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Calcineurin: Form and Function

FRANK RUSNAK AND PAMELA MERTZ

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 1483II. A Brief History and Overview of Calcineurin 1484

A. Calcineurin: the early years 1484B. Calcineurin properties 1484

III. Physiological Roles for Calcineurin 1490A. Lower eukaryotes 1490B. Higher eukaryotes 1494C. Inhibitors of calcineurin 1497

IV. Calcineurin Structure 1500A. A dinuclear metal-binding phosphoesterase motif 1500B. Three-dimensional structure 1500C. Active site architecture 1502D. Metal ion requirements 1503

V. Enzymatic Mechanism 1504A. Mechanism of phosphoryl group transfer: evidence for direct transfer to water 1504B. Catalytic role of the dinuclear metal center 1505C. Conserved active site residues 1505D. A model for the calcineurin catalytic mechanism 1508

VI. Regulation 1510

Rusnak, Frank, and Pamela Mertz. Calcineurin: Form and Function. Physiol Rev 80: 1483–1521, 2000.—Cal-cineurin is a eukaryotic Ca21- and calmodulin-dependent serine/threonine protein phosphatase. It is a heterodimericprotein consisting of a catalytic subunit calcineurin A, which contains an active site dinuclear metal center, and atightly associated, myristoylated, Ca21-binding subunit, calcineurin B. The primary sequence of both subunits andheterodimeric quaternary structure is highly conserved from yeast to mammals. As a serine/threonine proteinphosphatase, calcineurin participates in a number of cellular processes and Ca21-dependent signal transductionpathways. Calcineurin is potently inhibited by immunosuppressant drugs, cyclosporin A and FK506, in the presenceof their respective cytoplasmic immunophilin proteins, cyclophilin and FK506-binding protein. Many studies haveused these immunosuppressant drugs and/or modern genetic techniques to disrupt calcineurin in model organismssuch as yeast, filamentous fungi, plants, vertebrates, and mammals to explore its biological function. Recentadvances 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 esterhydrolysis including the importance of the dinuclear metal ion cofactor and metal ion redox chemistry, studieswhich may lead to new calcineurin inhibitors. This review provides a comprehensive examination of the biologicalroles of calcineurin and reviews aspects related to its structure and catalytic mechanism.

I. INTRODUCTION

The year 1999 marked the 20th anniversary of theisolation of the Ca21- and calmodulin-dependent proteinserine/threonine phosphatase calcineurin (206). Duringthe past 20 years, the biological roles of calcineurin haveprogressed from a putative inhibitor of the calmodulin-

dependent phosphodiesterase (444) to the ground-breakingdiscovery that it is the target of the immunosuppressantdrugs cyclosporin A (CsA) and FK506, pharmacological re-agents that have been used to demonstrate it as a majorplayer in Ca21-dependent eukaryotic signal transductionpathways (238). In recent years, several milestones regard-ing calcineurin structure have been achieved including the

PHYSIOLOGICAL REVIEWS

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determination of the three-dimensional structure by X-raydiffraction methods (124, 197) and biochemical, spectro-scopic, and physical studies that are beginning to unravel itscatalytic mechanism (150, 259, 261, 262, 470, 471). Insightinto its physiological functions include mapping its subcel-lular localization (10, 106, 175, 219, 286, 306); the discoveryof its colocalization with other important signaling proteins(365); and, aside from Ca21/calmodulin, the finding of pos-sible endogenous regulators of its activity including redoxand/or oxidative stress (45, 111, 345, 447, 470, 471) as well asinteracting proteins (224, 234, 277, 359, 400). The next gen-eration of studies, which includes the use of transgenicmouse technology, is beginning to reveal interesting yetsometimes subtle roles for this enzyme in the whole organ-ism (181, 254, 281, 320, 460, 477).

It will not be the attempt of this review to provide anall-encompassing survey of calcineurin. In fact, severalexcellent review articles on calcineurin and other proteinserine/threonine phosphatases are available, some quitecomprehensive (60, 130, 203, 205, 207, 208, 316, 371). Inaddition, numerous specialized articles focusing on par-ticular aspects of either calcineurin structure or functionhave been published. A list of these appears in Table 1 forthe benefit of the reader who would prefer to be directedto specific calcineurin-related topics. Rather, we focus ona comprehensive treatise of some of the recent develop-ments of calcineurin since the last major review waspublished (371) (ca. 1990 to present).

II. A BRIEF HISTORY AND OVERVIEW

OF CALCINEURIN

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. Indepen-dently, Watterson and Vanaman (452) also obtainedhighly purified fractions of calcineurin from bovine brainextract by use of calmodulin-affinity chromatography buterroneously referred to the 58- and 18-kDa subunits ofcalcineurin as “affinity-purified phosphodiesterase.” Kleeand Krinks (206) are credited with the first purification ofcalcineurin and hypothesized that it might be a regulatorysubunit of phosphodiesterase since it was demonstratedto inhibit phosphodiesterase activity. Other groups sub-sequently showed that calcineurin inhibited the Ca21/calmodulin-dependent isozymes of cyclic nucleotidephosphodiesterase and adenylate cyclase by competingfor calmodulin in a Ca21-dependent fashion, and theyspeculated that its function may be regulatory (435, 436,445). Shortly thereafter, Klee et al. (204) coined the de-scriptive label “calcineurin” on the basis of its Ca21-bind-ing properties and localization to neuronal tissue (204), apopularized name which is widely used to date and whichwe will use throughout this review. At that time, the truefunction of calcineurin had yet to be revealed. It was notuntil pioneering work in the early 1980s in Philip Cohen’slab, investigating cellular extracts capable of dephosphor-ylating the a- and b-subunits of phosphorylase kinase,that a fraction represented as protein phosphatase 2B(PP2B) was demonstrated to be identical to Klee’s cal-cineurin (390, 391).

B. Calcineurin Properties

Biochemical studies during the 1980s continued anddetermined many of the physical properties listed in Table2 (61, 130, 208). Purified calcineurin is a heterodimerconsisting of a catalytic subunit, calcineurin A, and a“regulatory” subunit, calcineurin B.

TABLE 1. Calcineurin reviews listed according to subject

Review Topic Reference No.

General aspects of calcineurin 60, 61, 130, 145, 167, 202–205, 207, 208, 316, 371Control of adenylyl cyclase 13, 369Neural roles for immunosuppressive drugs, immunophilins,

and calcineurin59, 119, 160, 253, 299, 333, 353, 384, 385, 434, 465, 474

Role of calcineurin in brain ischemia and injury 285Calcineurin and NF-AT family of transcription factors 173, 338–340Calcineurin, Ca21 signaling, and the cell cycle 266, 276, 357, 358, 405Calcineurin in yeast and other lower eukaryotic organisms 47, 68, 71, 141–143, 303, 368, 387Calcineurin, T lymphocyte activation, and the mechanism of

action of immunosuppressive agents30, 46, 55, 133, 134, 155, 179, 189, 192, 222, 236, 237, 249, 255, 274,

311, 361, 363, 370, 378, 412, 413, 439, 455, 458, 472Structure of calcineurin and related phosphatases 22–24, 199, 215, 223, 244, 307, 351, 392, 411, 428, 430, 432, 433, 457Calcineurin in the mammalian nephron 418Calcineurin localization via AKAP 57, 103, 323, 366Calcineurin and ion channel regulation 404, 465Calcineurin and apoptosis 170Calcineurin regulation of dystrophin 275Calcineurin and protein phosphatases in plants 245, 368, 383Role of calcineurin in hypertension 356Regulation of microtubules by calcineurin: tau phosphorylation 253

NF-AT, nuclear factor of activated T cells; AKAP, protein kinase A anchoring protein.

1484 FRANK RUSNAK AND PAMELA MERTZ Volume 80

Cloning efforts have provided evidence that all eu-karyotic organisms possess one or more genes for eachsubunit; Table 3 is a compilation of known calcineurin Aand calcineurin B gene sequences to date. Genes forcalcineurin A and B subunits have been identified in yeast,filamentous fungi, protozoa, insects, and mammals. Theab-quaternary structure of calcineurin observed in mam-mals is conserved in lower eukaryotic organisms. Thesesubunits are tightly associated and can only be dissoci-ated by use of denaturants (271).

1. Calcineurin A

A) CLASSIFICATION. In addition to calcineurin, the serine/threonine protein phosphatase family members includeprotein phosphatases 1 (PP1), 2A (PP2A), and 2C (PP2C),phosphatases essential for a number of signal transduc-tion pathways in eukaryotic cells (61, 371). The originalclassification of this family was proposed by Ingebritsenand Cohen (167), separating almost all the serine/threo-nine phosphatase activity in mammalian tissue extractsinto two classes (60, 61, 167, 371). Type 1 protein phos-phatases were found to dephosphorylate the b-subunit ofphosphorylase kinase, whereas type 2 protein phospha-tases dephosphorylate the a-subunit of phosphorylasekinase. Differences between the two types are also foundwith inhibitors; type 1 is inhibited by phosphopeptideinhibitors 1 and 2, whereas the type 2 class is not affectedby these inhibitors.

A difference in divalent metal ion dependence led tothe resolution of the type 2 enzymes into PP2A, PP2B(calcineurin), and PP2C (60). PP2A was originally de-scribed as having no requirement for divalent metal ion,calcineurin is regulated by Ca21/calmodulin, and PP2C isMg21 dependent.1 A more detailed discussion regardingthe importance of metal ion cofactors of these phospha-

tases is described in section IVD. Differences among type2 members are also found with regard to sensitivity toinhibition by macrolide inhibitors (see sect. IIIC1). PP2Aand PP1 are inhibited by okadaic acid, whereas cal-cineurin is specifically inhibited by the immunosuppres-sant drugs CsA and FK506, in the presence of cyclophilinand FK506-binding protein (FKBP), respectively (238,361).

Although different in metal ion dependence, sensitiv-ity to inhibitors, and substrate specificity, PP1, PP2A, andcalcineurin have homologous amino acid sequences andare evolutionarily related (26, 61, 371). In fact, the PP1/PP2A/calcineurin superfamily ranks among one of themost highly conserved enzyme families encountered (61).Within the active site domain, PP1 shares 49% amino acididentity with PP2A and 39% identity with calcineurin.Recent work has found homologs of this family in cya-nobacteria (373, 473) and the archea (229, 251, 386). Withthe determination of the amino acid sequences of thesephosphatases, it was found that the original classificationof PP2C along side PP1, PP2A, and calcineurin does nothold at the primary sequence level. Thus PP2C does notshare any homology with PP1/2A/calcineurin and is con-sidered to be in a separate superfamily (33).

B) DOMAIN STRUCTURE. The active site of calcineurin islocated on the A subunit which, in mammals, is 57–59 kDadepending on the isoform. The size of the catalytic sub-unit 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 discoi-

deum, 71 kDa (75, 310)]. Nevertheless, there is strictconservation throughout all eukaryotic organisms suchthat all calcineurin A genes encode for a polypeptideconsisting of a catalytic domain homologous to otherserine/threonine protein phosphatases and three regula-tory domains at the COOH terminus that distinguish cal-cineurin from other family members (Fig. 1). These do-mains have been identified as the calcineurin B binding

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

TABLE 2. Physical properties of calcineurin

Physical Properties Reference No.

Subunit structure and size ab 205, 371Calcineurin A 57–59 kDa (mammals)

57–71 kDa (lower eukaryotes) 72, 75, 153, 242, 310, 343, 467, 468Calcineurin B 19–20 kDa 213, 221, 346

Domain structure 61, 130, 208Calcineurin A NH2 terminus, catalytic, calcineurin B binding,

calmodulin binding, autoinhibitoryCalcineurin B four Ca21-binding EF-hand motifs, myristoylation motif

Activators Ca21 and calmodulin 206, 390, 391Cofactors Fe31, Zn21 124, 195, 471Co-/posttranslational modification Phosphorylation of calcineurin A 44, 136, 138, 257, 382, 420

Myristoylation of calcineurin B 1

October 2000 CALCINEURIN: FORM AND FUNCTION 1485

TABLE 3. A comprehensive list of published calcineurin A and calcineurin B gene sequences

Organism GeneAlternative

Name Tissue/Stage Genbank EntryChromosomal

Location Reference No.

Calcineurin A

Saccharomyces cerevisiae CNA1a CMP1 YSCCALA1.Gb_pl1 XII 72, 242CMP2a CNA1 YSCCALA2.Gb_pl2 XIII

Schizosaccharomyces

pombe

ppb11 b Q12705.SWISSPROT I or IIc 327, 468

Dictyostelium discoideum canA Vegetative growth DDCALNRNA.Gb_in 75Aspergillus nidulans cnaA1 Cell cycle G1/S

transitionP2b_Emeni.SW 343

Neurospora crassa cna-1 Early mycelia NEUCAM.Gb_pl 153Caenorhabditis elegansd CeCnA Neuronal, vulva 328, 456Cryptococcus neoformanse AF042082.Gb_pl1 310Drosophila melanogaster CnnA21EF ? DMCNA.Seq Chromosome 2,

position 21EF132

CnnA14Df PP2B 14D Neuronal,embryonic

DMU30493.Gb_in X chromosome,position 14D

38, 158

Xenopus laevisg AF019569.GB_OVMouse Ppp3cah,i PP2BAa,

PP2Ba1

Brain MUSCALCAT.Gb_ro 190, 191

P2BA_MOUSE.SWPpp3cbh PP2BAb Brain P2BB_MOUSE.SW 117Ppp3cch PP2BAg Testis 291

Rat Ppp3cah,i PP2BAa,

Calna1

Brain RATCNRAA.Gb_ro Chromosome 15 169, 354, 466

RNCALCA.Gb_ro (D 10aa) 354Ppp3cbh PP2BAb,

Calna2

Brain RATCNRAB.Gb_ro Chromosome 15 220, 466

RATCALAB.Gb_roRabbit PP2Bw RABPP2BWS.Gb_om 74Bovine PPP3CAh PP2BAa BTU33868.Gb_om 124

P2BA_BOVIN.SWHuman PPP3CAh,i PP2BAa,

PP2Ba1

Brain HUMCALCAT.Gb_pr1 (part.) 4q21 3 q24j 129, 190, 293

E07798.Gb_pr1PPP3CBh,k PP2BAb,

PP2Ba2

Brain HUMCNRA1.Gb_pr1 10q21 3 q22j 129

HUMCRNAB.Gb_pr1PP2Bb3

k Teratocarcinoma 265PPP3CCh PP2BAg,

PP2Ba3

Testis S46622.Gb_pr1 Chromosome 8l 292

Calcineurin B

Saccharomyces cerevisiae CNB1a YSCCNB.Gb_pl1 XI 221Arabidopsis thaliana SOS3 AF060553 239

CBL1 AF076251.Gb_pl2 218CBL2 AF076252.Gb_pl2 218CBL3 AF076253.Gb_pl2 218

Caenorhabditis elegansd CeCnB 328, 456Neurospora crassa cnb-1 Asexual

developmentAF034089.Gb_pl1 213

NCCALCINB.Gb_pl1Naegleria gruberi CNB1 Flagellar

differentiationNGU04380.Pep 346

Drosophila melanogaster dCNB1 DROCALCB.Gb_in X chromosome,position 4F

CALB_DROME.Sw 132dCNB2 U56245.Gb_In Chromosome 2,

position 43E450

Mouse Ppp3r1h PP2Bb1 Brain CALB_MOUSE.SW 424Ppp3r2h PP2Bb2 Testis 424

Rat Ppp3r1h CNBa1,

Pp2bb1

Brain RATCALCB.GB_RO 48

Alt. Splice CNBa2,

Pp2bb2

Testis RATCALNB.GB_RO 48

Ppp3r2h TestisCBLP Testis RATCBLP.Gb_ro 289


domain (56, 132, 164, 379, 451), the calmodulin-bindingdomain (132, 191), and the “autoinhibitory” domain (137,326), which binds in the active site cleft in the absence ofCa21/calmodulin (197) and inhibits the enzyme, acting inconcert with the calmodulin binding domain to confercalmodulin regulation.

