues of the NH₂ terminus of calcineurin B as disordered, suggesting increased mobility of the NH₂ terminus relative to the acylated protein (197). Thus the myristoyl group appears to be anchored via multiple hydrophobic contacts and may contribute to the overall structural stability of the enzyme and may explain why the acylated protein has increased thermal stability compared with the unmodified enzyme (182).

C.  Active Site Architecture

The dinuclear metal cofactor of calcineurin has been modeled as an Fe³⁺-Zn²⁺ cluster based on the presence of near-stoichiometric quantities of Fe³⁺ and Zn²⁺ (195, 471), electron paramagnetic resonance (EPR) spectroscopic experiments (471), and X-ray diffraction studies which show a dinuclear metal center separated by 3.14 Å in a coordination environment shown in Figure 7A (197). A similar coordination environment and metal-metal distance is also found in PP1 (120), although in that enzyme, the metal ions could not be identified with certainty and were therefore referred to as M1 and M2. In the X-ray structure of calcineurin, the Fe³⁺ was modeled in the M1 site largely based on a comparison to the Fe³⁺-Zn²⁺ active site metal cofactor of kidney bean purple acid phosphatase. In purple acid phosphatase, a tyrosine residue coordinates to the Fe³⁺ (M1 site) and gives rise to a tyrosine-to-iron charge transfer band at 510-550 nm, responsible for the purple color of these enzymes (Fig. 7B) (223, 433). As can be seen in Figure 7, this tyrosine ligand is missing in calcineurin and is replaced by a histidine, thus explaining why calcineurin does not exhibit any appreciable absorbance in the visible region of the optical spectrum (471). The additional substitution of a histidine ligand with a water molecule results in a net water-for-tyrosine substitution at the Fe³⁺ site in calcineurin compared with purple acid phosphatase. The coordination of the Zn²⁺ (M2) provided by amino acid side chains in these enzymes is identical, with ligands provided by two histidines, an aspartic acid that bridges to the Fe³⁺ and an asparagine residue (compare Fig. 7, A and B). Figure 8 shows a comparison of the active sites of kidney bean purple acid phosphatase and calcineurin that illustrates the remarkable superposition of both metal ions and protein ligands in these two enzymes.

View larger version (21K): [in this window] [in a new window]   Fig. 7. A: schematic of the active site of human calcineurin based on the 2.1-Å resolution structure described by Kissinger et al. (197). B: schematic of the active site of kidney bean purple acid phosphatase based on the 2.65-Å resolution structure described by Klabunde et al. (201).

View larger version (98K): [in this window] [in a new window]   Fig. 8. Comparison of the active sites of calcineurin and kidney bean purple acid phosphatase. An alignment of the active sites of calcineurin and purple acid phosphatase was performed based on the conserved His-Asp (calcineurin His-151/Asp-121) pair using the program Quanta (Quanta 97 Molecular Modeling Software Package from Molecular Simulations, 1997). The X-ray crystal structures used were Protein Data Bank entries 1AUI and 1KBP for human calcineurin (197) and kidney bean purple acid phosphatase (201), respectively. The A subunit of calcineurin and a monomer form of purple acid phosphatase were used to generate the alignments. Only the metal ions and the active site residues shown in the figure were used for the alignment. Calcineurin and purple acid phosphatase residues are indicated in red and yellow, respectively. The Zn atom is green, and the Fe atom is purple. The numbers denote the following residues: 1) His-92 in calcineurin, Tyr-167 in purple acid phosphatase; 2) Asp-121 in calcineurin, Asp-169 in purple acid phosphatase; 3) His-151 in calcineurin, His-202 in purple acid phosphatase; 4) Asn-150 in calcineurin, Asn-201 in purple acid phosphatase; 5) His-199 in calcineurin, His-286 in purple acid phosphatase; 6) His-281 in calcineurin, His-323 in purple acid phosphatase; 7) His-325 in purple acid phosphatase; 8) Asp-90 in calcineurin, Asp-135 in purple acid phosphatase; 9) Asp-118 in calcineurin, Asp-164 in purple acid phosphatase.

In addition to coordination provided by protein side chains, the metal ions in calcineurin and purple acid phosphatase have one or more solvent molecules as additional ligands. All enzymes show bridging and terminal solvent molecules, but at least in one X-ray structure of calcineurin, an additional terminal solvent molecule was modeled into the coordination sphere of the M1 site as shown in Figure 7A (197). From the iron-oxygen bond length (2.1 Å in purple acid phosphatase, Ref. 128), the bridging solvent molecule is most likely a hydroxo group based on distances obtained from model compounds (223).

D.  Metal Ion Requirements

It has been known for some time that divalent cations in assay buffers are necessary to achieve the high activities of purified calcineurin, with the best activators being Mn²⁺ and Ni²⁺ (194, 230, 315). Mn²⁺ and Ni²⁺ have been shown to increase the activity of calcineurin in the absence of Ca²⁺/calmodulin (315), to prevent inactivation, or to restore activity following inactivation by exposure to Ca²⁺/calmodulin (194). Similar divalent metal ion effects have been observed with PP1 and PP2A (3, 28, 43, 53, 54, 94, 478) and with -protein phosphatase (486). It is unclear why these divalent metal ions are potent activators. One possibility is that Mn²⁺ or Ni²⁺ are native metal ions that become lost during purification. Pallen and Wang (317) incubated calcineurin with Ni²⁺ or Mn²⁺ and showed that these metal ions are not dissociable by extensive dialysis or gel filtration but can be released after prolonged exposure to Ca²⁺/calmodulin or by the use of chelating reagents, both of which occur during a typical purification protocol. To address this issue, Rao and Wang (341) used an anticalcineurin immunoaffinity matrix to rapidly purify calcineurin from crude bovine brain extract in the absence of calmodulin. Analysis showed that the immunoprecipitated calcineurin did not contain significant amounts of Ni²⁺ and Mn²⁺. Although no mention was made of the Fe content, ~1 equivalent of Zn²⁺ was found in all samples, suggesting that Zn²⁺ is an intrinsic metal ion but not Ni²⁺ or Mn²⁺.

An alternative hypothesis to explain the mechanism of divalent metal ion activation is that prolonged exposure of calcineurin to Ca²⁺/calmodulin promotes the release of the intrinsic Fe and Zn metal ions and subsequent replacement by Mn²⁺ or Ni²⁺. The most efficient method to purify calcineurin utilizes Ca²⁺/calmodulin affinity chromatography. It is possible that calmodulin binding exposes the active site and thereby promotes loss of Fe and/or Zn while calcineurin is adsorbed to the matrix. Alternatively, because elution of calcineurin from calmodulin-Sepharose requires the use of a metal chelator (e.g., EDTA), it is possible that the Fe³⁺ and/or Zn²⁺ may be removed during elution. Thus far, neither hypothesis has been rigorously tested. However, King and Huang (195) demonstrated that there was no correlation between the loss of enzymatic activity after calmodulin-dependent inactivation and the iron and zinc content. Both metal ions remained tightly bound during prolonged exposure to calmodulin. Nevertheless, additional studies have demonstrated that up to two equivalents each of Mn²⁺ and Ni²⁺ could bind to calcineurin (317, 482), a result consistent with these metal ions occupying the sites of the dinuclear metal cluster. In support of this was an EPR study following Mn²⁺ binding to -protein phosphatase which found that a dinuclear Mn²⁺-Mn²⁺ cluster was formed upon addition of two equivalents of Mn²⁺ to the apoenzyme (352). The situation with calcineurin is not as straightforward due to the presence of calmodulin and the calcineurin B subunit, each which can provide additional divalent metal ion binding sites. Indeed, an EPR study following Mn²⁺ binding to calcineurin in the presence of calmodulin demonstrated 10 Mn²⁺ sites and attributed 4 each to calmodulin and calcineurin B and 2 to the catalytic subunit (453).

Clearly further work is needed to understand the mechanism whereby exogenous Ni²⁺ and Mn²⁺ activate calcineurin after prolonged exposure to Ca²⁺/calmodulin in vitro and whether or not this activation also occurs in vivo.

	V. ENZYMATIC MECHANISM

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A.  Mechanism of Phosphoryl Group Transfer: Evidence for Direct Transfer to Water

Much of the initial work on the mechanism of calcineurin focused on determining whether a phosphoenzyme intermediate was formed during catalysis, since other phosphatases are known to proceed by this mechanism (Fig. 9). For example, E. coli alkaline phosphatase catalyzes phosphate ester hydrolysis by first transferring the phosphoryl group to an active site serine residue to form a transient phosphoenzyme intermediate (63, 64). In the next step, the enzyme is regenerated for another round of catalysis by hydrolysis of the phosphoenzyme intermediate. Alkaline phosphatase is a metalloenzyme containing a Mg²⁺ and a Zn-Zn dinuclear center reminiscent of the dinuclear metal sites of the serine/threonine phosphatases and purple acid phosphatases (187). A phosphoenzyme intermediate has also been demonstrated in the protein tyrosine phosphatases (52, 127, 479), enzymes that function without active site metal ions.

View larger version (13K): [in this window] [in a new window]   Fig. 9. Phosphatase catalysis: phosphoenzyme intermediate versus direct transfer mechanisms. Shown are two possibilities by which phosphatases catalyze phosphoryl group transfer to water. Escherichia coli alkaline phosphatase and protein tyrosine phosphatases represent two classes of phosphatases that utilize the phosphoenzyme intermediate mechanism. The metallophosphatases appear to proceed by direct transfer of the phosphoryl group to a metal-coordinated water molecule, without the formation of a phosphoenzyme intermediate. The "X" symbol represents an active nucleophile, e.g., a cysteine residue in the protein tyrosine phosphatases and a serine residue in alkaline phosphatase.

Steady-state experiments of calcineurin carried out by Graves and colleagues (262) suggested that a phosphoenzyme intermediate was not formed during catalysis. A linear relationship between log (V/K) and the pK_a of the leaving group was observed for a set of four substrates, with a trend toward increasing velocity as the pK_a of the leaving group decreased (262). This result is consistent with a direct transfer to water without formation of a phosphoenzyme intermediate (Fig. 9). Additional experiments failed to demonstrate phosphotransferase activity in the presence of alternate nucleophiles, which also is consistent with a direct transfer mechanism (262). In a subsequent study using p-NPP as substrate, product inhibition studies showed that both phosphate and the phenol were competitive inhibitors of calcineurin (259). These inhibition patterns are consistent with a random uni-bi mechanism. In contrast, an ordered uni-bi mechanism is expected for a phosphoenzyme intermediate, since the product alcohol would be released before the phosphoenzyme intermediate is hydrolyzed. Although these results are consistent with a direct transfer mechanism, they are not conclusive, since they can also be interpreted to indicate a mechanism involving a transient phosphoenzyme intermediate whose breakdown is not rate limiting.

The most definitive work that addressed whether a phosphoenzyme intermediate was formed during catalysis for the metallophosphatases was performed with bovine spleen purple acid phosphatase. Using a chiral [¹⁸O,¹⁷O]phosphorothioate ester and carrying out the hydrolysis in [¹⁶O]H₂O, Knowles and co-workers (288) demonstrated that purple acid phosphatase carries out hydrolysis with net inversion of configuration at the phosphorus center, thus indicating that the phosphoryl group is transferred directly to water. Because of the similarity of the active sites of calcineurin and PP1 to purple acid phosphatases, it is thought that the catalytic mechanism of the serine/threonine phosphatases also proceeds by a similar mechanism.

B.  Catalytic Role of the Dinuclear Metal Center

Several pieces of data indicate that the dinuclear metal center is a key component of the active site of calcineurin. 1) As already mentioned, the dinuclear metal center has a ligand environment similar to purple acid phosphatases, enzymes which contain dinuclear Fe-Fe or Fe-Zn centers previously demonstrated to be essential for catalytic activity (223, 433). 2) Crystallographic (91, 124, 201) and spectroscopic (80, 334, 416, 448, 449, 470) studies indicate that the product of the reaction, phosphate, and product analogs such as tungstate, molybdate, and arsenate, coordinate the metal ions. 3) Redox titrations of either the Fe³⁺-Zn²⁺ or Fe³⁺-Fe²⁺ forms of calcineurin and purple acid phosphatase indicate a correlation between enzyme activity and the oxidation state of the bound metal ions (8, 9, 18, 77, 470, 471).

The metal ions of the dinuclear center could function in numerous ways to catalyze phosphate ester hydrolysis. The Lewis acidity of the metal ion(s) could serve to activate a solvent molecule, a well-known mechanism in several metalloenzymes such as carbonic anhydrase (132a) and adenosine deaminase (459). A metal-activated water molecule has been proposed for purple acid phosphatases (85, 432). Alternatively, a metal-coordinated solvent molecule could serve as a general acid to donate a proton to the leaving group, as has been proposed for inorganic pyrophosphatase (140, 355). In addition to a role in activation of the solvent nucleophile, the metal ions in the serine/threonine phosphatases could be involved in other aspects of the catalytic mechanism. Metal coordination of the phosphate ester could have several positive effects acting to accelerate hydrolysis. Neutralization of the negative charge on the oxygen atoms of the phosphate ester oxygen atoms would increase the electrophilicity of the phosphorus atom, making it more prone to nucleophilic attack. During P-O bond scission, the metal ions could stabilize the developing charge on the leaving group. However, another possible role for the metal ions could be to orient the substrate for in-line attack. These are discussed in section VC in the context of specific active site residues and the effect that mutagenesis of these residues has on catalytic efficiency.

C.  Conserved Active Site Residues

In addition to the metal ions and their cognate protein ligands, several conserved residues within 4-9 Å of the dinuclear metal cofactor are involved in catalysis. One of these is a histidine residue that is not a metal ligand but is within 5 Å of either metal ion. The conserved histidine in calcineurin, His-151, is also conserved in PP1 (His-125), purple acid phosphatases (His-202, kidney bean enzyme numbering), and -protein phosphatase (His-76). It is represented in the phosphoesterase motif as the underlined residue of Figure 5 (244). This histidine is hydrogen bonded to an aspartic acid that is also part of the phosphoesterase motif, Asp-121 in calcineurin, Asp-95 in PP1, Asp-169 in kidney bean purple acid phosphatases, and Asp-52 in -protein phosphatase. Two arginines, Arg-122 and Arg-254, are also present in the active site of calcineurin. These arginines are also conserved in other metallophosphatases and correspond to Arg-96 and Arg-221 in PP1 (120) and Arg-53 and possibly Arg-162 in -protein phosphatase. Figure 10 depicts the active site of calcineurin in the phosphate-inhibited form showing the conserved nonligand residues. The corresponding residues in kidney bean purple acid phosphatases are depicted in Figure 11. In purple acid phosphatase, two histidines (His-295 and His-296) are substituted for two arginines in the serine/threonine phosphatase family.

View larger version (20K): [in this window] [in a new window]   Fig. 10. Schematic of the active site of calcineurin complexed with product phosphate. Shown is the dinuclear metal center of calcineurin, along with the conserved His-Asp pair (His-151/Asp-121) and two arginine residues (Arg-122/Arg-254) that form a region of positive electrostatic potential in the active site.

View larger version (20K): [in this window] [in a new window]   Fig. 11. Schematic of the active site of purple acid phosphatase complexed with product phosphate. Shown is the active site of purple acid phosphatases with the conserved His-Asp pair (His-202/Asp-169) and two histidines (His-296/His-295) thought to be important for the enzyme's mechanism (91, 124).

The importance of these residues and their potential role in catalysis has in most cases been addressed by enzymatic and spectroscopic studies of enzymes in which these residues have been altered by site-directed mutagenesis. The interpretation of these data assumes that mutagenesis alters only the physical-chemical features of the residue of interest and does not alter some other requisite structural feature, (e.g., tertiary structure, metal binding, etc). A review of the primary literature indicates that this assumption has not always been defended by rigorous structural studies. The reader is therefore cautioned that the roles ascribed to some of these active site residues are still tenuous.

1.  Role of the histidine/aspartate pair in the metallophosphatases

Studies have shown that mutation of His-151 in calcineurin, or its analog in -protein phosphatase, His-76, leads to significant reductions in enzyme activity (272, 487). When its hydrogen-bonding partner (corresponding to Asp-121 in calcineurin) is mutated, a 10¹- to 10³-fold decrease in the rate constant for catalysis (k_cat) occurs with little effect on K_m (161, 475, 487). One role considered for this histidine has been as an active site nucleophile. As discussed above, the data argue against this since a phosphoenzyme intermediate probably does not form during the catalytic cycle. Alternatively, this histidine may have a role in binding substrate, orienting the nucleophilic water molecule, and/or in acid/base catalysis. These possibilities are discussed in the following sections.

A) ACID/BASE CATALYSIS. A series of kinetic studies were undertaken to study the effect of mutating the conserved histidine in calcineurin (His-151) and -protein phosphatase (His-76) (272, 283). In both mutant enzymes there were significant reductions in enzyme activity but only small effects on K_m (272, 283). This was the case using p-NPP and the [P]-R_II peptide as substrates for calcineurin, and p-NPP and phenyl phosphate for -protein phosphatase. These substrates were chosen to explore the role of this histidine as a proton donor. A phosphate ester such as p-NPP has a better leaving group (pK_a of p-nitrophenol = 7.2) compared with either phenyl phosphate or a phosphoseryl peptide (pK_a of phenol = 10, pK_a serine side chain -OH ~14). Therefore, if protonation of the leaving group occurs in the transition state, hydrolysis of p-NPP will require less catalytic assistance than either phenyl phosphate or a phosphoseryl peptide such as [P]-R_II peptide. As a result, one would expect larger effects on relative k_cat (wild-type k_cat/mutant k_cat) for substrates with poorer leaving groups. The results showed less than a threefold difference in relative k_cat for calcineurin using p-NPP versus the [P]-R_II peptide, despite a difference in pK_a for the respective leaving groups of >10⁶ (272). With -protein phosphatase, mutagenesis of His-76 to Asn (H76N) resulted in the same 500- to 600-fold reduction in k_cat using either p-NPP or phenyl phosphate as substrates (leaving group pK_a difference >10³) (272). Ideally, more than two substrates with varying pK_a values should be used in this type of Brønsted analysis.

A useful method to determine if a residue is acting as an acid or base in catalysis is to follow the pH dependence of the rate for wild-type and mutant enzymes. Often one arm of the usual bell-shaped dependence curve is missing for the mutant if the residue is acting as a general acid or base. Using p-NPP as a substrate, pH dependence studies for wild-type and H76N -protein phosphatase were performed (156). The mutant enzyme had a lower catalytic rate at every pH but still exhibited a bell-shaped pH curve. However, kinetic data were difficult to obtain at low pH due to substrate inhibition that necessitated higher metal ion concentrations. In summary, the difficulties encountered were such that it was not possible to conclude whether this histidine serves as a general acid in catalysis. It was concluded that this residue may function more decisively as a general acid in reactions of phosphoprotein substrates that have a much poorer leaving group. It is noteworthy that the pH optimum for the mutant appeared to be shifted to pH 7.0, compared with 7.8 for the wild-type enzyme. Also the K_m for substrate increased at high pH for the native enzyme to values that were similar to those of the mutant enzyme at all pH values, indicating that protonation of this residue may assist in substrate binding.

