MINIREVIEW
Regulation of the Calmodulin-stimulated Protein Phosphatase, Calcineurin*

Claude B. KleeDagger , Hao Ren, and Xutong Wang

From the Laboratory of Biochemistry, NCI, National Institutes of Health, Bethesda, Maryland 20892

    INTRODUCTION
Top
Introduction
References

The role of protein phosphatases in the regulation of cellular processes is now well established (1, 2). Calcineurin (also called protein phosphatase 2B), a major calmodulin-binding protein in brain and the only serine/threonine protein phosphatase under the control of Ca2+/calmodulin, plays a critical role in the coupling of Ca2+ signals to cellular responses (3-6). Its stimulation by the multifunctional protein, calmodulin, ensures the coordinated regulation of its protein phosphatase activity with the activities of the many other enzymes, including a large number of protein kinases, under Ca2+ and calmodulin control. Despite its special abundance in neural tissues, calcineurin is broadly distributed, and its structure is highly conserved from yeast to man (6). Its resistance to the endogenous phosphatase inhibitor 1 and inhibitor 2 and to the potent inhibitors of protein phosphatase 1 and 2A, okadaic acid, calyculin, and microcystin (1, 2) made it difficult to identify its functions until it was identified as the target of the immunosuppressive drugs, FK506 and cyclosporin A (CsA).1 Calcineurin was thus shown to play an essential role in T cell activation (7). The demonstration that FK506 and CsA, when bound to their respective binding proteins, FKBP12 and cyclophilin A, are specific inhibitors of calcineurin provided the tools needed to reveal its many other roles in the transduction of Ca2+ signals (8). Its calmodulin dependence distinguishes it from two other known Ca2+-regulated protein phosphatases, the insulin-sensitive pyruvate dehydrogenase phosphatase of mitochondria (9) and a family of protein phosphatases homologous to the product of the Drosophila retinal degeneration C (rdgC) gene (10-12).

    Substrate Specificity and Mechanism of Action

Calcineurin has a relatively narrow substrate specificity. Phosphoproteins listed in Table I are preferentially dephosphorylated by calcineurin whereas others such as casein, synapsin 1, and calmodulin kinase II are dephosphorylated at much slower rates or not at all (5). Other potentially physiological substrates, whose kinetic characteristics have not been determined, include NO synthase, a GTPase involved in endocytosis (dynamin, previously called dephosphin), the transcription factor Elk-1, and the heat shock protein, hsp25 (13-16). The substrate specificity of calcineurin is not due only to a specific sequence but rather is determined by both primary and higher order structural features (17, 18). The phosphorylation-independent tight binding of substrates, such as described for the transcription factor NF-ATp (nuclear factor-activated T cells), may allow calcineurin to dephosphorylate proteins whose intracellular concentration is very low (19, 20). Calcineurin also dephosphorylates phosphotyrosine, but the Kcat, except when determined in the presence of Ni2+, is 2 orders of magnitude lower than that for phosphoserine (Table I).

                              
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Table I
Substrate specificity of calcineurin
Except when indicated kinetic constants were measured in the presence of Mn2+ or Mg2+.

The synthetic peptide corresponding to residues 81-99 of the RII subunit of cAMP-dependent protein kinase (Table I) is most commonly used to measure calcineurin phosphatase activity (17). Because it is a poor substrate for protein phosphatases 1, 2A, and 2C it is well suited to quantitate the Ca2+/calmodulin-dependent, metal-independent, okadaic acid-insensitive calcineurin activity in crude tissue extracts provided that the incubation time is reduced to 1-2 min to minimize Ca2+-dependent calcineurin inactivation (21-24). The conveniently measurable p-nitrophenyl-phosphatase activity has been employed to study its catalytic mechanism and to propose a catalysis involving the protonation of the phosphoester bond by a metal-activated water molecule followed by the cleavage of the bond by a second metal-activated water molecule, without the formation of a covalent intermediate (25). This mechanism is consistent with the metal requirement for calcineurin activity (5), the identification of calcineurin as an iron-zinc enzyme (26), and the demonstration that calcineurin contains a binuclear [Fe3+-Zn2+] metal center (27). In the recently published crystal structures of calcineurin (28, 29), these two metal ions are modeled on the structure of the [Fe3+-Zn2+] kidney bean purple acid phosphatase. The high specific activity of calcineurin in crude extracts in the absence of added metals suggests that the crude enzyme has retained its natural cofactors (22). Inactivation of crude calcineurin by the superoxide anion and its protection and reactivation by ascorbate strongly suggest that reduced iron is required for activity (23).