These domains have been identified through stan-dard biochemical mapping procedures including primarysequence comparisons, partial proteolysis experiments,peptide interaction studies, site-directed mutagenesisstudies, and cross-linking studies. The X-ray structures ofcalcineurin (see sect. IVB) confirm the identification ofresidues involved in these regulatory domains and alsoindicate that the autoinhibitory domain forms an a-helixthat binds to the substrate-binding cleft of the enzyme(197). It has been shown that when Ca21/calmodulinbinds to the enzyme, inhibition ceases and likely involvesa conformational change that exposes the active site.Interestingly, Perrino (324) has provided recent evidencefor additional autoinhibitory elements within residuesthat are situated between the calmodulin-binding and au-toinhibitory domains noted in Figure 1.

The NH2 and COOH termini are highly variable be-tween species as well as between calcineurin A geneswithin the same organism (130, 188, 208). Particularlystriking are unusual amino acid compositions and se-quences of particular isoforms. For example, the mam-malian calcineurin Ab-isoform contains the sequenceMAAPEPARAAPPPPPPPPPPPGAD. . . at the NH2 termi-nus (117, 129, 220). The COOH terminus of the canA geneproduct from Dictyostelium contains a fourfold repeat ofthe sequence RXNSX(G/A)(E/D)LX as well as the repeti-tious sequence ..TTNNINPNSITTNENNSNEQLQQQQQ-QQQQQQPPTTTSTTT.. and, along with the NH2 terminus,is enriched in glutamine and asparagine (75). The COOH

terminus of calcineurin A from C. neoformans is highlyenriched in proline, serine, threonine, and glycine (310).The function of these variable domains is unknown, butthey may play a role in substrate recognition and/or lo-calization.

C) CALCINEURIN PHOSPHORYLATION. Purified calcineurin frombovine 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 caseinkinase II has been determined to be the serine in thesequence -RVFS(p)VLR- near the COOH terminus of thecalmodulin-binding domain (Fig. 1) (138, 257). Althoughphosphorylation could be blocked by Ca21/calmodulin,the kinetic properties of the phosphorylated and dephos-phorylated forms are similar (136, 138). Furthermore,phosphorylated calcineurin still binds and is activated bycalmodulin. Whether phosphorylation represents a meansof regulating calcineurin activity in vivo remains to bedemonstrated.

2. Calcineurin B

A) SEQUENCE DIVERSITY AND ISOFORMS. The calcineurin B sub-unit is also highly conserved throughout evolution, withmammalian calcineurin B showing 86% amino acid se-quence identity with insect calcineurin B (i.e., Drosoph-

ila) and 54% identity with calcineurin B from S. cerevisiae

(Fig. 2). This high degree of conservation allows func-tional interchange of calcineurin B subunits betweenmammalian and N. crassa catalytic subunits (423). Thegene for mammalian calcineurin B encodes a protein of170 amino acids containing four Ca21-binding EF-handmotifs (Fig. 2) (2).

TABLE 3. (Continued)

Organism GeneAlternative

Name Tissue/Stage Genbank EntryChromosomal

Location Reference No.

Bovine PPP3R1h Brain BBCALCB.GB_OM 2, 304CALB_BOVIN.SW

Human PPP3R1h Brain HUMCNR.Pr 2p16 3 p15j 131

Tissue/stage refers to tissue or developmental stage of highest abundance. a Gene locus and chromosomal location according toinformation posted at http://genome-www.stanford.edu/Saccharomyces/. b Sequence available at http://www.sanger.ac.uk/Projects/S_pombe/.c There is still an unresolved discrepancy whether the S. pombe ppb11 gene resides on chromosome I (468) or chromosome II (327). d A completeanalysis of the C. elegans genome had not yet been completed at the time this review went to press. Evidence for both calcineurin A- and calcineurin B-likegenes has been found. Future information can likely be found at one of the C. elegans genome databases: http://stein.cshl.org/elegans/ or http://wormsrv1.sanger.ac.uk/cgi-bin/ace/simple/worm. e Filobasidiella neoformans. f Two splice variants were identified (158). g GeneBank entry.h Gene nomenclature of human protein serine/threonine phosphatase genes according to Cohen (62). PP3CA corresponds to a-isoforms, PP3CBcorresponds to b-isoforms, and PP3CC corresponds to g-isoforms. Similar nomenclature has been adopted for mouse (http://www.informatics.jax.org/)and rat (http://ratmap.gen.gu.se/) calcineurin genes. i Two major splice variants have been identified, both identical with the exception of a 10-aminoacid deletion between calmodulin-binding domain and autoinhibitory domain (see References 190, 354.) These have been referred to as PP2Ba1 andPP2Ba2; note numerical subscript in Reference 265. j Chromosomal location according to References 117, 446. k Three major splice variants havebeen identified. Two isozymes were designated CNA1 and CNA2 in the original report (129). CNA1 and CNA2 are referred to as PP2Bb1 and PP2Bb2,respectively, while a third alternatively spliced isozyme was designated as PP2Bb3 in Reference 265 [note use of subscript to designate splice variant, incontrast to the gene names used by Kincaid et al. to describe the a (PP2Ba1), b (PP2Ba2), and g (PP2Ba3) isoforms of the catalytic subunit (118,292)]. l Chromosomal location according to Reference 292.


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

B) NH2-TERMINAL MYRISTOYLATION. The mature calcineurin Bprotein is missing the initiator methionine, and the newa-amino group of glycine at position 2 is acylated withmyristic acid (1). This modification has been conservedthroughout evolution from yeast to mammals, suggestinga crucial physiological role (71). To explore possible bi-ological roles for calcineurin myristoylation, Heitman andcolleagues (483) generated a mutant of calcineurin B inwhich glycine at position 2 was mutated to alanine,thereby preventing myristoylation. Surprisingly, expres-sion of the wild-type and mutant proteins in S. cerevisiae

demonstrated that myristoylation was not required formembrane association nor for interaction with immuno-suppressant drug complexes. Indeed, the nonmyristoy-lated protein exhibited full biological function. These re-sults were subsequently confirmed in biochemicalexperiments with purified myristoylated and nonmyris-toylated calcineurin heterodimer which showed equiva-lent enzymatic activities, inhibition by the CsA/cyclophilinimmunosuppressant drug complex, and interactions with

a synthetic phospholipid monolayer (182). Interestingly,the myristoylated protein exhibited substantial thermalstability (;12°C) relative to the nonmyristolyated protein(182). At present, it is unknown whether the biologicalrole of calcineurin B myristoylation is to impart increasedstability to the protein or whether there is another role yetto be identified.

C) CALCIUM BINDING PROPERTIES. Klee et al. (204) were thefirst to discover that calcineurin binds Ca21. With the useof flow dialysis, it was demonstrated that four Ca21 bindwith high affinity [dissociation constant (Kd) #1026 M]and that the Ca21-binding sites were localized to thecalcineurin B subunit. The complete primary sequencedetermination of calcineurin B revealed homology withcalmodulin (35% identity) and troponin C (29% identity)(2), most of which was confined to four Ca21-binding“EF-hand” motifs. More detailed thermodynamic aspectsof Ca21 binding became possible when the recombinantcalcineurin B subunit was obtained via heterologous ex-pression in Escherichia coli. Using the purified recombi-nant protein, Burroughs et al. (40) studied the metal bind-ing properties using Eu31 and Tb31 luminescencespectroscopy. Four Eu31-binding sites were revealed, two

FIG. 1. Primary sequence and domain structure of calcineurin A. The amino acid sequence represents the ratcalcineurin A a-isoform reported by Saitoh et al. (354). CaM, calmodulin; CNB, calcineurin B; AI, autoinhibitory.


with relatively low affinity (Kd values of 1 6 0.2 and1.6 6 0.5 mM) and two with relatively high affinity (Kd

values of 0.14 6 0.020 and 0.020 6 0.010 mM). Tb31 alsobound but with slightly weaker affinities (Kd values of0.04 6 0.01 and 0.17 6 0.02 mM for the COOH-terminalsites and 1–3 mM for the NH2-terminal sites). Direct Ca21

binding to calcineurin B has also been studied by flow

dialysis, which found one high-affinity (Kd 5 0.024 mM)and three lower affinity sites (Kd 5 15 mM) (176). TheNMR-active isotope 113Cd has been used as a Ca21 surro-gate to identify four similar but distinct metal bindingsites consisting of all-oxygen coordination of pentagonalbipyramidal geometry as expected for an EF-hand Ca21-binding site (176), later confirmed in the X-ray structure

FIG. 2. Multiple sequence alignment of calcineurin B sequences from diverse organisms. Calcineurin B sequences ofSaccharomyces 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 alignmenteditor of the Wisconsin Package version 9.0, Genetics Computer Group (Madison, WI). The four Ca21-binding EF-handmotifs are indicated. The residues that participate in Ca21 coordination are noted by an asterisk. The consensussequence is defined in which a residue is conserved in all 8 sequences.


(124, 197). Ca21 binding to individual sites of calcineurinB has been studied using point mutants of this subunitaltered in each of the four EF-hands (104). This studyconfirmed the higher Ca21 affinity for COOH-terminalEF-hand sites and also found that Ca21 binding at thesesites is likely to be structural.

D) CALCINEURIN B HOMOLOGS. EF-hand proteins have beenclassified into 39 distinct subfamilies containing any-where from two to eight EF-hand domains (178). Cal-cineurin B proteins represent one subfamily of EF-handproteins based on sequence alignments and congruenceof domain and interdomain regions (302). In recent years,a number of Ca21-binding proteins containing EF-handdomains have been identified from cloning studies andshown to be homologous to calcineurin B. These includeNCS-1, a neuronal calcium sensor that inhibits rhodopsinphosphorylation in a Ca21-dependent fashion (81), mod-ulates calmodulin targets (359), and may regulate exocy-tosis (264); a protein p22/CHP (for calcineurin homolo-gous protein) that is required for constitutive membranetraffic (25) and inhibits serum and GTPase stimulation ofthe Na1/H1 exchanger NHE1 (234); CIB (for calcium- andintegrin-binding protein), a 22-kDa Ca21-binding proteinthat interacts with the cytoplasmic tail of the integrin aIIb

portion of the GPIIb/IIIa fibrinogen receptor (297); and aprotein product of the SOS3 gene of Arabidopsis thali-

ana involved in tolerance to the ionic component of saltstress in plants (239). Homology to calcineurin B rangesfrom 27 to 31% for NCS-1, CIB, and SOS3 and to up to 43%for p22/CHP. Like calcineurin B, p22/CHP is myristoylatedwhile NCS-1, CIB, and SOS3 contain the requisite consen-sus sequences for myristoylation. The fact that a consti-tutively active form of yeast calcineurin in transgenictobacco plants resulted in increased salt tolerance pro-vides evidence for a possible functional overlap betweenSOS3 and calcineurin B (320). On the contrary, biochem-ical studies with NCS-1 suggested that it could replacecalmodulin rather than calcineurin B in activating cal-cineurin and other calmodulin-dependent enzymes (359).Furthermore, although p22/CHP is completely congruentwith the calcineurin B family of proteins, arguments havebeen forwarded that it is more appropriate to place it in aseparate subfamily (25). Undoubtedly, further studies are

required to determine whether any or all of these proteinscan function in a fashion comparable to calcineurin B.

III. PHYSIOLOGICAL ROLES FOR CALCINEURIN

A. Lower Eukaryotes

Genetic methods for the selective deletion of one orboth calcineurin subunits to assess biological function bynoting the phenotype of the mutant strain are now possi-ble in several eukaryotic organisms. In addition, the im-munosuppressant drugs CsA and FK506, as specific cal-cineurin inhibitors, have provided complementary toolsfor discerning the role of calcineurin in many eukaryoticorganisms (71, 142, 303, 387). Some of the most thoroughwork investigating biological roles for calcineurin haveused the yeast S. cerevisiae as a model system. There aretwo genes for the catalytic subunit of calcineurin in S.

cerevisiae (CNA1/CMP1 and CNA2/CMP2) and only onegene for the B subunit (CNB1). Calcineurin is essential inCsA- and FK506-sensitive yeast strains (36). Recent workhas begun to explore the role of calcineurin in slightlymore complex organisms such as N. crassa (153, 213, 335)and D. discoideum (75, 159, 252). Furthermore, cal-cineurin has either been isolated or detected from thehuman pathogens C. neoformans (310), Leishmania spe-cies (21, 342), the malarial parasite Plasmodium falcipa-

rum (87), helminth parasites (347), and schistosomes(186). In some of these, growth can be inhibited by theimmunosuppressive agents FK506 and its analogs as wellas CsA (16, 87, 186, 309, 342), thus raising the possibilitythat novel calcineurin inhibitors might be developed asspecific antifungal and antiparasitic agents. The followingsections detail what has been learned regarding physio-logical roles for calcineurin in lower eukaryotic organ-isms and is summarized in Table 4.