B) KINETIC ISOTOPE EFFECTS. Kinetic isotope effect studies have been performed with both calcineurin (150, 260, 261) and -protein phosphatase (156) to study the chemistry of the transition state. In the case of calcineurin, the use of D₂O to measure a solvent isotope effect found no effect on k_cat but a modest isotope effect (1.35) on k_cat/K_m (260). The lack of a solvent isotope effect on k_cat may be due to having some other step in the reaction mechanism other than the proton transfer step be rate limiting, a situation that was confirmed in a subsequent study that used heavy atom isotopes of p-NPP (150). In the latter study it was shown that P-O bond cleavage was partially rate limiting at the pH optimum, and therefore, the isotope effects were suppressed, a situation which could be improved upon raising the pH from 7.0 to 8.5. For the -protein phosphatase reaction, P-O bond cleavage was fully rate limiting, and the measured isotope effects represented the intrinsic isotope effects on the bond-breaking step of the catalytic mechanism. With both enzymes, the data indicate that the substrate of the reaction is the p-NPP dianion, the predominant form at neutral pH (pK_a2 = 4.96). The isotope effect studies also provide evidence for a dissociative mechanism, in which the transition state has substantial P-O bond cleavage before bond formation to the nucleophilic water molecule. A dissociative mechanism has also been observed for the protein tyrosine phosphatases (148, 151, 152) and for uncatalyzed reactions in solution (149). This result is important because it demonstrates that the metal ions do not change the transition state of the phosphoryl transfer reaction to become more associative, a hypothesis that was previously forwarded based on the idea that metal ions could stabilize the extra negative charge in the transition state that results from bond formation to the nucleophile (139). Instead, the transition state of the phosphoryl group looks very much the same as in the solution reaction.

The ¹⁵N kinetic isotope effects indicate that in calcineurin and wild-type -protein phosphatase, there is substantial charge neutralization of the leaving group in the transition state (150, 156). In contrast, the magnitude of this charge increases when His-76 of -protein phosphatase is mutated to asparagine. This result suggests that this histidine may be protonating the leaving group in the transition state; its absence would lead to a greater charge accumulation on the leaving group compared with the wild-type enzyme. Interestingly, the magnitude of negative charge on the leaving group is smaller than expected for the mutant enzyme compared with comparable studies with protein tyrosine phosphatases that have the active site general acid removed by mutagenesis. Indeed, the magnitude of the isotope effect in the mutant is significantly less than would result from a full negative charge on the departing p-nitrophenol product. One explanation that was forwarded is that the metal ions may participate in stabilizing the transition state of the reaction by neutralizing the developing negative charge on the leaving group.

C) POTENTIAL ROLE IN SUBSTRATE BINDING. Another possible role of His-76/His-151 could be to assist in substrate binding. With -protein phosphatase, the K_m for p-NPP increased from 1 to 70 mM with increasing pH (156). In contrast, the K_m for p-NPP in the -PP(H76N) mutant was higher at low pH compared with wild type. As the pH was increased to neutral pH and greater, the K_m values for both enzymes became similar. It is possible that at acidic and neutral pH, where the histidine would be protonated, this residue assists in substrate binding via electrostatic interactions. For example, a proton on His-76 may form a hydrogen bond with one of the substrate oxygen atoms of the phosphorylated substrate. In X-ray structures of other serine/threonine phosphatases with bound inhibitors, phosphate or tungstate, this histidine is within hydrogen bonding distance of the most solvent exposed oxygen atom of the inhibitor (91, 124). A similar orientation is observed with purple acid phosphatases with phosphate or tungstate bound (128, 201, 425). This type of electrostatic interaction will not affect the isotope effect on ¹⁸(V/K)_nonbridge. However, if the substrate were actually protonated in the transition state, this would have been revealed in the ¹⁸(V/K)_nonbridge isotope effect; this was not observed (156). It is not clear if His-76 really does participate in substrate binding, since at basic pH it would be deprotonated. The pH optimum for the wild-type enzyme is ~8.0, where both the wild-type enzyme and mutant have similar K_m values for p-NPP.

D) GENERAL BASE CATALYSIS. Another possibility for His-151/His-76 would be to act as a general base to deprotonate the metal-bound water molecule that is the nucleophile in the mechanism. In one X-ray structure of calcineurin, the N of His-151 is hydrogen-bonded to one of two terminal solvent molecules coordinated to the Fe ion. Considering also Asp-121, which is hydrogen bonded to His-151, the interaction of this histidine/aspartate pair with the metal-coordinated solvent molecule is analogous to the Asp-His-Ser catalytic triad of serine proteases, where an Asp/His pair is important for interacting with the nucleophilic serine residue. The "Asp-His-HO-metal" motif in the serine/threonine phosphatases can be thought of as a catalytic tetrad, with the metal ion serving to lower the pK_a of the nucleophilic water molecule while the Asp-His functions as a catalytic base to assist in hydroxide formation. Histidines in other metalloproteins are thought to perform a similar role by acting as a base to deprotonate a metal-bound water molecule or by assisting in stabilizing a metal-bound hydroxyl group. For example, His-372 in E. coli alkaline phosphatase forms a hydrogen bond with Asp-327 (a bidentate Zn ligand) and is thought to lower the pK_a of a zinc-bound water molecule (464). Mutagenesis studies of Asp-327 to asparagine result in increased enzyme activity, since the negative charge of the hydroxyl group is more stable due to the loss of the aspartate side chain and replacement with a neutral residue. However, this comes at the expense of Zn binding since a carboxylate is a better metal ligand than a carboxamide, and thus a higher concentration of Zn in the assay is needed to achieve this increased activity. Examples of other enzymes where a histidine is postulated to deprotonate a metal-coordinated water molecule include His-320 in Klebsiella aerogenes urease (172, 321), His-231 in thermolysin (27), and His-89 in Serratia nuclease (109).

E) ORIENTATION OF THE NUCLEOPHILIC HYDROXIDE SOLVENT MOLECULE. Another potential role of His-76/His-151 could be to position the metal-coordinated hydroxide for optimal in-line attack of substrate, a mechanism analogous to the concept of "orbital steering" proposed by Storm and Koshland (393) almost 30 years ago to provide an explanation for the great rate accelerations seen in enzyme catalysis. The theory of orbital steering postulates that a major factor in catalytic enhancement is that an enzyme arranges the reaction trajectory to optimize the overlap of attractive (bonding) orbitals and minimize the overlap of repulsive (nonbonding) orbitals. This concept has been debated and contested by numerous groups (39, 174, 270). Nevertheless, in a recent study of isocitrate dehydrogenase by Koshland and co-workers (273), small structural perturbations in isocitrate dehydrogenase were created to evaluate the contribution of precise substrate alignment to the catalytic rate of an enzyme. They found that small changes in the orientation of substrates had large effects on reaction velocity (10³- to 10⁵-fold decreases). Their main conclusion was that orbital steering is an important contribution to the catalytic power of enzymes.

A role in orientation of metal-bound water was proposed for His-238 of murine adenosine deaminase (459) and is possible for the conserved histidine in the serine/threonine phosphatases. Interestingly, small perturbations in the EPR spectra of the dinuclear metal center in CN(H151Q) and -PP(H76N) enzymes were observed compared with the wild-type enzymes (272), indicating subtle perturbations to the geometry of the dinuclear metal center. X-ray structures of these mutants would be valuable to obtain better information on whether substrate orientation may be affected.

2.  Role of arginines in the active site

As shown in Figure 10, there are two arginine residues in the active site of calcineurin, Arg-122 and Arg-254. These arginine residues are conserved in other phosphatases. Mutagenesis of Arg-122 in calcineurin or its homologs in other phosphatases (Arg-96 in PP1 and Arg-53 in -protein phosphatase) resulted in 10²- to 10³-fold decreases in k_cat and only slight changes in K_m (161, 283, 475, 487). An exception to this was a 20-fold increase in K_m when Arg-53 in -protein phosphatase was mutated to an alanine and assayed in the presence of Ni²⁺ (487). However, when this mutant was assayed in the presence of Mn²⁺, there were no significant changes in K_m. Site-directed mutagenesis studies of Arg-254 in calcineurin or Arg-221 in PP1 resulted in an 200-fold reduction in k_cat, while values for K_m increased 2- to 10-fold (161, 283). In the X-ray structures of wild-type calcineurin and PP1, the guanidinium groups of these arginine residues form salt bridges with the oxygen atoms of bound phosphate or tungstate and stabilize the anion inhibitor (91, 120, 124, 197). The purpose of these arginine residues may be to provide electrostatic stabilization for binding the negatively charged phosphate ester. In addition, they may help neutralize developing negative charge in the transition state. Because the mutation of Arg-122 led to greater decreases in k_cat than mutation of Arg-254, it may be that Arg-122 has a more important role in catalysis, perhaps involving stabilization of the transition state. To note, in the active site of kidney bean purple acid phosphatase (Fig. 11), two histidine residues, His-295 and His-296, appear in place of these arginine residues, and it has been suggested that these substitutions might explain the lower pH optimum of purple acid phosphatases (201). Krebs and colleagues (201) have hypothesized that one of these histidines may be the active site residue with an apparent pK_a of 6.9 that produces the basic pH portion of the bell-shaped pH/kinetic profile. In an analogous manner, it is possible that Arg-53 in -protein phosphatase produces the basic pH arm observed in the pH dependence studies (156). In mammalian purple acid phosphatases, there is a histidine residue (His-195) corresponding to His-296 of kidney bean purple acid phosphatase, but Glu-194 substitutes for His-295 in the mammalian enzyme and is oriented away from the phosphate in the oxidized enzyme-phosphate complex.

D.  A Model for the Calcineurin Catalytic Mechanism

Taking into account the available biochemical and chemical data, we propose a model for the catalytic mechanism of calcineurin and other members of this family (Fig. 12). The first step of the mechanism involves association of the phosphate monoester, as the dianion, with the enzyme (Fig. 12A). In this step, neutralization of negative charge by the metal ions may occur. X-ray structures of calcineurin and PP1 show phosphate and other anionic product inhibitors coordinated to both metal ions, inferring that the substrate phosphoryl group might also coordinate to one or both metal ions at some point during catalysis. In addition, the conserved arginines, Arg-122 and Arg-254, may also play a role in binding substrate and neutralizing charge by forming hydrogen bonds with the oxygen atoms of the phosphoryl group. The intermediate in Figure 12A shows the Zn atom and the two conserved arginines assisting in substrate binding/neutralization of charge. This neutralization of charge would make the substrate more electrophilic and ready for attack by a nucleophile.

View larger version (18K): [in this window] [in a new window]   Fig. 12. Proposed mechanism for calcineurin. The mechanism shown in this figure poses His-151 acting as a general base and a general acid. A: substrate binds to the active site, and His-151 deprotonates a Fe3+-bound water molecule to form hydroxide. B: bond cleavage to the leaving group occurs to a greater extent than bond formation to the hydroxyl group in the transition state since the transition states for both -protein phosphatase and calcineurin were shown to be dissociative. The Zn atom may aid in neutralization of the negative charge on the leaving group in the transition state. The leaving group is protonated by His-151 and then leaves the active site. C: the product-inhibited state is shown with phosphate bridging the two metal ions. D: a water molecule displaces bound phosphate, and the enzyme is ready for another round of turnover. Another substrate then enters the active site and displaces the bridging water molecule, which then becomes a terminal water molecule that must be deprotonated to a hydroxide molecule as is shown in A.

The interaction between the metal-bound water molecule and the conserved histidine His-151 in calcineurin is also depicted in Figure 12. In the first step of this mechanism (Fig. 12A), His-151 is functioning as a general base to remove the proton from the metal-bound water molecule. Alternatively, it may orient the solvent nucleophile for optimal nucleophilic attack of the phosphate ester.

Kinetic isotope studies of calcineurin and -protein phosphatase show that the transition state of the reaction is dissociative. A dissociative transition state is represented in Figure 12B, where bond cleavage to the leaving group has occurred before bond formation to the nucleophile. The phosphoryl group in a dissociative mechanism resembles the metaphosphate anion (147).

A metal-bound water hydroxide coordinated to Fe³⁺ is shown as the attacking nucleophile in Figure 12B, with the Fe³⁺ functioning as a Lewis acid to lower the pK_a of the water molecule. Extensive redox studies by Yu and co-workers (470, 471) have demonstrated a requirement for Fe³⁺ by calcineurin and a loss of activity upon reduction to Fe²⁺, a result consistent with a decreased Lewis acidity of Fe²⁺ versus Fe³⁺.

P-O bond scission in the transition state results in a significant negative charge on the leaving group. Neutralization of the charge by protonation (general acid catalysis) or coordination to a metal ion would lower the energy of the transition state and increase the rate of the reaction. Kinetic isotope studies indicate that considerable charge is neutralized in the transition states of calcineurin and -protein phosphatase. His-151 may play a role in this charge neutralization (Fig. 12B). It is also possible that one of the metal ions (e.g., Zn²⁺ in Fig. 12B) neutralizes the charge to the leaving group by coordination. Another possibility is that a metal-bound solvent molecule acts as a general acid as has been proposed for the mechanism of inorganic pyrophosphatase (140, 355). In addition, the two conserved arginines in the active site may also be important for charge neutralization and transition state stabilization. After bond cleavage and proton transfer to the leaving group occur, the result is the product-inhibited state that has a molecule of orthophosphate bridging the two metal ions of the dinuclear center (Fig. 12C). Evidence for this intermediate is obtained in the X-ray structure of the product-inhibited enzyme which shows phosphate coordinated to both metal ions as depicted (124). An identical coordination is observed in phosphate and tungstate complexes of PP1 (91, 395) and the phosphate complex of purple acid phosphatase (128, 201). Phosphate release, perhaps by exchange with a solvent molecule, regenerates the enzyme for another turnover.

	VI. REGULATION

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The classical mechanism by which calcineurin is regulated in vivo is via changes in intracellular Ca²⁺. Thus, in a resting cell where [Ca²⁺] is low, calcineurin is unable to bind calmodulin, and the enzyme exists in an inactive form. In signaling pathways that lead to a rise in intracellular Ca²⁺, Ca²⁺ binding to calmodulin results in a conformational change, thereby allowing it to bind to calcineurin and activate its phosphatase activity (203). Ca²⁺ binding to calcineurin B also appears to play a role (389).

Calcineurin activity has also been shown to be affected by phospholipids, with either activation or inhibition resulting depending on the phospholipid and substrate investigated (163, 329, 330). Recently, recombinant Dictyostelium calcineurin has been shown to be activated by arachidonic acid and unsaturated, long-chain fatty acids (184). These effects may be physiologically significant given the fact that calcineurin is found associated with membranes during fractionation.

Recently, an additional mechanism for regulating calcineurin involving redox reactions of active site metal ions has been considered (350, 447). Previous studies demonstrated that calcineurin is susceptible to redox regulation in vitro (470, 471), a process that may also occur in vivo. Wang et al. (447) found that superoxide dismutase protects calcineurin from inactivation and hypothesized that this might occur by preventing oxidation of active site metals ions. Recently, a number of groups have begun testing this hypothesis and have provided evidence that calcineurin activity can be affected by extracellular oxidants, in particular, H₂O₂ (45, 111, 345). Thus exposure of cells to micromolar concentrations of H₂O₂ results in inhibition of NF-AT (111, 345) or NFB-mediated processes (45) and appears to be mediated by calcineurin.

The mechanism for this regulation may reside in the redox-active Fe³⁺ of the active site dinuclear metal center, which can toggle between reduced (Fe²⁺) and oxidized (Fe³⁺) states. Indeed, redox titrations by Yu and co-workers (470, 471) previously demonstrated that the mixed-valence oxidation state, either Fe³⁺-Zn²⁺ or Fe³⁺-Fe²⁺, is required for enzyme activity; reduction to the Fe²⁺-M²⁺ (M = Zn, Fe) state led to loss of activity (470, 471). At first glance, the in vivo results, which suggest that oxidation (i.e., treatment with H₂O₂) results in inactivation, appear contrary to the in vitro results. One hypothesis that has been forwarded to reconcile this discrepancy is that calcineurin may exist in different forms containing either Fe-Zn or Fe-Fe dinuclear metal centers. Because the Fe³⁺-Fe²⁺ form of calcineurin can lose activity by oxidation to the Fe³⁺-Fe³⁺ state, it may be that calcineurin exists in oxidation-sensitive (Fe³⁺-Fe²⁺) and oxidation-inert (Fe³⁺-Zn²⁺) states in vivo.

Further work is obviously necessary to determine whether calcineurin is a specific mediator for changes in the redox state of the cytosol. If the dinuclear center of calcineurin has a standard redox potential near the physiological state of the cytosol, a mechanism whereby small alterations in the redox state could mediate changes in enzyme activity would be highly tenable.

	ACKNOWLEDGMENTS

We apologize for any oversight that has resulted in a key reference on calcineurin structure/function being excluded from the bibliography. We gratefully acknowledge Drs. Robert Abraham, Ian Armitage, Howard Brockman, Alvan Hengge, and Ron Victor for their collaborations. Also acknowledged are Timothy Born, Alice Haddy, Michael Kennedy, Nicholas Reiter, Tiffany Reiter, Robert Sikkink, Selene Swanson, Smilja Todorovic, Daniel White, Janet Yao, and Lian Yu for their significant contributions during their tenure in F. Rusnak's laboratory. We thank Dr. Elizabeth Kurian for assistance with the program Quanta. We are indebted to the editorial and review staff of Physiological Reviews, in particular Dr. Susan Hamilton, for support, helpful suggestions, and the opportunity to write this review.

Financial support from National Institute of General Medical Sciences Grant GM-46865 and the Mayo Clinic Eagles Cancer Fund is noted.

	FOOTNOTES

Address for reprint requests and other correspondence: F. Rusnak, Sect. of Hematology Research, Dept. of Biochemistry and Molecular Biology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: rusnak{at}mayo.edu).

¹ Although PP2A was originally characterized as having no divalent metal ion dependence (60), more recently it has also been found to be stabilized or reactivated by divalent metal ions (43).