    Subunit Structure and Isoforms

Regardless of its source, calcineurin is always a heterodimer of a 58-64-kDa catalytic and calmodulin-binding subunit, calcineurin A, tightly bound (even in the presence of only nanomolar concentrations of Ca2+) to a regulatory, 19-kDa Ca2+-binding regulatory subunit, calcineurin B (5). This two-subunit structure, unique among the protein phosphatases, is conserved from yeast to man and is essential for activity. Also highly conserved are the amino acid sequences of the catalytic and regulatory domains of calcineurin A isoforms from different organisms (2, 6). The primary structure of the alpha , beta , and gamma  isoforms of mammalian calcineurin A, products of three different genes,2 is shown in Fig. 1. With the exception of variable N- and C-terminal tails, whose functions are not known, the three enzymes exhibit 83-89% identity over the remaining 90% of their sequence. An N-terminal polyproline motif is a conserved feature of the beta  isoform, whereas several additional basic residues in the C-terminal tail are responsible for the high pI (7.1) of the testis-specific gamma -isoform, as opposed to pIs of 5.6 and 5.8 for the neural alpha  and the broadly distributed beta  isoforms (5, 6, 30, 31).


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Fig. 1.   Functional domain organization of calcineurin A. A, schematic representation of the three mammalian isoforms of calcineurin A. The variable regions and 10-amino acid insert, resulting from alternative splicing, in the alpha  and beta  isoforms of mammalian calcineurin A are shown in black. B, extended representation of the regulatory domain; the amino acid sequences of the calcineurin B-binding helix, the calmodulin-binding domain, and the autoinhibitory peptide are boxed. The numbering of the amino acids is that of calcineurin Aalpha . Residues critical for interaction with cyclophilin and FKBP are represented by white on black letters.

Calcineurin B is a highly conserved protein originally identified as an "EF-hand" Ca2+-binding protein on the basis of its amino acid sequence (32). Its dumbbell structure, determined by multidimensional NMR, is similar to that of calmodulin with two lobes, each composed of two adjacent Ca2+-binding loops connected by a flexible helix linker (33). As predicted from its sequence, it binds 4 mol of Ca2+, one with high affinity (kd < 10-7 M) and three with affinities in the micromolar range (34). Equally conserved from yeast to man is the myristoylation of the N-terminal glycine (35, 36). There are two mammalian isoforms of calcineurin B,2 CNB1 originally found associated with calcineurin Aalpha and beta  and CNB2, which is expressed only in testis; only one form has been reported in fruit flies and the budding yeast (6).

    Functional Domain Organization and Crystal Structure

The highly conserved multidomain structure of calcineurin A, illustrated in Fig. 1, was first revealed by limited proteolysis (37). The catalytic domain (residues 70-328 of calcineurin Aalpha ), followed by the calcineurin B-binding domain localized by site-directed mutagenesis and binding of synthetic peptides to calcineurin B between residues 333 and 390, is resistant to proteolysis (37-43). These domains, still associated with calcineurin B, are often referred to as the Ca2+-independent form of calcineurin. The enzymatic activity of calcineurin is repressed in the native protein, but it becomes fully active when severed by proteases from the regulatory domain (residues 390-521). The regulatory domain, readily susceptible to proteolysis in the absence of calmodulin, contains two subdomains: a calmodulin-binding and an autoinhibitory subdomain (37, 44, 45).

