C ALCIUM DEPENDENCE

The phosphatase activity of calcineurin is dependent on both Ca²⁺ binding to calcineurin B and Ca²⁺-dependent binding of one molecule of calmodulin. Calcineurin B contains four characteristic EF-hand Ca²⁺-binding motifs [Aitken et al., 1984]. They have different affinities for calcium: a carboxy-terminal pair of sites with high (nanomolar) affinity, and an amino-terminal pair with (micromolar) affinity [Feng and Stemmer, 1999;

Gallagher et al., 2001]. The carboxy-terminal sites also exhibit very slow rates of calcium-exchange, retaining calcium constitutively and presumably playing a structural role [Feng and Stemmer, 1999]. In contrast, binding of calcium to low-affinity sites evokes conformational changes in both calcineurin subunits [Yang and Klee, 2000], modulates the enzyme affinity for calmodulin [Feng and Stemmer, 2001] and calcineurin activation by calmodulin [Stemmer and Klee, 1994; Feng and Stemmer, 2001]. Calmodulin binds calcineurin in the presence of calcium with very high affinity (Kd<10^-10M) [Hubbard and Klee, 1987]. Calcium-bound calmodulin increases calcineurin phosphatase activity 10-100 fold [Stewart et al., 1982;

Tallant and Cheung, 1984] by increasing Vmax of the enzyme. Calcium-dependence of calcineurin activation by calmodulin is highly cooperative (Hill coefficient 2.8-3) [Stemmer and Klee, 1994], reflecting binding of calcium to more than two sites on calmodulin. This

cooperativity allows a narrow threshold for calcium stimulation of calcineurin. Binding to calcineurin also greatly increases calmodulin affinity for calcium [Stemmer and Klee, 1994], and allows optimal stimulation by levels of calcium achieved in stimulated cells.

1.2.5. MODULATION OF CALCINEURIN ACTIVITY IN VITRO.

The phosphatase activity of calcineurin was initially assayed by measuring release of radioactive phosphate from ³²P-labeled protein substrates (α-subunit of phosphorylase kinase, myosin light chain, histone H1) [Stewart et al., 1983; King and Huang, 1983]. Calcineurin was later found to hydrolyze a range of low-molecular range substrates, including p-nitrophenylphosphate (pNPP) [Pallen and Wang, 1983; Li, 1984; Pallen et al., 1985]. The latter became the main substrate in studies of calcineurin catalytic mechanism. Subsequently, the serine-phosphorylated RII regulatory subunit of protein kinase A was shown to be a good calcineurin substrate [Blumenthal et al., 1986]. A 19-aminoacid part of RII, which contained phosphorylated residue and retained substrate properties of the entire protein, provided conventional substrate for determination of calcineurin serine/threonine phosphatase activity.

Use of ³²P-labeled RII peptide allowed for a reliable assay of calcineurin activity in cell and tissue extracts.

Apart from calcium, other divalent metal ions can modulate calcineurin phosphatase activity. The best activators at physiological pH values were shown to be Mn²⁺ and Ni²⁺

[King and Huang, 1983; Pallen and Wang, 1984], and Mg²⁺ at pH 8 [Li, 1984]. In initial work on calcineurin isolation Cohen and co-workers showed that Mn²⁺-dependence is acquired only after a calmodulin affinity chromatography step [Stewart et al., 1982]. Later, King and Huang [King and Huang, 1984] described rapid inactivation of calcineurin in the presence of calcium and calmodulin. Mn²⁺ and Ni²⁺ prevented and reversed this inactivation.

More detailed studies showed some difference in the activation mode between these two metals [Pallen and Wang 1984; Pallen and Wang 1986] and found up to two binding sites for each of the metal ions, one of which was competitive.

Phosphorylation affects the functional properties of many cellular proteins.

Calcineurin was shown to be phosphorylated in vitro by protein kinase C and calmodulin-dependent kinase II [Hashimoto and Soderling, 1989; Martensen et al., 1989] and casein kinase I [Singh and Wang, 1987]. The kinetic properties of phosphorylated and unphosphorylated forms are similar [Hashimoto and Soderling, 1989], and the relevance of calcineurin phosphorylation for its regulation in vivo is not clear.