	REFERENCES

Top Previous

1.	Aitken A, Cohen P, Santikarn S, Williams DH, Calder AG, Smith A, and Klee CB. Identification of the NH2-terminal blocking group of calcineurin B as myristic acid. FEBS Lett 150: 314-318, 1982[Web of Science][Medline].
2.	Aitken A, Klee CB, and Cohen P. The structure of the B subunit of calcineurin. Eur J Biochem 139: 663-671, 1984[Web of Science][Medline].
3.	Alessi DR, Street AJ, Cohen P, and Cohen PTW. Inhibitor-2 functions like a chaperone to fold three expressed isoforms of mammalian protein phosphatase-1 into a conformation with the specificity and regulatory properties of the native enzyme. Eur J Biochem 213: 1055-1066, 1993[Web of Science][Medline].
4.	Alexander DR, Hexham JM, and Crumpton MJ. The association of type 1, type 2A, and type 2B phosphatases with the human T lymphocyte plasma membrane. Biochem J 256: 885-892, 1988[Web of Science][Medline].
5.	Alspaugh JA, Perfect JR, and Heitman J. Signal transduction pathways regulating differentiation and pathogenicity of Cryptococcus neoformans. Fungal Genet Biol 25: 1-14, 1998[Web of Science][Medline].
6.	Ample NM. Emerging disease issues and fungal pathogens associated with HIV infection. Emerg Infect Dis 2: 109-116, 1996[Web of Science][Medline].
7.	Ankarcrona M, Dypbukt JM, Orrenius S, and Nicotera P. Calcineurin and mitochondrial function in glutamate-induced neuronal cell death. FEBS Lett 394: 321-324, 1996[Web of Science][Medline].
8.	Antanaitis BC, Aisen P, and Lilienthal HR. Physical characterization of two-iron uteroferrin: evidence for a spin-coupled binuclear iron cluster. J Biol Chem 258: 3166-3172, 1983[Free Full Text].
9.	Antanaitis BC, Aisen P, Lilienthal HR, Roberts RM, and Bazer FW. The Novel "g = 1.74" EPR spectrum of pink and purple uteroferrin. J Biol Chem 255: 11204-11209, 1980[Abstract/Free Full Text].
10.	Anthony FA, Winkler MA, Edwards HH, and Cheung WY. Quantitative subcellular localization of calmodulin-dependent phosphatase in chick forebrain. J Neurosci 8: 1245-1253, 1988[Abstract].
11.	Antoni FA, Barnard RJO, Shipston MJ, Smith SM, Simpson J, and Paterson JM. Calcineurin feedback inhibition of agonist-evoked cAMP formation. J Biol Chem 270: 28055-28061, 1995[Abstract/Free Full Text].
12.	Antoni FA, Shipston MJ, and Smith SM. Inhibitory role for calcineurin in stimulus-secretion coupling revealed by FK506 and cyclosporin A in pituitary corticotrope tumor cells. Biochem Biophys Res Commun 194: 226-233, 1993[Web of Science][Medline].
13.	Antoni FA, Smith SM, Simpson J, Rosie R, Fink G, and Paterson JM. Calcium control of adenylyl cyclase: the calcineurin connection. Adv Second Messenger Phosphoprot Res 32: 153-172, 1998[Medline].
14.	Aperia A, Ibarra R, Svensson L-B, Klee C, and Greengard P. Calcineurin mediates -adrenergic stimulation of Na+,K+-ATPase activity in renal tubule cells. Proc Natl Acad Sci USA 89: 7394-7397, 1992[Abstract/Free Full Text].
15.	Aramburu J, Yaffe MB, López-Rodríguez C, Cantley LC, Hogan PG, and Rao A. Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A. Science 285: 2129-2133, 1999[Abstract/Free Full Text].
16.	Arndt C, Cruz MC, Cardenas ME, and Heitman J. Secretion of FK506/FK520 and rapamycin by Streptomyces inhibits the growth of competing Saccharomyces cerevisiae and Cryptococcus neoformans. Microbiology 145: 1989-2000, 1999[Abstract/Free Full Text].
17.	Asai A, Qui J-H, Narita Y, Chi S, Saito N, Shinoura N, Hamada H, Kuchino Y, and Kirino T. High level calcineurin activity predisposes neuronal cells to apoptosis. J Biol Chem 274: 34450-34458, 1999[Abstract/Free Full Text].
18.	Averill BA, Davis JC, Burman S, Zirino T, Sanders-Loehr J, Loehr TM, Sage JT, and Debrunner PG. Spectroscopic and magnetic studies of the purple acid phosphatase from bovine spleen. J Am Chem Soc 109: 3760-3767, 1987.
19.	Awumey EM, Moonga BS, Sodam BR, Koval AP, Adebanjo OA, Kumegawa M, Zaidi M, and Epstein S. Molecular and functional evidence for calcineurin-A  and  isoforms in the osteoclast: novel insights into cyclosporin A action on bone resorption. Biochem Biophys Res Communun 254: 248-252, 1999[Web of Science][Medline].
20.	Bacon DJ, and Anderson WF. A fast algorithm for rendering space-filling molecule pictures. J Mol Graph 6: 219-220, 1988.
21.	Banerjee C, Sarkar D, and Bhaduri A. Ca2+ and calmodulin-dependent protein phosphatase from Leishmania donovani. Parasitology 118: 567-573, 1999.
22.	Barford D. Protein phosphatases. Curr Opin Struct Biol 5: 728-734, 1995[Web of Science][Medline].
23.	Barford D. Molecular mechanisms of the protein serine/threonine phosphatases. Trends Biochem Sci 21: 407-412, 1996[Web of Science][Medline].
24.	Barford D, Das AK, and Egloff M-P. Structure and mechanism of protein phosphatases: insights into catalysis and regulation. Annu Rev Biophys Biomol Struct 27: 133-164, 1998[Web of Science][Medline].
25.	Barroso MR, Bernd KK, DeWitt ND, Chang A, Mills K, and Sztul ES. A novel Ca2+-binding protein, p22, is required for constitutive membrane traffic. J Biol Chem 271: 10183-10187, 1996[Abstract/Free Full Text].
26.	Barton GJ, Cohen PTW, and Barford D. Conservation analysis and structure prediction of the protein serine/threonine phosphatases. Eur J Biochem 220: 225-237, 1994[Web of Science][Medline].
27.	Beaumont A, O'Donohue MJ, Paredes N, Rousselet N, Assicot M, Bohuon C, Fournie-Zaluski M-C, and Roques BP. The role of histidine 231 in thermolysin-like enzymes. J Biol Chem 270: 16803-16808, 1995[Abstract/Free Full Text].
28.	Berndt N, and Cohen PTW. Renaturation of protein phosphatase 1 expressed at high levels in insect cells using a baculovirus vector. Eur J Biochem 190: 291-297, 1990[Web of Science][Medline].
29.	Bialojan C, and Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Biochem J 256: 283-290, 1988[Web of Science][Medline].
30.	Bierer BE, Holländer G, Fruman D, and Burakoff SJ. Cyclosporin A and FK506: molecular mechanisms of immunosuppression and probes for transplantation biology. Curr Opin Immunol 5: 763-773, 1993[Web of Science][Medline].
31.	Blumenthal DK, Takio K, Hansen RS, and Krebs EG. Dephosphorylation of cAMP-dependent protein kinase regulatory subunit (type II) by calmodulin-dependent protein phosphatase. J Biol Chem 261: 8140-8145, 1986[Abstract/Free Full Text].
32.	Bonnefoy-Berard N, Genestier L, Flacher M, and Revillard JP. The phosphoprotein phosphatase calcineurin controls calcium-dependent apoptosis in B cell lines. Eur J Immunol 24: 325-329, 1994[Web of Science][Medline].
33.	Bork P, Brown NP, Hegyi H, and Schultz J. The protein phosphatase 2C (PP2C) superfamily: detection of bacterial homologues. Protein Sci 5: 1421-1425, 1996[Web of Science][Medline].
34.	Bosser R, Aligué R, Guerini D, Agell N, Carafoli E, and Bachs O. Calmodulin can modulate protein phosphorylation in rat liver cells nuclei. J Biol Chem 268: 15477-15483, 1993[Abstract/Free Full Text].
35.	Boxer AL, Moreno H, Rudy B, and Ziff EB. FGF-2 potentiates Ca2+-dependent inactivation of NMDA receptor currents in hippocampal neurons. J Neurophysiol 82: 3367-3377, 1999[Abstract/Free Full Text].
36.	Breuder T, Hemenway CS, Movva NR, Cardenas ME, and Heitman J. Calcineurin is essential in cyclosporin A- and FK506-sensitive yeast strains. Proc Natl Acad Sci USA 91: 5372-5376, 1994[Abstract/Free Full Text].
37.	Brill GM, Premachandran U, Karwowski JP, Henry R, Cwik DK, Traphagen LM, Humphrey PE, Jackson M, Clement JJ, Burres NS, Kadam S, Chen RH, and McAlpine JB. Dibefurin, a novel fungal metabolite inhibiting calcineurin phosphatase activity. J Antibiot 49: 124-128, 1996[Medline].
38.	Brown L, Chen MX, and Cohen PTW. Identification of a cDNA encoding a Drosophila calcium/calmodulin regulated protein phosphatase, which has its most abundant expression in the early embryo. FEBS Lett 339: 124-128, 1994[Web of Science][Medline].
39.	Bruice TC, Brown A, and Harris DO. On the concept of orbital steering in catalytic reactions. Proc Natl Acad Sci USA 68: 658-661, 1971[Abstract/Free Full Text].
40.	Burroughs SE, Horrocks WD Jr, Ren H, and Klee CB. Characterization of the lanthanide ion-binding properties of calcineurin-B using laser-induced luminescence spectroscopy. Biochemistry 33: 10428-10436, 1994[Medline].
41.	Butcher SP, Henshall DC, Teramura Y, Iwasaki K, and Sharkey J. Neuroprotective actions of FK506 in experimental stroke: in vivo evidence against an antiexcitotoxic mechanism. J Neurosci 17: 6939-6946, 1997[Abstract/Free Full Text].
42.	Buttini M, Limonta S, Luyten M, and Boddeke H. Distribution of calcineurin A isoenzyme mRNAs in rat thymus and kidney. Histochem J 27: 291-299, 1995[Web of Science][Medline].
43.	Cai L, Chu Y, Wilson SE, and Schlender KK. A metal-dependent form of protein phosphatase 2A. Biochem Biophys Res Commun 208: 274-279, 1995[Web of Science][Medline].
44.	Calalb MB, Kincaid RL, and Soderling TR. Phosphorylation of calcineurin: effect on calmodulin binding. Biochem Biophys Res Commun 172: 551-556, 1990[Web of Science][Medline].
45.	Carballo M, Márquez G, Conde M, Martín-Nieto J, Monteseirín J, Conde J, Pintado E, and Sobrino F. Characterization of calcineurin in human neutrophils. J Biol Chem 274: 93-100, 1999[Abstract/Free Full Text].
46.	Cardenas ME, and Heitman J. Role of calcium in T-lymphocyte activation. Adv Second Messenger Phosphoprotein Res 30: 281-298, 1995[Web of Science][Medline].
47.	Cardenas ME, Lorenz M, Hemenway C, and Heitman J. Yeast as model T cells. Perspect Drug Disc Design 2: 103-126, 1994.
48.	Chang C-D, Mukai H, Kuno T, and Tanaka C. cDNA cloning of an alternatively spliced isoform of the regulatory subunit of Ca2+/calmodulin-dependent protein phosphatase (calcineurin B2). Biochim Biophys Acta 1217: 174-180, 1994[Medline].
49.	Chang HY, Takei K, Sydor AM, Born T, Rusnak F, and Jay DG. Asymmetric retraction of growth cone filopodia following focal inactivation of calcineurin. Nature 376: 686-690, 1995[Medline].
50.	Chantler PD. Calcium-dependent association of a protein complex with the lymphocyte plasma membrane: probable identity with calmodulin-calcineurin. J Cell Biol 101: 207-216, 1985[Abstract/Free Full Text].
51.	Chen T-C, Law B, Kondratyuk T, and Rossie S. Identification of soluble protein phosphatases that dephosphorylate voltage-sensitive sodium channels in rat brain. J Biol Chem 270: 7750-7756, 1995[Abstract/Free Full Text].
52.	Cho H, Krishnaraj R, Kitas E, Bannwarth W, Walsh CT, and Anderson KS. Isolation and structural elucidation of a novel phosphocysteine intermediate in the LAR protein tyrosine phosphatase enzymatic pathway. J Am Chem Soc 114: 7296-7298, 1992.
53.	Chu Y, Lee EYC, and Schlender KK. Activation of protein phosphatase 1: formation of a metalloenzyme. J Biol Chem 271: 2574-2577, 1996[Abstract/Free Full Text].
54.	Chu Y, Wilson SE, and Schlender KK. A latent form of protein phosphatase 1 associated with bovine heart myofibrils. Biochim Biophys Acta 1208: 45-54, 1994[Medline].
55.	Clipstone NA, and Crabtree GR. Calcineurin is a key signaling enzyme in T lymphocyte activation and the target of the immunosuppressive drugs cyclosporin A and FK506. Ann NY Acad Sci 696: 20-30, 1993[Web of Science][Medline].
56.	Clipstone NA, Fiorentino DF, and Crabtree GR. Molecular analysis of the interaction of calcineurin with drug-immunophilin complexes. J Biol Chem 269: 26431-26437, 1994[Abstract/Free Full Text].
57.	Coghlan VM, Bergeson SE, Langeberg L, Nilaver G, and Scott JD. A-kinase anchoring proteins: a key to selective activation of cAMP-responsive events? Mol Cell Biochem 127/128: 309-319, 1993.
58.	Coghlan VM, Perrino BA, Howard M, Langeberg LK, Hicks JB, Gallatin WM, and Scott JD. Association of protein kinase A and protein phosphatase 2B with a common anchoring protein. Science 267: 108-111, 1995[Abstract/Free Full Text].
59.	Cohen P. The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature 296: 613-620, 1982[Medline].
60.	Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem 58: 453-508, 1989[Web of Science][Medline].
61.	Cohen P, and Cohen PTW. Protein phosphatases come of age. J Biol Chem 264: 21435-21438, 1989[Free Full Text].
62.	Cohen PTW. Nomenclature and chromosomal localization of human protein serine/threonine phosphatase Genes. Adv Prot Phosphatases 8: 371-376, 1994.
63.	Coleman JE. Structure and mechanism of alkaline phosphatase. Annu Rev Biophys Biomol Struct 21: 441-483, 1992[Web of Science][Medline].
64.	Coleman JE, and Gettins P. Alkaline phosphatase p, solution structure, and mechanism. Adv Enzymol 55: 381-452, 1983.
65.	Conboy IM, Manoli D, Mhaiskar V, and Jones PP. Calcineurin and vacuolar-type H+-ATPase modulate macrophage effector functions. Proc Natl Acad Sci USA 96: 6324-6329, 1999[Abstract/Free Full Text].
66.	Cooper NGF, McLaughlin BJ, Tallant EA, and Cheung WY. Calmodulin-dependent protein phosphatase: immunocytochemical localization in chick retina. J Cell Biol 101: 1212-1218, 1985[Abstract/Free Full Text].
67.	Cruz MC, Del Poeta M, Wang P, Wenger R, Zenke G, Quesniaux VFJ, Movva NR, Perfect JR, Cardenas ME, and Heitman J. Immunosuppressive and nonimmunosuppressive cyclosporine analogs are toxic to the opportunistic fungal pathogen Cryptoccus neoformans via cyclophilin-dependent inhibition of calcineurin. Antimicrobial Agents Chemother 44: 143-149, 2000[Abstract/Free Full Text].
68.	Cunningham KW, and Fink GR. Ca2+ transport in Saccharomyces cerevisiae. J Exp Biol 196: 157-166, 1994[Abstract/Free Full Text].
69.	Cunningham KW, and Fink GR. Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae. Mol Cell Biol 16: 2226-2237, 1996[Abstract].
70.	Cunningham KW, and Kink GR. Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases. J Cell Biol 124: 351-363, 1994[Abstract/Free Full Text].
71.	Cyert MS. The function of Ca2+/calmodulin-regulated phosphatase in yeast. Adv Protein Phosphatases 7: 429-443, 1993.
72.	Cyert MS, Kunisawa R, Kaim D, and Thorner J. Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase. Proc Natl Acad Sci USA 88: 7376-7380, 1991[Abstract/Free Full Text].
73.	Cyert MS, and Thorner J. Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone. Mol Cell Biol 12: 3460-3469, 1992[Abstract/Free Full Text].
74.	Da Cruz e Silva E, and Cohen PTW. Isolation of a cDNA likely to encode a novel Ca2+-dependent/calmodulin-stimulated protein phosphatase. Biochim Biophys Acta 1009: 293-296, 1989[Medline].
75.	Dammann H, Hellstern S, Husain Q, and Mutzel R. Primary structure, expression and developmental regulation of a Dictyostelium calcineurin A homolog. Eur J Biochem 238: 391-399, 1996[Web of Science][Medline].
76.	Danielsson A, Larsson C, Larsson K, Gustafsson L, and Adler L. A genetic analysis of the role of calcineurin and calmodulin in Ca2+-dependent improvement of NaCl tolerance of Saccharomyces cerevisiae. Curr Genet 30: 476-484, 1996[Web of Science][Medline].
77.	Davis JC, and Averill BA. Evidence for a spin-coupled binuclear iron unit at the active site of the purple acid phosphatase from beef spleen. Proc Natl Acad Sci USA 79: 4623-4627, 1982[Abstract/Free Full Text].
78.	Davis TN. Calcium in Saccharomyces cerevisiae. Adv Second Messenger Phosphoprotein Res 30: 339-358, 1995[Web of Science][Medline].
79.	Dawson TM, Steiner JP, Dawson VL, Dinerman JL, Uhl GR, and Snyder SH. Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity. Proc Natl Acad Sci USA 90: 9808-9812, 1993[Abstract/Free Full Text].
80.	Day EP, David SS, Peterson J, Dunham WR, Bonvoisin JJ, Sands RH, and Que L Jr. Magnetization and electron paramagnetic resonance studies of reduced uteroferrin and its "EPR-silent" phosphate complex. J Biol Chem 263: 15561-15567, 1988[Abstract/Free Full Text].
81.	De Castro E, Nef S, Fiumelli H, Lenz SE, Kawamura S, and Nef P. Regulation of rhodopsin phosphorylation by a family of neuronal calcium sensors. Biochem Biophys Res Commun 216: 133-140, 1995[Web of Science][Medline].
82.	De la Pompa JL, Timmerman LA, Takimoto H, Yoshida H, Elia AJ, Samper E, Potter J, Wakeham A, Marengere L, Langille BL, Crabtree GR, and Mak TW. Role of the NF-ATc transcription factor in morphogenesis of cardiac valves and septum. Nature 392: 182-186, 1998[Medline].
83.	Desdouits F, Buxbaum JD, Desdouits-Magnen J, Nairn AC, and Greengard P. Amyloid  peptide formation in cell-free preparations. J Biol Chem 271: 24670-24674, 1996[Abstract/Free Full Text].