1. Saccharomyces cerevisiae

A) RECOVERY FROM PHEROMONE-INDUCED GROWTH ARREST. Hap-loid cells of S. cerevisiae produce one of two mating

TABLE 4. Physiological roles of calcineurin in lower eukaryotic organisms

Organism Function Reference No.

Saccharomyces cerevisiae Recovery from mating factor a-induced growth arrest, cation (e.g., Li1, Na1, Mn21)resistance, Ca21 homeostasis, Ca21-mediated G2 arrest, onset of mitosis

71, 72, 279, 461, 467

Schizosaccharomyces pombe Cytokinesis, mating, nuclear and spindle pole body positioning, polarized growth, properseptation, chloride homeostasis

327, 399, 468

Dictyostelium discoideum Differentiation, stalk cell/spore formation 159Neurospora crassa Hyphal growth/conidiation, maintenance of apical Ca21 gradient, proper septation 213, 335Cryptococcus neoformans Virulence, pH and CO2 homeostasis, temperature-sensitive growth, resistance to Li1 310Aspergillus nidulans Cell cycle progression through G1/S, nuclear division, polarized growth, proper septation 303, 343Paramecium tetraurelia Exocytosis 209, 282


pheromones, a-factor and a-factor. Exposure of haploidstrains to the opposite mating pheromone prepares cellsfor mating by inducing cell cycle arrest in G1. This ismediated by an elaborate signal transduction pathwayinvolving a rise in intracellular Ca21 and activation ofcalcineurin (47, 142). Growth arrest can be observed as azone of clearing surrounding a source of a-factor on alawn of cells and occurs within 24 h at 30°C. Escape froma-factor-induced cell cycle arrest involves three meta-bolic processes that have been referred to as recovery,adaptation, and survival (287). Recovery is defined as theability of cells to resume growth after removal of thepheromone, whereas adaptation is a process in whichcells eventually resume growth in the continuous pres-ence of pheromone. Both recovery and adaptation mayinvolve common signaling components and can be ob-served by a shrinking of the zone of clearing and increas-ing turbidity within it, usually within ;24 h at 30°C.Survival differs from recovery and adaptation in that itdescribes whether a cell remains viable after exposure topheromone.

Coincident with the cloning of genes for the twoyeast calcineurin A subunits (CNA1/CMP1 and CNA2/

CMP2, Table 3), strains deficient in either subunit wereviable but failed to recover from a-factor-induced growtharrest (72, 73, 242). Mata strains containing a single CNA1

or CNA2 mutation were twice as sensitive as wild type toa-factor-induced growth arrest, whereas the double mu-tant CNA1/CNA2 was four times as sensitive, as assessedby the size of the halo after 24 h at 30°C (72, 73). Further-more, once arrested, the double mutant failed to resumegrowth. In contrast, the CNB1 mutant did not show anincreased sensitivity compared with wild type, but likethe CNA1/CNA2 double mutant, it failed to recover fromgrowth arrest. In wild-type cells, the immunosuppressantdrugs CsA and FK506 also inhibited recovery from a-fac-tor-mediated growth arrest, and these required the pres-ence of their respective immunophilins cyclophilin andFKBP (107). In addition, expression of the CNA1/CMP1

gene increased in the presence of a-factor, the result of59-noncoding sequences in the CNA1/CMP1 gene match-ing closely the consensus sequence for the a-factor ele-ment (467).

As expected, the activator protein of calcineurin,calmodulin, has also been shown to be required for es-cape from cell cycle arrest after exposure to pheromone(287). Calmodulin mutants did not display increased sen-sitivity to a-factor, nor did these mutant strains appear tobe affected in either recovery or adaptation. Indeed, bothcalcineurin and calmodulin mutants adapted as well as awild-type strain to low concentrations of pheromone, andboth mutants recovered after pheromone removal withthe same kinetics as the wild-type strain. The process thatappeared to be affected was survival, a result consistentwith previous work indicating that Ca21 is also essential

for survival after exposure to a-factor (166). Interestingly,in addition to calcineurin, the Ca21, calmodulin-depen-dent protein kinases (CMK1 and CMK2), yeast proteinkinase C (PKC1), and a mitogen-activated protein (MAP)kinase (MPK1) are also required for recovery fromgrowth arrest, thus indicating that enzymes of opposingfunction are required for surviving exposure to a-factor(287, 301, 461).

One downstream signaling component in S. cerevi-

siae regulated by calcineurin is the yeast transcriptionfactor Crz1p/Tcn1p. Crz1p/Tcn1p is required for cal-cineurin-dependent induction of genes for the vacuolarand secretory Ca21 pumps, Pmc1p and Pmr1p, respec-tively; one of two genes encoding b-1,3 glucan synthase,FKS2; and the gene for the plasma membrane Na1 pump,PMR2 (Fig. 3) (263, 388). In addition, calcineurin has beenshown to regulate the high-/low-affinity state of theplasma membrane K1 channel, Trk1p (269), and inhibitthe vacuolar H1/Ca21 exchanger Vcx1p (69) by posttran-lational mechanisms. Some of these are presented belowin more detail.

B) ADAPTATION TO SALT STRESS. The search for additionalphenotypes found that calcineurin-deficient yeast exhib-ited decreased tolerance to the monovalent cations Na1

and Li1, but not K1, Ca21, and Mg21 (269, 300). The roleof calcineurin in Na1/Li1 tolerance is thought to be me-diated by transcriptional and posttranslational mecha-nisms. Adaptation to high salt stress requires the presenceof a plasma membrane Na1-ATPase involved in Na1 andLi1 efflux, Pmr2p. Cells deficient in calcineurin accumu-late Na1 and Li1 due to decreased expression of Pmr2p(269). Although no changes in intracellular Ca21 havebeen observed after induction of the high-salt response,evidence indicates that Ca21 mediates this response.Ca21, via calmodulin activation of calcineurin, regulatesadaptation to high salt stress by induced expression ofPmr2p (76, 154, 268), mediated by the transcription factorCrz1p/Tcn1p (Fig. 3) (263, 388). The activity of Pmr2p isalso stimulated by Ca21/calmodulin, thereby providingboth transcriptional and posttranslational regulation ofNa1 efflux mediated by Ca21 (349, 454).

Cells deficient in CNB1 are unable to convert the K1

transport system (Trk1p, a K1 channel) to a high-affinitystate. In the high-affinity state, this pump has increasedaffinity for K1, but the Michaelis constant (Km) for Na1 orLi1 is unaffected, thereby resulting in increased Na1 up-take in calcineurin-deficient cells. The mechanism of thisregulation has been hypothesized to be direct or indirectdephosphorylation of Trk1p by calcineurin (269).

Other proteins in addition to calcineurin are requiredfor salt tolerance such as the gene products of PDE1, alow-affinity cAMP-dependent phosphodiesterase (154);URE2, a regulator of nitrogen catabolite repression (462);PMA1, the plasma membrane H1-ATPase (462); HAL3, aprotein involved in cell cycle control and ion homeostasis


(105); and STD1, a protein that interacts with the SNF1protein kinase in two-hybrid and in vitro binding studies(113). Thus multiple parallel pathways are necessary forfull induction of this response.

C) CALCIUM HOMEOSTASIS. Calcineurin is involved in theregulation of Ca21 pumps and exchangers responsible forCa21 homeostasis in yeast (Fig. 3). These maintain cyto-plasmic [Ca21] in the range of 100–300 nM (68, 78). Inaddition, other ion transporters indirectly influence intra-cellular [Ca21]. One of these is the vacuolar H1-ATPase,which provides the driving force for Ca21 sequestrationby the Ca21/H1 exchanger encoded for by the VCX1 gene(114, 144, 408). Two Ca21-ATPases, Pmc1p and Pmr1p,are responsible for depleting the cytosol of Ca21. Theformer is localized to the vacuole (70), while the latter is

important in the secretory pathway and localizes to theGolgi (349). Mutants deleted in either Pmc1p or Pmr1pcannot grow in media containing high Ca21. Deletion ofthe gene for either calcineurin subunit, or treatment ofcells with CsA or FK506, restores growth to either singlePMC1 or double PMC1/PMR1 mutants in high Ca21 media(70), indicating that calcineurin activation can have anegative effect on growth. As noted above, activation ofcalcineurin leads to transcriptional induction of the PMC1

and PMR1 genes via Crz1p/Tcn1p (263, 388).Calcineurin mutants are also sensitive to extracellu-

lar Mn21. Wild-type strains are able to prevent Mn21

entry, whereas mutants exhibit an increased uptake phe-notype (100), therefore indicating that the regulation ofMn21 homeostasis by calcineurin follows a different

FIG. 3. Physiological roles of calcineurin in Saccharomyces cerevisiae. Calcineurin has numerous roles in buddingyeast including recovery from a-factor-induced growth arrest, salt and temperature tolerance, Ca21 homeostasis, andMn21 tolerance (387). Many of these are mediated by Crz1p/Tcn1p, a calcineurin-dependent transcription factornecessary for the transcriptional induction of Pmc1p, Pmr1p, Pmr2p, and Fks2 (heavy arrows) (263, 388). In addition,calcineurin inhibits the activity of the vacuolar H1/Ca21 exchanger (Vcx1p) and causes conversion of the K1 channel,Trk1p, to the high-affinity state. The latter occur independently of Crz1p probably by posttranslational processes (narrowarrows) (69). Cch1p, Ca21 channel (313); Crz1p, calcineurin-responsive zinc finger transcription factor, product of theCRZ1 gene; Pmcp1, high-affinity vacuolar Ca21 pump, product of the PMC1 gene (69); Pmr1p: secretory Ca21 pump,product of the PMR1 gene; Tcn1p: alternative name for Crz1p protein; Vcx1p: low-affinity vacuolar H1/Ca21 exchanger(69). Mid1p is an alternative name for the plasma membrane Ca21 channel.


mechanism than monovalent cation transport, in whichexport is regulated by a P-type ATPase (269, 300). Analternative hypothesis has been proposed in whichPmr1p, the Golgi-localized Ca21 pump, plays a role inMn21 tolerance by sequestering Mn21 to late compart-ments in the secretory pathway (263). Mn21 may also betransported into the vacuole via the Ca21/H1 exchangerVcx1p (332).

D) B-GLUCAN SYNTHASE AND CELL WALL SYNTHESIS. Calcineurin isresponsible for transcriptional regulation of FKS2, one oftwo genes encoding b-1,3-glucan synthase (Fig. 3) (95,480). Calcineurin-dependent regulation occurs throughCrz1p/Tcn1p (263, 388). The Fks1p protein is the predom-inant synthase expressed during optimum growth, butexpression of Fks2p is induced upon treatment of cellswith mating pheromone, high Ca21, or growth on poorcarbon sources. Deletion of the FKS1 and CNB1 genesresults 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 inregulating cell wall structure.

2. Schizosaccharomyces pombe

Like the budding yeast, treatment of S. pombe withFK506 or deletion of the ppb11 gene, encoding for thecalcineurin A subunit (Table 3), is not lethal. However,calcineurin in fission yeast appears to have distinct func-tions. Calcineurin-deficient S. pombe cells exhibit drasticCl2-sensitive growth (399) and are defective in cytokine-sis, transport, nuclear and spindle pole body positioning,cell shape (468), and sporulation (327). One function forcalcineurin in S. pombe that appears to overlap with S.

cerevisiae is the mating process, although the roles forcalcineurin in mating appear to be distinct in these twoorganisms. In S. cerevisiae, calcineurin is required for thecell to recover from or survive growth arrest after expo-sure to pheromone. It thus may function to assist cells toreenter the cell cycle if they respond to a-factor but fail tomate (see sect. IIIA1A). In S. pombe, calcineurin is requiredfor the mating response, and calcineurin mutants in thisorganism are sterile (327, 468). Northern analysis indi-cates that the transcript for calcineurin varies during thecell cycle and can be induced by nitrogen limitation, acondition that favors mating in S. pombe (327). The lattereffect was dependent on the transcription factor ste1.

3. Neurospora crassa

N. crassa has been widely used as a model system forstudying eukaryotic gene expression. In this fungus, cal-cineurin is thought to play a major role in hyphal exten-sion during mycelial growth and in determining apicalorientation. Thus calcineurin mRNA exhibited the highestexpression during early mycelial logarithmic growth butwas repressed before conidiation upon entry into station-

ary phase (153). A similar pattern of protein expressionwas observed but with about a 12-h lag behind messageexpression. Expression of antisense mRNA to the cata-lytic subunit, treatment with CsA and FK506, or disrup-tion of the calcineurin B gene caused growth arrest pre-ceded by aberrant and increased hyphal branching andentry into conidiation, consistent with a role in apicalgrowth (213, 335). Growth on two different carbonsources, glutamate and sucrose, did not influence thelevel of expression, indicating that calcineurin is not in-volved in mechanisms related to catabolite repression(153).

4. Aspergillus nidulans

Similar to N. crassa, calcineurin A mRNA levels varyduring the cell cycle in A. nidulans, and disruption of thecnaA1 gene resulted in growth arrest (303, 343). Aftergrowth arrest in metaphase upon treatment with nocoda-zole followed by resuspension in fresh media to allow forsynchronous growth, it was shown that calcineurin Amessage levels peak after the end of mitosis before DNAreplication, with the highest expression appearing at theG1/S boundary. Inducible gene disruption by homologousrecombination revealed that the calcineurin A gene wasessential for normal growth, and disruption was lethal.Further results indicated that the cnaA1 gene was re-quired for early cell cycle events before DNA replication.Taken together with other data, this study suggested thatcalcineurin as well as other calmodulin targets may berequired during different periods of the cell cycle.