Calcineurin B is N-terminally myristoylated [Aitken et al., 1982]. A mutant, in which myristoylation is abolished retains the major properties of wild-type enzyme, except for decreased thermal stability [Kennedy et al., 1996]. It has been shown recently that myristoylation is also required for calcium-dependent calcineurin binding to phosphatidylserine vesicles, indicating contribution of myristoylation to membrane-binding properties of calcineurin [Perrino and Martin, 2001].

Phospholipids could modulate calcineurin activity with both activation and inhibition reported for different lipid types and substrates [Huang et al., 1983; Politino and King, 1987].

In addition, arachidonic acid and other unsaturated fatty acids activated recombinant calcineurin from Dictyostelium discoideum with no effect on bovine enzyme [Kessen et al., 1999]. Since part of calcineurin in vivo is localized to cell membranes this kind of modulation is probably of physiological significance.

Some natural compounds exhibit potent inhibition of calcineurin activity. The most potent inhibitors are the immunosuppressive drugs CsA and FK506 [Liu et al., 1991a;

Swanson et al., 1992]. They are effective only when bound to their respective binding proteins, cyclophilin and FKBP, and do not inhibit activity towards low molecular weight substrates like pNPP. Okadaic acid, a potent inhibitor of related phosphatases PP1 and PP2A, can also inhibit calcineurin with IC50~4 µM compared to 300 nM for PP1 and 1 nM for PP2A [Bialojan and Takai, 1988]. Pyrethroid insecticides (cypermethrin, deltamethrin) potently inhibited calcineurin activity towards pNPP [Enan and Matsumura, 1992], but later proved ineffective towards RII and phosphoproteins [Enz and Pombo-Villar, 1997; Fakata et al., 1998]. A 25-residue peptide corresponding to the sequence of calcineurin autoinhibitory domain (residues 457-481) also potently inhibits the enzyme activity [Hashimoto et al., 1990].

Similar to other phosphatases, calcineurin is inhibited by orthovanadate [Morioka et al., 1998], phosphate and pyrophosphate [King and Huang, 1984] and fluoride [Tallant and Cheung, 1984]. In addition, calmodulin antagonists such as trifluoroperazine [Stewart et al., 1983], W-7 and calmidazolium [Mukai et al., 1991] prevent calcineurin activation by calmodulin and thus serve as calcineurin inhibitors. Recently, calcineurin activity towards both RII and pNPP has been found to be inhibited by the polyphenolic aldehyde gossypol and its analogues with effective concentrations in the micromolar range [Baumgrass et al., 2001].

1.2.6. CALCINEURIN X-RAY STRUCTURE.

Based on homology of serine/threonine phosphatases to purple acid phosphatases, enzymes with well-characterized catalytic binuclear metal center, it was predicted that serine/threoninie phosphatases might also contain this center at their active site [Vincent and Averill, 1990]. Later, sequence alignments of serine/threonine phosphatases identified a

“phosphodiesterase motif” conserved in calcineurin, PP1, PP2A and in other enzymes cleaving phosphoester bonds such as bacterial exonucleases, alkaline and acid phosphatase, λ-phosphatase, phosphodiesterase and 5´-nucleotidase [Koonin, 1994; Lohse et al., 1995] (Fig.

1.2).

Figure 1.2. Phosphodiesterase motif in calcineurin and purple acid phosphatases.

Residues identified as metal ligands are shown in bold, and conserved non-ligand residues of the active site are underlined.

The phosphodiesterase motif presumably provides a scaffold for the binuclear metal center in each member of the phosphodiesterase family, and the existence of such a center was proved for λ-phosphatase [Rusnak et al., 1999b] and 5´-nucleotidase [Knöfel and Ströter, 1999]. The conservancy of this motif suggests a common catalytic mechanism for the enzymes involved in phosphotransfer reactions.