84.	Desdouits F, Siciliano JC, Nairn AC, Greengard P, and Girault J-A. Dephosphorylation of Ser-137 in DARRP-32 by protein phosphatases 2A and 2C: different roles in vitro and in striatonigral neurons. Biochem J 330: 211-216, 1998.
85.	Dietrich M, Münstermann D, Suerbaum H, and Witzel H. Purple acid phosphatase from bovine spleen: interactions at the active site in relation to the reaction mechanism. Eur J Biochem 199: 105-113, 1991[Web of Science][Medline].
86.	Ding B, Price RL, Borg TK, Weinberg EO, Halloran PF, and Lorell BH. Pressure overload induces severe hypertrophy in mice treated with cyclosporine, an inhibitor of calcineurin. Circ Res 84: 729-734, 1999[Abstract/Free Full Text].
87.	Dobson S, May T, Berriman M, Vecchio CD, Fairlamb AH, Chakrabarti D, and Barik S. Characterization of protein Ser/Thr phosphatases of the malaria parasite, Plasmodium falciparum: inhibition of the parasitic calcineurin by cyclophilin-cyclosporin complex. Mol Biochem Parasitol 99: 167-181, 1999[Web of Science][Medline].
88.	Dunn SE, Burns JL, and Michel RN. Calcineurin is required for skeletal muscle hypertrophy. J Biol Chem 274: 21908-21912, 1999[Abstract/Free Full Text].
89.	Dutz JP, Fruman DA, Burakoff SJ, and Beirer BE. A role of calcineurin in degranulation of murine cytotoxic T lymphocytes. J Immunol 150: 2591-2598, 1993[Abstract].
90.	Earnest S, Khokhlatchev A, Albanesi JP, and Barylko B. Phosphorylation of dynamin by ERK2 inhibits the dynamin-microtubule interaction. FEBS Lett 396: 62-66, 1996[Web of Science][Medline].
91.	Egloff M-P, Cohen PTW, Reinemer P, and Barford D. Crystal structure of the catalytic subunit of human protein phosphatase 1 and its complex with tungstate. J Mol Biol 254: 942-959, 1995[Web of Science][Medline].
92.	Ek-Rylander B, Bill P, Norgård M, Nilsson S, and Andersson G. Cloning, sequence, and developmental expression of a type 5, tartarate-resistant, acid phosphatase from rat bone. J Biol Chem 266: 24684-24689, 1991[Abstract/Free Full Text].
93.	Enan E, and Matsumura F. Specific inhibition of calcineurin by type II synthetic pyrethroid insecticides. Biochem Pharmacol 43: 1777-1784, 1992[Web of Science][Medline].
94.	Endo S, Connor JH, Forney B, Zhang L, Ingebritsen TS, Lee EYC, and Shenolikar S. Conversion of protein phosphatase 1 catalytic subunit to a Mn2+-dependent enzyme impairs its regulation by inhibitor 1. Biochemistry 36: 6986-6992, 1997[Medline].
95.	Eng W-K, Faucette L, McLaughlin MM, Cafferkey R, Koltin Y, Morris RA, Young PR, Johnson RK, and Livi GP. The yeast FKS1 gene encodes a novel membrane protein, mutations in which confer FK506 and cyclosporin A hypersensitivity and calcineurin-dependent growth. Gene 151: 61-71, 1994[Web of Science][Medline].
96.	Enslen H, and Soderling TR. Roles of calmodulin-dependent protein kinases and phosphatase in calcium-dependent transcription of immediate early genes. J Biol Chem 269: 20872-20877, 1994[Abstract/Free Full Text].
97.	Enz A, and Pombo-Villar E. Class II pyrethroids: noninhibitors of calcineurin. Biochem Pharmacol 54: 321-323, 1997[Web of Science][Medline].
98.	Enz A, Zenke G, and Pombo-Villar E. 7-Oxa[221]bicycloheptane-2,3-dicarboxylic acid derivatives as phosphatase inhibitors. Bioorg Med Chem Lett 7: 2513-2518, 1997.
99.	Fakata KL, Swanson SA, Vorce RL, and Stemmer PM. Pyrethroid insecticides as phosphatase inhibitors. Biochem Pharmacol 55: 2017-2022, 1998[Web of Science][Medline].
100.	Farcasanu IC, Hirata D, Tsuchiya E, Nishiyama F, and Miyakawa T. Protein phosphatase 2B of Saccharomyces cerevisiae is required for tolerance to manganese, in blocking the entry of ions into the cells. Eur J Biochem 232: 712-717, 1995[Web of Science][Medline].
101.	Farghali H, and Masek K. Immunopharmacologic agents in the amelioration of hepatic injuries. Int J Immunopharm 20: 125-139, 1998[Web of Science][Medline].
102.	Fast DJ, Lynch RC, and Leu RW. Cyclosporin A inhibits nitric oxide production by L929 cells in response to tumor necrosis factor and interferon-. J Interferon Res 13: 235-240, 1993[Web of Science][Medline].
103.	Faux MC, and Scott JD. More on target with protein phosphorylation: conferring specificity by location. Trends Biochem Sci 21: 312-315, 1996[Web of Science][Medline].
104.	Feng B, and Stemmer PM. Interactions of calcineurin A, calcineurin B, and Ca2+. Biochemistry 38: 12481-12489, 1999[Medline].
105.	Ferrando A, Kron SJ, Rios G, Fink GR, and Serrano R. Regulation of cation transport in Saccharomyces cerevisiae by salt tolerance gene HAL3. Mol Cell Biol 15: 5470-5481, 1995[Abstract].
106.	Ferreira A, Kincaid R, and Kosik KS. Calcineurin is associated with the cytoskeleton of cultured neurons and has a role in the acquisition of polarity. Mol Biol Cell 4: 1225-1238, 1993[Abstract].
107.	Foor F, Parent SA, Morin N, Dahl AM, Ramadan N, Chrebet G, Bostian KA, and Nielsen JB. Calcineurin mediates inhibition by FK506 and cyclosporin of recovery from -factor arrest in yeast. Nature 360: 682-684, 1992[Medline].
108.	Force T, Rosenzweig A, Choukroun G, and Hajjar R. Calcineurin inhibitors and cardiac hypertrophy. Lancet 353: 1290-1292, 1999[Web of Science][Medline].
109.	Friedhoff P, Kolmes B, Gimadutdinow O, Wende W, Krause KL, and Pingoud A. Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis. Nucleic Acid Res 24: 2632-2639, 1996[Abstract/Free Full Text].
110.	Fruman DA, Mather PE, Burakoff SJ, and Bierer BE. Correlation of calcineurin phosphatase activity and programmed cell death in murine T cell hybridomas. Eur J Immunol 22: 2513-2517, 1992[Web of Science][Medline].
111.	Furuke K, Shiraishi M, Mostowski HS, and Bloom ET. Fas ligand induction in human NK cells is regulated by redox through a calcineurin-nuclear factors of activated T cell-dependent pathway. J Immunol 162: 1988-1993, 1999[Abstract/Free Full Text].
112.	Gagliardino JJ, Krinks MH, and Gagliardino EE. Identification of the calmodulin-regulated protein phosphatase, calcineurin, in rat pancreatic islets. Biochim Biophys Acta 1091: 370-373, 1991[Medline].
113.	Ganster RW, McCartney RR, and Schmidt MC. Identification of a calcineurin-independent pathway required for sodium ion stress response in Saccharomyces cerevisiae. Genetics 150: 31-42, 1998[Abstract/Free Full Text].
114.	Garrett-Engele P, Moilanen B, and Cyert MS. Calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is essential in yeast mutants with cell integrity defects and in mutants that lack a functional vacuolar H+-ATPase. Mol Cell Biol 15: 4103-4114, 1995[Abstract].
115.	Garver TD, Kincaid RL, Conn RA, and Billingsley ML. Reduction of calcineurin activity in brain by antisense oligonucleotides leads to persistent phosphorylation of  protein at Thr181 and Thr231. Mol Pharmacol 55: 632-641, 1999[Abstract/Free Full Text].
116.	Genestier L, Dearden-Badet MT, Bonnefoy-Berard N, Lizard G, and Revillard JP. Cyclosporin A and FK506 inhibit activation-induced cell death in the murine WEHI-231 B cell line. Cell Immunol 155: 283-291, 1994[Web of Science][Medline].
117.	Giri PR, Higuchi S, and Kincaid RL. Chromosomal mapping of the human genes for the calmodulin-dependent protein phosphatase (calcineurin) catalytic subunit. Biochem Biophys Res Commun 181: 252-258, 1991[Web of Science][Medline].
118.	Giri PR, Marietta CA, Higuchi S, and Kincaid RL. Molecular and phylogenetic analysis of calmodulin-dependent protein phosphatase (calcineurin) catalytic subunit genes. DNA Cell Biol 11: 415-424, 1992[Web of Science][Medline].
119.	Gold BG. FK506 and the role of immunophilins in nerve regeneration. Mol Neurobiol 15: 285-306, 1997[Web of Science][Medline].
120.	Goldberg J, Huang H-B, Kwon Y-G, Greengard P, Nairn AC, and Kuriyan J. Three dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1. Nature 376: 745-753, 1995[Medline].
121.	Goto S, and Ushio Y. Immunostaining for calcineurin, a Ca2+/calmodulin-regulated protein phosphatase, in the diagnostic tumor pathology. Brain Tumor Pathol 12: 23-30, 1995.
122.	Goto S, Yamamoto H, Fukunaga K, Iwasa T, Matsukado Y, and Miyamoto E. Dephosphorylation of microtubule-associated protein 2,  factor, and tubulin by calcineurin. J Neurochem 45: 276-283, 1985[Web of Science][Medline].
123.	Graef IA, Mermelstein PG, Stankunas K, Neilson JR, Deisseroth K, Tsein RW, and Crabtree GR. L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in hippocampal neurons. Nature 401: 703-708, 1999[Medline].
124.	Griffith JP, Kim JL, Kim EE, Sintchak MD, Thomson JA, Fitzgibbon MJ, Fleming MA, Caron PR, Hsiao K, and Navia MA. X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex. Cell 82: 507-522, 1995[Web of Science][Medline].
125.	Groblewski GE, Wagner ACC, and Williams JA. Cyclosporin A inhibits Ca2+/calmodulin-dependent protein phosphatase and secretion in pancreatic acinar cells. J Biol Chem 269: 15111-15117, 1994[Abstract/Free Full Text].
126.	Gualberto A, Marquez G, Carballo M, Youngblood GL, Hunt SW III, Baldwin AS, and Sobrino F. p53 transactivation of the HIV-1 long terminal repeat is blocked by PD 144795, a calcineurin-inhibitor with anti-HIV properties. J Biol Chem 273: 7088-7093, 1998[Abstract/Free Full Text].
127.	Guan K, and Dixon JE. Evidence for protein-tyrosine-phosphatase catalysis proceeding via a cysteine-phosphate intermediate. J Biol Chem 266: 17026-17030, 1991[Abstract/Free Full Text].
128.	Guddat LW, McAlpine AS, Hume D, Hamilton S, de Jersey J, and Martin JL. Crystal structure of mammalian purple acid phosphatase. Structure 7: 757-767, 1999[Medline].
129.	Guerini D, and Klee CB. Cloning of human calcineurin A: evidence for two isozymes and identification of a proline structural domain. Proc Natl Acad Sci USA 86: 9183-9187, 1989[Abstract/Free Full Text].
130.	Guerini D, and Klee CB. Structural diversity of calcineurin, a Ca2+ and calmodulin-stimulated protein phosphatase. Adv Protein Phosphatases 6: 391-410, 1991.
131.	Guerini D, Krinks MH, Sikela JM, Hahn WE, and Klee CB. Isolation and sequence of a cDNA clone for human calcineurin B, the Ca2+-binding subunit of the Ca2+/calmodulin-stimulated protein phosphatase. DNA 8: 675-682, 1989[Web of Science][Medline].
132.	Guerini D, Montell C, and Klee CB. Molecular cloning and characterization of the genes encoding the two subunits of Drosophila melanogaster calcineurin. J Biol Chem 267: 22542-22549, 1992[Abstract/Free Full Text].
132a.	Håkansson K, Carlsson M, Svensson LA, and Liljas A. Structure of native and Apo carbonic anhydrase II and structure of some of its anion-ligand complexes. J Mol Biol 227: 1192-1204, 1992[Web of Science][Medline].
133.	Halloran PF. Molecular mechanisms of new immunosuppressants. Clin Transplant 10: 118-123, 1996[Web of Science][Medline].
134.	Halloran PF, Kung L, and Noujaim J. Calcineurin and the biological effect of cyclosporine and tacrolimus. Transplant Proc 30: 2167-2170, 1998[Web of Science][Medline].
135.	Hanley RM, Dedman JR, and Shenolikar S. Identification of high-affinity calmodulin-binding proteins in rat liver. Am J Physiol Cell Physiol 252: C272-C284, 1987.
136.	Hashimoto Y, King MM, and Soderling TR. Regulatory interactions of calmodulin-binding proteins: phosphorylation of calcineurin by autophosphorylated Ca2+/calmodulin-dependent protein kinase II. Proc. Natl Acad Sci USA 85: 7001-7005, 1988[Abstract/Free Full Text].
137.	Hashimoto Y, Perrino BA, and Soderling TR. Identification of an autoinhibitory domain in calcineurin. J Biol Chem 265: 1924-1927, 1990[Abstract/Free Full Text].
138.	Hashimoto Y, and Soderling TR. Regulation of calcineurin by phosphorylation. Identification of the regulatory site phosphorylated by Ca2+/calmodulin-dependent protein kinase II and protein kinase C. J Biol Chem 264: 16524-16529, 1989[Abstract/Free Full Text].
139.	Hassett A, Blättler W, and Knowles JR. Pyruvate kinase: is the mechanism of phospho transfer associative or dissociative? Biochemistry 21: 6335-6340, 1982[Medline].
140.	Heikinheimo P, Pohjanjoki P, Helminen A, Tasanen M, Cooperman BS, Goldman A, Baykov A, and Lahti R. A site-directed mutagenesis study of Saccharomyces cerevisiae pyrophosphatase. Functional conservation of the active site of soluble inorganic pyrophosphatases. Eur J Biochem 239: 138-143, 1996[Web of Science][Medline].
141.	Heitman J, Cardenas ME, Breuder T, Hemenway C, Muir RS, Lim E, Goetz L, Zhu D, Lorenz M, and Dolinski K. Antifungal effects of cyclosporine and FK 506 are mediated via immunophilin-dependent calcineurin inhibition. Transplant Proc 26: 2833-2834, 1994[Web of Science][Medline].
142.	Heitman J, Koller A, Cardenas ME, and Hall MN. Identification of immunosuppressive drug targets in yeast. Methods: A Companion to Methods Enzymol 5: 176-187, 1993.
143.	Hemenway C, and Heitman J. Proline isomerases in microorganisms and small eukaryotes. Ann NY Acad Sci 696: 38-46, 1993[Web of Science][Medline].
144.	Hemenway CS, Dolinski K, Cardenas ME, Hiller MA, Jones EW, and Heitman J. vph6 mutants of Saccharomyces cerevisiae require calcineurin for growth and are defective in vacuolar H+-ATPase assembly. Genetics 141: 833-844, 1995[Abstract].
145.	Hemenway CS, and Heitman J. Calcineurin: structure, function, and inhibition. Cell Biochem. Biophys 30: 115-151, 1999[Medline].
146.	Hendey B, Klee CB, and Maxfield FR. Inhibition of neutrophil chemokinesis on vitronectin by inhibitors of calcineurin. Science 258: 296-299, 1992[Abstract/Free Full Text].
147.	Hengge AC. Transfer of the PO32 group. In: Comprehensive Biological Catalysis: A Mechanistic Reference. Reactions of Electrophilic Carbon, Phosphorus and Sulfur, edited by Sinnott M. San Diego, CA: Academic, 1998, vol. 1, p. 517-542.
148.	Hengge AC, Denu JM, and Dixon JE. Transition-state structures for the native dual-specific phosphatase VHR and D92N and S131A mutants. contributions to the driving force for catalysis. Biochemistry 35: 7084-7092, 1996[Medline].
149.	Hengge AC, Edens WA, and Elsing H. Transition-state structures for phosphoryl-transfer reactions of p-nitrophenyl phosphate. J Am Chem Soc 116: 5045-5049, 1994.
150.	Hengge AC, and Martin BL. Isotope effect studies on the calcineurin phosphoryl-transfer reaction: transition state structure and effect of calmodulin and Mn2+. Biochemistry 36: 10185-10191, 1997[Medline].
151.	Hengge AC, Sowa GA, Wu L, and Zhang Z-Y. Nature of the transition state of the protein-tyrosine phosphatase-catalyzed reaction. Biochemistry 34: 13982-13987, 1995[Medline].
152.	Hengge AC, Zhao Y, Wu L, and Zhang Z-Y. Examination of the transition state of the low-molecular mass small tyrosine phosphatase. 1. Comparison with other protein phosphatases. Biochemistry 36: 7928-7936, 1997[Medline].
153.	Higuchi S, Tamura J, Giri PR, Polli JW, and Kincaid RL. Calmodulin-dependent protein phosphatase from Neurospora crassa. J Biol Chem 266: 18104-18112, 1991[Abstract/Free Full Text].
154.	Hirata D, Harada S-I, Namba H, and Miyakawa T. Adaptation to high-salt stress in Saccharomyces cerevisiae is regulated by Ca2+/calmodulin-dependent phosphoprotein phosphatase (calcineurin) and cAMP-dependent protein kinase. Mol Gen Genet 249: 257-264, 1995[Web of Science][Medline].
155.	Ho S, Clipstone N, Timmermann L, Northrop J, Graef I, Fiorentino D, Nouse J, and Crabtree G. The mechanism of action of cyclosporin A and FK506. Clin Immunol Immunopathol 80: S40-S45, 1996[Web of Science][Medline].
156.	Hoff RH, Mertz P, Rusnak F, and Hengge AC. The transition state of the phosphoryl-transfer reaction catalyzed by the lambda Ser/Thr protein phosphatase. J Am Chem Soc 121: 6382-6390, 1999.
157.	Holtz-Heppelmann CJ, Algeciras A, Badley AD, and Paya CV. Transcriptional regulation of the human FasL promoter-enhancer region. J Biol Chem 273: 4416-4423, 1998[Abstract/Free Full Text].
158.	Hong C-S, and Ganetzky B. Molecular characterization of neurally expressing genes in the para sodium channel gene cluster of Drosophila. Genetics 142: 879-892, 1996[Abstract].
159.	Horn F, and Gross J. A role for calcineurin in Dictyostelium discoideum development. Differentiation 60: 269-275, 1996[Web of Science][Medline].
160.	Huang EP. Synaptic plasticity: going through phases with LTP. Curr Biol 8: R350-R352, 1998[Web of Science][Medline].
161.	Huang H-B, Horiuchi A, Goldberg J, Greengard P, and Nairn AC. Site-directed mutagenesis of amino acid residues of protein phosphatase 1 involved in catalysis and inhibitor binding. Proc Natl Acad Sci USA 94: 3530-3535, 1997[Abstract/Free Full Text].