The crystal structures of the recombinant alpha  isoform of human calcineurin and of its complex with FKBP12-FK5063 (29) and that of the complex with FKBP12-FK506 (28) of the proteolytic fragment of bovine calcineurin, lacking the regulatory domain and the N-terminal 16 residues, have been determined at 2.1, 3.5, and 2.5 Å, respectively. With the exception of the N-terminal residues 1-16 and the regulatory domain of calcineurin A, missing in the bovine protein, the crystal structure of the Ca2+-saturated form of the truncated bovine calcineurin shown in Fig. 2 is similar to that of the full-length recombinant protein. The catalytic domain, similar to that of protein phosphatase 1 (46), consists of a sandwich of a sheet of six beta  strands covered by three alpha  helices and three beta  strands and a sheet of five beta  strands covered by an all helical structure. The two metal ions, iron and zinc, bound to residues provided by the two faces of the beta sandwich, define the catalytic center. The last beta  sheet extends into a five-turn amphipathic alpha  helix (residues 350-370) whose top face, completely non-polar, is covered by a 33-Å groove formed by the N- and C-terminal lobes and the C-terminal strand of calcineurin B. The contacts between the two subunits are in good agreement with the regions of calcineurin B involved in the interaction with calcineurin A (linkers between helix 1 and the Ca2+-binding loop 1, Ca2+-binding loops 3 and 4, the central helix linker, and the C-terminal tail) determined in solution (40, 41). Interaction of residues 14-23 of calcineurin A with the C-terminal lobe of calcineurin B may provide the additional binding energy to account for the very high affinity of calcineurin B for calcineurin A (kd < 10-13 M)4 as opposed to the relatively low affinity of the calcineurin B-binding peptides of calcineurin A for calcineurin B (41, 43). In the bovine protein, myristic acid, covalently linked to the N-terminal glycine of calcineurin B, lies parallel to the hydrophobic face of the N-terminal helix of calcineurin B whereas the non-myristoylated N terminus of the recombinant protein is disordered. This perfectly conserved post-translational modification of calcineurin B, apparently not involved in membrane association, is not required for activity but may serve as a stabilizing structural element (47, 48). In the full-length protein, with the exception of two short alpha helices corresponding to the inhibitory domain that block the catalytic center, the regulatory domain is not visible in the electron density map (29). The disordered structure of this domain is consistent with its extreme sensitivity to proteolytic attack (37).


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Fig. 2.   Ribbon representation of the crystal structure of truncated calcineurin complexed with FKBP12-FK506. Calcineurin A is shown in red and calcineurin B in purple with myristic acid covalently linked to the N-terminal glycine shown in pink. Iron and zinc in the active site are shown as yellow and green spheres respectively, and the bound phosphate is shown in purple. The four Ca2+ in the calcineurin B sites are shown as pink spheres. FKBP12 is shown in green, and FK506 (yellow) is shown in ball and stick representation (Protein Data Bank code 1TCO (28)).

The polar bottom face of the calcineurin B-binding helix of calcineurin A, exposed to solvent, constitutes, together with calcineurin B, the binding domain of the FK506-FKBP12 complex. FKBP12 interacts with calcineurin B, the catalytic and the calcineurin B-binding domain of calcineurin A, whereas the interface of the calcineurin B-binding domain of calcineurin A and calcineurin B forms the binding site of FK506 (Fig. 2). Two-thirds of the surface contact between FKBP12-FK506 and calcineurin B comes from the latch region identified as the major site of interaction of calcineurin B with cyclophilin-CsA (40). This latch region formed by calcineurin B upon binding to calcineurin A may be recognized by each of the two immunosuppressive complexes, explaining their competitive binding to calcineurin (7). Thus, the conserved structural features of calcineurin are responsible for the unique ability of calcineurin to interact specifically with two classes of immunosuppressive drugs, CsA and FK506, complexed with their respective binding proteins (as reviewed in Ref. 49).

    Calcium Regulation

The Ca2+ dependence of the phosphatase activity of calcineurin is controlled by two structurally similar but functionally different Ca2+-binding proteins, calmodulin and calcineurin B (5). At less than 10-7 M Ca2+, calcineurin B, with its high affinity site occupied, remains bound to calcineurin A, but the enzyme is inactive. Occupancy of the low affinity sites (kd between 0.5 and 1 µM) results in a small activation. The basal activity is stimulated more than 20-fold by the addition of an equimolar amount of calmodulin and is strictly the result of an increased Vmax. Consistent with the fact that activation is the result of the Ca2+-dependent binding of calmodulin to calcineurin, the concentration of Ca2+ needed for activation decreases with increasing concentrations of calmodulin, and the calmodulin concentration needed for activation decreases with increasing Ca2+ concentrations (50). The highly cooperative Ca2+ dependence of the calmodulin stimulation of calcineurin (Hill coefficient of 2.5-3) allows the enzyme to respond to narrow Ca2+ thresholds following cell stimulation. As with most calmodulin-regulated enzymes, the mechanism of calmodulin activation is believed to involve binding to the calmodulin-binding domain, resulting in a displacement of an autoinhibitory domain (5, 51). The flexible structure of the calmodulin-binding domain revealed in the crystal structure of calcineurin and the blocking of the catalytic center by the autoinhibitory domain is compatible with this mechanism, but definitive proof of this mechanism depends on the elucidation of the structure of the calcineurin-calmodulin complex.