The list of metallophosphatases whose X-ray structures have been solved up to date includes not only calcineurin [Griffith et al., 1995; Kissinger et al., 1995], but also PP1 [Egloff et al., 1995; Goldberg et al., 1995], kidney bean purple acid phosphatase [Strater et al., 1995], mammalian purple acid phosphatase [Guddat et al., 1999; Lindquist et al., 1999;

Uppenberg et al., 1999] and λ-phosphatase [Voegtli et al., 2000]. In these structures the phosphodiesterase motif forms a α-α-β scaffold for a binuclear metal center. The three β-strands form a parallel pleated sheet capped by intervening α-helices. Two metal ions are

Phosphoesterase Consensus: DXH (X)n GDXXDR (X)m GNHD/E

Calcineurin and PP1: DIH (X)23 GDYVDR (X)27 GNHE

Purple Acid Phosphatase: DXG (X)n GDXXYD (X)m GNHD/E

situated at the apex of this fold with a distance between them 3-4 Å. The residues in loops between β-strands and α-helices provide metal ligands.

The crystal structure of recombinant human calcineurin [Kissinger et al., 1995] is shown on Fig. 1.3. The calcineurin B-binding domain forms an amphipatic α-helix, whose top nonpolar face contacts calcineurin B subunit. The autoinhibitory domain folds into an α-helix that occupies the substrate-binding cleft. The calmodulin-binding domain is disordered and not visible on the electron density map, consistent with the sensitivity of this region to proteolytic degradation. Therefore, the structural details of calmodulin regulation remain obscure.

Figure 1.3. Crystal structure of recombinant human calcineurin. Diagram showing the three-dimensional structure of calcineurin using the X-ray coordinates of Kissinger et al.

([Kissinger et al., 1995], protein data bank file 1AUI). The figure was created using public domain software Cn3D (http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml).

Calcineurin A catalytic domain is shown in cyan, calcineurin B-binding domain and part of the autoinhibitory domain are in gray, and N- and C-terminal lobes of calcineurin B are in dark blue and green, respectively. Spheres at the catalytic domain represent iron and zinc, and those at B subunit bound calcium ions.

Calcineurin B contacts calcineurin A by a groove formed by its carboxy- and amino-terminal lobes and its C-terminal strand. In addition to amphipatic α-helix residues 14-23 of

calcineurin A provide a contact surface with carboxy-terminal lobe of calcineurin B. In the other structure [Griffith et al., 1995] the myristoyl group of calcineurin B lies in the hydrophobic cleft between amphipatic α-helices in the N-terminal part of calcineurin B and forms multiple hydrophobic contacts with the protein.

The active site of calcineurin is formed around a binuclear metal center (Fig. 1.4). The presence of Fe and Zn in the center has been verified by atomic absorption spectrometry [King and Huang, 1984; Yu et al., 1995]. A similar metal coordination and environment was found in PP1 [Goldberg et al., 1995], but the nature of metals in its active site remains unclear. The positions of Fe and Zn were attributed according to the positions of Fe and Zn in kidney bean purple acid phosphatase, where a tyrosine residue coordinates Fe³⁺ at the M1 site (left on the Fig. 1.4) and provides a characteristic charge transfer band at 510-550 nm in the enzyme optical spectrum [Strater et al., 1995]. In calcineurin this tyrosinate is replaced by a histidine, and the enzyme is colorless.

O

Fig. 1. 4. Schematic view of the active site of human calcineurin [Kissinger et al., 1995].

In addition to a histidine (His92 in human calcineurin) the M1 iron is coordinated by two aspartates, Asp90 and Asp118. The latter is a bridging ligand also coordinating the M2 zinc atom. This atom is additionally coordinated by side chains of Asn150, His199 and His281. Apart from protein aminoacid residues solvent molecules coordinate the metals of the active center, one of them in a µ-bridging mode. His151 and Asp121, although not binding metals directly, participate in catalysis as discussed below.

1.2.7. CALCINEURIN CATALYTIC MECHANISM.

Two major mechanisms of phosphate ester hydrolysis are discussed in literature: one involving formation of the phosphoenzyme intermediate and the other involving direct attack of the solvent nucleophile on the ester bond (Fig.1.5). The former occurs in catalysis by alkaline phosphatase, where an active site serine accepts a phosphoryl group during enzymatic turnover [Coleman, 1992]. Another example is provided by tyrosine phosphatases, where an active site cysteine plays the analogous role [Zhang 1998].