162.	Huang R-Q, and Dillon GH. Maintenance of recombinant type A -aminobutyric acid receptor function: role of protein tyrosine phosphorylation and calcineurin. J Pharmacol Exp Ther 286: 243-255, 1998[Abstract/Free Full Text].
163.	Huang S, Merat D, and Cheung WY. Phosphatidylinositol modulates the response of calmodulin-dependent phosphatase to calmodulin. Arch Biochem Biophys 270: 42-49, 1989[Web of Science][Medline].
164.	Husi H, Luyten MA, and Zurini MGM. Mapping of the immunophilin-immunosuppressant site of interaction on calcineurin. J Biol Chem 269: 14199-14204, 1994[Abstract/Free Full Text].
166.	Iida H, Yagawa Y, and Anraku Y. Essential role for induced Ca2+ influx followed by [Ca2+]i rise in maintaining viability of yeast cells late in the mating pheromone response pathway. J Biol Chem 265: 13391-13399, 1990[Abstract/Free Full Text].
167.	Ingebritsen TS, and Cohen P. Protein phosphatases: properties and role in cellular regulation. Science 221: 331-338, 1983[Abstract/Free Full Text].
168.	Ingebritsen TS, Stewart AA, and Cohen P. The protein phosphatases involved in cellular regulation: measurement of type-1 and type-2 phosphatases in extracts of mammalian tissues; assessment of their physiological roles. Eur J Biochem 132: 297-307, 1983[Web of Science][Medline].
169.	Ito A, Hashimoto T, Hirai M, Takeda T, Shuntoh H, Kuno T, and Tanaka C. The complete primary structure of calcineurin A, a calmodulin binding protein homologous with protein phosphatases 1 and 2A. Biochem Biophys Res Commun 163: 1492-1497, 1989[Web of Science][Medline].
170.	Iwata M, Ohoka Y, Kuwata T, and Asada A. Regulation of T cell apoptosis via T cell receptors and steroid receptors. Stem Cells 14: 632-641, 1996[Web of Science][Medline].
171.	Izumo S, and Aoki H. Calcineurinthe missing link in cardiac hypertrophy. Nature Med 4: 661-662, 1998[Web of Science][Medline].
172.	Jabri E, Carr MB, Hausinger RP, and Karplus PA. The crystal structure of urease from Klebsiella aerogenes. Science 268: 998-1004, 1995[Abstract/Free Full Text].
173.	Jain J, Loh C, and Rao A. Transcriptional regulation of the IL-2 gene. Curr Opin Immunol 7: 333-342, 1995[Web of Science][Medline].
174.	Jencks WP, and Page MI. "Orbital steering," entropy, and rate accelerations. Biochem Biophys Res Commun 57: 887-892, 1974[Web of Science][Medline].
175.	Jiang H, Xiong F, Kong S, Ogawa T, Kobayasi M, and Liu JO. Distinct tissue and cellular distribution of two major isoforms of calcineurin. Mol Immunol 34: 663-669, 1997[Web of Science][Medline].
176.	Kakalis LT, Kennedy M, Sikkink R, Rusnak F, and Armitage IM. Characterization of the calcium-binding sites of calcineurin B. FEBS Lett 362: 55-58, 1995[Web of Science][Medline].
177.	Kashishian A, Howard M, Loh C, Gallatin WM, Hoekstra MF, and Lai T. AKAP79 inhibits calcineurin through a site distinct from the immunophilin-binding region. J Biol Chem 273: 27412-27419, 1998[Abstract/Free Full Text].
178.	Kawasaki H, and Kretsinger RH. Calcium-binding proteins 1: EF-hands. Protein Profile 2: 1-490, 1995[Web of Science][Medline].
179.	Kay JE, Doe SEA, and Benzie CR. The mechanism of action of the immunosuppressive drug FK-506. Cell Immunol 124: 175-181, 1989[Web of Science][Medline].
180.	Kaye RE, Fruman DA, Bierer BE, Albers MW, Zydowsky LD, Ho SI, Jin Y-J, Castells MC, Schreiber SL, Walsh CT, Burakoff SJ, Austen KF, and Katz HR. Effects of cyclosporin A and FK506 on Fc receptor type I-initiated increases in cytokine mRNA in mouse bone marrow-derived progenitor mast cells: resistance to FK506 is associated with a deficiency in FK506-binding protein FKBP12. Proc Natl Acad Sci USA 89: 8542-8546, 1992[Abstract/Free Full Text].
181.	Kayyali US, Zhang W, Yee AG, Seidman JG, and Potter H. Cytoskeletal changes in the brains of mice lacking calcineurin A. J Neurochemistry 68: 1668-1678, 1997[Web of Science][Medline].
182.	Kennedy MT, Brockman H, and Rusnak F. Contributions of myristoylation to calcineurin structure/function. J Biol Chem 271: 26517-26521, 1996[Abstract/Free Full Text].
183.	Kennedy MT, Brockman H, and Rusnak F. Determinants of calcineurin binding to model membranes. Biochemistry 36: 13579-13585, 1997[Medline].
184.	Kessen U, Schaloske R, Aichem A, and Mutzel R. Ca2+/calmodulin-independent activation of calcineurin from Dictyostelium by unsaturated long chain fatty acids. J Biol Chem 274: 37821-37826, 1999[Abstract/Free Full Text].
185.	Ketcham CM, Roberts RM, Simmen RCM, and Nick HS. Molecular cloning of the type 5, iron-containing, tartarate-resistant acid phosphatase from human placenta. J Biol Chem 264: 557-563, 1989[Abstract/Free Full Text].
186.	Khattab A, Pica-Mattoccia L, Wenger R, Cioli D, and Klinkert M-Q. Assay of Schistosoma mansoni calcineurin phosphatase activity and assessment of its role in parasite survival. Mol Biochem Parasitol 99: 269-273, 1999[Web of Science][Medline].
187.	Kim EE, and Wyckoff HW. Reaction mechanism of alkaline phosphatase based on crystal structures: two-metal ion catalysis. J Mol Biol 218: 449-464, 1991[Web of Science][Medline].
188.	Kincaid R. Calmodulin-dependent protein phosphatases from microorganisms to man: a study in structural conservatism and biological diversity. Adv Second Messenger Phosphoprotein Res 27: 1-23, 1993[Web of Science][Medline].
189.	Kincaid RL. The role of calcineurin in immune system responses. J Allergy Clin Immunol 95: 1170-1177, 1995.
190.	Kincaid RL, Giri PR, Higuchi S, Tamura J, Dixon SC, Marietta CA, Amorese DA, and Martin BM. Cloning and characterization of molecular isoforms of the catalytic subunit of calcineurin using nonisotopic methods. J Biol Chem 265: 11312-11319, 1990[Abstract/Free Full Text].
191.	Kincaid RL, Nightingale MS, and Martin BM. Characterization of a cDNA clone encoding the calmodulin-binding domain of mouse brain calcineurin. Proc Natl Acad Sci USA 85: 8983-8987, 1988[Abstract/Free Full Text].
192.	Kincaid RL, and O'Keefe SJ. Calcineurin and immunosuppression: a calmodulin-dependent protein phosphatase acts as the "gatekeeper" to interleukin-2 gene transcription. Adv Protein Phosphatases 7: 543-583, 1993.
193.	Kincaid RL, Takayama H, Billingsley ML, and Sitkovsky MV. Differential expression of calmodulin-binding proteins in B, T lymphocytes and thymocytes. Nature 330: 176-178, 1987[Medline].
194.	King MM, and Huang CY. Activation of calcineurin by nickel ions. Biochem Biophys Res Commun 114: 955-961, 1983[Web of Science][Medline].
195.	King MM, and Huang CY. The calmodulin-dependent activation and deactivation of the phosphoprotein phosphatase, calcineurin, and the effect of nucleotides, pyrophosphate, and divalent metal ions. J Biol Chem 259: 8847-8856, 1984[Abstract/Free Full Text].
196.	King MM, Huang CY, Chock PB, Nairn AC, Hemmings HC Jr, Chan K-FJ, and Greengard P. Mammalian brain phosphoproteins as substrates for calcineurin. J Biol Chem 259: 8080-8083, 1984[Abstract/Free Full Text].
197.	Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, Kalish VJ, Tucker KD, Showalter RE, Moomaw EW, Gastinel LN, Habuka N, Chen X, Maldonado F, Barker JE, Bacquet R, and Villafranca JE. Crystal structure of human calcineurin and the human FKBP12-FK506-calcineurin complex. Nature 378: 641-644, 1995[Medline].
198.	Kissmehl R, Treptau T, Hofer HW, and Plattner H. Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia. Biochem J 317: 65-76, 1996.
199.	Klabunde T, and Krebs B. The dimetal center in purple acid phosphatase. Struct Bonding 89: 177-198, 1997.
200.	Klabunde T, Stahl B, Suerbaum H, Hahner S, Karas M, Hillenkamp F, Krebs B, and Witzel H. The amino acid sequence of red kidney bean Fe(III)-Zn(II) purple acid phosphatase. Eur J Biochem 226: 369-375, 1994[Web of Science][Medline].
201.	Klabunde T, Sträter N, Frälich R, Witzel H, and Krebs B. Mechanism of Fe(III)-Zn(II) purple acid phosphatase based on crystal structure. J Mol Biol 259: 737-748, 1996[Web of Science][Medline].
202.	Klee CB. Concerted regulation of protein phosphorylation and dephosphorylation by calmodulin. Neurochem Res 16: 1059-1065, 1991[Web of Science][Medline].
203.	Klee CB, and Cohen P. The calmodulin-regulated protein phosphatase. In: Molecular Aspects of Cellular Regulation: Calmodulin, edited by Cohen P, and Klee CB. Amsterdam: Elsevier, 1988, vol. 5, p. 225-248.
204.	Klee CB, Crouch TH, and Krinks MH. Calcineurin: a calcium- and calmodulin-binding protein of the nervous system. Proc Natl Acad Sci USA 76: 6270-6273, 1979[Abstract/Free Full Text].
205.	Klee CB, Draetta GF, and Hubbard MJ. Calcineurin. Adv Enzymol Relat Areas Mol Biol 61: 149-200, 1988[Web of Science][Medline].
206.	Klee CB, and Krinks MH. Purification of cyclic 3',5'-nucleotide phosphodiesterase inhibitory protein by affinity chromatography on activator protein coupled to Sepharose. Biochemistry 17: 120-126, 1978[Medline].
207.	Klee CB, Krinks MH, Manalan AS, Draetta GF, and Newton DL. Control of calcineurin protein phosphatase activity. Adv Protein Phosphatases 1: 135-146, 1985.
208.	Klee CB, Ren H, and Wang X. Regulation of the calmodulin-stimulated protein phosphatase calcineurin. J Biol Chem 273: 13367-13370, 1998[Free Full Text].
209.	Klumpp S, Steiner AL, and Schultz JE. Immunocytochemical localization of cyclic GMP, cGMP-dependent protein kinase, calmodulin and calcineurin in Paramecium tetraurelia. Eur J Cell Biol 32: 164-170, 1983[Web of Science][Medline].
210.	Knapp J, Bokník P, Huke S, Bombosová I, Linck B, Lüss H, Müller FU, Müller T, Nacke P, Schmitz W, Vahlensieck U, and Neumann J. Contractility and inhibition of protein phosphatases by cantharidin. Gen Pharmacol 31: 729-733, 1998[Web of Science][Medline].
211.	Knöfel T, and Ströter N. X-ray structure of the Escherichia coli periplasmic 5'-nucleotidase containing a dimetal catalytic site. Nature Struct Biol 6: 448-453, 1999[Web of Science][Medline].
212.	Koonin EV. Conserved sequence pattern in a wide variety of phosphoesterases. Protein Sci 3: 356-358, 1994[Web of Science][Medline].
213.	Kothe GO, and Free SJ. Calcineurin subunit B is required for normal vegetative growth in Neurospora crassa. Fungal Genet Biol 23: 248-258, 1998[Web of Science][Medline].
214.	Kraulis P. MOLSCRIPT: a program to produce both detail and schematic plots of protein structure. J Appl Crystallogr 24: 946-950, 1991.
215.	Krebs B. Purple acid phosphatases and catechol oxidase and their model compounds. Crystal structure studies of purple acid phosphatase from red kidney beans. In: Bioinorganic Chemistry: An Inorganic Perspective of Life, edited by Kessissoglou DP. Dordrecht, Germany: Kluwer, 1995, p. 371-384.
216.	Krinks MH, Manalan AS, and Klee CB. Calcineurin: a brain-specific isozyme of protein phosphatase-2B (Abstract). Federation Proc 44: 707a, 1985.
217.	Krupp JJ, Vissel B, Heinemann SF, and Westbrook GL. Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific. Mol Pharmacol 50: 1680-1688, 1996[Abstract].
218.	Kudla J, Xu Q, Harter K, Gruissem W, and Luan S. Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals. Proc Natl Acad Sci USA 96: 4718-4723, 1999[Abstract/Free Full Text].
219.	Kuno T, Mukai H, Ito A, Chang C-D, Kishima K, Saito N, and Tanaka C. Distinct cellular expression of calcineurin A and A in rat brain. J Neurochemistry 58: 1643-1651, 1992[Web of Science][Medline].
220.	Kuno T, Takeda T, Hirai M, Ito A, Mukai H, and Tanaka C. Evidence for a second isoform of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A). Biochem Biophys Res Commun 165: 1352-1358, 1989[Web of Science][Medline].
221.	Kuno T, Tanaka H, Mukai H, Chang C-D, Hiraga K, Miyakawa T, and Tanaka C. cDNA cloning of a calcineurin B homolog in Saccharomyces cerevisiae. Biochem Biophys Res Commun 180: 1159-1163, 1991[Web of Science][Medline].
222.	Kunz J, and Hall MN. Cyclosporin A, FK506 and rapamycin: more than just immunosuppression. Trends Biochem Sci 18: 334-338, 1993[Web of Science][Medline].
223.	Kurtz DM Jr. Oxo- and hydroxo-bridged diiron complexes: a chemical perspective on a biological unit. Chem Rev 90: 585-606, 1990.
224.	Lai MM, Burnett PE, Wolosker H, Blackshaw S, and Snyder SH. Cain, a novel physiological protein inhibitor of calcineurin. J Biol Chem 273: 18325-18331, 1998[Abstract/Free Full Text].
225.	Lai MM, Hong JJ, Ruggiero AM, Burnett PE, Slepnev VI, De Camilli P, and Snyder SH. The calcineurin-dynamin I complex as a calcium sensor for synaptic vesicle endocytosis. J Biol Chem 274: 25963-25966, 1999[Abstract/Free Full Text].
226.	Latinis KM, Norian LA, Eliason SL, and Koretzky GA. Two NFAT transcription factor binding sites participate in the regulation of CD95 (Fas) ligand expression in activated human T cells. J Biol Chem 272: 31427-31434, 1997[Abstract/Free Full Text].
227.	Lawen A, Baker MA, and Malik S. Apoptosis and redox homeostasis: on a possible mechanism of action of Bcl-2. Protoplasma 205: 10-20, 1998[Web of Science].
228.	Lawson MA, and Maxfield FR. Ca2+- and calcineurin-dependent recycling of an integrin to the front of migrating neutrophils. Nature 377: 75-79, 1995[Medline].
229.	Leng J, Cameron AJM, Buckel S, and Kennelly PJ. Isolation and cloning of a protein-serine/threonine phosphatase from an archaeon. J Bacteriol 177: 6510-6517, 1995[Abstract/Free Full Text].
230.	Li H-C. Activation of brain calcineurin phosphatase towards nonprotein phosphoesters by Ca2+, calmodulin, and Mg2+. J Biol Chem 259: 8801-8807, 1984[Abstract/Free Full Text].
231.	Lieberman DN, and Mody I. Regulation of NMDA channel function by endogenous Ca2+-dependent phosphatase. Nature 369: 235-239, 1994[Medline].
232.	Lim HW, and Molkentin JD. Calcineurin and human heart failure. Nature Med 5: 246-247, 1999[Web of Science][Medline].
233.	Lim HW, and Molkentin JD. Reply to revisiting calcineurin in human heart failure. Nature Med 6: 3, 2000[Web of Science][Medline].
234.	Lin X, and Barber DL. A calcineurin homologous protein inhibits GTPase-stimulated Na-H exchange. Proc Natl Acad Sci USA 93: 12631-12636, 1996[Abstract/Free Full Text].
235.	Lin X, Sikkink RA, Rusnak F, and Barber DL. Inhibition of calcineurin phosphatase activity by a calcineurin B homologous protein. J Biol Chem 274: 36125-36131, 1999[Abstract/Free Full Text].
236.	Liu J. FK506 and cyclosporin, molecular probes for studying intracellular signal transduction. Immunol Today 14: 290-295, 1993[Web of Science][Medline].
237.	Liu J. FK506 and cyclosporin: molecular probes for studying intracellular signal transduction. Trends Pharmacol Sci 14: 182-188, 1993[Medline].
238.	Liu J, Farmer JD Jr, Lane WS, Friedman J, Weissman I, and Schreiber SL. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66: 807-815, 1991[Web of Science][Medline].
239.	Liu J, and Zhu J-K. A calcium sensor homolog required for plant salt tolerance. Science 280: 1943-1945, 1998[Abstract/Free Full Text].
240.	Liu J-P, Sim ATR, and Robinson PJ. Calcineurin inhibition of dynamin I GTPase activity coupled to nerve terminal depolarization. Science 265: 970-973, 1994[Abstract/Free Full Text].
241.	Liu Q-S, and Berg DK. Actin filaments and the opposing actions of CaM kinase II and calcineurin in regulating 7-containing nicotinic receptors on chick ciliary ganglion neurons. J Neurosci 19: 10280-10288, 1999[Abstract/Free Full Text].
242.	Liu Y, Ishii S, Tokai M, Tsutsumi H, Ohki O, Akada R, Tanaka K, Tsuchiya E, Fukui S, and Miyakawa T. The Saccharomyces cerevisiae genes (CMP1 and CMP2) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B. Mol Gen Genet 227: 52-59, 1991[Web of Science][Medline].
243.	Lobo FM, Zanjani R, Ho N, Chatila TA, and Fuleihan RL. Calcium-dependent activation of TNF family gene expression by Ca2+/calmodulin kinase type IV/GR and calcineurin. J Immunol 162: 2057-2063, 1999[Abstract/Free Full Text].
244.	Lohse DL, Denu JM, and Dixon JE. Insights derived from the structures of the Ser/Thr phosphatases calcineurin and protein phosphatase 1. Structure 3: 987-990, 1995[Medline].
245.	Luan S. Protein phosphatases and signaling cascades in higher plants. Trends Plant Sci 3: 271-275, 1998[Web of Science].
246.	Luan S, Li W, Rusnak F, Assmann SM, and Schreiber SL. Immunosuppressants implicate protein phosphatase regulation of K+ channels in guard cells. Proc Natl Acad Sci USA 90: 2202-2206, 1993[Abstract/Free Full Text].