5. Cryptococcus neoformans

Fungal diseases are becoming an increasing healthproblem, most notably in individuals infected with humanimmunodeficiency virus (HIV); C. neoformans representsone of these life-threatening infectious agents (6). Heit-man and colleagues (5, 67, 309, 310) have begun to ex-plore the role of calcineurin in this pathogen with the goalof using novel CsA and FK506 analogs that have increasedspecificity toward the fungal calcineurin compared withthe host (human) enzyme. Such compounds may eventu-ally prove useful as antifungal agents with reduced toxic-ity and immunosuppressive effect toward the host. Onesuch candidate is the FK506 analog L-685,818 (18-hy-droxy, 21-ethyl-FK506). L-685,818 is toxic to C. neofor-

mans, mediated by binding and inhibiting the fungal cal-cineurin, but has reduced immunosuppressive activity inhumans (309). Growth studies in the presence of CsA andFK506 indicate that these drugs are growth inhibitory at37°C but not at 24°C and that they inhibit a commontarget. Disruption of the calcineurin A gene resulted inmutant strains that are viable at 24°C but do not surviveunder conditions that mimic the host environment includ-ing elevated temperature, 5% CO2, or alkaline pH (310).


These mutant strains are no longer pathogenic, thus indi-cating that calcineurin is necessary for virulence in thisorganism.

6. Dictyostelium discoideum

Calcineurin in the slime mold D. discoideum exhib-ited the familiar developmental pattern of expression asnoted above for the filamentous fungi, with the highestlevel of expression during vegetative growth and decreas-ing expression during multicellular development (75).CsA and FK506 had no effect on growth, a process thatcan be separated from development in this organism(159). However, these drugs do inhibit developmentalprocesses such as stalk cell spore formation and expres-sion of prestalk and prespore developmental markers.

7. Other lower eukaryotic organisms

A gene for calcineurin B has been isolated in theamoeboflagellate N. gruberi (346). mRNA levels are de-tectable in the amoebae and are cyclic, with peak abun-dance during flagellar formation, followed by a gradualdecline. In the unicellular organism Paramecium tetra-

urelia, calcineurin localization was investigated by use ofa specific antibody and immunocytochemical methods(209). Calcineurin was largely localized to the cilia andcell membrane, with only a diffuse staining pattern ob-served within the cell body. Further staining indicatedthat there was no difference in either localization or abun-dance in cells prepared either in logarithmic or stationaryphase. Thus calcineurin abundance does not appear tochange during the cell cycle as it does in the simple fungi.To further explore calcineurin’s role in P. tetraurelia,anticalcineurin antibody or Ca21/calmodulin-calcineurinwas microinjected into cells. Anticalcineurin antibodyblocked exocytosis after treatment with the exocytosistrigger agent, aminoethyldextran, while microinjection ofa complex of Ca21/calmodulin-calcineurin induced exo-cytosis. These results implicate calcineurin as the phos-phatase previously shown to dephosphorylate a 63-kDaprotein hypothesized to be involved in trichocyst exocy-tosis (198).

Recently, calcineurin has been isolated from Leish-

mania major (342) and Leishmania donovani (21). Cal-cineurin was isolated by chromatographic separation ofcytosol from promastigotes where it was hypothesized tobe a key regulatory component in the life cycle of thisparasite. Interestingly, in L. major, extracellular growth isnot inhibited by CsA, and in fact, a high-affinity complexof CsA with L. major cyclophilin forms [inhibitory con-stant (Ki) 5 5.2 nM] but does not inhibit or form a tightcomplex with calcineurin from that organism, suggestinga possible mechanism for this organism’s resistance toCsA (342). Interestingly, a complex between CsA, recom-binant human cyclophilin, and L. major calcineurin was

formed indicating that the parasitic calcineurin is func-tionally and structurally equivalent to mammalian cal-cineurin. A similar phenomenon was observed with cal-cineurin from the tapeworms Hymenolepsis microstoma

and Hymenolepsis diminuta such that calcineurin fromboth organisms was inhibited by CsA complexed withmammalian cyclophilin but not H. microstoma cyclophi-lin (347). This was not the case with calcineurin fromSchistoma mansoni (186) and Plasmodium falciparum

(87). One hypothesis to explain the lack of complex for-mation with calcineurin is that parasitic cyclophilins arestructurally different from mammalian cyclophilins, suchthat cyclophilin residues surrounding the CsA binding sitethat interact with calcineurin are not conserved in para-sitic cyclophilins. Further studies are necessary to resolvethese interesting findings.

B. Higher Eukaryotes

1. Calcineurin in plants

Evidence for a plant homolog of calcineurin was firstobtained by Luan et al. (246) who demonstrated, usingpatch-clamp techniques, that CsA and FK506 blockedCa21-dependent inactivation of K1 channels in Vicia

faba. A partially proteolyzed and constituitively activeform of calcineurin also inhibited K1 channel activity.Furthermore, both CsA and FK506 inhibited a Ca21-de-pendent phosphatase activity in cellular extract. Subse-quent studies have provided additional evidence for cal-cineurin function in plants (reviewed in Ref. 245).

To date, however, calcineurin has not been success-fully purified to homogeneity from plant tissue nor havebona fide genes for either subunit been cloned. The clos-est contenders are two EF-hand Ca21-binding proteinsencoded for by the SOS3 and AtCBL genes that are ho-mologous to the calcineurin B subunit (218, 239). Theprotein encoded by the SOS3 gene is 30% identical tocalcineurin B from various organisms, and mutations inSOS3 render A. thaliana sensitive to Na1 (239). The SOS3protein is also homologous to NCS-1 (30% identity), aneuronal Ca21 sensor in the recoverin family of EF-handproteins (see sect. IIB2D). The AtCBL proteins are mosthomologous to calcineurin B (32% identity to rat cal-cineurin B) and can complement a yeast calcineurin Bmutation, indicating a calcineurin B-like physiologicalfunction (218). Recent work, however, indicates that theAtCBL proteins interact with a novel group of proteinkinases in a Ca21-dependent fashion (372). A. thaliana

contains at least six AtCBL genes. The AtCBL and SOS3proteins clearly play different roles since they are unableto complement each other (218). It is intriguing that SOS3

and AtCBL encode for Ca21-binding proteins, indicatingthat salt stress in plants may be regulated by Ca21-depen-dent signaling pathways (possibly via calcineurin) as has


been found in S. cerevisiae (see sect. IIIA1B). Furtherevidence for this hypothesis was obtained in a studydemonstrating that overexpression of an activated formof yeast calcineurin conferred salt tolerance in transgenictobacco plants (320). Similarly, genes for three of theAtCBL isoforms appear to be stress regulated. WhetherSOS3 or the AtCBL proteins represent plant calcineurin Bhomologs or just close relatives will hopefully be resolvedif a protein corresponding to plant calcineurin can beisolated and/or cloned and shown to be a functional phos-phatase.

2. Calcineurin in mammals

A) TISSUE DISTRIBUTION. Calcineurin is widely distributedin mammalian tissues, with the highest levels found inbrain (168, 175, 216, 437). In addition, calcineurin A and Bsubunits have been observed in adipose tissue, adrenalcells (318, 319), bone osteoclasts (19), heart, hindbrainand spinal cord (394), kidney (42, 418, 419), liver (135), Band T lymphocytes (4, 50, 193), lung, medulla, olfactorybulb, 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 vari-ous isoforms of each subunit (42, 175, 219). An isoform ofthe catalytic subunit encoded for by the PPP3CC gene(g-isoform, see Table 3) is testes specific (291, 292), as isthe product of the PPP3R2 gene encoding an isoform ofthe regulatory subunit (48, 424). With the use of poly-clonal antibodies that distinguish between the a- andb-isoforms of calcineurin A (encoded by the PPP3CA andPPP3CB genes, respectively; Table 3), it was found thatcalcineurin Aa was more abundant than Ab in the ratbrain and heart, but the relative abundance is reversed inspleen, thymus, and lymphocytes (175, 219). These resultspartly explain the recent finding that PPP3CA knock-outmice produce T and B cells that mature normally, respondto mitogenic stimulation, and remain sensitive to bothCsA and FK506, but are defective in in vivo antigen-specific T-cell responses (477). These PPP3CA-deficientmice also accumulate hyperphosphorylated tau proteinand exhibit cytoskeletal changes in the hippocampus as aresult of reduced calcineurin Aa activity (181). More re-cent studies indicate that synaptic depotentiation is com-pletely abolished, indicating that calcineurin Aa may playa role in the learning and memory process (181, 485).

B) SUBCELLULAR DISTRIBUTION. Using a radioimmunoassay,Cheung and colleagues (10) measured the subcellulardistribution of calcineurin in chick forebrain homogenate.In that study, calcineurin was highly enriched in the cy-toplasmic and microsomal fractions as well as synapto-somes. Subsequent studies have confirmed its predomi-nance in the cytoplasm and synaptosomal cytosol (219,

306). Politino and King (329) explored the physical asso-ciation of calcineurin with synthetic phospholipid vesi-cles and showed that calcineurin binds small, acidic,unilamellar vesicles in a Ca21-dependent fashion. Further-more, the phosphatase activity of calcineurin was pro-foundly affected by phosphatidylglycerol or phosphatidyl-serine, with up to a 23-fold increase in activity towardphosphorylated histone, but inhibition using p-nitrophe-nylphosphate (p-NPP) or tyrosine phosphate. In a subse-quent study it was hypothesized that the phospholipid-binding site was located on the calcineurin B subunit(330). Using synthetic phospholipid monolayers, Kennedyet al. (183) investigated the factors that contributed tocalcineurin-phospholipid interactions and found that cal-cineurin binding is myristoyl independent, mediated byanionic phospholipids and/or diacylglycerol, and also af-fected by the presence of calmodulin.

There is overwhelming evidence for calcineurin inthe nucleus along with other calmodulin-binding proteinssuch as casein kinase-2 and myosin light-chain kinase (34,286, 336, 376, 426). In spermatids, calcineurin was local-ized to the nucleus, and its levels were most abundantduring the initial stage of nuclear elongation, with almostno signal present in the cytoplasm (286). In the context ofsignaling pathways that activate nuclear factor of acti-vated T cells (NF-AT), Shibasaki et al. (376) have shownthat calcium induces an association between calcineurinand NF-AT that results in colocalization of both moleculesto the nucleus.

Calcineurin has also been shown to be associatedwith the cytoskeleton (106). The latter finding is of inter-est given that several substrates of calcineurin are colo-calized 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 beenidentified for calcineurin in higher eukaryotic organisms,and it is beyond the scope of this review to cover all ofthem comprehensively. Klee et al. (208) have provided arecent update and cited a number of specific reviewsregarding calcineurin function (208). Table 5 is an attemptto summarize a number of tissues, systems, and specificsubstrates that are implicated to be regulated by cal-cineurin. In sections IIIB2D and IIIB2E, we provide a shortreview of the role of calcineurin on two key systems ofimportance in modern biology, apoptosis and cardiac hy-pertrophy.

D) CALCINEURIN AND APOPTOSIS. It has been recognized forsome time that calcineurin plays a role in programmedcell death of T and B lymphocytes (32, 110, 116, 481).Recently, it has also been shown that calcineurin plays arole in apoptosis in neuronal cells via the cytochromec/caspase-3 pathway (17). In T-cell hybridomas, apoptosiscan be stimulated by ligation of the T-cell receptor/CD3complex and has provided a useful in vitro model to


investigate signaling pathways responsible for this biolog-ical phenomenon. Both CsA and FK506 inhibit this pro-cess, implicating calcineurin in the signaling pathway ofapoptosis which is known to involve a rise in intracellularCa21 (110). Similarly, in the B-cell lymphoma cell linesWEHI-231, B104, and BL60, apoptosis induced by cross-linking of surface immunoglobulin receptors was inhib-ited by these immunosuppressant drugs (32, 116).

In lymphocytes, calcineurin and NF-AT appear toparticipate in apoptosis, in part by mediating the induc-

tion of Fas and Fas ligand which then interact and trans-duce the apopototic signal after T-cell receptor ligation(157, 226, 243, 421). Using a constituitive (Ca21- and cal-modulin-independent) form of calcineurin, Shibasaki andMcKeon (375) demonstrated that calcineurin functions incalcium-induced apoptosis in mammalian cells deprivedof growth factors and that this was a direct consequenceof calcineurin’s phosphatase activity. Interestingly, coex-pression of Bcl-2 blocked calcineurin-induced apoptosis.At least one mechanism for how this occurs was provided

TABLE 5. Physiological roles of calcineurin in higher eukaryotic organisms

Organ or System Function Reference No.