The displacement of the inhibitory domain upon calmodulin binding can also explain the role of calmodulin in the oxidative inactivation of calcineurin. In crude tissue extracts, calcineurin exhibits a high phosphatase activity that is almost completely dependent on calmodulin and does not depend on added metals for activity but is subject to a time- and Ca2+/calmodulin-dependent inactivation facilitated by small heat-stable inactivators (22). The search for factors responsible for the high phosphatase activity and instability of crude calcineurin led to the finding that, in crude extracts, calcineurin is protected against inactivation by superoxide dismutase (23). The displacement of the autoinhibitory domain upon binding of Ca2+/calmodulin may expose the metal cofactors in the active site of the enzyme to the damaging effects of superoxide anion. The reversibility of calcineurin inactivation by ascorbate suggests that it is the result of the oxidation of Fe2+ at the active site. This protection of calcineurin activity by superoxide dismutase has also been observed in yeast cells and in hippocampal neurons after prolonged Ca2+ stimulation (23, 52). The modulation of calcineurin activity by the oxidation of iron provides a reversible mechanism to desensitize the enzyme and to couple Ca2+-dependent protein dephosphorylation to the redox state of the cells.

The clarification of the role of calcineurin B in the Ca2+ regulation of calcineurin has been more elusive. Calcineurin B is required for the reconstitution of an active enzyme from its subunits separated under denaturing conditions (53) or expressed in Escherichia coli and SF-9 cells (40, 42, 43, 54). The irreversible inactivation and dissociation of the two subunits accompanying the complete decalcification of calcineurin indicate that Ca2+ binding to the high affinity site of calcineurin B plays a structural rather than a regulatory role (50). Ca2+ binding to the low affinity sites is apparently not only responsible for the small calmodulin-independent activation but also for calmodulin activation (50). The mechanism of calcineurin B activation has not yet been elucidated. It is not clear why the catalytic domain of calcineurin, whose structure is similar to the catalytic subunit of other protein phosphatases, is inactive or why the calmodulin-independent form of calcineurin, which still binds calcineurin B but lacks the calmodulin-binding and autoinhibitory domain, has an affinity for Ca2+ 10 times greater than that of the native enzyme (50).

    Target Proteins

The crystal structure of the calcineurin-FKBP12-FK506 complex identified the interface between calcineurin B and calcineurin A as the binding site of the FK506-FKBP complex, providing an explanation for the requirement of both subunits for interaction first proposed on the basis of cross-linking experiments (55) and confirmed by affinity labeling and site-directed mutagenesis (38-40, 56-58). It also confirmed the identification of the calcineurin-binding domain of FK506 predicted on the basis of the structural and functional differences between FK506 and rapamycin and the preferential binding of the FKBP12 among FK506-binding proteins (49). Residues of FKBP12 and cyclophilin A involved in interaction with calcineurin are distinct from the isomerase catalytic and drug-binding sites in agreement with the lack of correlation between isomerase activity and calcineurin inhibition (59-62). The apparent competitive binding of two structurally different drug complexes, FKBP-FK506 and cyclophilin-CsA, to the same site on calcineurin remained a puzzle until the isolation of calcineurin A mutants that are resistant to either FK506 or CsA, indicating that the interaction sites are overlapping but not identical (57, 58).

The conservation from yeast to man of the drug-binding domains of calcineurin raises the possibility that these domains interact with natural ligands. Although no small endogenous analogs of FK506 and CsA have been detected, the disruption of the FKBP12-mediated anchoring of calcineurin to the ryanodine and IP3 receptors by FK506 suggests that these receptors may be such analogs (63, 64). The FKBP-mediated targeting of calcineurin to the receptors may ensure a rapid modulation of Ca2+ release from internal stores by protein dephosphorylation (65, 66). Another potentially important partner of calcineurin is AKAP79 (A-kinase anchoring protein), a member of the family of proteins whose function is to bring kinases or phosphatases close to their substrates (67). Like the IP3 and ryanodine receptors, AKAP79 requires neither FK506 nor Ca2+ to bind calcineurin. The tight, phosphorylation-independent binding of the transcription factor NF-AT to calcineurin may be another example of calcineurin targeting mediated by the substrate itself (20). It remains to be shown if the binding sites for these target proteins overlap with the drug-binding site.