Kinetic experiments on calcineurin gave evidence supporting direct transfer of the phosphoryl group to water without formation of the phosphoenzyme intermediate. First, a direct relationship

Fig.1.5. Mechanisms of phosphate ester hydrolysis.

between log (V/K) and the pKa of the calcineurin substrates was observed [Martin et al., 1985] with increase of the rate following decrease of the leaving group pKa. Second, there was no phosphotransferase activity towards alternative nucleophiles [Martin et al., 1985].

Both phosphate and phenol were competitive inhibitors of p-NPP hydrolysis by calcineurin, consistent with a random uni-bi mechanism (in contrast to an ordered uni-bi mechanism for phosphoenzyme intermediates) [Martin and Graves, 1986]. Most convincing evidence for the direct phosphoryl group transfer to water came from the work on spleen purple acid phosphatase [Mueller et al., 1993]. Using a chiral [¹⁸O, ¹⁷O]phosphorothiolate ester the authors showed that catalytic hydrolysis of the ester bond occurred with a net inversion of configuration at the phosphorus atom, directly indicating the phosphoryl transfer to water without formation of the phosphoenzyme intermediate. Since the active site of PAPs is similar

to calcineurin, it is possible to presume that catalysis by calcineurin follows the same mechanism.

The role of metal ions in the catalytic mechanism of calcineurin is still not clear. Metal ions could serve as Lewis acids in activating a solvent nucleophile like in carbonic anhydrase [Hakansson et al., 1992]. They could neutralize the negative charge on the phosphate oxygen atoms making the phosphorus atom more suitable for nucleophilic attack, and also stabilize the developing charge on the leaving group during the ester bond cleavage. Additionally, metals could help orient the substrate for in-line attack of the nucleophile.

Several conserved aminoacid residues are situated within 4-9 Å of the binuclear metal center of calcineurin. The contribution of these residues to calcineurin catalysis was investigated by site-directed mutagenesis. These studies highlighted the importance of the conserved histidine residue (His151) (Fig.1.4) in a couple with an equally conserved aspartate (Asp121). These residues form part of a phosphodiesterase motif shown in Fig. 1.2 and are present in all metallophosphatases. Mutation of His151 causes ~10³-fold reduction of rate constant (kcat) [Mertz et al., 1997], and a similar decrease occurred after mutation of Asp121 analogue in PP1 [Zhang et al., 1996; Huang et al., 1997] without greatly affecting KM in both cases. His151 could assist in substrate binding or orient the nucleophilic solvent molecule, as well as participate in acid/base catalysis. Similar differences in kcat for wild-type and His151 mutant enzyme with substrates having very different pKa of their leaving groups [Mertz et al., 1997] argue against a role of that residue as acid catalyst. On the other hand, this histidine could serve as a general base deprotonating nucleophilic water molecule. With Asp121 it might form an Asp-His-HO triad similar to the Asp-His-Ser triad of serine proteases. Apart from His-Asp pair two conserved arginines, Arg122 and Arg254, are in the vicinity of the calcineurin active site. Mutation of these arginines caused a 10²-10³-fold reduction of kcat and a 2-10-fold KM increase for Arg254 [Mondragon et al., 1997]. These residues contact oxygen atoms of phosphate and may provide stabilization for binding negatively charged phosphoester and also neutralize developing charge in the transition state. In purple acid phosphatases these arginines are substituted by histidines, probably explaining the lower pH optimum of PAPs [Klabunde et al., 1996].