Bone Osteoclast bone resorption 19Brain Amyloid b-peptide formation 83

Apoptosis 17Dephosphorylation of DARPP-32 84, 196Dephosphorylation of nitric oxide synthase 79, 102, 474Endocytosis of synaptic vesicles via dynamin I 90, 225, 240Gene regulation 96, 123, 364, 465Hormonal control 59, 434Ischemia 41, 285, 422Long-term potentiation 460, 485Memory 123, 181, 254Neuritogenesis 49, 106Regulation of Ion Channels 299, 465

Glutamatergic receptors 7, 35, 79, 217, 231, 284, 415Glutamate release 308, 427g-Aminobutyric acid receptor 162M channel modal gating 256Nicotinic receptors 241Voltage-gated Ca21 channels 465, 484Voltage-gated Na1 channels 51, 294Voltage-gated K1 channels 348

Gastric chief cells Exocytosis and pepsinogen secretion 344Heart Cardiac hypertrophy 82, 86, 108, 171, 232, 233, 247, 267, 281, 290,

312, 337, 377, 397, 401, 402, 417, 441Hematopoetic system Progenitor mast cell IgE-dependent cytokine transcription 180

Thymic selection 442Erythropoietin-dependent c-myb regulation 250, 360

Kidney Immunosuppressant-induced hypertension 356Simulation of Na1-K1-ATPase activity in renal tubule cells 14Adrenergic, dopamanergic, and angiotensin receptor signaling 418Glucocorticoid and mineralocorticoid signaling 418

Liver Protein phosphorylation 34, 135Hepatoprotection 101

Lymphocytes T-lymphocyte activation and cytokine signaling 46, 340Neutrophil chemokinesis 146Lymphocyte degranulation 89Apoptosis and FasL gene transcription 32, 110, 116, 157, 226, 243, 374, 375, 421,

429, 443, 463, 481Angiotensin II regulation of immune responses 305Macrophage effector function 65

Pancreas Acinar cell amylase secretion 125b-Cell insulin secretion 407

Pituitary Regulation of adenylyl cyclase 11, 13, 322Hormone secretion 11, 12

Placenta Epidermal growth factor urogastrone receptor dephosphorylation 314Skeletal muscle Skeletal muscle hypertrophy 88, 295, 367Smooth muscle Inhibition of L-type Ca21 channels 362Other functions Spermatid motility 409

Integrin recycling, integrin/fibronectin interaction 228, 331Tumor cell autocrine growth 298Dephosphorylation of Elk-1 398, 414DNA binding of p53 to HIV-1 long terminal repeat 126

HIV, human immunodeficiency virus.


subsequently by experiments which showed that Bcl-2forms a complex with calcineurin that targets it to thecytoplasmic membrane (374). Although still maintainingphosphatase activity, calcineurin bound to Bcl-2 is unableto promote nuclear translocation of NF-AT. Furthermore,BAD, a proapoptotic member of the Bcl-2 family, is asubstrate of calcineurin. Dephosphorylation of BAD bycalcineurin enhances BAD heterodimerization with Bcl-xL

and apoptosis (443).However, another intriguing hypothesis is that apo-

ptosis is linked to cellular redox homeostasis. Wolvetanget al. (463) showed that inhibitors of the plasma mem-brane NADH-oxidoreductase (PMOR) activity induce ap-optosis through a signaling pathway involving calcineurin(463). It was proposed that PMOR serves as a redoxsensor that can regulate the signals required for apoptosis(227). The finding that calcineurin activity is sensitive toredox state changes (45, 111, 345, 447, 470, 471) providessupport for this hypothesis and a means by which apo-ptosis could be regulated by the cellular redox potential.

E) IMPORTANCE OF CALCINEURIN IN CARDIOVASCULAR FUNCTION.

Recently, calcineurin and NF-AT have been implicated intransducing signals responsible for cardiac morphogene-sis 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 cardiacvalves and septa, and the transgenic mice die from con-gestive heart failure in utero (82, 337). Overexpression ofcalcineurin has also been shown to induce cardiac hyper-trophy and heart failure in transgenic mice that could beblocked by the immunosuppressant drug CsA (281). Fur-thermore, a transgenic mouse model for hypertrophy inwhich tropomodulin-overexpressing transgenic mice de-velop progressive dilated cardiomyopathy has providedevidence for increased calcineurin protein levels beforethe onset of the hypertrophic phenotype, suggesting thatcalcineurin may play an early regulatory role in this pro-cess (402). Similar results were found in skeletal musclefrom mice subject to overload (88) and confirmed later inskeletal muscle cells virally transfected with insulin-likegrowth factor I (295, 367). Some of these studies haveeven proposed that immunosuppressant drugs such asCsA 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 hypertrophyand showed that treatment with CsA, albeit an excessivedose, resulted in significantly less hypertrophy. However,although a few studies have confirmed this finding (267,402), several other groups examining calcineurin’s role inthis process have failed to demonstrate any efficacy ofCsA (86, 247, 290, 476) and, in fact, Molkentin (280) hasresponded by reporting that CsA protected against pres-sure-overload hypertrophy after 7 days but not after 21days. Although the reason for some of these discrepanciesmay be due to the dose of immunosuppressant drug used,current hypotheses suggest that multiple signaling path-

ways might be recruited to participate in the hypertrophicresponse and that inhibition of one parallel pathway (i.e.,calcineurin) might delay but not prevent hypertrophy(108, 397, 441). Nevertheless, they implicate a possiblerole for calcineurin and NF-AT in cardiac function.

Oxidative stress is also thought to play a role incardiomyopathy and heart failure (381). The possibilitythat calcineurin may be regulated by oxidative stressindicates that signaling pathways in which it is involvedmay be important in mediating processes that lead tocardiac dysfunction.

C. Inhibitors of Calcineurin

1. Natural product and synthetic inhibitors

A number of natural products have been isolated thatare potent inhibitors of calcineurin and other serine/thre-onine protein phosphatases. The most potent, specific,and well-known inhibitors of calcineurin are the immuno-suppressant drugs CsA and FK506 (Fig. 4), which inhibitcalcineurin when complexed with their respective cyto-plasmic receptors cyclophilin and FKBP (see Table 1entry for a number of reviews on these drugs). Interest-ingly, in vitro calcineurin inhibition by these immunosup-pressant drug complexes only occurs when a physiologi-cal substrate is used to assay the enzyme such asphosphocasein or phospho-RII peptide, a peptide whosesequence represents the phosphorylation site of the reg-ulatory subunit of cAMP-dependent protein kinase, a well-characterized and more physiological phosphopeptidesubstrate (31). The use of p-NPP as substrate results inactivation of calcineurin by these immunosuppressantdrug complexes (238, 403).

A number of other compounds have demonstratedinhibitory activity against calcineurin and other serine/threonine protein phosphatases. Okadaic acid, often usedas a potent and specific inhibitor of PP2A, can also inhibitPP1 and calcineurin at higher concentrations. The ID50 ofokadaic acid for PP2A has been measured to be ;1 nM,while the ID50 values for PP1 and calcineurin are ;300 nMand ;4 mM, respectively (29). The cyclic peptide micro-cycstin LR is a potent inhibitor of PP1 and PP2A, with a Ki

value ,0.1 nM. Although the inhibition of calcineurin bymicrocystin LR occurs at over 1,000-fold higher concen-trations, microcystin LR still is a relatively potent inhibi-tor of calcineurin (IC50 5 0.2 mM) (248). Dibefurin, a novelfungal metabolite, also has modest inhibitory activityagainst calcineurin (37).

Since the discovery of these natural product inhibi-tors, several new synthetic compounds have been foundto be reasonable inhibitors of calcineurin and other phos-phatases. Tatlock et al. (410) utilized computational dock-ing 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 ligandbinding to calcineurin (410). Endothall is structurally re-lated 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). Substitu-tion at the 5-endo position was hypothesized to providereasonable binding interactions that mimicked the inter-action between the active site of calcineurin and its auto-inhibitory domain. Incorporation of a trans-cyclopropyl-phenyl group at this position afforded the most potentinhibitor, with an apparent Ki of 0.5 mM. Interestingly, thetethered dicarboxylic acid moiety and bridgehead oxygenatom of endothall and cantharidin derivatives were mod-eled to interact with the active site dinuclear metal center(see sect. IVC ) (410).

A similar approach to inhibitor design incorporat-ing pendant metal-coordinating groups that could an-chor the inhibitor to the active site metal ions has been

introduced by Widlanski and colleagues (296). A varietyof alkylphosphonic acid derivatives containing an addi-tional thiol or carboxylate group (Fig. 4) were exploredas inhibitors of alkaline phosphatase and purple acidphosphatase. Assuringly, nearly all bound more tightlythan substrate p-nitrophenol and up to 55-fold tighterthan ethanylphosphonic acid, indicating that these ad-ditional function groups could improve binding affinity.Whether binding occurs via direct metal ligation forendothall and/or alkylphosphonic acid derivatives re-mains to be demonstrated by spectroscopic means. Ifcorrect, these compounds could provide a route to thedesign of more potent and selective metallophos-phatase inhibitors.

Peptide inhibitors of calcineurin have been also beenintroduced. One of these, a 25-residue peptide based onthe sequence of the autoinhibitory domain of the cal-cineurin A subunit from residues 457–481 (Fig. 1), is a

FIG. 4. Natural product and synthetic inhibitors of calcineurin. For the metal-ligating phosphonate inhibitors, n

refers to the number of methylene units.


relatively potent inhibitor of calcineurin phosphatase ac-tivity (137, 325). Recently, a high-affinity calcineurin-bind-ing peptide was selected from a combinatorial peptidelibrary based on the calcineurin docking motif of NF-AT(15). The peptide inhibited NF-AT activation and expres-sion of NF-AT-dependent cytokine genes in T cells, butdid not inhibit calcineurin phosphatase activity towardphospho-RII peptide, and thus did not affect the expres-sion of other cytokines that require calcineurin but notNF-AT. The latter point is significant because compoundssuch as this peptide that selectively interfere with cal-cineurin-NF-AT interaction without disrupting calcineurinphosphatase activity may prove to be less toxic immuno-suppressants compared with CsA and FK506.

At least one other synthetic calcineurin inhibitor hasbeen reported, PD 144795, a benzothiophene derivativeshown to have anti-inflammatory and anti-HIV effects(126). Transcriptional activity mediated by p53 and NF-kBwere inhibited by both CsA and PD 144795. An in vitroassay of calcineurin activity from Jurkat cell lysate alsoindicates that PD 144795 led to dose-dependent inhibitionof calcineurin.

It was previously concluded by Enan and Matsumura(93) that class II pyrethroid insecticides were potent in-hibitors of bovine brain calcineurin, with IC50 values of1029 to 10211 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 IIpyrethroids was able to inhibit purified bovine calcineurinusing phospho-RII peptide (97). Calcineurin activity in ratbrain homogenate and in IMR-32 neuroblastoma cells inculture was also not affected by pyrethroids, indicatingthat these insecticides are not effective inhibitors of cal-cineurin (99).

The tyrphostins A8, A23, and A48, members of afamily of tyrosine kinase inhibitors, inhibited calcineurinwith IC50 values of ;1025 M (258). However, the use ofp-NPP as substrate in these studies should be questionedgiven the inhibition pattern of calcineurin noted above forCsA, FK506, and the pyrethroid insecticides. A follow-upstudy using phospho-RII peptide or other suitable phos-phoprotein substrate may confirm yet another class ofcalcineurin inhibitors.

2. Endogenous regulators

In addition to synthetic and natural product inhibi-tors of calcineurin, a number of endogenous cellular pro-teins have emerged as inhibitors of calcineurin proteinphosphatase activity and thus potential regulators of its invivo function. One of the first to be identified was a79-kDa protein kinase A anchoring protein (AKAP79)(58). AKAP79 associates with the regulatory subunit ofthe cAMP-dependent protein kinase and localizes it topostsynaptic densities. Using a yeast two-hybrid approach

to search for proteins that interacted with AKAP79,Scott and colleagues (58) identified a positive clone en-coding the calcineurin A subunit. Immunofluorescencestudies demonstrated that calcineurin and the regulatorysubunit of protein kinase A were colocalized in rat hip-pocampal neurons via AKAP79. Interestingly, AKAP79contained a domain homologous to FKBP, hypothe-sized to be the calcineurin binding domain. A syntheticpeptide based on this sequence was a noncompetitiveinhibitor of calcineurin activity (58). A subsequent study,however, suggests that AKAP79 interacts with calcineurinthrough a site distinct from the FKBP-homologous region(177).

Another potential calcineurin regulatory protein iscain/cabin 1, a 2,220-residue phosphoprotein isolated byyeast two-hybrid screens of either rat hippocampal ormouse T-cell cDNA libraries (224, 400). Cain/cabin 1 bindsto calcineurin and inhibits it in a noncompetitive fashion.The interaction between cain/cabin 1 and calcineurin wasdependent on protein kinase C activation, and over-expression inhibited the transcriptional activation ofthe interleukin-2 gene and prevented dephosphoryla-tion of the transcription factor NF-AT. Recently, cain/cabin 1 was found to regulate the transcription factorMEF2, itself regulated via calcineurin-dependent path-ways, by binding to MEF2 and sequestering it in an inac-tive state (469).

In an expression library screen searching for proteinsthat interact with the ubiquitously expressed Na1-H1 ex-changer NHE1, Lin and Barber (234) identified a novelprotein, CHP (see sect. IIB2D), that specifically boundNHE1 and was critical for growth factor stimulation ofexchange activity. Overexpression of CHP in Jurkat andHeLa cells resulted in inhibition of NF-AT nuclear trans-location and transcriptional activity that was hypothe-sized to be the result of calcineurin inhibition (235). In-deed, the phosphatase activity of immunoprecipitatedcalcineurin was inhibited 50% in cells overexpressingCHP, whereas in a reconstitution assay, the activity ofpurified calcineurin was inhibited nearly quantitatively ina dose-dependent fashion. These results indicate thatCHP could represent yet another member of this emerg-ing class of endogenous calcineurin regulators.

Recently, a protein of the African swine fever virus,A238L, was found to display immunosuppressive activityby inhibiting NF-AT-regulated gene transcription in vivo(277). A238L coimmunoprecipitated with calcineurin afterviral infection of Vero cells, and calcineurin phosphataseactivity was inhibited in cellular extracts from viral-in-fected cells. It was hypothesized that A238L may enablethe virus to evade host defense systems by preventingtranscriptional activation of genes important for host im-munity.

Although the classical mechanism for regulating cal-cineurin activity is via Ca21/calmodulin, it is intriguing to


speculate that these and possibly other proteins can in-teract with calcineurin to regulate subcellular targetingand/or activity toward specific substrates in novel yetundefined ways.

IV. CALCINEURIN STRUCTURE

A. A Dinuclear Metal-Binding

Phosphoesterase Motif

Before the availability of any structural data, Averilland colleagues (431, 432) predicted that the serine/threo-nine protein phosphatases were homologous to purpleacid phosphatases and therefore might contain an activesite dinuclear metal center. Their hypothesis was basedon a comparison of the primary sequences of serine/threonine protein phosphatases with human, porcine, andbovine purple acid phosphatases, enzymes which werealready well characterized and known to containdinuclear iron centers. Their prediction was correct, andthe authors were able to identify three of the metal li-gands.