247.	Luo Z, Shyu K-G, Gualberto A, and Walsh K. Calcineurin inhibitors and cardiac hypertrophy. Nature Med 4: 1092-1093, 1998[Web of Science][Medline].
248.	MacKintosh C, Beattie KA, Klumpp S, Cohen P, and Codd GA. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. FEBS Lett 264: 187-192, 1990[Web of Science][Medline].
249.	MacKintosh C, and MacKintosh RW. Inhibitors of protein kinases and phosphatases. Trends Biochem Sci 19: 444-448, 1994[Web of Science][Medline].
250.	Magósci M, Apáti A, Gáti R, and Kolonics A. Signalling mechanisms and the role of calcineurin in erythropoiesis. Immunol Lett 68: 187-195, 1999[Web of Science][Medline].
251.	Mai B, Frey G, Swanson RV, Mathur EJ, and Stetter KO. Molecular cloning and functional expression of a protein-serine/threonine phosphatase from the hyperthermophilic archaeon Pyrodictum abyssi TAG11. J Bacteriol 180: 4030-4035, 1998[Abstract/Free Full Text].
252.	Malchow D, Mutzel R, and Schlatterer C. On the role of calcium during chemotactic signalling and differentiation of the cellular slime mold Dictyostelium discoideum. Int J Dev Biol 40: 135-139, 1996[Web of Science][Medline].
253.	Mandelkow E-M, Biernat J, Drewes G, Gustke N, Trinczek B, and Mandelkow E. Tau domains, phosphorylation, and interactions with microtubules. Neurobiol Aging 16: 355-363, 1995[Web of Science][Medline].
254.	Mansuy IM, Mayford M, Jacob B, Kandel ER, and Bach ME. Restricted and regulated overexpression reveals calcineurin as a key component in the transition from short-term to long-term memory. Cell 92: 39-49, 1998[Web of Science][Medline].
255.	Marks AR. Cellular functions of immunophilins. Physiol Rev 76: 631-649, 1996[Abstract/Free Full Text].
256.	Marrion NV. Calcineurin regulates M channel modal gating in sympathetic neurons. Neuron 16: 163-173, 1996[Web of Science][Medline].
257.	Martensen TM, Martin BM, and Kincaid RL. Identification of the site on calcineurin phosphorylated by Ca2+/CaM-dependent kinase II: modification of the CaM-binding domain. Biochemistry 28: 9243-9247, 1989[Medline].
258.	Martin BL. Inhibition of calcineurin by the tyrphostin class of tyrosine kinase inhibitors. Biochem Pharmacol 56: 483-488, 1998[Web of Science][Medline].
259.	Martin BL, and Graves DJ. Mechanistic aspects of the low-molecular-weight phosphatase activity of the calmodulin-activated phosphatase, calcineurin. J Biol Chem 261: 14545-14550, 1986[Abstract/Free Full Text].
260.	Martin BL, and Graves DJ. Isotope effects on the mechanism of calcineurin catalysis: kinetic solvent isotope and isotope exchange studies. Biochim Biophys Acta 1206: 136-142, 1994[Medline].
261.	Martin BL, Jurado LA, and Hengge AC. Comparison of the reaction progress of calcineurin with Mn2+ and Mg2+. Biochemistry 38: 3386-3392, 1999[Medline].
262.	Martin BL, Pallen CJ, Wang JH, and Graves DJ. Use of fluorinated tyrosine phosphates to probe the substrate specificity of the low molecular weight phosphatase activity of calcineurin. J Biol Chem 260: 14932-14937, 1985[Abstract/Free Full Text].
263.	Matheos DP, Kingsbury TJ, Ahsan US, and Cunningham KW. Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae. Genes Dev 11: 3445-3458, 1997[Abstract/Free Full Text].
264.	McFerran BW, Graham ME, and Burgoyne RD. Neuronal Ca2+ sensor 1, the mammalian homologue of frequenin, is expressed in chromaffin and PC12 cells and regulates neurosecretion from dense-core granules. J Biol Chem 273: 22768-22772, 1998[Abstract/Free Full Text].
265.	McPartlin AE, Barker HM, and Cohen PTW. Identification of a third alternatively spliced cDNA encoding the catalytic subunit of protein phosphatase 2B. Biochim Biophys Acta 1088: 308-310, 1991[Medline].
266.	Means TR. Calcium, calmodulin, and cell cycle regulation. FEBS Lett 347: 1-4, 1994[Web of Science][Medline].
267.	Meguro T, Hong C, Asai K, Takagi G, McKinsey TA, Olson EN, and Vatner SF. Cyclosporine attenuates pressure-overload hypertrophy in mice while enhancing susceptibility to decompensation and heart failure. Circ Res 84: 735-740, 1999[Abstract/Free Full Text].
268.	Mendoza I, Quintero FJ, Bressan RA, Hasegawa PM, and Pardo JM. Activated calcineurin confers high tolerance to ion stress and alters the budding pattern and cell morphology of yeast cells. J Biol Chem 271: 23061-23067, 1996[Abstract/Free Full Text].
269.	Mendoza I, Rubio F, Rodriguez-Navarro A, and Pardo JM. The protein phosphatase calcineurin is essential for NaCl tolerance of Saccharomyces cerevisiae. J Biol Chem 269: 8792-8796, 1994[Abstract/Free Full Text].
270.	Menger FM, and Glass LE. Contribution of orbital alignment to organic and enzymatic reactivity. J Am Chem Soc 102: 5404-5406, 1980.
271.	Merat DL, and Cheung WY. Calmodulin-dependent protein phosphatase: isolation of subunits and reconstitution to holoenzyme. Methods Enzymol 139: 79-87, 1987[Web of Science][Medline].
272.	Mertz P, Yu L, Sikkink R, and Rusnak F. Kinetic and spectroscopic analyses of mutants of a conserved histidine in the metallophosphatases calcineurin and  protein phosphatase. J Biol Chem 272: 21296-21302, 1997[Abstract/Free Full Text].
273.	Mesecar AD, Stoddard BL, and Koshland DE Jr. Orbital steering in the catalytic power of enzymes: small structural changes seen with large catalytic consequences. Science 277: 202-206, 1997[Abstract/Free Full Text].
274.	Metcalfe SM, and Richards FM. Cyclosporine, FK506, and rapamycin. Transplantation 49: 798-802, 1990[Web of Science][Medline].
275.	Michalak M, Fu SY, Milner RE, Busaan JL, and Hance JE. Phosphorylation of the carboxyl-terminal region of dystrophin. Biochem Cell Biol 74: 431-437, 1996[Web of Science][Medline].
276.	Mikoshiba K. The InsP3 receptor and intracellular Ca2+ signaling. Curr Opin Neurobiol 7: 339-345, 1997[Web of Science][Medline].
277.	Miskin JE, Abrams CC, Goatley LC, and Dixon LK. A viral mechanism for inhibition of the cellular phosphatase calcineurin. Science 281: 562-565, 1998[Abstract/Free Full Text].
278.	Miyamoto K, Matsui H, Tomizawa K, Kuwata Y, Itano T, Tokuda M, and Hatase O. In situ localization of rat testis-specific calcineurin B subunit isoform 1 in the developing rat testis. Biochem Biophys Res Commun 203: 1275-1283, 1994[Web of Science][Medline].
279.	Mizunuma M, Hirata D, Miyahara K, Tsuchiya E, and Miyakawa T. Role of calcineurin and Mpk1 in regulating the onset of mitosis in budding yeast. Nature 392: 303-306, 1998[Medline].
280.	Molkentin JD. Calcineurin inhibition and cardiac hypertrophy. Science 282: 1007a, 1998[Free Full Text].
281.	Molkentin JD, Lu J-R, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, and Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93: 215-228, 1998[Web of Science][Medline].
282.	Momayezi M, Lumpert CJ, Kersken J, Gras U, Plattner H, Krinks MH, and Klee CB. Exocytosis induction in Paramecium tetraurelia cells by exogenous phosphoprotein phosphatase in vivo and in vitro: possible involvement of calcineurin in exocytotic membrane fusion. J Cell Biol 105: 181-189, 1987[Abstract/Free Full Text].
283.	Mondragon A, Griffith EC, Sun L, Xiong F, Armstrong C, and Liu JO. Overexpression and purification of human calcineurin  from Escherichia coli and assessment of catalytic functions of residues surrounding the binuclear metal center. Biochemistry 36: 4934-4942, 1997[Medline].
284.	Montoro RJ, Díaz-Nido J, Avila J, and López-Barneo J. N-methyl-D-aspartate stimulates the dephosphorylation of the microtubule-associated protein 2 and potentiates excitatory synaptic pathways in the rat hippocampus. Neuroscience 54: 859-871, 1993[Web of Science][Medline].
285.	Morioka M, Hamada J-I, Ushio Y, and Miyamoto E. Potential role of calcineurin for brain ischemia and traumatic injury. Prog Neurobiol 58: 1-30, 1999[Web of Science][Medline].
286.	Moriya M, Fujinaga K, Yazawa M, and Katagiri C. Immunohistochemical localization of the calcium/calmodulin-dependent protein phosphatase calcineurin, in the mouse testis: its unique accumulation in spermatid nuclei. Cell Tissue Res 281: 273-281, 1995[Web of Science][Medline].
287.	Moser MJ, Geiser JR, and Davis TN. Ca2+-calmodulin promotes survival of pheromone-induced growth arrest by activation of calcineurin and Ca2+-calmodulin-dependent protein kinase. Mol Cell Biol 16: 4824-4831, 1996[Abstract].
288.	Mueller EG, Crowder MW, Averill BA, and Knowles JR. Purple acid phosphatase: a diiron enzyme that catalyzes a direct phospho group transfer to water. J Am Chem Soc 115: 2974-2975, 1993.
289.	Mukai H, Chang C-D, Tanaka H, Ito A, Kuno T, and Tanaka C. cDNA cloning of a novel testis-specific calcineurin B-like protein. Biochem Biophys Res Commun 179: 1325-1330, 1991[Web of Science][Medline].
290.	Müller JG, Nemoto S, Laser M, Carabello BA, and Menick DR. Calcineurin inhibition and cardiac hypertrophy. Science 282: 1007, 1998.
291.	Muramatsu T, Giri PR, Higuchi S, and Kincaid RL. Molecular cloning of a calmodulin-dependent phosphatase from murine testis: identification of a developmentally expressed nonneural isoenzyme. Proc Natl Acad Sci USA 89: 529-533, 1992[Abstract/Free Full Text].
292.	Muramatsu T, and Kincaid RL. Molecular cloning and chromosomal mapping of the human gene for the testis-specific catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A). Biochem Biophys Res Commun 188: 265-271, 1992[Web of Science][Medline].
293.	Muramatsu T, and Kincaid RL. Molecular cloning of a full-length cDNA encoding the catalytic subunit of human calmodulin-dependent protein phosphatase (calcineurin A). Biochim Biophys Acta 1178: 117-120, 1993[Medline].
294.	Murphy BJ, Rossie S, De Jongh KS, and Catterall WA. Identification of the sites of selective phosphorylation and dephosphorylation of the rat brain Na+ channel  subunit by cAMP-dependent protein kinase and phosphoprotein phosphatases. J Biol Chem 268: 27355-27362, 1993[Abstract/Free Full Text].
295.	Musarò A, McCullagh KJA, Naya FJ, Olson EN, and Rosenthal N. IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1. Nature 400: 581-585, 1999[Medline].
296.	Myers JK, Antonelli SM, and Widlanski TS. Motifs for metallophosphatase inhibition. J Am Chem Soc 119: 3163-3164, 1997.
297.	Naik UP, Patel PM, and Parise LV. Identification of  novel calcium-binding protein that interacts with the integrin iib cytoplasmic domain. J Biol Chem 272: 4651-4654, 1997[Abstract/Free Full Text].
298.	Nair APK, Hahn S, Banholzer R, Hirsch HH, and Moroni C. Cyclosporin A inhibits growth of autocrine tumour cell lines by destabilizing interleukin-3 mRNA. Nature 369: 239-242, 1994[Medline].
299.	Nairn AC, and Shenolikar S. The role of protein phosphatases in synaptic transmission, plasticity, and neuronal development. Curr Opin Neurobiol 2: 296-301, 1992[Medline].
300.	Nakamura T, Liu Y, Hirata D, Namba H, Harada S-I, Hirokawa T, and Miyakawa T. Protein phosphatase type 2B (calcineurin)-mediated, FK506-sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions. EMBO J 12: 4063-4071, 1993[Web of Science][Medline].
301.	Nakamura T, Ohmoto T, Hirata D, Tsuchiya E, and Miyakawa T. Genetic evidence for the functional redundancy of the calcineurin- and Mpk1-mediated pathways in the regulation of cellular events important for growth in Saccharomyes cerevisiae. Mol Gen Genet 251: 211-219, 1996[Web of Science][Medline].
302.	Nakayama S, and Kretsinger RH. Evolution of the EF-hand family of proteins. Annu Rev Biophys Biomol Struct 23: 473-507, 1994[Web of Science][Medline].
303.	Nanthakumar NN, Dayton JS, and Means AR. Role of Ca2+/calmodulin binding proteins in Aspergillus nidulans cell cycle regulation. Prog Cell Cycle Res 2: 217-228, 1996[Medline].
304.	Nargang CE, Bottorff DA, and Adachi Z. Isolation and characterization of a cDNA clone coding for the calcium-binding subunit of calcineurin from bovine brain: an identical amino acid sequence to the human protein. DNA Sequence 4: 313-318, 1994[Web of Science][Medline].
305.	Nataraj C, Oliverio MI, Mannon RB, Mannon PJ, Audoly LP, Amuchastegui CS, Ruiz P, Smithies O, and Coffman TM. Angiotensin II regulates cellular immune responses through a calcineurin-dependent pathway. J Clin Invest 104: 1693-1701, 1999[Web of Science][Medline].
306.	Natarajan K, Ness J, Wooge CH, Janovick JA, and Conn PM. Specific identification and subcellular localization of three calmodulin-binding proteins in the rat gonadotrope: spectrin, caldesmon, and calcineurin. Biol Reprod 44: 43-52, 1991[Abstract].
307.	Navia MA. Protein-drug complexes important for immunoregulation and organ transplantation. Curr Opin Struct Biol 6: 838-847, 1996[Web of Science][Medline].
308.	Nichols RA, Suplick GR, and Brown JM. Calcineurin-mediated protein dephosphorylation in brain nerve terminals regulates the release of glutamate. J Biol Chem 269: 23817-23823, 1994[Abstract/Free Full Text].
309.	Odom A, Del Poeta M, Perfect J, and Heitman J. The immunosuppressant FK506 and its nonimmunosuppressive analog L-685,818 are toxic to Cryptococcus neoformans by inhibition of a common target protein. Antimicrobial Agents Chemother 41: 156-161, 1997[Abstract].
310.	Odom A, Muir S, Liu E, Toffaletti DL, Perfect J, and Heitman J. Calcineurin is required for virulence of Cryptococcus neoformans. EMBO J 16: 2576-2589, 1997[Web of Science][Medline].
311.	O'Keefe SJ, and O'Neill EA. Cyclosporin A and FK-506: immunosuppression, inhibition of transcription and the role of calcineurin. Perspect Drug Disc Design 2: 85-102, 1994.
312.	Olson EN, and Molkentin JD. Prevention of cardiac hypertrophy by calcineurin inhibition. Hype or hope? Circ Res 84: 623-632, 1999[Free Full Text].
313.	Paidhungat M, and Garrett S. A homolog of mammalian, voltage-gated calcium channels mediates yeast pheromone-stimulated Ca2+ uptake and exacerbates the cdc1(ts) growth defect. Mol Cell Biol 17: 6339-6347, 1997[Abstract].
314.	Pallen CJ, Valentine KA, Wang JH, and Hollenberg MD. Calcineurin-mediated dephosphorylation of the human placental membrane receptor for epidermal growth factor urogastrone. Biochemistry 24: 4727-4730, 1985[Medline].
315.	Pallen CJ, and Wang JH. Regulation of calcineurin by metal ions. J Biol Chem 259: 6134-6141, 1984[Abstract/Free Full Text].
316.	Pallen CJ, and Wang JH. A multifunctional calmodulin-stimulated phosphatase. Arch Biochem Biophys 237: 281-291, 1985[Web of Science][Medline].
317.	Pallen CJ, and Wang JH. Stoichiometry and dynamic interaction of metal ion activators with calcineurin phosphatase. J Biol Chem 261: 16115-16120, 1986[Abstract/Free Full Text].
318.	Papadopoulos V, Brown AS, and Hall PF. Isolation and characterization of calcineurin from adrenal cell cytoskeleton: identification of substrates of Ca2+-calmodulin-dependent phosphatase activity. Mol Cell Endocrinol 63: 23-38, 1989[Web of Science][Medline].
319.	Papadopoulos V, Brown AS, and Hall PF. Calcium-calmodulin-dependent phosphorylation of cytoskeletal proteins from adrenal cells. Mol Cell Endocrinol 74: 109-123, 1990[Web of Science][Medline].
320.	Pardo JM, Reddy MP, Yang S, Maggio A, Huh G-H, Matsumoto T, Coca MA, Paino-D'Urzo M, Koiwa H, Yun D-J, Watad AA, Bressan RA, and Hasegawa PM. Stress signaling through Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates salt adaption in plants. Proc Natl Acad Sci USA 95: 9681-9686, 1998[Abstract/Free Full Text].
321.	Park I-S, and Hausinger RP. Site-directed mutagensis of Klebsiella aerogenes urease: identification of histidine residues that appear to function in nickel ligation, substrate binding, and catalysis. Protein Sci 2: 1034-1041, 1993[Web of Science][Medline].
322.	Paterson JM, Smith SM, Harmar AJ, and Antoni FA. Control of a novel adenylyl cyclase by calcineurin. Biochem Biophys Res Commun 214: 1000-1008, 1995[Web of Science][Medline].
323.	Pawson T, and Scott JD. Signaling through scaffold, anchoring, and adaptor proteins. Science 278: 2075-2080, 1997[Abstract/Free Full Text].
324.	Perrino BA. Regulation of calcineurin phosphatase activity by its autoinhibitory domain. Arch Biochem Biophys 372: 159-165, 1999[Web of Science][Medline].
325.	Perrino BA, Fong Y-L, Brickey DA, Saitoh Y, Ushio Y, Fukunaga K, Miyamoto E, and Soderling TR. Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform. J Biol Chem 267: 15965-15969, 1992[Abstract/Free Full Text].
326.	Perrino BA, Ng LY, and Soderling TR. Calcium regulation of calcineurin phosphatase activity by its B subunit and calmodulin. J Biol Chem 270: 340-346, 1995[Abstract/Free Full Text].