The model for the phosphate ester hydrolysis by calcineurin proposed by Rusnak and Mertz [Rusnak and Mertz, 2000] is presented in Fig. 1.6. According to this model upon substrate binding the negative charge on phosphate is neutralized by Arg122 and Arg254 together with Zn²⁺, while His151 removes the proton from the metal-bound water and orients the solvent nucleophile for optimal attack on the phosphate ester (Fig. 1.6.A). A dissociative

transition state then forms (Fig. 1.6.B), where bond cleavage to the leaving group occurs before bond formation to the solvent nucleophile. The negative charge on the leaving group is neutralized by His151 and Zn²⁺. After bond cleavage and proton transfer to the leaving group the result is the product-inhibited state with phosphate bridging two metals as was shown for the X-ray structure of calcineurin [Griffith et al., 1995] (Fig. 1.6.C). Exchange of the phosphate for solvent molecule restores the active enzyme form.

Fig.1.6. Mechanism of calcineurin catalysis. In this model His151 acts as a general base and a general acid. A. Substrate binds to the active site and His151 assists in deprotonating water bound to iron with hydroxide formation. B. The transition state is dissociative, zinc helps to neutralize charge on the leaving group, which is protonated by His151. C. The product-inhibited state with phosphate bridging two metal ions. D. A water molecule displaces the phosphate, the enzyme is ready for the next catalytic turn.

1.2.8. PHYSIOLOGICAL FUNCTIONS. ROLE OF CALCINEURIN IN YEAST.

Saccharomyces cerevisiae provides an important model system for studying calcineurin function, especially by using genetic manipulation of its expression.

Saccharomyces cerevisiae have two isoforms of calcineurin catalytic subunit (CNA1/CMP1 and CNA2/CMP2) and one gene encoding regulatory subunit (CNB1) [Cyert et al., 1991;

Kuno et al., 1991; Liu et al., 1991b]. Strains deficient in both isoforms of calcineurin A or in calcineurin B failed to recover from α-factor-induced growth arrest [Cyert et al., 1991; Cyert and Thorner, 1992] and were hypersensitive to salt stress (induced by Na⁺ and Li⁺) [Mendoza et al., 1994] as well as to Mn²⁺ [Farcasanu et al., 1995]. Similar phenotypic responses were observed after treatment of the cells with the immunosuppressants FK506 and CsA [Foor et al., 1992; Nakamura et al., 1993]. Calcineurin was shown to influence the expression of several yeast genes, including vacuolar and secretory Ca²⁺-pumps (Pmc1p and Pmr1p), β-1,3 glucan synthase (Fks2p) and a plasma membrane Na⁺ pump (Pmr2p) [Mendoza et al., 1994;

Matheos et al., 1997; Zhao et al., 1998]. The latter is involved in salt stress resistance [Mendoza et al., 1994], while Pmr1p is linked to Mn²⁺-homeostasis [Matheos et al., 1997], and Fks2p is the putative calcineurin-responsive element in the response to α-factor [Zhao et al., 1998]. The transcription factor Crz1p/Tcn1p was identified as a mediator of calcineurin-dependent gene expression [Matheos et al., 1997; Stathopolous and Cyert, 1997]. Mechanism of Crz1p/Tcn1p activation involves calcineurin-mediated dephosphorylation of the Crz1p/Tcn1p region having similarity to the mammalian NFAT transcription factor family (reviewed later in the text) followed by the nuclear translocation mediated by the nuclear import protein Nmd5p [Stathopoulos-Gerontides et al., 1999; Polizotto and Cyert, 2001].

Apart from the Crz1p/Tcn1p vacuolar H⁺/Ca²⁺ exchanger Vcx1p [Cunningham and Fink, 1996] as well as Hsl1, a kinase participating in yeast cell cycle regulation [Mizunuma et al., 2001] appeared to be directly modified by calcineurin.

Although biochemical properties of the yeast calcineurin seem to be identical to those of mammalian calcineurin, the later mutagenesis work identified a new region essential for the activity of yeast calcineurin [Jiang and Cyert, 1999]. This region is situated between catalytic and calcineurin B-binding domains, and mutation of several residues within this domain (S373P, H375L, and L379S) dramatically decreased the enzyme phosphatase activity.

It is not clear whether the same mutations impair the activity of mammalian calcineurin.

In Schizosaccharomyces pombe calcineurin serves functions distinct of those in budding yeast. Calcineurin mutants are defect in cytokinesis, polarity, mating and spindle body positioning [Yoshida et al., 1994] and are Cl^--hypersensitive [Sugiura et al., 1998].