With increasing sequence data available, severalgroups have completed comprehensive sequence align-ments of serine/threonine protein phosphatases and haveidentified a number of residues that are conserved in allmembers of this family (26, 132, 212, 244, 371, 486). Thesestudies identified a “phosphoesterase motif” (Fig. 5) thatis conserved not only in PP1, PP2A, and calcineurin,but in many other enzymes involved in the cleavage ofphosphoester bonds, including acid and alkaline phospha-tases, bacterial exonucleases, diadenosine tetraphos-phatase, 59-nucleotidase, phosphodiesterase, sphingomy-elin phosphodiesterase, an enzyme involved in RNAdebranching, and a phosphatase in the bacteriophage lgenome, l protein phosphatase (244).

Four of the residues in the phosphoesterase motif(bold letters, Fig. 5) are ligands to a dinuclear metalcofactor in PP1 and calcineurin. Thus it has been hypoth-esized that this motif provides a scaffold for an active sitedinuclear metal center in every member of the phosphoes-terase family (244, 351). Support for this hypothesis wasprovided in studies of l protein phosphatase which dem-onstrated a spin-coupled dinuclear metal binding site byspectroscopic means (272, 352), and in the recent deter-mination of the three-dimensional structure of E. coli

59-nucleotidase, a distant member of the metallophos-phatase superfamily that has an active site containing twoZn21 separated by 3.3 Å (211). The conserved phosphoes-terase motif suggests a common catalytic mechanism forenzymes involved in phosphotransfer reactions, a hypoth-esis that seems to be the case for at least two of these,calcineurin and l protein phosphatase (see below).

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

B. Three-Dimensional Structure

The three-dimensional structures of several enzymesin the metallophosphatase family have been solved. X-raystructures (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 59-nucleotidase from E. coli (1.7Å) (211) have been solved. In these structures, the phos-phoesterase motif described in the previous section isrepresented as a b-a-b-a-b scaffold for an active sitedinuclear metal center. The three b-strands of this motifform a parallel pleated sheet that is capped by interveninga-helices. Two metal ions are positioned at the apex ofthis fold forming a dinuclear metal center with 3.0–4.0 Åbetween metal ions, with four of the metal ligands pro-vided by residues in loops between b-sheets and a-heli-ces.

A ribbon diagram representing the X-ray structure ofphosphate-inhibited calcineurin, complexed with the im-munosuppressant drug complex FK506zFKBP, is shown inFigure 6. The overall structure of the catalytic subunit(shown in gray) is ellipsoidal and consists of a mixture ofa-helices and b-sheets. The metal ions of the dinuclearmetal center are obscured in this diagram by the orangevan der Waals spheres of phosphate that form a bridgebetween the two metal ions (see Fig. 10, below). Thecalcineurin B-binding domain (cf. Fig. 1) is evident in thisstructure as an a-helix that protrudes from the core of the

FIG. 5. The phosphoesterase motif as found in calcineurin andpurple acid phosphatase. The original motif was identified by Koonin(212) and modified by Zhuo et al. (486). Residues that have been iden-tified as metal ligands are shown in bold (for calcineurin Aa, theserepresent residues Asp-90, His-92, Asp-118, and Asn-150; cf. Figs. 7A and10). Conserved, nonligand residues also found in the active site (Asp-121, Arg-122, and His-151) are underlined. The phosphoesterase motif ofplant (200) and mammalian purple acid phosphatases (92, 185, 380) isalso shown (bold residues are represented as Asp-135, Asp-164, Y-167,and Asn-201, while the underlined residues are represented as Asp-169and His-202 in the kidney bean enzyme; cf. Figs. 7B and 11). The motifin the plant and mammalian purple acid phosphatases represents avariation of the phosphoesterase motif. PP1, protein phosphatase 1.


molecule, forming the binding site for the calcineurin Bsubunit (Fig. 6, yellow). Absent in this structure are thecalmodulin-binding and autoinhibitory domains, since atruncated form of calcineurin missing these domains wasthe source of protein for crystallization studies (124). In asubsequent study that determined the structure of theholoenzyme, it was found that the autoinhibitory domainfolds into an a-helix that binds to the substrate-bindingcleft of the catalytic domain, with one of the glutamateresidues forming hydrogen bonds to metal-coordinatedwater molecules (197). Interestingly, the calmodulin do-main was disordered in the X-ray structure, and therefore,our knowledge of how this domain interacts with theactive site and autoinhibitory domains to confer calmod-ulin-regulation remains rudimentary.

The calcineurin B subunit in Figure 6 is colored in

yellow and is seen forming a complex with calcineurin Avia the calcineurin B-binding helix. Four Ca21, shown asblue spheres, are bound in the EF-hand domains of the Bsubunit. The NH2-terminal myristoyl group of calcineurinB (red van der Waals spheres) is situated in a hydrophobiccleft between two amphipathic helices that are integralcomponents of two EF-hands (124). In contrast, the X-raystructure of the nonmyristoylated protein shows the firstfive residues of the NH2 terminus of calcineurin B asdisordered, suggesting increased mobility of the NH2 ter-minus relative to the acylated protein (197). Thus themyristoyl group appears to be anchored via multiple hy-drophobic contacts and may contribute to the overallstructural stability of the enzyme and may explain whythe acylated protein has increased thermal stability com-pared with the unmodified enzyme (182).

FIG. 6. Ribbon diagram showing the three-dimensional structure of calcineurin complexed with FK506zFK506binding 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 calcineurinB subunit is yellow, FKBP is green, and the immunosuppressant drug FK506 is shown in ball-and-stick fashion in gray.The four Ca21 of the B subunit are represented as blue spheres, and the myristoyl group of the B subunit is colored asred van der Waals spheres. The phosphate molecule bound to the active site metal ions of the A subunit is representedas orange van der Waals spheres.


C. Active Site Architecture

The dinuclear metal cofactor of calcineurin has beenmodeled as an Fe31-Zn21 cluster based on the presence ofnear-stoichiometric quantities of Fe31 and Zn21 (195,471), electron paramagnetic resonance (EPR) spectro-scopic experiments (471), and X-ray diffraction studieswhich show a dinuclear metal center separated by 3.14 Åin a coordination environment shown in Figure 7A (197).A similar coordination environment and metal-metal dis-tance is also found in PP1 (120), although in that enzyme,the metal ions could not be identified with certainty andwere therefore referred to as M1 and M2. In the X-raystructure of calcineurin, the Fe31 was modeled in the M1site largely based on a comparison to the Fe31-Zn21 activesite metal cofactor of kidney bean purple acid phospha-tase. In purple acid phosphatase, a tyrosine residue coor-dinates to the Fe31 (M1 site) and gives rise to a tyrosine-to-iron charge transfer band at 510–550 nm, responsiblefor the purple color of these enzymes (Fig. 7B) (223, 433).As can be seen in Figure 7, this tyrosine ligand is missingin calcineurin and is replaced by a histidine, thus explain-ing why calcineurin does not exhibit any appreciableabsorbance in the visible region of the optical spectrum

(471). The additional substitution of a histidine ligandwith a water molecule results in a net water-for-tyrosinesubstitution at the Fe31 site in calcineurin compared withpurple acid phosphatase. The coordination of the Zn21

(M2) provided by amino acid side chains in these enzymesis identical, with ligands provided by two histidines, anaspartic acid that bridges to the Fe31 and an asparagineresidue (compare Fig. 7, A and B). Figure 8 shows acomparison of the active sites of kidney bean purple acidphosphatase and calcineurin that illustrates the remark-able superposition of both metal ions and protein ligandsin these two enzymes.

In addition to coordination provided by protein sidechains, the metal ions in calcineurin and purple acidphosphatase have one or more solvent molecules as ad-ditional ligands. All enzymes show bridging and terminalsolvent molecules, but at least in one X-ray structure ofcalcineurin, an additional terminal solvent molecule wasmodeled into the coordination sphere of the M1 site asshown in Figure 7A (197). From the iron-oxygen bondlength (2.1 Å in purple acid phosphatase, Ref. 128), thebridging solvent molecule is most likely a hydroxo groupbased on distances obtained from model compounds(223).

FIG. 7. A: schematic of the active site of humancalcineurin based on the 2.1-Å resolution structure de-scribed by Kissinger et al. (197). B: schematic of theactive site of kidney bean purple acid phosphatasebased on the 2.65-Å resolution structure described byKlabunde et al. (201).


D. Metal Ion Requirements

It has been known for some time that divalent cationsin assay buffers are necessary to achieve the high activi-ties of purified calcineurin, with the best activators beingMn21 and Ni21 (194, 230, 315). Mn21 and Ni21 have beenshown to increase the activity of calcineurin in the ab-sence of Ca21/calmodulin (315), to prevent inactivation,or to restore activity following inactivation by exposure toCa21/calmodulin (194). Similar divalent metal ion effectshave been observed with PP1 and PP2A (3, 28, 43, 53, 54,94, 478) and with l-protein phosphatase (486). It is un-clear why these divalent metal ions are potent activators.One possibility is that Mn21 or Ni21 are native metal ionsthat become lost during purification. Pallen and Wang(317) incubated calcineurin with Ni21 or Mn21 andshowed that these metal ions are not dissociable by ex-tensive dialysis or gel filtration but can be released afterprolonged exposure to Ca21/calmodulin or by the use ofchelating reagents, both of which occur during a typicalpurification protocol. To address this issue, Rao and Wang

(341) used an anticalcineurin immunoaffinity matrix torapidly purify calcineurin from crude bovine brain extractin the absence of calmodulin. Analysis showed that theimmunoprecipitated calcineurin did not contain signifi-cant amounts of Ni21 and Mn21. Although no mentionwas made of the Fe content, ;1 equivalent of Zn21 wasfound in all samples, suggesting that Zn21 is an intrinsicmetal ion but not Ni21 or Mn21.

An alternative hypothesis to explain the mechanismof divalent metal ion activation is that prolonged expo-sure of calcineurin to Ca21/calmodulin promotes the re-lease of the intrinsic Fe and Zn metal ions and subsequentreplacement by Mn21 or Ni21. The most efficient methodto purify calcineurin utilizes Ca21/calmodulin affinitychromatography. It is possible that calmodulin bindingexposes the active site and thereby promotes loss of Feand/or Zn while calcineurin is adsorbed to the matrix.Alternatively, because elution of calcineurin from calmod-ulin-Sepharose requires the use of a metal chelator (e.g.,EDTA), it is possible that the Fe31 and/or Zn21 may beremoved during elution. Thus far, neither hypothesis has

FIG. 8. Comparison of the active sites of calcineurinand kidney bean purple acid phosphatase. An alignmentof the active sites of calcineurin and purple acid phos-phatase was performed based on the conserved His-Asp(calcineurin His-151/Asp-121) pair using the programQuanta (Quanta 97 Molecular Modeling Software Pack-age from Molecular Simulations, 1997). The X-ray crystalstructures used were Protein Data Bank entries 1AUIand 1KBP for human calcineurin (197) and kidney beanpurple acid phosphatase (201), respectively. The A sub-unit of calcineurin and a monomer form of purple acidphosphatase were used to generate the alignments. Onlythe metal ions and the active site residues shown in thefigure were used for the alignment. Calcineurin and pur-ple acid phosphatase residues are indicated in red andyellow, respectively. The Zn atom is green, and the Featom is purple. The numbers denote the following resi-dues: 1) His-92 in calcineurin, Tyr-167 in purple acidphosphatase; 2) Asp-121 in calcineurin, Asp-169 in pur-ple acid phosphatase; 3) His-151 in calcineurin, His-202in purple acid phosphatase; 4) Asn-150 in calcineurin,Asn-201 in purple acid phosphatase; 5) His-199 in cal-cineurin, His-286 in purple acid phosphatase; 6) His-281in calcineurin, His-323 in purple acid phosphatase; 7)His-325 in purple acid phosphatase; 8) Asp-90 in cal-cineurin, Asp-135 in purple acid phosphatase; 9) Asp-118in calcineurin, Asp-164 in purple acid phosphatase.


been rigorously tested. However, King and Huang (195)demonstrated that there was no correlation between theloss of enzymatic activity after calmodulin-dependent in-activation and the iron and zinc content. Both metal ionsremained tightly bound during prolonged exposure tocalmodulin. Nevertheless, additional studies have demon-strated that up to two equivalents each of Mn21 and Ni21

could bind to calcineurin (317, 482), a result consistentwith these metal ions occupying the sites of the dinuclearmetal cluster. In support of this was an EPR study follow-ing Mn21 binding to l-protein phosphatase which foundthat a dinuclear Mn21-Mn21 cluster was formed uponaddition of two equivalents of Mn21 to the apoenzyme(352). The situation with calcineurin is not as straightfor-ward due to the presence of calmodulin and the cal-cineurin B subunit, each which can provide additionaldivalent metal ion binding sites. Indeed, an EPR studyfollowing Mn21 binding to calcineurin in the presence ofcalmodulin demonstrated 10 Mn21 sites and attributed 4each to calmodulin and calcineurin B and 2 to the cata-lytic subunit (453).

Clearly further work is needed to understand the mech-anism whereby exogenous Ni21 and Mn21 activate cal-cineurin after prolonged exposure to Ca21/calmodulin invitro and whether or not this activation also occurs in vivo.

V. ENZYMATIC MECHANISM

A. Mechanism of Phosphoryl Group Transfer:

Evidence for Direct Transfer to Water

Much of the initial work on the mechanism of cal-cineurin focused on determining whether a phosphoen-zyme intermediate was formed during catalysis, sinceother phosphatases are known to proceed by this mech-anism (Fig. 9). For example, E. coli alkaline phosphatasecatalyzes phosphate ester hydrolysis by first transferringthe phosphoryl group to an active site serine residue to

form a transient phosphoenzyme intermediate (63, 64). Inthe next step, the enzyme is regenerated for anotherround of catalysis by hydrolysis of the phosphoenzymeintermediate. Alkaline phosphatase is a metalloenzymecontaining a Mg21 and a Zn-Zn dinuclear center reminis-cent of the dinuclear metal sites of the serine/threoninephosphatases and purple acid phosphatases (187). Aphosphoenzyme intermediate has also been demon-strated in the protein tyrosine phosphatases (52, 127,479), enzymes that function without active site metal ions.

