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).

	II. A BRIEF HISTORY AND OVERVIEW OF CALCINEURIN

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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).

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. 

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).

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.

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.

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.

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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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.

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.

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).

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).

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.

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.

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).

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.

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.

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.

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).

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.

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.

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).

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.

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.

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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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). Conserv