    Calcineurin Functions

The complex regulation of calcineurin is expected from an enzyme, which has now been shown to be a major player in the regulation of cellular processes. None is better understood than the Ca2+-dependent, calcineurin-mediated regulation of transcription of the T cell growth factor, interleukin-2 (49, 68-70). The translocation of the transcription factor, NF-ATp, in response to an increase of intracellular Ca2+ induced by the occupancy of the T cell receptor, is dependent upon its dephosphorylation by calcineurin. It was the first example of the transduction of a signal at the plasma membrane to the nucleus (7, 19). A prolonged Ca2+ signal and the cotranslocation of calcineurin and NF-ATp to the nucleus ensure the sustained activation of gene expression, which is reversed by the glycogen synthase kinase, GSK3, following a decrease of intracellular Ca2+ (71-73). The involvement of calcineurin in the regulation of the expression of an array of growth factors, kappa 3, TNFalpha , NFkappa B, NF(P), and TGFbeta , is reviewed in Refs. 68-70 and 74.

As it does in T cells, calcineurin plays an important role in the regulation of gene expression in response to Ca2+ signals in yeast (70, 75-79). Two major sites of action of calcineurin in this organism are the pheromone response pathway (36) and the adaptation to high salt stress (75). The induction of genes involved in these two pathways has now been shown to be regulated differentially by the Ca2+-dependent and FK506-sensitive interaction of a single transcription factor (Tcn1p also named Crz1p) with calcineurin (76, 77). Other processes under calcineurin control include Ca2+ sequestration, cytokinesis, sporulation, and mating (78-80).

Identifying the sites of action of calcineurin in striatal and hippocampal neurons, which are particularly rich in calcineurin, continues to be a major challenge. The dephosphorylation of DARPP-32 by calcineurin in striatal neurons was the first evidence for a protein phosphatase cascade involving calcineurin responsible for the opposite effects of glutamate and cAMP on neuronal excitability (81). In hippocampal neurons activation of calcineurin not only results in inhibition of the release of the neurotransmitters, glutamate and gamma -aminobutyric acid (82, 83), but is also involved in the desensitization of the postsynaptic NMDA receptor-coupled Ca2+ channels (84). The complex regulation of the function of the NMDA receptors may be the basis for the proposed role of calcineurin in long term potentiation and depression and long term memory (85, 86). Calcineurin-mediated activation of nitric oxide synthase has also been invoked to explain glutamate neurotoxicity (87).

A role for calcineurin has also been proposed in apoptosis (88) and in the redistribution of integrins required for the migration of neutrophils on vibronectin in response to Ca2+ transients (89, 90). The inhibition of the calcineurin-mediated regulation of the Na+,K+-ATPase by the immunosuppressive drugs in the kidney (91) may be responsible for their nephrotoxicity, whereas in cerebellar neurons, the calcineurin activation of the Na+,K+-ATPase is required to prevent neurotoxicity because of excessive Na+ entry induced by glutamate binding to NMDA receptors (92). Regulation of two intracellular Ca2+ channels (the ryanodine receptor involved in excitation-contraction coupling in skeletal muscle and other excitable cells and the IP3 receptor involved in Ca2+ release by hormones and neurotransmitters) can potentially affect all cellular processes under Ca2+ control.

    ACKNOWLEDGEMENTS

We thank May Liu for expert editorial assistance and regret the omission of many relevant references because of space constraints.

    FOOTNOTES

* This minireview will be reprinted in the 1997 Minireview Compendium, which will be available in December, 1997. 

Dagger To whom correspondence should be addressed. Tel.: 301-496-3038; Fax: 301-402-3095; E-mail: ckl{at}helix.nih.gov.

1 The abbreviations used are: CsA, cyclosporin A; IP3, inositol trisphosphate; FKBP, FK506-binding protein; NMDA, N-methyl-D-aspartate.

2 The gene symbols are PPP3CAa, PPP3CAb, PPP3R1, and PPP3R2 for human calcineurin Aalpha , calcineurin Abeta , calcineurin B1, and calcineurin B2, respectively.

3 No changes in the structure of the recombinant calcineurin were detected upon complex formation with FKBP-FK506 (29).

4 H. Ren, X. Wang, and C. Klee, unpublished observations.

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