Among other lower eukaryotes, calcineurin is essential for growth of Aspergillus nidulans and Neurospora crassa [Higuchi et al., 1991; Rasmussen et al., 1994].

1.2.9. CALCINEURIN IN T-CELL ACTIVATION.

The role of calcineurin in T-cell signaling was thoroughly investigated during the last 10 years and is discussed in several specialized reviews [Rao et al., 1997; Crabtree, 1999;

Crabtree, 2001; Macian et al., 2001]. T-cell activation cascade is initiated upon the binding of antigen-MHC complexes on the surface of the antigen-presenting cell to the T-cell receptor (TCR) on the reactive T-lymphocyte, and results in the complex response marked by the up-regulation of expression levels of a plentitude of lymphocyte genes, including IL-2. IL-2 then activates lymphocyte proliferation. The involvement of calcineurin in T-cell activation was revealed by the discovery of its inhibition by immunosuppressants CsA and FK506 [Liu et al., 1991]. Another critical component of the activation cascade was identified as transcription factor NFAT (nuclear factor of activated T-cells) [Shaw et al., 1988; Flanagan et al., 1991;

Clipstone and Crabtree, 1992]. NFAT comprises a family of 5 proteins (NFAT1-5), four of which are regulated in a calcineurin-dependent manner [Macian et al., 2001]. Two of them, NFAT1 and NFAT2, are critical for the T-lymphocyte cytokine production [Peng et al., 2001], with NFAT4 also contributing to T-cell function [Oukka et al., 1998]. The activation of the calcineurin-NFAT pathway begins with a receptor-initiated release of calcium from intracellular stores followed by calcium influx through store-operated channels (capacitative calcium entry). Patients having a defect of capacitative calcium entry suffer from severe immunodeficiency associated with a failure of NFAT activation [Feske et al., 2000]. Low sustained increases of intracellular calcium preferably activate NFAT, while shorter calcium spikes could activate other signaling components (e.g. NFκB) without affecting NFAT [Dolmetsch et al., 1997]. These calcium signals cause optimal activation of the calcineurin phosphatase activity. Calcineurin is constitutively bound to NFAT through the PxIxIT consensus motif in the regulatory domain [Aramburu et al., 1998], and peptides modeled on this site are potent inhibitors of NFAT activation by calcineurin [Aramburu et al., 1998;

Aramburu et al., 1999]. Some other parts of NFAT could also contribute to the binding [Liu et al., 1999; Park et al., 2000]. Calcineurin activation results in dephosphorylation of multiple serine residues in the NFAT regulatory domain [Ruff and Leach, 1995; Park et al., 1995].

Detailed analysis of NFAT1 [Okamura et al., 2000] identified 14 phosphoserine in a resting state, 13 of which are dephosphorylated upon stimulation. In NFAT1 first removal of five phosphates from a serine-rich sequence adjacent to PxIxIT motif exposes nuclear localization signal (NLS) in the NFAT regulatory domain and renders other eight phosphoserines more accessible to calcineurin. Dephosphorylation causes NFAT translocation into the nucleus

[Shaw et al., 1995; Shibasaki et al., 1996; Timmerman et al., 1996]. This translocation is mediated by NFAT NLS exposed after dephosphorylation as well as its nuclear export signal (NES), which is masked upon dephosphorylation [Beals et al., 1997a; Okamura et al., 2000].

Apart from its phosphatase activity, calcineurin possibly regulates nuclear export of NFAT by competing with the nuclear export factor Crm1 for a common binding site [Zhu and McKeon, 1999]. The action of calcineurin is opposed by several nuclear kinases, particularly by GSK3 [Beals et al., 1997b], casein kinase I [Zhu et al., 1998] and JNK [Chow et al., 1997].

Calcineurin-mediated dephosphorylation affects not only nuclear localization of

Calcineurin-mediated dephosphorylation affects not only nuclear localization of

Im Dokument Redox regulation of protein serine/threonine phosphatase calcineurin (Seite 21-0)