327.	Plochocka-Zulinska D, Rasmussen G, and Rasmussen C. Regulation of calcineurin gene expression in Schizosaccharomyces pombe. J Biol Chem 270: 24794-24799, 1995[Abstract/Free Full Text].
328.	Plowman GD, Sudarsanam S, Bingham J, Whyte D, and Hunter T. The protein kinases of Caenorhabditis elegans: a model for signal transduction in multicellular organisms. Proc Natl Acad Sci USA 96: 13603-13610, 1999[Abstract/Free Full Text].
329.	Politino M, and King MM. Calcium- and calmodulin-sensitive interactions of calcineurin with phospholipids. J Biol Chem 262: 10109-10113, 1987[Abstract/Free Full Text].
330.	Politno M, and King MM. Calcineurin-phospholipid interactions: identification of the phospholipid-binding subunit and analyses of a two-stage binding process. J Biol Chem 265: 7619-7622, 1990[Abstract/Free Full Text].
331.	Pomiés P, Frachet P, and Block MR. Control of the 51 integrin/fibronectin interaction in vitro by the serine/threonine protein phosphatase calcineurin. Biochemistry 34: 5104-5112, 1995[Medline].
332.	Pozos TC, Sekler I, and Cyert MS. The product of HUM1, a novel yeast gene, is required for vacuolar Ca2+/H+ exchange and is related to mammalian Na+/Ca2+ exchangers. Mol Cell Biol 16: 3730-3741, 1996[Abstract/Free Full Text].
333.	Price NE, and Mumby MC. Brain protein serine/threonine phosphatases. Curr Opin Neurobiol 9: 336-342, 1999[Web of Science][Medline].
334.	Priggemeyer S, Eggers-Borkenstein P, Ahlers F, Henkel G, Körner M, Witzel H, Nolting H-F, and Krebs B. XAS investigations on the iron-zinc center of purple acid phosphatase from red kidney beans. Inorg Chem 34: 1445-1454, 1995.
335.	Prokisch H, Yarden O, Dieminger M, Tropschug M, and Barthelmess IB. Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2+ gradient. Mol Gen Genet 256: 104-114, 1997[Web of Science][Medline].
336.	Pujol MJ, Bosser R, Vendrell M, Serratosa J, and Bachs O. Nuclear calmodulin-binding protiens in rat neurons. J Neurochem 60: 1422-1428, 1993[Web of Science][Medline].
337.	Ranger AM, Grusby MJ, Hodge MR, Gravallese EM, de la Brousse FC, Hoey T, Mickanin C, Baldwin HS, and Glimcher LH. The transcription factor NF-ATc is essential for cardiac valve formation. Nature 392: 186-190, 1998[Medline].
338.	Rao A. NF-ATp: a transcription factor required for the co-ordinate induction of several cytokine genes. Immunol Today 15: 274-280, 1994[Web of Science][Medline].
339.	Rao A. NFATp, a cyclosporin-sensitive transcription factor implicated in cytokine gene induction. J Leukoc Biol 57: 536-542, 1995[Abstract].
340.	Rao A, Luo C, and Hogan PG. Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol 15: 707-747, 1997[Web of Science][Medline].
341.	Rao J, and Wang JH. Calcineurin immunoprecipitated from bovine brain extract contains no detectable Ni2+ or Mn2+. J Biol Chem 264: 1058-1061, 1989[Abstract/Free Full Text].
342.	Rascher C, Pahl A, Pecht A, Brune K, Solbach W, and Bang H. Leishmania major parasites express cyclophilin isoforms with an unusual interaction with calcineurin. Biochem J 334: 659-667, 1998.
343.	Rasmussen C, Garen C, Brining S, Kincaid RL, Means RL, and Means AR. The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans. EMBO J 13: 2545-2552, 1994[Web of Science][Medline].
344.	Raufman J-P, Malhotra R, and Rafaniello RD. Regulation of calcium-induced exocytosis from gastric chief cells by protein phosphatase-2B (calcineurin). Biochim Biophys Acta 1357: 73-80, 1997[Medline].
345.	Reiter TA, Abraham RT, Choi M, and Rusnak F. Redox regulation of calcineurin in T-lymphocytes. J Biol Inorg Chem 4: 632-644, 1999[Web of Science][Medline].
346.	Remillard SP, Lai EY, Levy YY, and Fulton C. A calcineurin-B-encoding gene expressed during differentiation of the amoeboflagellate Naegleria gruberi contains two introns. Gene 154: 39-45, 1995[Web of Science][Medline].
347.	Roberts HC, Sternberg JM, and Chappell LH. Characterization of calcineurin from Hymenolepsis microstoma and H. diminuta and its interactions with cyclosporin A. Parasitology 114: 279-283, 1997.
348.	Roeper J, Lorra C, and Pongs O. Frequency-dependent inactivation of mammalian A-type K+ channel kv1.4 regulated by Ca2+/calmodulin-dependent protein kinase. J Neurosci 17: 3379-3391, 1997[Abstract/Free Full Text].
349.	Rudolph HK, Antebi A, Fink GR, Buckley CM, Dorman TE, LeVitre J, Davidow LS, Mao J-I, and Moir DT. The yeast secretory pathway is perturbed by mutations in Pmr1, a member of the Ca2+ ATPase family. Cell 58: 133-145, 1989[Web of Science][Medline].
350.	Rusnak F, Mertz P, Reiter T, and Yu L. Regulation of the protein phosphatase calcineurin by redox: implications for catalysis and signal transduction. In: Iron Metabolism: Inorganic Biochemistry and Regulatory Mechanisms, edited by Ferreira GC, Moura JJG, and Franco R. New York: Wiley-VCH, 1999, p. 275-302.
351.	Rusnak F, Yu L, and Mertz P. Metalloenzymes and signal transduction: the protein serine/threonine phosphatases, a novel class of binuclear metal-containing enzymes. J Biol Inorg Chem 1: 388-396, 1996[Web of Science].
352.	Rusnak F, Yu L, Todorovic S, and Mertz P. The interaction of bacteriophage  protein phosphatase with Mn(II): evidence for the formation of a [Mn(II)2] cluster. Biochemistry 38: 6943-6952, 1999[Medline].
353.	Sabatini DM, Lai MM, and Snyder SH. Neural roles of immunophilins and their ligands. Mol Neurobiol 15: 223-239, 1997[Web of Science][Medline].
354.	Saitoh Y, Maeda S, Fukunaga K, Yasugawa S, Sakamoto Y, Uyemura K, Shimada K, Ushio Y, and Miyamoto E. Nucleotide sequence of a rat calcineurin A cDNA lacking a specific 30-base pair region. Biomed Res 12: 215-218, 1991.
355.	Salminen T, Käpylä J, Heikinheimo P, Kankare J, Goldman A, Heinonen J, Baykov AA, Cooperman BS, and Lahti R. Structure and function analysis of Escherichia coli inorganic pyrophosphatase: is a hydroxide ion the key to catalysis? Biochemistry 34: 782-791, 1995[Medline].
356.	Sander M, Lyson T, Thomas GD, and Victor RG. Sympathetic neural mechanisms of cyclosporine-induced hypertension. Am J Hypertens 9: 121S-138S, 1996[Medline].
357.	Santella L. The role of calcium in the cell cycle: facts and hypotheses. Biochem Biophys Res Commun 244: 317-324, 1998[Web of Science][Medline].
358.	Santella L, and Carafoli E. Calcium signaling in the cell nucleus. FASEB J 11: 1091-1109, 1997[Abstract].
359.	Schaad NC, De Castro E, Nef S, Hegi S, Hinrichsen R, Martone ME, Ellisman MH, Sikkink R, Rusnak F, Sygush J, and Nef P. Direct modulation of calmodulin targets by the neuronal calcium sensor NCS-1. Proc Natl Acad Sci USA 93: 9253-9258, 1996[Abstract/Free Full Text].
360.	Schaefer A, Magócsi M, Stöcker U, Fandrich A, and Marquardt H. Ca2+/calmodulin-dependent and -independent down-regulation of c-myb mRNA levels in erythropoietin-responsive murine erythroleukemia cells. J Biol Chem 271: 13484-13490, 1996[Abstract/Free Full Text].
361.	Schreiber SL, and Crabtree GR. The mechanism of action of cyclosporin A and FK506. Immunol Today 13: 136-142, 1992[Web of Science][Medline].
362.	Schuhmann K, Romanin C, Baumgartner W, and Groschner K. Intracellular Ca2+ inhibits smooth muscle L-type Ca2+ channels by activation of protein phosphatase type 2B and by direct interaction with the channel. J Gen Physiol 110: 503-513, 1997[Abstract/Free Full Text].
363.	Schumacher A, and Nordheim A. Progress towards a molecular understanding of cyclosporin A-mediated immunosuppression. Clin Invest 70: 773-779, 1992[Web of Science][Medline].
364.	Schwaninger M, Blume R, Oetjen E, Lux G, and Knepel W. Inhibition of cAMP-responsive element-mediated gene transcription by cyclosporin A and FK506 after membrane depolarization. J Biol Chem 268: 23111-23115, 1993[Abstract/Free Full Text].
365.	Scott JD. Dissection of protein kinase and phosphatase targeting interactions. Soc Gen Phys Ser 52: 227-239, 1997.
366.	Scott JD, and McCartney S. Localization of A-kinase through anchoring proteins. Mol Endocrinol 8: 5-11, 1994[Free Full Text].
367.	Semsarian C, Wu M-J, Ju Y-K, Marciniec T, Yeoh T, Allen DG, Harvey RP, and Graham RM. Skeletal muscle hypertrophy is mediated by a Ca2+-dependent calcineurin signalling pathway. Nature 400: 576-581, 1999[Medline].
368.	Serrano R. Salt tolerance in plants and microorganisms: toxicity targets and defense responses. In: International Review of Cytology: A Survey of Cell Biology, edited by Jeon KW. San Diego, CA: Academic, 1996, p. 1-52.
369.	Sharma RK, and Kalra J. Molecular interaction between cAMP and calcium in calmodulin-dependent cyclic nucleotide phosphodiesterase system. Clin Invest Med 17: 374-382, 1994[Web of Science][Medline].
370.	Shenolikar S. Protein phosphatase regulation by endogenous inhibitors. Semin Cancer Biol 6: 219-227, 1995[Web of Science][Medline].
371.	Shenolikar S, and Nairn AC. Protein phosphatases: recent progress. Adv Second Messenger Phosphoprotein Res 23: 3-121, 1991.
372.	Shi J, Kim KN, Ritz O, Albrecht V, Gupta R, Harter K, Luan S, and Kudla J. Novel protein kinases associated with calcineurin-B-like calcium sensors in arabidopsis. Plant Cell 11: 2393-2406, 1999[Abstract/Free Full Text].
373.	Shi L, Carmichael WW, and Kennelly PJ. Cyanobacterial PPP family protein phosphatases possess multifunctional capabilities and are resistant to microcystin-LR. J Biol Chem 274: 10039-10046, 1999[Abstract/Free Full Text].
374.	Shibasaki F, Kondo E, Akagi T, and McKeon F. Suppression of signalling through transcription factor NF-AT by interactions between calcineurin and Bcl-2. Nature 386: 728-731, 1997[Medline].
375.	Shibasaki F, and McKeon F. Calcineurin functions in Ca2+-activated cell death in mammalian cells. J Cell Biol 131: 735-743, 1995[Abstract/Free Full Text].
376.	Shibasaki F, Price ER, Milan D, and McKeon F. Role of kinases and the phosphatase calcineurin in the nuclear shuttling of transcription factor NF-AT4. Nature 382: 370-373, 1996[Medline].
377.	Shimoyama M, Hayashi D, Takimoto E, Zou Y, Oka T, Uozumi H, Kudoh S, Shibasaki F, Yazaki Y, Nagai R, and Komuro I. Calcineurin plays a critical role in pressure overload-induced cardiac hypertrophy. Circulation 100: 2449-2454, 1999[Abstract/Free Full Text].
378.	Siekierka JJ, and Sigal NH. FK-506 and cyclosporin A: immunosuppressive mechanism of action and beyond. Curr Opin Immunol 4: 548-552, 1992[Web of Science][Medline].
379.	Sikkink R, Haddy A, MacKelvie S, Mertz P, Litwiller R, and Rusnak F. Calcineurin subunit interactions: mapping the calcineurin B binding domain on calcineurin A. Biochemistry 34: 8348-8356, 1995[Medline].
380.	Simmen RCM, Srinivas V, and Roberts RM. cDNA sequence, gene organization, and progesterone induction of mRNA for uteroferrin, a porcine uterine iron transport protein. DNA 8: 543-554, 1989[Web of Science][Medline].
381.	Singal PK, Khaper N, Palace V, and Kumar D. The role of oxidative stress in the genesis of heart disease. Cardiovasc Res 40: 426-432, 1998[Abstract/Free Full Text].
382.	Singh TJ, and Wang JH. Phosphorylation of calcineurin by glycogen synthase (casein) kinase-1. Biochem Cell Biol 65: 917-921, 1987[Web of Science][Medline].
383.	Smith RD, and Walker JC. Plant protein phosphatases. Annu Rev Plant Physiol Plant Mol Biol 47: 101-125, 1996[Web of Science].
384.	Snyder SH, Lai MM, and Burnett PE. Immunophilins in the nervous system. Neuron 21: 283-294, 1998[Web of Science][Medline].
385.	Snyder SH, Sabatini DM, Lai MM, Steiner JP, Hamilton GS, and Suzdak PD. Neural actions of immunophilin ligands. Trends Pharmacol Sci 19: 21-26, 1998[Medline].
386.	Solow B, Young JC, and Kennelly PJ. Gene cloning and expression and characterization of a toxin-sensitive protein phosphatase from the methanogenic archeon Methanosarcina thermophila TM-1. J Bacteriol 179: 5072-5075, 1997[Abstract/Free Full Text].
387.	Stark MJR. Yeast protein serine/threonine phosphatases: multiple roles and diverse regulation. Yeast 12: 1647-1675, 1996[Web of Science][Medline].
388.	Stathopoulos AM, and Cyert MS. Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast. Genes Dev 11: 3432-3444, 1997[Abstract/Free Full Text].
389.	Stemmer PM, and Klee CB. Dual calcium ion regulation of calcineurin by calmodulin and calcineurin B. Biochemistry 33: 6859-6866, 1994[Medline].
390.	Stewart AA, Ingebritsen TS, and Cohen P. The protein phosphatases involved in cellular regulation. 5. Purification and properties of a Ca2+/calmodulin-dependent protein phosphatase (2B) from rabbit skeletal muscle. Eur J Biochem 132: 289-295, 1983[Web of Science][Medline].
391.	Stewart AA, Ingebritsen TS, Manalan A, Klee CB, and Cohen P. Discovery of a Ca2+- and calmodulin-dependent protein phosphatase: probable identity with calcineurin (CaM-BP80). FEBS Lett 137: 80-84, 1982[Web of Science][Medline].
392.	Stoddard BL, and Flick KE. Calcineurin-immunosupressor complexes. Curr Opin Struct Biol 6: 770-775, 1996[Web of Science][Medline].
393.	Storm DR, and Koshland DE Jr. A source for the special catalytic power of enzymes: orbital steering. Proc Natl Acad Sci USA 66: 445-452, 1970[Abstract/Free Full Text].
394.	Strack S, Wadzinski BE, and Ebner FF. Localization of the calcium/calmodulin-dependent protein phosphatase, calcineurin, in the hindbrain and spinal cord of the rat. J Comp Neurol 375: 66-76, 1996[Web of Science][Medline].
395.	Sträter N, Klabunde T, Tucker P, Witzel H, and Krebs B. Crystal structure of a purple acid phosphatase containing a dinuclear Fe(III)-Zn(II) active site. Science 268: 1489-1492, 1995[Abstract/Free Full Text].
396.	Su Q, Zhao M, Weber E, Eugster H-P, and Ryffel B. Distribution and activity of calcineurin in rat tissues. Evidence for post-transcriptional regulation of testis-specific calcineurin B. Eur J Biochem 230: 469-474, 1995[Web of Science][Medline].
397.	Sugden PH. Signaling in myocardial hypertrophy. Life after calcineurin? Circ Res 84: 633-646, 1999[Free Full Text].
398.	Sugimoto T, Stewart S, and Guan K-L. The calcium/calmodulin-dependent protein phosphatase calcineurin is the major Elk-1 phosphatase. J Biol Chem 272: 29415-29418, 1997[Abstract/Free Full Text].
399.	Sugiura R, Toda T, Shuntoh H, Yanagida M, and Kuno T. Pmp1+, a suppressor of calcineurin deficiency, encodes a novel MAP kinase phosphatase in fission yeast. EMBO J 17: 140-148, 1998[Web of Science][Medline].
400.	Sun L, Youn H-D, Loh C, Stolow M, He W, and Liu JO. Cabin 1, a negative regulator for calcineurin signalling in T lymphocytes. Immunity 8: 703-711, 1998[Web of Science][Medline].
401.	Sussman MA, Lim HW, Gude N, Taigen T, Olson EN, Robbins J, Colbert MC, Gualberto A, Wieczorek DF, and Molkentin JD. Prevention of cardiac hypertrophy in mice by calcineurin inhibition. Science 281: 1690-1693, 1998[Abstract/Free Full Text].
402.	Sussman MA, Welch S, Gude N, Khoury PR, Daniels SR, Kirkpatrick D, Walsh RA, Price RL, Lim HW, and Molkentin JD. Pathogenesis of dilated cardiomyopathy: molecular, structural, and population analyses in tropomodulin-overexpressing transgenic mice. Am J Pathol 155: 2101-2113, 1999[Abstract/Free Full Text].
403.	Swanson SK-H, Born T, Zydowsky LD, Cho H, Chang HY, Walsh CT, and Rusnak F. Cyclosporin-mediated inhibition of bovine calcineurin by cyclophilins A and B. Proc Natl Acad Sci USA 89: 3741-3745, 1992[Abstract/Free Full Text].
404.	Swope SL, Moss SJ, Raymond LA, and Huganir RL. Regulation of ligand-gated ion channels by protein phosphorylation. Adv Second Messenger Phosphoprotein Res 33: 49-78, 1999[Web of Science][Medline].
405.	Takuwa N, Zhou W, and Takuwa Y. Calcium, calmodulin and cell cycle progression. Cell Signal 7: 93-104, 1995[Web of Science][Medline].
406.	Tallant EA, Brumley LM, and Wallace RW. Activation of a calmodulin-dependent phosphatase by a Ca2+-dependent protease. Biochemistry 27: 2205-2211, 1988[Medline].
407.	Tamura K, Fujimura T, Tsutsumi T, Nakamura K, Ogawa T, Atumaru C, Hirano Y, Ohara K, Ohtsuka K, Shimomura K, and Kobayashi M. Transcriptional inhibition of insulin by FK506 and possible involvement of FK506 binding protein-12 in pancreatic  cells. Transplantation 59: 1606-1613, 1995[Web of Science][Medline].
408.	Tanida I, Hasegawa A, Iida H, Ohya Y, and Anraku Y. Cooperation of calcineurin and vacuolar H+-ATPase in intracellular Ca2+ homeostasis of yeast cells. J Biol Chem 270: 10113-10119, 1995[Abstract/Free Full Text].
409.	Tash JS, Krinks M, Patel J, Means RL, Klee CB, and Means AR. Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm. J Cell Biol 106: 1625-1633, 1988[Abstract/Free Full Text].