Steady-state experiments of calcineurin carried outby Graves and colleagues (262) suggested that a phos-phoenzyme intermediate was not formed during catalysis.A linear relationship between log (V/K) and the pKa of theleaving group was observed for a set of four substrates,with a trend toward increasing velocity as the pKa of theleaving group decreased (262). This result is consistentwith a direct transfer to water without formation of aphosphoenzyme intermediate (Fig. 9). Additional experi-ments failed to demonstrate phosphotransferase activityin the presence of alternate nucleophiles, which also isconsistent with a direct transfer mechanism (262). In asubsequent study using p-NPP as substrate, product inhi-bition studies showed that both phosphate and the phenolwere competitive inhibitors of calcineurin (259). Theseinhibition patterns are consistent with a random uni-bimechanism. In contrast, an ordered uni-bi mechanism isexpected for a phosphoenzyme intermediate, since theproduct alcohol would be released before the phosphoen-zyme intermediate is hydrolyzed. Although these resultsare consistent with a direct transfer mechanism, they arenot conclusive, since they can also be interpreted toindicate a mechanism involving a transient phosphoen-zyme intermediate whose breakdown is not rate limiting.

The most definitive work that addressed whether aphosphoenzyme intermediate was formed during cataly-sis for the metallophosphatases was performed with bo-

FIG. 9. Phosphatase catalysis: phos-phoenzyme intermediate versus directtransfer mechanisms. Shown are twopossibilities by which phosphatases cat-alyze phosphoryl group transfer to water.Escherichia coli alkaline phosphataseand protein tyrosine phosphatases repre-sent two classes of phosphatases that uti-lize the phosphoenzyme intermediatemechanism. The metallophosphatasesappear to proceed by direct transfer ofthe phosphoryl group to a metal-coordi-nated water molecule, without the forma-tion of a phosphoenzyme intermediate.The “X” symbol represents an active nu-cleophile, e.g., a cysteine residue in theprotein tyrosine phosphatases and aserine residue in alkaline phosphatase.


vine spleen purple acid phosphatase. Using a chiral[18O,17O]phosphorothioate ester and carrying out the hy-drolysis in [16O]H2O, Knowles and co-workers (288) dem-onstrated that purple acid phosphatase carries out hydro-lysis with net inversion of configuration at the phosphoruscenter, thus indicating that the phosphoryl group is trans-ferred directly to water. Because of the similarity of theactive sites of calcineurin and PP1 to purple acid phos-phatases, it is thought that the catalytic mechanism of theserine/threonine phosphatases also proceeds by a similarmechanism.

B. Catalytic Role of the Dinuclear Metal Center

Several pieces of data indicate that the dinuclearmetal center is a key component of the active site ofcalcineurin. 1) As already mentioned, the dinuclear metalcenter has a ligand environment similar to purple acidphosphatases, enzymes which contain dinuclear Fe-Fe orFe-Zn centers previously demonstrated to be essential forcatalytic activity (223, 433). 2) Crystallographic (91, 124,201) and spectroscopic (80, 334, 416, 448, 449, 470) stud-ies indicate that the product of the reaction, phosphate,and product analogs such as tungstate, molybdate, andarsenate, coordinate the metal ions. 3) Redox titrations ofeither the Fe31-Zn21 or Fe31-Fe21 forms of calcineurinand purple acid phosphatase indicate a correlation be-tween enzyme activity and the oxidation state of thebound metal ions (8, 9, 18, 77, 470, 471).

The metal ions of the dinuclear center could functionin numerous ways to catalyze phosphate ester hydrolysis.The Lewis acidity of the metal ion(s) could serve toactivate a solvent molecule, a well-known mechanism inseveral metalloenzymes such as carbonic anhydrase(132a) and adenosine deaminase (459). A metal-activatedwater molecule has been proposed for purple acid phos-phatases (85, 432). Alternatively, a metal-coordinated sol-vent molecule could serve as a general acid to donate aproton to the leaving group, as has been proposed forinorganic pyrophosphatase (140, 355). In addition to arole in activation of the solvent nucleophile, the metalions in the serine/threonine phosphatases could be in-volved in other aspects of the catalytic mechanism. Metalcoordination of the phosphate ester could have severalpositive effects acting to accelerate hydrolysis. Neutral-ization of the negative charge on the oxygen atoms of thephosphate ester oxygen atoms would increase the elec-trophilicity of the phosphorus atom, making it more proneto nucleophilic attack. During P-O bond scission, themetal ions could stabilize the developing charge on theleaving group. However, another possible role for themetal ions could be to orient the substrate for in-lineattack. These are discussed in section VC in the context ofspecific active site residues and the effect that mutagen-esis of these residues has on catalytic efficiency.

C. Conserved Active Site Residues

In addition to the metal ions and their cognate pro-tein ligands, several conserved residues within 4–9 Å ofthe dinuclear metal cofactor are involved in catalysis. Oneof these is a histidine residue that is not a metal ligand butis within 5 Å of either metal ion. The conserved histidinein calcineurin, His-151, is also conserved in PP1 (His-125),purple acid phosphatases (His-202, kidney bean enzymenumbering), and l-protein phosphatase (His-76). It is rep-resented in the phosphoesterase motif as the underlinedresidue of Figure 5 (244). This histidine is hydrogenbonded to an aspartic acid that is also part of the phos-phoesterase motif, Asp-121 in calcineurin, Asp-95 in PP1,Asp-169 in kidney bean purple acid phosphatases, andAsp-52 in l-protein phosphatase. Two arginines, Arg-122and Arg-254, are also present in the active site of cal-cineurin. These arginines are also conserved in othermetallophosphatases and correspond to Arg-96 and Arg-221 in PP1 (120) and Arg-53 and possibly Arg-162 inl-protein phosphatase. Figure 10 depicts the active site ofcalcineurin in the phosphate-inhibited form showing theconserved nonligand residues. The corresponding resi-dues in kidney bean purple acid phosphatases are de-picted in Figure 11. In purple acid phosphatase, two his-tidines (His-295 and His-296) are substituted for twoarginines in the serine/threonine phosphatase family.

The importance of these residues and their potentialrole in catalysis has in most cases been addressed by

FIG. 10. Schematic of the active site of calcineurin complexed withproduct phosphate. Shown is the dinuclear metal center of calcineurin,along with the conserved His-Asp pair (His-151/Asp-121) and two argi-nine residues (Arg-122/Arg-254) that form a region of positive electro-static potential in the active site.


enzymatic and spectroscopic studies of enzymes in whichthese residues have been altered by site-directed mu-tagenesis. The interpretation of these data assumes thatmutagenesis alters only the physical-chemical features ofthe residue of interest and does not alter some otherrequisite structural feature, (e.g., tertiary structure, metalbinding, etc). A review of the primary literature indicatesthat this assumption has not always been defended byrigorous structural studies. The reader is therefore cau-tioned that the roles ascribed to some of these active siteresidues are still tenuous.

1. Role of the histidine/aspartate pair

in the metallophosphatases

Studies have shown that mutation of His-151 in cal-cineurin, or its analog in l-protein phosphatase, His-76,leads to significant reductions in enzyme activity (272,487). When its hydrogen-bonding partner (correspondingto Asp-121 in calcineurin) is mutated, a 101- to 103-folddecrease in the rate constant for catalysis (kcat) occurswith little effect on Km (161, 475, 487). One role consid-ered for this histidine has been as an active site nucleo-phile. As discussed above, the data argue against thissince a phosphoenzyme intermediate probably does notform during the catalytic cycle. Alternatively, this histi-dine may have a role in binding substrate, orienting thenucleophilic water molecule, and/or in acid/base cataly-sis. These possibilities are discussed in the followingsections.

A) ACID/BASE CATALYSIS. A series of kinetic studies wereundertaken to study the effect of mutating the conservedhistidine in calcineurin (His-151) and l-protein phospha-tase (His-76) (272, 283). In both mutant enzymes therewere significant reductions in enzyme activity but onlysmall effects on Km (272, 283). This was the case usingp-NPP and the [P]-RII peptide as substrates for cal-cineurin, and p-NPP and phenyl phosphate for l-proteinphosphatase. These substrates were chosen to explorethe role of this histidine as a proton donor. A phosphateester such as p-NPP has a better leaving group (pKa ofp-nitrophenol 5 7.2) compared with either phenyl phos-phate or a phosphoseryl peptide (pKa of phenol 5 10, pKa

serine side chain -OH ;14). Therefore, if protonation ofthe leaving group occurs in the transition state, hydrolysisof p-NPP will require less catalytic assistance than eitherphenyl phosphate or a phosphoseryl peptide such as [P]-RII peptide. As a result, one would expect larger effects onrelative kcat (wild-type kcat/mutant kcat) for substrateswith poorer leaving groups. The results showed less thana threefold difference in relative kcat for calcineurin usingp-NPP versus the [P]-RII peptide, despite a difference inpKa for the respective leaving groups of .106 (272). Withl-protein phosphatase, mutagenesis of His-76 to Asn(H76N) resulted in the same 500- to 600-fold reduction inkcat using either p-NPP or phenyl phosphate as substrates(leaving group pKa difference .103) (272). Ideally, morethan two substrates with varying pKa values should beused in this type of Brønsted analysis.

A useful method to determine if a residue is acting asan acid or base in catalysis is to follow the pH dependenceof the rate for wild-type and mutant enzymes. Often onearm of the usual bell-shaped dependence curve is missingfor the mutant if the residue is acting as a general acid orbase. Using p-NPP as a substrate, pH dependence studiesfor wild-type and H76N l-protein phosphatase were per-formed (156). The mutant enzyme had a lower catalyticrate at every pH but still exhibited a bell-shaped pH curve.However, kinetic data were difficult to obtain at low pHdue to substrate inhibition that necessitated higher metalion concentrations. In summary, the difficulties encoun-tered were such that it was not possible to concludewhether this histidine serves as a general acid in catalysis.It was concluded that this residue may function moredecisively as a general acid in reactions of phosphopro-tein substrates that have a much poorer leaving group. Itis noteworthy that the pH optimum for the mutant ap-peared to be shifted to pH 7.0, compared with 7.8 for thewild-type enzyme. Also the Km for substrate increased athigh pH for the native enzyme to values that were similarto those of the mutant enzyme at all pH values, indicatingthat protonation of this residue may assist in substratebinding.

B) KINETIC ISOTOPE EFFECTS. Kinetic isotope effect studieshave been performed with both calcineurin (150, 260, 261)

FIG. 11. Schematic of the active site of purple acid phosphatasecomplexed with product phosphate. Shown is the active site of purpleacid phosphatases with the conserved His-Asp pair (His-202/Asp-169)and two histidines (His-296/His-295) thought to be important for theenzyme’s mechanism (91, 124).


and l-protein phosphatase (156) to study the chemistry ofthe transition state. In the case of calcineurin, the use ofD2O to measure a solvent isotope effect found no effecton kcat but a modest isotope effect (1.35) on kcat/Km (260).The lack of a solvent isotope effect on kcat may be due tohaving some other step in the reaction mechanism otherthan the proton transfer step be rate limiting, a situationthat was confirmed in a subsequent study that used heavyatom isotopes of p-NPP (150). In the latter study it wasshown that P-O bond cleavage was partially rate limitingat the pH optimum, and therefore, the isotope effectswere suppressed, a situation which could be improvedupon raising the pH from 7.0 to 8.5. For the l-proteinphosphatase reaction, P-O bond cleavage was fully ratelimiting, and the measured isotope effects represented theintrinsic isotope effects on the bond-breaking step of thecatalytic mechanism. With both enzymes, the data indi-cate that the substrate of the reaction is the p-NPP dian-ion, the predominant form at neutral pH (pKa2 5 4.96).The isotope effect studies also provide evidence for adissociative mechanism, in which the transition state hassubstantial P-O bond cleavage before bond formation tothe nucleophilic water molecule. A dissociative mecha-nism has also been observed for the protein tyrosinephosphatases (148, 151, 152) and for uncatalyzed reac-tions in solution (149). This result is important because itdemonstrates that the metal ions do not change the tran-sition state of the phosphoryl transfer reaction to becomemore associative, a hypothesis that was previously for-warded based on the idea that metal ions could stabilizethe extra negative charge in the transition state that re-sults from bond formation to the nucleophile (139). In-stead, the transition state of the phosphoryl group looksvery much the same as in the solution reaction.

The 15N kinetic isotope effects indicate that in cal-cineurin and wild-type l-protein phosphatase, there issubstantial charge neutralization of the leaving group inthe transition state (150, 156). In contrast, the magnitudeof this charge increases when His-76 of l-protein phos-phatase is mutated to asparagine. This result suggests thatthis histidine may be protonating the leaving group in thetransition state; its absence would lead to a greater chargeaccumulation on the leaving group compared with thewild-type enzyme. Interestingly, the magnitude of nega-tive charge on the leaving group is smaller than expectedfor the mutant enzyme compared with comparable stud-ies with protein tyrosine phosphatases that have the ac-tive site general acid removed by mutagenesis. Indeed, themagnitude of the isotope effect in the mutant is signifi-cantly less than would result from a full negative chargeon the departing p-nitrophenol product. One explanationthat was forwarded is that the metal ions may participatein stabilizing the transition state of the reaction by neu-tralizing the developing negative charge on the leavinggroup.

C) POTENTIAL ROLE IN SUBSTRATE BINDING. Another possiblerole of His-76/His-151 could be to assist in substrate bind-ing. With l-protein phosphatase, the Km for p-NPP in-creased from 1 to 70 mM with increasing pH (156). Incontrast, the Km for p-NPP in the l-PP(H76N) mutant washigher at low pH compared with wild type. As the pH wasincreased to neutral pH and greater, the Km values forboth enzymes became similar. It is possible that at acidicand neutral pH, where the histidine would be protonated,this residue assists in substrate binding via electrostaticinteractions. For example, a proton on His-76 may form ahydrogen bond with one of the substrate oxygen atoms ofthe phosphorylated substrate. In X-ray structures of otherserine/threonine phosphatases with bound inhibitors,phosphate or tungstate, this histidine is within hydrogenbonding distance of the most solvent exposed oxygenatom of the inhibitor (91, 124). A similar orientation isobserved with purple acid phosphatases with phosphateor tungstate bound (128, 201, 425). This type of electro-static interaction will not affect the isotope effect on18(V/K)nonbridge. However, if the substrate were actuallyprotonated in the transition state, this would have beenrevealed in the 18(V/K)nonbridge isotope effect; this was notobserved (156). It is not clear if His-76 really does partic-ipate in substrate binding, since at basic pH it would bedeprotonated. The pH optimum for the wild-type enzymeis ;8.0, where both the wild-type enzyme and mutanthave similar Km values for p-NPP.