410.	Tatlock JH, Linton MA, Hou XJ, Kissinger CR, Pelletier LA, Showlater RE, Tempczyk A, and Villafranca JE. Structure-based design of novel calcineurin (PP2B) inhibitors. Bioorg Med Chem Lett 7: 1007-1012, 1997.
411.	Taylor WP, and Widlanski TS. Charged with meaning: the structure and mechanism of phosphoprotein phosphatases. Chem Biol 2: 713-718, 1995[Web of Science][Medline].
412.	Thomson AW, Bonham CA, and Zeevi A. Mode of action of tacrolimus (FK506): molecular and cellular mechanisms. Ther Drug Monit 17: 584-591, 1995[Web of Science][Medline].
413.	Thomson AW, and Starzl TE. New immunosuppressive drugs: mechanistic insights and potential therapeutic advances. Immunol Rev 136: 71-98, 1993[Web of Science][Medline].
414.	Tian J, and Karin M. Stimulation of Elk1 transcriptional activity by mitogen-activated protein kinases is negatively regulated by protein phosphatase 2B (calcineurin). J Biol Chem 274: 15173-15180, 1999[Abstract/Free Full Text].
415.	Traynelis SF, and Wahl P. Control of rat GluR6 glutamate receptor open probability by protein kinase A and calcineurin. J Physiol (Lond) 503: 513-531, 1997[Abstract/Free Full Text].
416.	True AE, Scarrow RC, Randall CR, Holz RC, and Que L Jr. EXAFS studies of uteroferrin and its anion complexes. J Am Chem Soc 115: 4246-4255, 1993.
417.	Tsao L, Neville C, Muraro A, McCullagh KJA, and Rosenthal N. Revisiting calcineurin and human heart failure. Nature Med 6: 2-3, 2000[Web of Science][Medline].
418.	Tumlin JA. Expression and function of calcineurin in mammalian nephron: physiological roles, receptor signaling, and ion transport. Am J Kidney Dis 30: 884-895, 1997[Web of Science][Medline].
419.	Tumlin JA, Someren JT, Swanson CE, and Lea JP. Expression of calcineurin activity and -subunit isoforms in specific segments of the rat nephron. Am J Physiol Renal Fluid Electrolyte Physiol 269: F558-F563, 1995[Abstract/Free Full Text].
420.	Tung HYL. Phosphorylation of the calmodulin-dependent protein phosphatase by protein kinase C. Biochem Biophys Res Commun 138: 783-788, 1986[Web of Science][Medline].
421.	Tóth R, Szegezdi E, Molnár G, Lord JM, Fésüs L, and Zsuzsa S. Regulation of cell surface expression of Fas (CD95) ligand and susceptibility to Fas (CD95)-mediated apoptosis in activation-induced T cell death involves calcineurin and protein kinase C, respectively. Eur J Immunol 29: 383-393, 1999[Web of Science][Medline].
422.	Uchino H, Elmér E, Uchino K, Li P-A, He Q-P, Smith M-L, and Seisjö BK. Amelioration by cyclosporin A of brain damage in transient forebrain ischemia in the rat. Brain Res 812: 216-226, 1998[Web of Science][Medline].
423.	Ueki K, and Kincaid RL. Interchangeable associations of calcineurin regulatory subunit isoforms with mammalian and fungal catalytic subunits. J Biol Chem 268: 6554-6559, 1993[Abstract/Free Full Text].
424.	Ueki K, Muramatsu T, and Kincaid RL. Structure and expression of two isoforms of the murine calmodulin-dependent protein phosphatase regulatory subunit (calcineurin B). Biochem Biophys Res Commun 187: 537-543, 1992[Web of Science][Medline].
425.	Uppenberg J, Lindqvist F, Svensson C, Ek-Rylander B, and Andersson G. Crystal structure of mammalian purple acid phosphatase. J Mol Biol 290: 201-211, 1999[Web of Science][Medline].
426.	Usuda N, Arai H, Sasaki H, Hanai T, Nagata T, Muramatsu T, Kincaid RL, and Higuchi S. Differential subcellular localization of neural isoforms of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin) in central nervous system neurons: immunohistochemistry on Formalin-fixed paraffin sections employing antigen retrieval by microwave irradiation. J Histochem Cytochem 44: 13-18, 1996[Abstract].
427.	Victor RG, Thomas GD, Marban E, and O'Rourke B. Presynaptic modulation of cortical synaptic activity by calcineurin. Proc Natl Acad Sci USA 92: 6269-6273, 1995[Abstract/Free Full Text].
428.	Villafranca JE, Kissinger CR, and Parge HE. Protein serine/threonine phosphatases. Curr Opin Biotechnol 7: 397-402, 1996[Web of Science][Medline].
429.	Villalba M, Kasibhatla S, Genestier L, Mahboubi A, Green DR, and Altman A. Protein kinase C cooperates with calcineurin to induce Fas ligand expression during activation-induced T cell death. J Immunol 163: 5813-5819, 1999[Abstract/Free Full Text].
430.	Vincent JB, and Averill BA. An enzyme with a double identity: purple acid phosphatase and tartarate-resistant acid phosphatase. FASEB J 4: 3009-3014, 1990[Abstract].
431.	Vincent JB, and Averill BA. Sequence homology between purple acid phosphatases and phosphoprotein phosphatases: are phosphoprotein phosphatases metalloproteins containing oxide-bridged dinuclear metal centers? FEBS Lett 263: 265-268, 1990[Web of Science][Medline].
432.	Vincent JB, Crowder MW, and Averill BA. Hydrolysis of phosphate monoesters: a biological problem with multiple chemical solutions. Trends Biochem Sci 17: 105-110, 1992[Web of Science][Medline].
433.	Vincent JB, Olivier-Lilley GL, and Averill BA. Proteins containing oxo-bridged dinuclear iron centers: a bioinorganic perspective. Chem Rev 90: 1447-1467, 1990[Web of Science].
434.	Walaas SI, and Greengard P. Protein phosphorylation and neuronal function. Pharmacol Rev 43: 299-349, 1991[Abstract].
435.	Wallace RW, Lynch TJ, Tallant EA, and Cheung WY. Purification and characterization of an inhibitor protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase. J Biol Chem 254: 377-382, 1978[Abstract/Free Full Text].
436.	Wallace RW, Lynch TJ, Tallant EA, and Cheung WY. An endogenous inhibitor protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase. Arch Biochem Biophys 187: 328-334, 1978[Web of Science][Medline].
437.	Wallace RW, Tallant EA, and Cheung WY. High levels of a heat-labile calmodulin-binding protein (CaM-BP80) in bovine neostriatum. Biochemistry 19: 1831-1837, 1980[Medline].
438.	Wallace RW, Tallant EA, and McManus MC. Human platelet calmodulin-binding proteins: identification and Ca2+-dependent proteolysis upon platelet activation. Biochemistry 26: 2766-2773, 1987[Medline].
439.	Walsh CT, Zydowsky LD, and McKeon FD. Cyclosporin A, the cyclophilin class of peptidylprolyl isomerases, and blockade of T cell signal transduction. J Biol Chem 267: 13115-13118, 1992[Abstract/Free Full Text].
440.	Walsh MP, Busaan JL, Fraser ED, Fu SY, Pato MD, and Michalak M. Characterization of the recombinant C-terminal domain of dystrophin: phosphorylation by calmodulin-dependent protein kinase ii and dephosphorylation by type 2B protein phosphatase. Biochemistry 34: 5561-5568, 1995[Medline].
441.	Walsh RA. Calcineurin inhibition as therapy for cardiac hypertrophy and heart failure. Circ Res 84: 741-743, 1999[Free Full Text].
442.	Wang C-R, Hashimoto K, Kubo S, Yokochi T, Kubo M, Suzuki M, Suzuki K, Tada T, and Nakayama T. T cell receptor-mediated signaling events in CD4+ CD8+ thymocytes undergoing thymic selection: requirement of calcineurin activation for thymic positive selection but not negative selection. J Exp Med 181: 927-941, 1995[Abstract/Free Full Text].
443.	Wang H-G, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, McKeon F, Bobo T, Franke TF, and Reed JC. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science 284: 339-343, 1999[Abstract/Free Full Text].
444.	Wang JH, and Desai R. A brain protein and its effect on the Ca2+- and protein modulator-activated cyclic nucleotide phosphodiesterase. Biochem Biophys Res Commun 72: 926-932, 1976[Web of Science][Medline].
445.	Wang JH, and Desai R. Modulator binding protein: bovine brain protein exhibiting the Ca2+-dependent association with the protein modulator of cyclic nucleotide phosphodiesterase. J Biol Chem 252: 4175-4184, 1977[Free Full Text].
446.	Wang MG, Yi H, Guerini D, Klee CB, and McBride OW. Calcineurin A alpha (PPP3CA), calcineurin A beta (PPP3CB) and calcineurin B (PPP3R1) are located on human chromosomes 4, 10q21q22 and 2p16p15, respectively. Cytogenet Cell Genet 72: 236-241, 1996[Web of Science][Medline].
447.	Wang X, Culotta VC, and Klee CB. Superoxide dismutase protects calcineurin from inactivation. Nature 383: 434-437, 1996[Medline].
448.	Wang X, and Que L Jr. Extended X-ray absorption fine structure studies of the anion complexes of FeZn uteroferrin. Biochemistry 37: 7813-7821, 1998[Medline].
449.	Wang X, Randall CR, True AE, and Que L Jr. X-ray absorption spectroscopic studies of the FeZn derivative of uteroferrin. Biochemistry 35: 13946-13954, 1996[Medline].
450.	Warren WD, Phillips AM, and Howells AJ. Drosophila melanogaster contains both X-linked and autosomal homologues of the gene encoding calcineurin B. Gene 177: 149-153, 1996[Web of Science][Medline].
451.	Watanabe Y, Perrino BA, Chang BH, and Soderling TR. Identification in the calcineurin A subunit of the domain that binds the regulatory B subunit. J Biol Chem 270: 456-460, 1995[Abstract/Free Full Text].
452.	Watterson DM, and Vanaman TC. Affinity chromatrography purification of a cyclic nucleotide phosphodiesterase using immobilized modulator protein, a troponin C-like protein from brain. Biochem Biophys Res Commun 73: 40-46, 1976[Web of Science][Medline].
453.	Wei Q, Xiao F, Lu J, and Zhou J. ESR study on calcineurin. Sci China (Ser B) 38: 1117-1122, 1995.
454.	Weiland J, Nitsche AM, Strayle J, Steiner H, and Rudolph HK. The PMR2 gene cluster encodes functionally distinct isoforms of a putative Na+ pump in the yeast plasma membrane. EMBO J 14: 3870-3882, 1995[Web of Science][Medline].
455.	Weiss A, and Littman DR. Signal transduction by lymphocyte antigen receptors. Cell 76: 263-274, 1994[Web of Science][Medline].
456.	Wheelan SJ, Boguski MS, Duret L, and Makalowski W. Human and nematode orthologs: lessons from the analysis of 1800 human genes and the proteome of Caenorhabditis elegans. Gene 238: 163-170, 1999[Web of Science][Medline].
457.	Widlanski TS, Myers JK, Stec B, Holtz KM, and Kantrowitz ER. The road less travelled: taming phosphatases. Chem Biol 4: 489-492, 1997[Web of Science][Medline].
458.	Wiederrecht G, Lam E, Hung S, Martin M, and Sigal N. The mechanism of action of FK-506 and cyclosporin A. Ann NY Acad Sci 696: 9-19, 1993[Web of Science][Medline].
459.	Wilson DK, Rudolph FB, and Quiocho FA. Atomic structure of adenosine deaminase complexed with a transition-state analog: understanding catalysis and immunodeficiency mutations. Science 252: 1278-1284, 1991[Abstract/Free Full Text].
460.	Winder DG, Mansuy IM, Osman M, Moallem TM, and Kandel ER. Genetic and pharmacological evidence for a novel, intermediate phase of long-term potentiation suppressed by calcineurin. Cell 92: 25-37, 1998[Web of Science][Medline].
461.	Withee JL, Mulholland J, Jeng R, and Cyert MS. An essential role of the yeast pheromone-induced Ca2+ signal is to activate calcineurin. Mol Biol Cell 8: 263-277, 1997[Abstract].
462.	Withee JL, Sen R, and Cyert MS. Ion tolerance of Saccharomyces cerevisiae lacking the Ca2+/CaM-dependent phosphatase (calcineurin) is improved by mutations in URE2 or PAM1. Genetics 149: 865-878, 1998[Abstract/Free Full Text].
463.	Wolvetang EJ, Larm JA, Moutsoulas P, and Lawen A. Apoptosis induced by inhibitors of the plasma membrane NADH-oxidase involves Bcl-2 and calcineurin. Cell Growth Differ 7: 1315-1325, 1996[Abstract].
464.	Xu X, Qin X-Q, and Kantrowitz ER. Probing the role of histidine-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase by site-specific mutagenesis. Biochemistry 33: 2279-2284, 1994[Medline].
465.	Yakel JL. Calcineurin regulation of synaptic function: from ion channels to transmitter release and gene expression. Trends Pharmacol Sci 18: 124-134, 1997[Medline].
466.	Yamada T, Kim JK, Muramatsu Y, Serikawa T, and Matsumoto K. Chromosomal assignments of the genes for the calcineurin a (Calna1) and a subunits (Calna2) in the rat. Cytogenet Cell Genet 67: 55-57, 1994[Web of Science][Medline].
467.	Ye RR, and Bretscher A. Identification and molecular characterization of the calmodulin-binding subunit gene (CMP1) of protein phosphatase 2B from Saccharomyces cerevisiae. Eur J Biochem 204: 713-723, 1992[Web of Science][Medline].
468.	Yoshida T, Toda T, and Yanagida M. A calcineurin-like gene ppb1+ in fission yeast: mutant defects in cytokinesis, cell polarity, mating and spindle pole body positioning. J Cell Sci 107: 1725-1735, 1994[Abstract].
469.	Youn H-D, Sun L, Prywes R, and Liu JO. Apoptosis of T cells mediated by Ca2+-induced release of the transcription factor MEF2. Science 286: 790-793, 1999[Abstract/Free Full Text].
470.	Yu L, Golbeck J, Yao J, and Rusnak F. Spectroscopic and enzymatic characterization of the active site dinuclear metal center of calcineurin: implications for a mechanistic role. Biochemistry 36: 10727-10734, 1997[Medline].
471.	Yu L, Haddy A, and Rusnak F. Evidence that calcineurin accommodates an active site binuclear metal center. J Am Chem Soc 117: 10147-10148, 1995.
472.	Zav'Yalov VP, Denesyuk A, Lundell J, and Korpela T. Some new aspects of molecular mechanisms of cyclosporin A effect on immune response. APMIS 103: 401-415, 1995[Web of Science][Medline].
473.	Zhang C-C, Friry A, and Peng L. Molecular and genetic analysis of two closely linked genes that encode, respectively, a protein phosphatase 1/2A/2B homolog and a protein kinase homolog in the Cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 180: 2616-2622, 1998[Abstract/Free Full Text].
474.	Zhang J, and Steiner JP. Nitric oxide synthase, immunophilins and poly(ADP-ribose) synthetase: novel targets for the development of neuroprotective drugs. Neurol Res 17: 285-288, 1995[Web of Science][Medline].
475.	Zhang J, Zhang Z, Brew K, and Lee EYC. Mutational analysis of the catalytic subunit of muscle protein phosphatase-1. Biochemistry 35: 6276-6282, 1996[Medline].
476.	Zhang W, Kowal RC, Rusnak F, Sikkink RA, Olson EN, and Victor RG. Failure of calcineurin inhibitors to prevent pressure-overload left ventricular hypertrophy in rats. Circ Res 84: 722-728, 1999[Abstract/Free Full Text].
477.	Zhang W, Zimmer G, Chen J, Ladd D, Li E, Alt FW, Wiederrecht G, Cryan J, O'Neill EA, Seidman CE, Abbas AK, and Seidman JG. T cell responses in calcineurin A-deficient mice. J Exp Med 183: 413-420, 1996[Abstract/Free Full Text].
478.	Zhang Z, Bai G, Deans-Zirattu S, Browner MF, and Lee EYC. Expression of the catalytic subunit of phosphorylase phosphatase (protein phosphatase-1) in Escherichia coli. J Biol Chem 267: 1484-1490, 1992[Abstract/Free Full Text].
479.	Zhang Z-Y, and VanEtten RL. Pre-steady-state and steady-state kinetic analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart. J Biol Chem 266: 1516-1525, 1991[Abstract/Free Full Text].
480.	Zhao C, Jung US, Garrett-Engele P, Roe T, Cyert MS, and Levin DE. Temperature-induced expression of yeast FKS2 is under the dual control of protein kinase C and calcineurin. Mol Cell Biol 18: 1013-1022, 1998[Abstract/Free Full Text].
481.	Zhao Y, Tozawa Y, Iseki R, Mukai M, and Iwata M. Calcineurin activation protects T cells from glucocorticoid-induced apoptosis. J Immunol 154: 6346-6354, 1995[Abstract].
482.	Zhou J, Xiang B, Wei Q, and Lu J. Interaction of metal ions, Mn2+ and Ni2+, with calcineurin. Chinese Sci Bull 41: 1029-1032, 1996.
483.	Zhu D, Cardenas ME, and Heitman J. Myristoylation of calcineurin B is not required for function or interaction with immunophilin-immunosuppressant complexes in the yeast Saccharomyces cerevisiae. J Biol Chem 270: 24831-24838, 1995[Abstract/Free Full Text].
484.	Zhu Y, and Yakel JL. Calcineurin modulates G protein-mediated inhibition of N-type calcium channels in rat sympathetic neurons. J Neurophysiol 78: 1161-1165, 1997[Abstract/Free Full Text].
485.	Zhuo M, Zhang W, Son H, Mansuy I, Sobel RA, Seidman J, and Kandel ER. A selective role of calcineurin A in synaptic depotentation in hippocampus. Proc Natl Acad Sci USA 96: 4650-4655, 1999[Abstract/Free Full Text].
486.	Zhuo S, Clemens JC, Hakes DJ, Barford D, and Dixon JE. Expression, purification, crystallization, and biochemical characterization of a recombinant protein phosphatase. J Biol Chem 268: 17754-17761, 1993[Abstract/Free Full Text].
487.	Zhuo S, Clemens JC, Stone RL, and Dixon JE. Mutational analysis of a Ser/Thr phosphatase. J Biol Chem 269: 26234-26238, 1994[Abstract/Free Full Text].

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