D) GENERAL BASE CATALYSIS. Another possibility for His-151/His-76 would be to act as a general base to deproto-nate the metal-bound water molecule that is the nucleo-phile in the mechanism. In one X-ray structure ofcalcineurin, the Ne of His-151 is hydrogen-bonded to oneof two terminal solvent molecules coordinated to the Feion. Considering also Asp-121, which is hydrogen bondedto His-151, the interaction of this histidine/aspartate pairwith the metal-coordinated solvent molecule is analogousto the Asp-His-Ser catalytic triad of serine proteases,where an Asp/His pair is important for interacting withthe nucleophilic serine residue. The “Asp-His-HO-metal”motif in the serine/threonine phosphatases can bethought of as a catalytic tetrad, with the metal ion servingto lower the pKa of the nucleophilic water molecule whilethe Asp-His functions as a catalytic base to assist inhydroxide formation. Histidines in other metalloproteinsare thought to perform a similar role by acting as a baseto deprotonate a metal-bound water molecule or by as-sisting in stabilizing a metal-bound hydroxyl group. Forexample, His-372 in E. coli alkaline phosphatase forms ahydrogen bond with Asp-327 (a bidentate Zn ligand) and isthought to lower the pKa of a zinc-bound water molecule(464). Mutagenesis studies of Asp-327 to asparagine resultin increased enzyme activity, since the negative charge ofthe hydroxyl group is more stable due to the loss of theaspartate side chain and replacement with a neutral res-


idue. However, this comes at the expense of Zn bindingsince a carboxylate is a better metal ligand than a carbox-amide, and thus a higher concentration of Zn in the assayis needed to achieve this increased activity. Examples ofother enzymes where a histidine is postulated to depro-tonate a metal-coordinated water molecule include His-320 in Klebsiella aerogenes urease (172, 321), His-231 inthermolysin (27), and His-89 in Serratia nuclease (109).

E) ORIENTATION OF THE NUCLEOPHILIC HYDROXIDE SOLVENT MOLE-

CULE. Another potential role of His-76/His-151 could be toposition the metal-coordinated hydroxide for optimal in-line attack of substrate, a mechanism analogous to theconcept of “orbital steering” proposed by Storm and Kosh-land (393) almost 30 years ago to provide an explanationfor the great rate accelerations seen in enzyme catalysis.The theory of orbital steering postulates that a majorfactor in catalytic enhancement is that an enzyme ar-ranges the reaction trajectory to optimize the overlap ofattractive (bonding) orbitals and minimize the overlap ofrepulsive (nonbonding) orbitals. This concept has beendebated and contested by numerous groups (39, 174, 270).Nevertheless, in a recent study of isocitrate dehydroge-nase by Koshland and co-workers (273), small structuralperturbations in isocitrate dehydrogenase were created toevaluate the contribution of precise substrate alignmentto the catalytic rate of an enzyme. They found that smallchanges in the orientation of substrates had large effectson reaction velocity (103- to 105-fold decreases). Theirmain conclusion was that orbital steering is an importantcontribution to the catalytic power of enzymes.

A role in orientation of metal-bound water was pro-posed for His-238 of murine adenosine deaminase (459)and is possible for the conserved histidine in the serine/threonine phosphatases. Interestingly, small perturba-tions in the EPR spectra of the dinuclear metal center inCN(H151Q) and l-PP(H76N) enzymes were observedcompared with the wild-type enzymes (272), indicatingsubtle perturbations to the geometry of the dinuclearmetal center. X-ray structures of these mutants would bevaluable to obtain better information on whether sub-strate orientation may be affected.

2. Role of arginines in the active site

As shown in Figure 10, there are two arginine resi-dues in the active site of calcineurin, Arg-122 and Arg-254.These arginine residues are conserved in other phospha-tases. Mutagenesis of Arg-122 in calcineurin or its ho-mologs in other phosphatases (Arg-96 in PP1 and Arg-53in l-protein phosphatase) resulted in 102- to 103-fold de-creases in kcat and only slight changes in Km (161, 283,475, 487). An exception to this was a 20-fold increase inKm when Arg-53 in l-protein phosphatase was mutated toan alanine and assayed in the presence of Ni21 (487).However, when this mutant was assayed in the presence

of Mn21, there were no significant changes in Km. Site-directed mutagenesis studies of Arg-254 in calcineurin orArg-221 in PP1 resulted in an 200-fold reduction in kcat,while values for Km increased 2- to 10-fold (161, 283). Inthe X-ray structures of wild-type calcineurin and PP1, theguanidinium groups of these arginine residues form saltbridges with the oxygen atoms of bound phosphate ortungstate and stabilize the anion inhibitor (91, 120, 124,197). The purpose of these arginine residues may be toprovide electrostatic stabilization for binding the nega-tively charged phosphate ester. In addition, they may helpneutralize developing negative charge in the transitionstate. Because the mutation of Arg-122 led to greaterdecreases in kcat than mutation of Arg-254, it may be thatArg-122 has a more important role in catalysis, perhapsinvolving stabilization of the transition state. To note, inthe 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 beensuggested that these substitutions might explain the lowerpH optimum of purple acid phosphatases (201). Krebs andcolleagues (201) have hypothesized that one of thesehistidines may be the active site residue with an apparentpKa of 6.9 that produces the basic pH portion of thebell-shaped pH/kinetic profile. In an analogous manner, itis possible that Arg-53 in l-protein phosphatase producesthe basic pH arm observed in the pH dependence studies(156). In mammalian purple acid phosphatases, there is ahistidine residue (His-195) corresponding to His-296 ofkidney bean purple acid phosphatase, but Glu-194 substi-tutes for His-295 in the mammalian enzyme and is ori-ented away from the phosphate in the oxidized enzyme-phosphate complex.

D. A Model for the Calcineurin

Catalytic Mechanism

Taking into account the available biochemical andchemical data, we propose a model for the catalytic mech-anism of calcineurin and other members of this family(Fig. 12). The first step of the mechanism involves asso-ciation of the phosphate monoester, as the dianion, withthe enzyme (Fig. 12A). In this step, neutralization of neg-ative charge by the metal ions may occur. X-ray structuresof calcineurin and PP1 show phosphate and other anionicproduct inhibitors coordinated to both metal ions, infer-ring that the substrate phosphoryl group might also coordi-nate to one or both metal ions at some point during cataly-sis. In addition, the conserved arginines, Arg-122 and Arg-254, may also play a role in binding substrate andneutralizing charge by forming hydrogen bonds with theoxygen atoms of the phosphoryl group. The intermediate inFigure 12A shows the Zn atom and the two conserved argi-nines assisting in substrate binding/neutralization of charge.


This neutralization of charge would make the substratemore electrophilic and ready for attack by a nucleophile.

The interaction between the metal-bound water mol-ecule and the conserved histidine His-151 in calcineurin isalso depicted in Figure 12. In the first step of this mech-anism (Fig. 12A), His-151 is functioning as a general baseto remove the proton from the metal-bound water mole-cule. Alternatively, it may orient the solvent nucleophilefor optimal nucleophilic attack of the phosphate ester.

Kinetic isotope studies of calcineurin and l-proteinphosphatase show that the transition state of the reactionis dissociative. A dissociative transition state is repre-sented in Figure 12B, where bond cleavage to the leavinggroup has occurred before bond formation to the nucleo-phile. The phosphoryl group in a dissociative mechanismresembles the metaphosphate anion (147).

A metal-bound water hydroxide coordinated to Fe31

is shown as the attacking nucleophile in Figure 12B, withthe Fe31 functioning as a Lewis acid to lower the pKa ofthe water molecule. Extensive redox studies by Yu andco-workers (470, 471) have demonstrated a requirementfor Fe31 by calcineurin and a loss of activity upon reduc-tion to Fe21, a result consistent with a decreased Lewisacidity of Fe21 versus Fe31.

P-O bond scission in the transition state results in asignificant negative charge on the leaving group. Neutral-ization of the charge by protonation (general acid cataly-sis) or coordination to a metal ion would lower the energyof the transition state and increase the rate of the reac-tion. Kinetic isotope studies indicate that considerablecharge is neutralized in the transition states of calcineurinand l-protein phosphatase. His-151 may play a role in thischarge neutralization (Fig. 12B). It is also possible thatone of the metal ions (e.g., Zn21 in Fig. 12B) neutralizes

FIG. 12. Proposed mechanism for calcineurin. The mechanism shown in this figure poses His-151 acting as a generalbase and a general acid. A: substrate binds to the active site, and His-151 deprotonates a Fe31-bound water molecule toform hydroxide. B: bond cleavage to the leaving group occurs to a greater extent than bond formation to the hydroxylgroup in the transition state since the transition states for both l-protein phosphatase and calcineurin were shown to bedissociative. 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 withphosphate bridging the two metal ions. D: a water molecule displaces bound phosphate, and the enzyme is ready foranother 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 charge to the leaving group by coordination. Anotherpossibility is that a metal-bound solvent molecule acts asa general acid as has been proposed for the mechanism ofinorganic pyrophosphatase (140, 355). In addition, thetwo conserved arginines in the active site may also beimportant for charge neutralization and transition statestabilization. After bond cleavage and proton transfer tothe leaving group occur, the result is the product-inhibitedstate that has a molecule of orthophosphate bridging thetwo metal ions of the dinuclear center (Fig. 12C). Evi-dence for this intermediate is obtained in the X-ray struc-ture of the product-inhibited enzyme which shows phos-phate coordinated to both metal ions as depicted (124).An identical coordination is observed in phosphate andtungstate complexes of PP1 (91, 395) and the phosphatecomplex of purple acid phosphatase (128, 201). Phos-phate release, perhaps by exchange with a solvent mole-cule, regenerates the enzyme for another turnover.

VI. REGULATION

The classical mechanism by which calcineurin is reg-ulated in vivo is via changes in intracellular Ca21. Thus, ina resting cell where [Ca21] is low, calcineurin is unable tobind calmodulin, and the enzyme exists in an inactiveform. In signaling pathways that lead to a rise in intracel-lular Ca21, Ca21 binding to calmodulin results in a con-formational change, thereby allowing it to bind to cal-cineurin and activate its phosphatase activity (203). Ca21

binding to calcineurin B also appears to play a role (389).Calcineurin activity has also been shown to be af-

fected by phospholipids, with either activation or inhibi-tion resulting depending on the phospholipid and sub-strate investigated (163, 329, 330). Recently, recombinantDictyostelium calcineurin has been shown to be acti-vated by arachidonic acid and unsaturated, long-chainfatty acids (184). These effects may be physiologicallysignificant given the fact that calcineurin is found associ-ated with membranes during fractionation.

Recently, an additional mechanism for regulating cal-cineurin involving redox reactions of active site metalions has been considered (350, 447). Previous studiesdemonstrated that calcineurin is susceptible to redox reg-ulation in vitro (470, 471), a process that may also occurin vivo. Wang et al. (447) found that superoxide dismutaseprotects calcineurin from inactivation and hypothesizedthat this might occur by preventing oxidation of activesite metals ions. Recently, a number of groups have beguntesting this hypothesis and have provided evidence thatcalcineurin activity can be affected by extracellular oxi-dants, in particular, H2O2 (45, 111, 345). Thus exposure ofcells to micromolar concentrations of H2O2 results ininhibition of NF-AT (111, 345) or NFkB-mediated pro-cesses (45) and appears to be mediated by calcineurin.

The mechanism for this regulation may reside in theredox-active Fe31 of the active site dinuclear metal cen-ter, which can toggle between reduced (Fe21) and oxi-dized (Fe31) states. Indeed, redox titrations by Yu andco-workers (470, 471) previously demonstrated that themixed-valence oxidation state, either Fe31-Zn21 or Fe31-Fe21, is required for enzyme activity; reduction to theFe21-M21 (M 5 Zn, Fe) state led to loss of activity (470,471). At first glance, the in vivo results, which suggest thatoxidation (i.e., treatment with H2O2) results in inactiva-tion, appear contrary to the in vitro results. One hypoth-esis that has been forwarded to reconcile this discrepancyis that calcineurin may exist in different forms containingeither Fe-Zn or Fe-Fe dinuclear metal centers. Becausethe Fe31-Fe21 form of calcineurin can lose activity byoxidation to the Fe31-Fe31 state, it may be that cal-cineurin exists in oxidation-sensitive (Fe31-Fe21) and ox-idation-inert (Fe31-Zn21) states in vivo.

Further work is obviously necessary to determinewhether calcineurin is a specific mediator for changes inthe redox state of the cytosol. If the dinuclear center ofcalcineurin has a standard redox potential near the phys-iological state of the cytosol, a mechanism whereby smallalterations in the redox state could mediate changes inenzyme activity would be highly tenable.

We apologize for any oversight that has resulted in a keyreference on calcineurin structure/function being excluded fromthe bibliography. We gratefully acknowledge Drs. Robert Abra-ham, Ian Armitage, Howard Brockman, Alvan Hengge, and RonVictor for their collaborations. Also acknowledged are TimothyBorn, Alice Haddy, Michael Kennedy, Nicholas Reiter, TiffanyReiter, Robert Sikkink, Selene Swanson, Smilja Todorovic,Daniel White, Janet Yao, and Lian Yu for their significant con-tributions during their tenure in F. Rusnak’s laboratory. Wethank Dr. Elizabeth Kurian for assistance with the programQuanta. We are indebted to the editorial and review staff ofPhysiological Reviews, in particular Dr. Susan Hamilton, forsupport, helpful suggestions, and the opportunity to write thisreview.

Financial support from National Institute of General Med-ical Sciences Grant GM-46865 and the Mayo Clinic Eagles Can-cer Fund is noted.

Address for reprint requests and other correspondence: F.Rusnak, Sect. of Hematology Research, Dept. of Biochemistryand Molecular Biology, Mayo Clinic, 200 First St. SW, Rochester,MN 55905 (E-mail: [email protected]).

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